Cancer Research: Intravenous Capsol-T

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Cancer Research: Tumor Treating Fields

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STEP TWO

Schedule consultation with R. Douglas Wichman, MD.

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Submit "New Patient Form", current labwork, & imaging results prior to consultation.

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*** SPECIAL NOTE FROM DR. WICHMAN ***

The following excellent article was reproduced from Neuro-Oncology at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035531/:

Neuro Oncol. 2016 Oct; 18(10): 1338–1349.

Published online 2016 Sep 23. doi: 10.1093/neuonc/now182

PMCID: PMC5035531

Tumor treating fields: a novel treatment modality and its use in brain tumors

Andreas F. Hottinger, corresponding author

Patricia Pacheco, and Roger Stupp corresponding author

Author information ► Article notes ► Copyright and License information ►

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Preclinical Data and Mechanism of Action

Living cells consist of charged or polar molecules and ions and thus are responsive to electrical fields and currents. Electric activity of cells plays a key role in many essential biological processes including cell division. Cellular processes can be influenced by electric fields. The overall effect will depend upon the magnitude of the potential difference between the 2 electrodes (field intensity) and the frequency: at very low frequencies (<1kHz) excitable cells such as neurons or myocytes will be depolarized.1 Cardiac pacemakers or deep brain stimulators work in this range of frequency. At very high frequencies (>MHz), heat is generated in the tissues due to dielectric loss. This property is mainly used for radiofrequency ablation or diathermy treatments.2 The alternating frequency of electrical fields at intermediate frequencies (range, 10–1000kHz) is too fast to induce cell depolarization and induces no or only minimal heat by dielectric loss. In the past, these frequencies were considered to have no interaction with biological processes.3 Nevertheless, a number of effects have been observed in biological tissues such as microscopic particle alignment,4 cell rotation,5 and transient pore formation in cell membranes.6 At low intensities (2V/cm) and intermediate alternating frequencies (between 100–300kHz), Kirson and Palti et al. demonstrated a specific inhibiting effect on cell division in cancer cell culture models.7

Tumor treating fields (TTFields) are alternating, low-intensity, intermediate frequency electric fields that aim to disrupt cell division and inhibit tumor growth. In initial experiments, exposure of a variety of tumor cell lines to TTFields was shown to exert a profound growth inhibitory effect by inducing cell cycle arrest and apoptosis, while no effect was induced on non-dividing cells.7 These in vitro observations could also be confirmed in vivo in mice and rabbit tumor models.8 Further studies demonstrated that the growth inhibitory effect is largely mediated by interference on the mitotic spindle apparatus. TTFields will target proteins with large dipole moments (ie, septins and the spindle microtubules, components essential in the metaphase and anaphase stages of the mitotic cycle for separation and equal distribution of chromosomes).9,10 Furthermore, inhibition of the polymerization of microtubules interferes with proper assembly of the mitotic spindle apparatus. In telophase, during cytokinesis the hourglass shape taken by the daughter cells that are about to separate induces a nonuniform electric field that is strongly enhanced at the level of the furrow region (→ fig. 1). This results in dielectrophoretic forces that may attract charged molecules from the cytosol and compromise normal cytokinesis.7 The antitumoral effect results from disruption of the microtubular assembly during mitosis, blocking formation of the mitotic spindle apparatus and blocking separation of the 2 daughter cells.9 This effect also results in abnormal chromosomal segregation and reduced clonogenic potential of the cell’s progeny.10

Fig. 1.

Mechanisms of action of tumor treating fields in and around quiescent and dividing cells. Inside quiescent cells (1A), the field is uniform, and the oscillating electric forces result only in ‘‘vibration’’ of ions and dipoles ...

Animal models of various tumors, including glioblastoma (GBM), non–small cell lung cancer, pancreatic cancer, and malignant melanoma confirmed the inhibition of tumor growth or metastatic seeding when externally applied TTFields were delivered at the appropriate frequencies.11 As an example, an experimental model of rats with intracranially inoculated GBM cells treated with TTFields at a frequency of 200kHz over 6 days showed smaller tumors compared with untreated rats.12 The inhibitory effect was significantly increased when 2 or more, rather than 1 field directions were used.12 Importantly, synergistic antitumor activity was demonstrated when TTFields were applied in conjunction with cytotoxic chemotherapy with paclitaxel, doxorubicin, cyclophosphamide, or dacarbazine (DTIC).13

In summary, TTFields will block the mitotic cell cycle, in particular during metaphase, anaphase, and telophase. This will result in cell cycle arrest or delay in cell division and interfere with organelle assembly, particularly the spindle apparatus (fig. 1 C). The consequences are inadequate cell division and unequal chromosome distribution. Ultimately, cells will die in apoptosis. In order to have an optimal treatment effect, the field intensity and frequency needs to be adapted to the tumor type and cell properties (eg, cell size). The optimal frequency to maximize the antitumor effect is inversely correlated with cell size and when the incident angle of the electrical field is perpendicular to the mitotic plate.7

As the cell division may occur at any time, prolonged exposure to the electrical fields is required for maximal effect. For the delivery of TTFields, a portable and battery-powered device has been developed (→ fig. 2). The electric field is applied to the brain through 4 transducer arrays with 9 insulated electrodes each and continuous temperature sensing fixed to the patient’s shaved scalp.

Fig. 2.

Tumor treating fields (TTFields) device (2nd generation Optune) and its clinical use TTFields are administered by 4 transducer arrays placed on the shaved scalp and connected to a portable device generating 200kHz electric fields within the brain. The ...

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Clinical Experience:

Glioblastoma as a Proof of Concept Model

In the very first clinical application, TTFields treatment was applied to patients with cutaneous metastases of melanoma or breast cancer. Tumor shrinkage or even complete disappearance was demonstrated.12,14However, metastatic cancer is a systemic disease, thus most effects of application of TTFields would be expected to be seen in diseases or situations where primarily locoregional control is warranted. Primary brain tumors—and notably glioma—rarely metastasize; recurrence in the brain is the predominant cause of treatment failure and was chosen as the first disease in which the effect of TTFields could be investigated prospectively.

In a computational model, the optimal frequency of TTFields for GBM was found to be in the range of 200kHz. The field strength should be ≥ 1V/cm. These TTFields are able to penetrate into the deep brain tissue from the surface of the scalp. The computational model also revealed inhomogeneous fields with intensification of the field strength near the ventricles as a result of the high conductivity of the cerebrospinal fluid. Necrotic areas and edematous regions also showed high conductivity of the TTFields. In contrast, areas with high cellularity showed lower conductivity.15

Following the demonstration of feasibility in a small pilot trial, the clinical merits of this innovative cancer treatment were evaluated in 2 pivotal randomized trials in recurrent (EF-11) and newly diagnosed (EF-14) GBM. In our summary, herein we always refer to the results of the intent-to-treat population (ITT); for details on the predefined per protocol populations, the reader is kindly directed to the respective original publications16,17 (→ table 1).

Table 1.

Results of EF-14 & EF-11 trials

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TTFields in Recurrent Glioblastoma

The EF-11 Trial

For this trial, GBM patients with progressive or recurrent disease after initial treatment with radiotherapy and TMZ chemotherapy were eligible. Patients may have received several lines of prior chemotherapy. A total of 237 patients were then randomized 1:1 to either the novel TTFields therapy (120 patients) or to the best available treatment (117 patients) according to the local oncologist’s best choice (active control, fig. 3A). The primary endpoint of the trial was OS. Patients’ median age was 54 years, with a median KPS of 80% (range, 50–100%). Eighty-eight percent of patients had received 2 or more lines of prior chemotherapy including prior bevacizumab in 18% of patients. With a median survival of only 6.6 and 6.0 months in the TTFields and the control arm, respectively (hazard ratio: 0.86 [95% CI, 0.66–1.12), P=0.27) and a 1-year survival rate of only 20% in both groups, the trial failed to demonstrate superiority over “established” or commonly used chemotherapy regimens. The median duration of TTFields administration was only 2.3 months (95% CI, 2.1– 2.4) with tumor progression as the primary reason for discontinuation of treatment. Still it showed objective responses with TTFields alone in 14% of patients compared with 9.6% in the control arm. The study demonstrated safety and feasibility of TTFields in a large multicenter setting.

Fig. 3.

Design of the pivotal trials in GBM. A: EF-11 Trial Design B: EF-14 Trial Design

The design and conduct of the EF-11 trial had some inherent limitations: numerous prior treatment lines were allowed and led to a heterogeneous patient population. More than 40% of patients were included after the third recurrence, including many patients who had failed prior bevacizumab therapy. Thus, most patients suffered from very advanced disease and had only few available treatment options and limited life expectancy. In the absence of a commonly accepted standard treatment for recurrent GBM, the control patients were to receive the best available systemic therapy according to the current local practice. The heterogeneity of treatments prescribed to the control patients reflects this fact: about one-third of the control patients received bevacizumab alone or in combination, 25% nitrosoureas, and 11% re-exposure to TMZ. A number of patients were discontinued from TTFields therapy very early (after <1 month of therapy), presumably due to absence of a treatment response (rather than true tumor progression) based on skepticism of the investigators. It is known that TTFields take a prolonged period of time until its effects can be clinically demonstrated or that delayed responses may occur after initial radiological tumor growth; thus, some patients may have discontinued TTFields treatment prematurely.

Based on the results of the EF-11 trial, the US Food and Drug Administration (FDA) felt that these results provided reasonable assurance that the benefits of TTFields outweigh its risks when administered as monotherapy in place of standard medical therapy and therefore approved TTFields in 2011 (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm251669.htm).

TTFields in Daily Practice: PriDE Dataset

After FDA approval, TTFields became commercially available in the United States, and Mrugala et al summarized the daily practice experience on 457 patients treated at 91 US institutions (Patient Registry Dataset; PRiDe).18 Interestingly, patients’ age and performance status were similar in this dataset and in the EF-11 trial; however, treatment with TTFields was initiated much earlier in the course of the disease. One-third of patients were treated at first recurrence (compared with 9% in the EF11 trial). Overall, median survival was 9.6 months, and the subgroup of patients who were treated at first recurrence (n=152) had a median survival of 20 months.18

TTFields in Newly Diagnosed GBM (EF-14 Trial)

This open label phase 3 study in patients with newly diagnosed GBM was initiated while the trial for recurrent disease was still ongoing. For practical reasons (interference of the electrodes during radiotherapy), patients had to be randomized only after completion of standard concomitant TMZ chemoradiotherapy to standard maintenance TMZ chemotherapy (for 6–12 cycles) with or without concomitant administration of TTFields (fig. 3B). The primary endpoint was PFS, and OS was a powered secondary endpoint. Eligible adult patients (KPS ≥ 70%, supratentorial tumor location) had to be progression-free after the end of chemoradiotherapy, thus excluding the patients with the worst prognosis. After stratification by extent of resection (biopsy, partial resection, gross total resection, determined on MRI 24–48 hours postsurgery) and MGMT status (methylated, unmethylated, unknown) patients were randomized at a ratio of 2:1 within 3–7 weeks from the last day of radiation. Patients assigned for TTFields therapy received additional instruction and technical support for the use of the device by a device specialist (technician) during the first weeks of treatment and thereafter with monthly visits. This support was limited to technical aspects of the device and assistance with the application of arrays.

A total of 695 patients from 83 centers across the world were included between July 2009 and November 2014. More than half of the patients came from the United States. The medical follow-up was similar in both treatment arms. It included monthly clinic visits for complete physical examination and blood hematology and chemistry analyses. A mini-mental status examination (MMSE), quality of life evaluation (EORTC QLQ-C30 questionnaire and the brain-specific module BN-20) were performed at baseline and every 3 months thereafter.19,20 MRI and disease assessment using the Macdonald criteria were to be performed every 2 months. All treatment-related clinical decisions were based on local interpretation of imaging; however, a blinded central imaging and disease assessment review determined the date of progression. Patients experiencing tumor progression were offered second-line treatment according to local practices. Patients were allowed to continue TTFields treatment beyond first progression based on the prior experience of pseudoprogression and delayed responses. For the purpose of the trial, the date of first progression, as assessed by the independent review panel, was considered the primary endpoint.

The baseline patient characteristics were well balanced between the 2 groups. In both groups, 66% were male, and the median age at inclusion was 57 years (range, 20–83y), and the median KPS was 90%. Sixty-four percent of patients underwent gross total resection, and 11% had biopsy only. Central MGMT gene promoter methylation analysis was available for 72% of patients. MGMT was methylated in 39% of patients in the TTFields/TMZ group and in 41% of the control TMZ group. The median time from end of radiation therapy to randomization was 36 and 38 days in the TTFields and control groups, respectively. The median time from randomization to initiation of TTFields was 5 days. Median time from diagnosis of GBM to randomization was 3.8 months in both groups (ranges, 2.0–5.7 and 1.4–5.7 months in the treatment and control groups, respectively). Of note, 53% of patients were randomized after initiation of the first cycle of TMZ (as allowed by the protocol). Per protocol, TTFields treatment was continued up to the second progression in two-thirds of the patients; the median duration of treatment with TTFields was 9 months (range, 1–58 mo). Three-quarters of patients receiving treatment with TTFields were adherent to therapy as prescribed (ie, wearing the device ≥ 18 hours per day on average during the first 3 treatment months) (n=157/210).

At a prespecified interim and futility analysis to be performed once the first 315 randomized patients reached a minimum follow-up of 18 months, a significant improvement in progression-free and overall survival was seen; consequently the independent data monitoring committee recommended that the trial be terminated early for success and that patients be allowed to cross over to the TTFields arm. In the interim analysis, the ITT median PFS was increased by 3.1 months in the TTFields group with a median PFS of 7.1 months (95% CI, 5.9–8.2 mo) compared with 4.0 months (95% CI, 3.3–5.2 mo) in the control group (HR, 0.62 [98.7% CI, 0.43–0.89]; P=.001). As a direct consequence, patients in the control group received a median of 4 cycles of TMZ (range, 1–24), whereas patients in the TTFields group received a median of 6 cycles (range,1–26) of TMZ. Median OS from randomization (ITT) was 19.6 months (95% CI, 16.6–24.4 mo) in the TTFields plus TMZ group compared with 16.6 months (95% CI, 13.6–19.2 mo) in the TMZ control group (HR: 0.74 [95% CI, 0.56–0.98]; P =.03). The percentage of patients alive at 2 years following enrollment was 43% in the TTFields/TMZ group and 29% in the TMZ alone group (P=.006)17 (→ fig. 4). At first progression, 67% of the patients in the TTFields/TMZ group received a second-line therapy compared with 57% of patients in the TMZ control group. The type of salvage chemotherapy offered was balanced between the 2 groups: about 40% of second-line therapies included bevacizumab and about 40% nitrosoureas.

Fig. 4.

Progression-free and overall survival in EF-11 (A&B) & EF-14 (C&D) trials. EF-11 trial: Progression-free survival (A) and overall survival (B) of the intent-to-treat population. Hazard ratio for overall survival: 0.86 (CI, 0.66–1.12, ...

Preliminary subgroup analyses showed that the positive effect observed on PFS and OS by the addition of TTFields was not restricted to any subgroup of patients: in particular neither age, performance status, MGMT methylation status nor extent of resection was predictive for a better treatment effect. However, the sample size of the interim dataset may not be large enough to identify meaningful subgroups, and detailed subgroup analyses are to be performed on the final and validated dataset. Due to the 2:1 randomization, the control arm comprised only 105 patients, which limited the ability to perform formal subgroup analyses. In the final dataset, the control group will comprise 229 patients. Before publication of the interim analysis, the overall dataset of 695 randomized patients was statistically scrutinized. It was concluded that the results are unlikely to change substantially once the whole dataset reaches a mature follow-up (see supplemental material, reference17). In October 2015, the FDA approved TTFields for use in newly diagnosed GBM patients.

Toxicity, Support, and Quality of Life with TTFields

Toxicity related to TTFields therapy consisted, by the nature of this treatment, mainly of local skin irritation. This is usually mild, self-limiting, easily manageable with local application of steroid-containing ointments, and may require an occasional treatment break for a few days. In the EF-11 trial, skin toxicity was reported in 16% of patients (grade 3 in only 2%). In the EF-14 trial for newly diagnosed GBM patients, where the treatment exposure was longer than that for recurrent disease, grades 1 and 2 skin reactions were reported in 43% of patients. Severe (grade 3) reactions were again seen in only 2% of patients. Examples of allergic contact dermatitis, irritant contact dermatitis, folliculitis, and erosions are shown in → fig. 5.21

Fig. 5.

Skin toxicities observed under tumor treating fields (TTFields). Some mild-moderate (grade 1–2) skin reaction is observed in up to half of patients (in EF-14 trial reported in 43%, grade 3 in 2%); however, it is usually self-limiting and resolves ...

Importantly, when compared with TMZ maintenance treatment alone, the addition of TTFields did not result in any modification of the overall incidence, severity, and distribution of side effects in patients with newly diagnosed GBM. The incidence of seizures was identical in both treatment groups (7% in the TTFields group vs 8% in the control group). Some nonspecific adverse effects including nervous system disorders such as grade 1–2 headache (21% in the experimental group vs 14% in patients with TMZ alone), mild anxiety, confusion, insomnia, and headaches were reported more frequently in the patients treated with TTFields plus TMZ and occurred mainly at the time of therapy initiation.

Given the need for continuous and long-term use of the TTFields device, the quality of life of patients has been a concern. In the EF-11 trial, there were no differences in global health and social functioning between patients treated with TTFields or chemotherapy. In fact, cognitive and emotional functioning was higher in the TTFields group than in the chemotherapy group.16 In the EF-14 trial, preliminary quality of life data showed identical scores at baseline and at 12 months for patients in the treatment and control groups at the levels of cognitive, emotional, physical, and social functioning.22 Moreover, the global health status showed an improvement at 3 and 6 months in comparison with baseline for patients treated with TTFields and TMZ, whereas patients in the control group showed a decrease in global health status over the same time period.

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Discussion

More than a decade ago, in vitro and in vivo experiments in tumor cell lines and in mouse, rat, and rabbit tumor models have demonstrated antitumor activity of low-intensity, intermediate-frequency alternating electric fields. Dividing cells are arrested in metaphase and anaphase, assembly and function of the mitotic spindle are perturbed, and cells ultimately undergo apoptosis. However, in order to translate these findings into a clinically useful treatment, certain conditions must be met: (1) TTFields need to be delivered in a continuous manner to achieve the expected cytotoxic effect; (2) TTFields can only be applied to certain areas of the body, and this (3) requires the possibility to affix transducer array to the skin of the patient over the area of the tumor. As GBM is a disease that remains confined to the CNS and the scalp offers an easy application site for long-term use of transducer arrays, it appeared to be the ideal candidate to serve as a proof of concept demonstration of TTFields.

Two pivotal randomized trials have been reported to date. In recurrent disease, the trial has not demonstrated improved outcome compared best physicians’ choice chemotherapy. However, TTFields when administered as part of the initial treatment in newly diagnosed patients showed a consistent prolongation of both PFS and OS (hazard ratio for death HR: 0.74 (95% CI, 0.56–0.98). Giving TTFields early in the disease course allows for prolonged exposure, and the in vitro observed synergy with TMZ may further enhance its efficacy. The median treatment duration in recurrent disease was only 2.3 months compared with 9 months in newly diagnosed GBM. Still, TTFields alone in recurrent disease have shown objective responses in 14% of patients, consistent or even numerically higher than that observed in other trials using alkylating agent chemotherapy with lomustine 23,24 or TMZ.25 In the PriDE dataset reflecting routine clinical use of the device, patients who received TTFields at first recurrence were treated for a median of 6.2 months and had a median survival of 20 months, comparing favorably with recent trial results investigating other novel agents. However, a strong selection bias and inclusion of patients with pseudoprogression after initial TMZ chemoradiotherapy cannot be ruled out in this uncontrolled routine practice patient population.26 The best results with this novel treatment modality have been achieved when TTFields were administered early in the disease course in combination with standard maintenance TMZ therapy,17 similar to that shown 10 years ago when TMZ was added to standard radiotherapy.

It may be scientifically regrettable that the trial had to be open label and did not include a double-blinded control group. However, a sham device would neither be practically feasible (some heating of the electrodes is inevitable; technically savvy patients would rapidly figure out whether there is any current flowing), nor acceptable for patients, caregivers, and ethics committees given the perceived burden of shaving the scalp and replacing transducer arrays every 3 days. Whereas some placebo effect might be expected on subjective endpoints, such as quality of life or cognitive function, it is difficult to envision an effect on objective endpoints such as OS or PFS (especially when progression was determined by blinded central radiologists).27Requiring a placebo or sham device would also mean a paradigm shift in conducting clinical trials with survival endpoints in oncology. Sham radiation therapy would be required for RT trials, and a placebo control would only be feasible for agents that have rare and mild toxicities.

Indeed, patients receiving TTField therapy received some additional assistance by the technical support team providing the TTFields device and arrays. However, this support was on average limited to 1 visit per month, and 1–2 extra visits or contacts at the initiation of treatment. Most patients became rapidly independent and self-proficient with the device; on average, there was a median of 1.12 visits per patient and month of treatment (range, 0.5–4 visits per month).

It is highly unlikely that this additional technical support would translate into a 3-month prolongation of median survival, which is in the range of the benefit seen with the introduction of TMZ.26 In a contemporary randomized open-label non-placebo-controlled trial, patients in the experimental arm received twice weekly i.v. administration of cilengitide. Still, this did not translate into any benefit in outcome (hazard ratio for death: 1·02; 95% CI 0·81–1·29).28

It might have been possible that the control arm in EF-14 performed exceptionally poorly. We thus scrutinized the data and compared the performance of the control arms of contemporary trials. The patient characteristics of both the TTFields and control groups were comparable with other clinical trials for newly diagnosed GBM patients in respect to all known prognostic factors (distribution of age, performance status, extent of resection, MGMT status).

One important difference in the EF-14 trial compared with many other reports is that patients were randomized only after the end of chemoradiotherapy, and for most patients the first cycle of maintenance TMZ had already been started at time of randomization. This implies that all patients with early tumor progression during the concomitant radiation and TMZ part of the treatment were excluded from this trial. On the other hand, the 3.8 months from diagnosis to randomization will need to be added to survival times in order to have an estimate of the individual patients’ effective outcome. The PFS of 4.0 months (from randomization) observed in the control group of the EF14 trial is numerically shorter (likely due to the independent imaging review and assessment of progression) but overall comparable to the observed PFS of 5.5 months observed in the RTOG 0525 trial,29 a trial that also randomized patients only after completion of the concomitant radiochemotherapy part of treatment. Moreover, in both trials, the OS was identical at 16.6 months for the control groups. It is therefore unlikely that the benefit observed in the treatment group of the EF14 trial can be attributed to patient selection and a poor outcome of patients in the control arm.

For many experts in neuro-oncology who were not involved in the trial, the most important criticism to TTFields was the requirement for patients to wear a device and their presumed stigmatization. Having to shave the scalp is indeed a psychological barrier, although in oncology, for decades we have been using cytotoxic agents that are inducing complete hair loss of all body hair and not only the scalp. In our experience, patients and families rapidly adapt to wearing the device and are able to continue their regular activities including work and even sports. Preliminary analysis of the self-reported quality of life data from the EF14 trial showed identical scores for both groups at baseline and at 12 months for patients in the treatment and control group at the levels of cognitive, emotional, physical, and social functioning.22 To reduce some of the burden of therapy, a second generation of the TTFields device with a reduction in size and weight by about 50% compared with the device used in the EF-11 and EF-14 devices has been developed.

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Conclusions & Future Perspectives

The results of the randomized phase III EF-14 trial provide level 1 evidence that alternating electric fields are able to positively impact tumor growth and significantly extend survival in GBM. As a logical consequence, TTFields were approved by the FDA for newly diagnosed GBM in October 2015. Nevertheless, numerous questions remain and need to be addressed, both within the EF-14 trial and in future studies; Notably, it will be essential to be able to (1) identify the patients most likely to respond to TTFields therapy, (2) further elucidate the mechanism of action of TTFields, (3) elucidate the mechanisms of resistance to and failure of TTFields therapy, and (4) elucidate the pattern and predictors of response.

In GBM we will need to integrate this novel treatment approach in the current standard of care, and ultimately novel clinical trials will also need to integrate TTFields (at least in the control arm). A pragmatic alternative is to stratify patients for the use of TTFields as part of their standard of care in both the standard and experimental arms.

TTFields is a locoregional treatment, and extending its use to other tumor types and metastatic disease is most promising in clinical situations where locoregional disease control is key for quality of life. The excellent compatibility between TTFields and various chemotherapeutic agents has already been demonstrated, not only in GBM patients in the EF-14 trial but also in lung cancer patients.30 TTFields may also be synergistic with immune therapy approaches. Senovilla et al showed that cells that cannot undergo mitotic exit show hallmarks of immunogenic cell death where the immune system induces a strong response against the dying cells.31

The positive results of the EF-14 demonstrate that neuro-oncology can lead the way to innovation. The results of the EF-14 trial paves the way to investigate the role of alternating electrical fields in other oncologic situations amenable to locoregional treatment such as brain metastases, ovarian carcinoma, mesothelioma, or pancreatic tumors (→ table 2). These trials are currently ongoing. For instance, in pancreatic cancer, TTfields therapy, in addition to gemcitabine resulted in a median PFS of 8.3 months (CI, 3–10.3 mo) in a phase 2 study on 20 patients. The partial response rate was 30% and another 30% stable disease. The median OS for all patients was promising for this disease at 14.9 months. It was not reached in patients with locally advanced disease, and 8.3 months (CI, 4.3–14.9 mo) for patients with metastatic disease with 1-year survival rates of 86% in locally advanced patients and 40% in patients with metastatic disease32 If those trials confirm the positive effects observed in GBM patients, a truly new cancer treatment modality has been born and will find multiple useful indications alone or in combination with other established or new treatments.

Table 2.

Ongoing clinical trials in solid tumors

Articles from Neuro-Oncology are provided here courtesy of Society for Neuro-Oncology and Oxford University Press

Cancer Research: Drug Repurposing / Metformin

STEP ONE

Be informed. Read information below.

STEP TWO

Schedule consultation with R. Douglas Wichman, MD.

STEP THREE

Submit "New Patient Form", current labwork, & imaging results prior to consultation.

STEP FOUR

Establish if you are a candidate for this therapy

in accordance with the Georgia

"Access to Medical Treatment Act".

Related image

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*** SPECIAL NOTE FROM DR. WICHMAN ***

The following excellent article was reproduced from Oncotarget at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130043/:

Oncotarget. 2016 Jun 28; 7(26): 40767–40780.

Published online 2016 Mar 19. doi: 10.18632/oncotarget.8194

PMCID: PMC5130043

Repurposing metformin for cancer treatment: current clinical studies

Young Kwang Chae,1,2,3 Ayush Arya,3 Mary-Kate Malecek,3 Daniel Sanghoon Shin,4 Benedito Carneiro,1,2,3Sunandana Chandra,1,2,3 Jason Kaplan,1,2,3 Aparna Kalyan,1,2,3 Jessica K. Altman,1,2,3 Leonidas Platanias,1,2,3,5 andFrancis Giles1,2,3

Author information ► Article notes ► Copyright and License information ►

This article has been cited by other articles in PMC.

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INTRODUCTION

Metformin is one of the most widely prescribed oral anti-diabetic medications. It is the first line therapy for type 2 diabetes mellitus [1]. It has an anti-hyperglycemic effect which is mediated by inhibiting gluconeogenesis, decreasing glucose absorption from the small intestine, increasing glucose uptake in cells, and decreasing plasma free fatty acid concentration [2]. Metformin also increases insulin induced translocation of glucose transporters to the cellular plasma membrane, thus reducing insulin resistance [3]. Use of metformin has been found to be generally safe, with mild gastrointestinal symptoms being the most common adverse effects [4].

There is substantial preclinical evidence suggesting that metformin has anti-cancer properties. In-vitro and in-vivo analysis of metformin has exhibited anti-proliferative activity by inhibiting intracellular pathways. It has also been observed that metformin activates the T cell mediated immune response against cancer cells.

Numerous retrospective studies have reported that metformin is associated with a reduced risk of developing cancer. Meta-analysis of data obtained from cohort and observational studies has revealed that metformin use was associated with a decrease in both cancer related and all-cause mortality.

Here we summarize the available evidence from clinical trials of metformin as part of cancer therapy. We review the landscape of current investigation and suggest directions for future investigation.

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PRECLINICAL DATA

Metformin has been extensively studied in preclinical models, which has revealed numerous molecular pathways that it modulates, either directly or through other downstream targets, contributing to reduction in growth and proliferation of tumor cells. The inhibition of mTOR (mammalian target of rapamycin) in tumor cells is one of the potential key mechanisms that facilitates the anti-cancer activity of metformin. Use of metformin in MCF-7 breast cancer cells exhibited reduction in phosphorylation of S6 kinase, ribosomal protein S6 and eIF4E binding protein, along with inhibition of mTOR and reduced translation initiation due to AMPK activation [5]. Animal models of pancreatic cancer fed with metformin showed inhibition of insulin like growth factor-1 (IGF-1) and mTOR, along with an increase in phosphorylated AMPK and tuberous sclerosis complex (TSC1, TSC2) [6]. The AMPK mediated phosphorylation of TSC2 has been observed to increase the activity of TSC2, leading to inactivation of mTOR [7, 8]. AMPK has been described to directly inhibit mTORC1 through phosphorylation of mTOR binding raptor as well [9]. Comparing the effects of metformin with rapamycin, a direct mTOR inhibitor, metformin decreases the activation of AKT in addition to AMPK dependent mTOR inhibition. Thus, metformin renders a better anti-tumor response than rapamycin in breast cancer cells [10]. Metformin has been found to decrease HER2 expression in human breast cancer cells by directly inhibiting p70S6K1, which is a downstream effector of mTOR [11]. In a study using nude mice with acute myeloid leukemia (AML), the use of metformin was correlated with a decrease in proliferation of AML cells. This action was characterized by the activation of LKB1/AMPK/TSC pathway, which led to mTOR inhibition and consequently suppression of mRNA translation [12]. In tobacco carcinogen induced lung cancer mice, the inhibition of insulin like growth factor 1 receptor/insulin receptor (IGF- 1R/IR) by metformin decreased the downstream signaling through Akt pathway. This reduced the activation of mTOR in lung tissue which corresponded to a 72% reduction in tumor burden [13].

Metformin can inhibit the activation of mTOR independent of AMPK pathway as well. Metformin has been shown to escalate the expression of REDD1 by a p53 mediated inhibition of mTOR in prostate cancer cells [14]. Another study described that metformin can prevent mTOR activation through Ras-related GTPase (RagGTPases), independent of AMPK, as well as TSC1/2 [15].

Tumors are known to exhibit the Warburg effect, where tumor cells generate ATP from glycolysis instead of oxidative phosphorylation secondary to low nutrient supply and hypoxia [16]. Metformin blunts the Warburg effect and consequently downregulates the growth of cancer stem cells [17]. In-vivo studies on hepatocellular carcinoma xenografts have shown that metformin improves cellular oxygenation ability and decreases mitochondrial oxygen consumption, thus suppressing hypoxia-induced HIF-1α accumulation. These effects form the basis of anti-cancer activity of metformin, particularly against hepatocellular carcinoma [18].

In addition to the above, various other mechanisms of metformin for cell growth inhibition have been identified in pre-clinical models. Metformin therapy in paired isogenic colon cancer cell lines (HCT116 p53 [+/+] and HCT116 p53 [−/−]) showed an increase in apoptosis of p53 deficient cells [19]. In-vitro and in-vivo analysis of metformin therapy showed that it can inhibit growth of ovarian stem cells [20], glioma initiating cells [21], breast cancer cells [22], endometrial cancer cells [23] and non-small cell lung cancer cells [24]. It has also exhibited synergistic action with VEGF inhibitors to inhibit proliferation of BRAF mutant melanoma cells [25]. Additionally, it has been noted that metformin increases radiosensitivity of cancer cells [22, 24].

Recent experiments on animal models have also suggested that metformin has immune modulatory properties. Metformin inhibits immune exhaustion of CD8+ tumor induced lymphocytes (TIL), thereby enhancing T cell mediated immune response to tumor tissue. It decreases apoptosis of CD8+ tumor infiltrating lymphocytes (TILs), and also shifts the phenotype of CD8+ TILs expressing exhaustion markers (especially PD1 negative Tim3 positive) from central memory T cells (TCM, inactive against tumor cells) to effector memory T cells (TEM, active against tumor cells). The increase in TEM cell population has been found to correlate with regression of tumor cells [26]. In a study evaluating an experimental cancer vaccine, administration of metformin after vaccination in animal models showed an increase in CD8+ memory T cells which conferred protective immunity upon subsequent tumor challenge [27]. Figure Figure11 summarizes the effects of metformin on various cellular pathways.

Figure 1

Possible mechanisms of anti-cancer activity of metformin

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EPIDEMIOLOGY

There are numerous epidemiological studies that have put forth evidence suggesting utility of metformin as an anti-cancer agent.

Several observational and cohort studies have been conducted to assess the influence of metformin on cancer. Survival analysis in a cohort study on diabetic patients comparing metformin users (n = 4,085) to non-users (n = 4,085) reported a reduced risk of cancer (hazard ratio, HR = 0.63; 7.3% diagnosed with cancer in metformin users versus 11.6% in non-users) [28]. Meta-analysis performed on 37 studies, with a total of 1,535,636 patients comparing metformin users and non-users reported overall cancer incidence summary relative risk (SRR) as 0.73. The results also noted a reduction of cancer incidence for liver (78%), breast (6%), pancreatic (46%) and colorectal cancer (23%). However, no significant correlation could be derived between the use of metformin and incidence of prostate cancer [29]. Meta-analysis of 6 case control studies comparing 39,787 participants that were on metformin to 177,752 participants that were not, exhibited a lower risk of developing lung cancer in the metformin group (odds ratio, OR = 0.55; p < 0.001) [30]. Likewise, another meta-analysis showed a reduction in incidence of prostate cancer (RR = 0.88; p = 0.03) in patients who were on metformin treatment [31]. Meta-analysis of 35 observational studies reported a considerable correlation with using metformin to reduce the risk of developing all-cancer (OR = 0.73), liver cancer (OR = 0.34), colorectal cancer (OR = 0.83), pancreatic cancer (OR = 0.56), gastric cancer (OR = 0.83), and esophageal cancer (OR = 0.90) [32]. A recent meta-analysis of 265 studies showed the use of metformin or thiazolidinediones was associated with a lower incidence of cancer (RR = 0.86 and 0.93 respectively). But interestingly, insulin, sulfonylureas, and alpha glucosidase inhibitor use was associated with an increased incidence of cancer (RR = 1.21, 1.20, 1.10 respectively) [33].

Meta-analysis of retrospective studies has provided substantial evidence associating the use of metformin with a decrease in cancer related mortality. A cohort study, investigating mortality due to cancer in type 2 diabetes patients, showed a decrease in cancer related mortality risk (HR = 0.56) with the use of metformin [34]. Data from meta-analysis of 6 observational studies noted a significant correlation between using metformin and risk of cancer related mortality (OR = 0.65) [32]. Retrospective analysis of data from over 350 primary care practices in the United Kingdom found a decrease in cancer related mortality (HR = 0.85) in diabetic patients who were on metformin monotherapy when compared with those on other drugs for diabetes [35]. Another meta-analysis showed that the use of metformin in diabetic patients diagnosed with cancer was associated with a decrease in risk of all-cause mortality in cancers of breast (pooled relative risk, RR = 0.70; p = 0.003), ovary (RR = 0.44; p < 0.001), endometrium (RR = 0.49; p = 0.001) and colorectal cancer (RR = 0.70; p < 0.001) [36].

In light of the available preclinical and retrospective data suggesting anti-cancer properties of metformin, clinical trials are necessary to further investigate its role in cancer therapy.

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METHODS

Information about the study drug was obtained from clinicaltrials.gov (a service of United States National Institutes of Health), using the search query “metformin” and “cancer”. Of the 223 results obtained from the search engine, relevant drug trials were selected. The published clinical trials were obtained from PubMed using the same search query and choosing the “clinical trial” filter. The publications that focused on use of metformin for treatment of cancer in human subjects through a prospective clinical trial were identified and selected. The results of completed clinical trials were obtained from PubMed and online abstract library for professional societies such as the American Association of Cancer Research (AACR) and American Society of Clinical Oncology (ASCO). The AACR and ASCO online libraries were explored using the NCT number associated with the clinical trial. The authors, enrollment number, primary & secondary outcomes and primary location of conducting the research mentioned in abstracts collected from ASCO and AACR database were matched with the information obtained from clinicaltrials.gov to avoid any discrepancies in associating the abstracts to the appropriate trial.

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CLINICAL TRIALS

Presently, there are 55 ongoing clinical trials in various stages that are evaluating metformin as a monotherapy (11 trials, 20% of all ongoing trials using metformin as an anti-cancer agent) or in combination with cytotoxic chemotherapy (38 trials, 69%) and/or radiotherapy (6 trials, 11%) for the treatment of cancer (Tables (Tables1,1, ,2,2, ,3,3, ,4).4). These trials primarily focus on establishing the effects of metformin on markers of cellular proliferation, pathological response rate, progression free survival, and recurrence free survival. Also, certain trials are directed towards determining the maximum tolerable dose of metformin in specific tumors.

Table 1

Proof of concept for anti-tumor activity of metformin

Table 2

Anti-tumor activity of metformin in locally advanced and hematologic cancers

Table 3

Anti-tumor activity of metformin in metastatic tumors

Table 4

Antitumor activity of metformin in combination with radiotherapy

A considerable amount of focus has been laid on investigating metformin as a potential anti-cancer agent for cases of breast cancer. Eleven trials (20% of all ongoing trials using metformin as an anti-cancer agent; Tables Tables1,1, ,2,2, ,3)3) are focused on evaluating metformin as a treatment for breast cancer. Of these, two trials are using metformin as monotherapy. There are 9 trials using metformin in combination with other anti-cancer agents. These include capecitabine, cyclophosphamide, docetaxel, doxorubicin, erlotinib, epirubicin, exemestane, ganitumab, letrozole, sirolimus and temsirolimus. One trial is exploring the use of metformin plus atorvastatin combination as a possible treatment for breast cancer. Two trials using metformin combination therapy are also evaluating pathological complete response as a primary endpoint. Apart from the ongoing trials, data obtained from 5 completed trials (all using metformin monotherapy in a pre-surgical window of opportunity trial design) has facilitated a better understanding regarding the effects of metformin in breast cancer. In addition to survival outcomes, several surrogate markers are also being employed to study the effects of metformin on breast cancer cell population. These include Ki67, S6K, 4E-BP-1, AMPK and effects on AMPK/mTOR pathway.

A phase 2 single arm window of opportunity trial of 39 breast cancer cases [37] showed significant reduction in Ki67 (36.5 to 33.5 %, p = 0.016) and an increase in TUNEL staining (0.56 to 1.05, p = 0.004) along with significant fall in HOMA (homeostatic model assessment, used for determining the status of insulin resistance) [38]. A recently published randomized control trial (RCT) also reported a decrease in Ki67 staining (mean = 3.4%, p = 0.027). Additionally, it noted an increase in mean AMPK score, fall in pAKT score and reduced caspase-3 staining in patient samples with the use of metformin when compared to placebo [39]. However, other trials have had conflicting outcomes. A phase II RCT with 200 participants recorded no significant changes in Ki67 on comparing metformin and placebo arms. But, interestingly, the cases with HOMA ≤ 2.8 showed a non-significant increase of Ki67 by 11.1% (95% Confidence interval (CI): −0.6% to 24.2%) and those with HOMA > 2.8 (implying a higher probability of insulin resistance) showed a non-significant mean proportional decrease in Ki67 by 10.5% (95% CI: −26.1% to 8.4%) [40]. Another phase II trial (non-randomized) examined effects of metformin in overweight/obese patients with stage 0-III breast cancer. Though noting a correlation of Ki67 with tumor growth, the calculated ln (Ki67) showed no significant changes when comparing metformin to placebo [41]. A different study with 200 participants randomized to metformin or placebo did not document any major difference in Ki67 and TUNEL levels (used for assessing cellular apoptosis) between the two arms. The study did note that TUNEL levels were higher in women without insulin resistance (metformin: +4%, interquartile range, IQR: 2-14, placebo: +2%, IQR: 0-7) as compared to those who had insulin resistance (metformin +2%, IQR: 0-6, placebo +5%, IQR: 0-15) [42]. The survival benefit with the use of metformin in breast cancer is being evaluated in 3 clinical trials, however there is no data presently available (NCT01627067, NCT0131023, NCT01885013).

Metformin is presently being evaluated as an anti-cancer agent for endometrial cancer as well. There are 6 ongoing trials (10.9% of all ongoing trials using metformin as an anti-cancer agent; Tables Tables1,1, ,2),2), with 3 each for monotherapy and combination chemotherapy. The drugs that are currently being assessed in combination with metformin for treatment of endometrial cancer are carboplatin, everolimus, letrozole, paclitaxel, and megestrol acetate. One trial is assessing the role of metformin as a maintenance therapy. In conjunction with clinical response, the effect of metformin on endometrial cancer is being studied through a wide variety of markers including Ki67, pS6, Akt, pAMPK, ERK1/2, histone H3, telomerase, topoisomerase IIα and p27. The effect of using metformin on expression of estrogen (ER) and progesterone (PR) receptors in cancer tissue of endometrial origin is also being investigated. Three completed trials, all pre-surgical window of opportunity trials using metformin monotherapy, have shown a significant decrease in Ki67 staining [43–45] and in pS6 staining [44, 45]. One trial also reported a reduction in topoisomerase IIα and ERK 1 / 2, along with significant elevation in pAMPK and p27 [45]. A different trial reported a decrease in tumor cell proliferation by 11.75% and a decrease in expression of ER with the use of metformin. PR expression, however, was not affected [43]. Thus far, no trial has provided any data on survival benefit with the use of metformin in endometrial cancer, though one trial is ongoing (NCT02065687).

Metformin is being assessed in combination with various anti-cancer agents for the treatment of pancreatic cancer. Presently, there are 7 ongoing and 2 completed trials (Tables (Tables2,2, ,3,3, ,4).4). With the exception of one trial where the treatment regimen involves using metformin together with stereotactic radiosurgery (Table (Table4),4), all others are using metformin in combination with different anti-cancer agents. These include cisplatin, capecitabine, epirubicin, erlotinib, everolimus, gemcitabine, octreotide, paclitaxel, rapamycin, and FOLFOX (fluorouracil, oxaliplatin, leucovorin). The effects of metformin are being assessed mostly through clinical outcomes including progression free survival (PFS), recurrence free survival (RFS) and toxicity due to chemotherapy combination. The results from a phase II non-randomized trial showed that the combination of metformin plus paclitaxel was not well tolerated, with 42.1% patients experiencing grade 3-4 toxicities. A total of 31.6% cases had to undergo metformin dose reduction secondary to development of diarrhea, while one case experienced febrile neutropenia which was attributed to paclitaxel. This trial reported a median overall survival (OS) of 133 days and median PFS as 43 days, but could not meet the disease control rate endpoint [46]. Another trial, consisting of 120 participants randomized to metformin or placebo arm, noted that although the combination of metformin, gemcitabine and erlotinib was well tolerated, the 6 month survival rate was 55% in metformin arm and 66% in placebo arm. Also, no significant difference was observed in PFS and median OS between metformin users and non-users [47].

Five clinical trials (Tables (Tables1,1, ,2,2, ,3)3) are presently working to evaluate if metformin may be of value in the treatment of prostate cancer. These include two trials that are using metformin as monotherapy and three in combination with different agents: abiraterone (NCT01677897), docetaxel (NCT01796028), and enzalutamide (NCT02339168). Data made available from one trial, a single arm window of opportunity study, showed a significant reduction in Ki67 index and 4E-BP-1 staining with no changes in pAMPK. Three of 24 patients developed grade 3-4 toxicities, indicating that the treatment was overall well tolerated [48]. The effect of metformin therapy on PFS for prostate cancer is being assessed in two trials (NCT01433913, NCT02339168), with one other trial evaluating PSA response (NCT01796028). However, presently there is no data available on survival benefit.

There is one phase II trial of metformin use in non-small cell lung cancer (NSCLC) combined with stereotactic body radiotherapy that is currently recruiting patients (NCT02285855).

There are two completed trials (Table (Table2,2, ,3)3) on multi-histology solid tumors assessing the dose limiting toxicity (DLT) of various treatment regimens that include metformin. One of the trials used metformin in combination with 26 chemotherapy regimens for 17 tumor types on a total of 100 participants. The study was divided in two stages. In stage one, participants were randomized to receive metformin or placebo with chemotherapy. In stage two, participants of delayed arm would be crossed over to receive metformin with chemotherapy. Results showed that 46% of participants documented stable disease. In the sub-set of patients having quantifiable tumor markers, 28% exhibited favorable changes. The participants receiving metformin together with chemotherapy showed a lower rate of DLT (6.1% in stage one of concurrent arm, including grade 3 anemia, decrease in albumin and elevation in ALT) compared to those who received just the chemotherapy (7.8% in stage one of delayed arm [including grade 3 syncope, dehydration and elevation of bilirubin] and 3.8 % in stage two [including dehydration, vomiting and proteinuria]). The participants reporting DLTs in stage two of delayed arm were known cases of adverse events with chemotherapy [49]. The other trial used metformin and temsirolimus combination in 11 patients. It reported that 100% of participants had grade 1 toxicity and 82% experienced grade 2 toxicity, with DLT being reported in all 3 patients of the first cohort (grade 4 pneumonitis, grade 3 fatigue and grade 3 thrombocytopenia). In the second cohort, the dose of temsirolimus and metformin was reduced and DLT was observed in only two of eight cases (grade 4 dyspnea and grade 3 thrombocytopenia). After 2 months of treatment, 5 patients had stable disease, 1 case had partial response and 2 showed progression [50].

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DISCUSSION

There is great excitement surrounding metformin as a potential anti-cancer agent. Epidemiological data has associated the use of metformin with a decrease in the risk of developing cancer and a reduced cancer related mortality. The information that has been gathered from preclinical studies has provided encouraging evidence for anticancer mechanisms of metformin. It has been suggested that metformin may well be used as a radiation sensitizer or an immunotherapy drug, in addition to a direct anti-proliferative agent for the treatment of cancer.

The anticancer mechanism of metformin has been extensively studied and attributed to mTOR inhibition. More recent data has revealed an immunomodulatory effect on cancer cells. Pre-clinical data has demonstrated that metformin can inhibit apoptosis of CD8+ TILs. In addition, it also increases the effector memory T cell population through phenotype switching of CD8+ TILs, thus enhancing the immune response against tumor cells [26]. The use of metformin with an experimental cancer vaccine (LmOVA) showed an increase in the number of CD8+ memory T cells that conferred immunity to cancer [27]. The recent breakthrough developments in immunotherapy for patients with advanced melanoma, triple negative breast cancer, and non-small cell lung cancer with check point blockade monoclonal antibodies (anti-PD-1/L1) have generated excitement in the oncology community [51–53]. Immunomodulatory properties of metformin have yet to be studied in combination with other forms of immunotherapy, in particular with check point blockade monoclonal antibodies. Further investigation into a possible synergistic effect is warranted.

The preliminary results from clinical trials assessing metformin as an anti-cancer agent have shown that metformin can significantly impact markers of tumor proliferation. A total of 19 ongoing and completed trials (Table (Table1)1) are using various surrogate markers to assess pro-apoptotic effects of metformin on cancer cells. Although majority of these trials are being performed on breast and endometrial cancer cases, a limited number of trials are also evaluating tumors of head & neck, prostate, bladder, lung, kidney and lymphoma.

Pre-surgical window of opportunity trials in endometrial and breast cancer showed that tumor markers such as Ki67 and TUNEL (indicative of changes in cell proliferation and apoptosis respectively) exhibited favorable anti-tumor effect. Two trials demonstrated favorable changes in Ki67 and TUNEL in a subset of women without insulin resistance as compared to those with insulin resistance. Although the findings were not statistically significant, the direction of change suggests an intriguing hypothesis. The use of metformin in patients without insulin resistance may offer more benefit as compared to those with insulin resistance.

Diabetes is known to be associated with insulin resistance [54] and an impaired immune response against various pathogens [55, 56]. The immune system responds to the proliferating tumor cells by increasing production of tumor specific lymphocytes which check tumor growth by various mechanisms [57]. Evidence from preclinical trials has described that metformin, at least in part, exerts an anti-cancer effect by inhibiting immune exhaustion of CD 8+ TILs [26], thus amplifying the existing immune action against cancer cells. Therefore, it may be hypothesized that the patients with insulin resistance have a compromised immune system, which consequently results in a sub-optimal anti-cancer effect of metformin. It might be rational to stratify outcomes according to the insulin resistance status of participants in future clinical trials in order to better appreciate the anti-cancer activity of metformin.

Fourteen ongoing trials (Tables (Tables1,1, ,2,2, ,3,3, ,4)4) are presently amassing evidence to ascertain if a survival benefit is associated with the use of metformin in various malignancies. These include tumors of the breast, pancreas, lung, endometrial, brain, prostate and gynecological cancers. Thus far, there are results available from two clinical trials on metastatic pancreatic cancer, neither of which had favorable outcomes. From this data, it can be speculated that metformin may not be viable option for the treatment of advanced pancreatic cancer. The upcoming clinical trials may need to shift focus towards treating earlier stages of pancreatic cancer or using a different combination of agents with metformin to have better outcomes in advanced disease. Additional data on survival indices from multiple ongoing trials will be pivotal to draw a better conclusion.

One concern with the clinical utility of metformin is its side effect profile, particularly in combination with cytotoxic chemotherapy. Metformin is well known for causing GI upset, sometimes limiting patient compliance due to discomfort. Clinical trials have revealed a low incidence of DLTs with metformin in combination with a wide variety of chemotherapy regimens [48]. With this data, clinicians can be reassured that metformin will most likely be a tolerable addition to a chemotherapy regimen, and should not limit its practical utility.

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CONCLUSIONS

A strong base of epidemiological and pre-clinical data has prompted attempts to probe the anti-cancer effects of metformin through clinical trials. Metformin has been shown to have a favorable effect on markers of tumor proliferation but it remains to be seen if that translates to benefit in survival rates. It is prudent to find better histology and the appropriate stage of tumors for utilizing metformin therapy. The potential use of metformin as an immunotherapy agent needs to be substantiated with further evidence to ascertain possible benefits in future.

Articles from Oncotarget are provided here courtesy of Impact Journals, LLC

Cancer Research: NORI Methionine Protocol

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*** SPECIAL NOTE FROM DR. WICHMAN ***

The following excellent article was reproduced from BMC Cancer at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891836/:

BMC Cancer. 2016; 16: 349.

Published online 2016 Jun 3. doi: 10.1186/s12885-016-2367-1

PMCID: PMC4891836

Methionine-restricted diet inhibits growth of MCF10AT1-derived mammary tumors by increasing cell cycle inhibitors in athymic nude mice

J. R. Hens, corresponding author

I. Sinha, F. Perodin, T. Cooper, R. Sinha, J. Plummer, C. E. Perrone, and D. Orentreich

Author information ► Article notes ► Copyright and License information ►

This article has been corrected. See BMC Cancer. 2016 July 14; 16: 474.

This article has been cited by other articles in PMC.

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Abstract

Background

Dietary methionine restriction (MR) improves healthspan in part by reducing adiposity and by increasing insulin sensitivity in rodent models. The purpose of this study was to determine whether MR inhibits tumor progression in breast cancer xenograft model and breast cancer cell lines.

Methods

Athymic nude mice were injected with MCF10AT1 cells in Matrigel® and fed a diet containing either 0.86 % methionine (control fed, CF), or 0.12 % methionine (MR) for 12 weeks. Plasma amino acid concentrations were measured by UPLC, and proliferation and apoptosis were examined using RT-PCR, immunohistochemistry, and Cell Titer 96® Aqueous One Solution Cell Proliferation assay.

Results

Mice on the MR diet had reduced body weight and decreased adiposity. They also had smaller tumors when compared to the mice bearing tumors on the CF diet. Plasma concentrations of the sulfur amino acids (methionine, cysteine, and taurine) were reduced, whereas ornithine, serine, and glutamate acid were increased in mice on the MR diet. MR mice exhibited decreased proliferation and increased apoptosis in cells that comprise the mammary glands and tumors of mice. Elevated expression of P21 occurred in both MCF10AT1-derived tumor tissue and endogenously in mammary gland tissue of MR mice. Breast cancer cell lines MCF10A and MDA-MB-231 grown in methionine-restricted cysteine-depleted media for 24 h also up-regulated P21 and P27 gene expression, and MDA-MB-231 cells had decreased proliferation.

Conclusion

MR hinders cancer progression by increasing cell cycle inhibitors that halt cell cycle progression. The application of MR in a clinical setting may provide a delay in the progression of cancer, which would provide more time for conventional cancer therapies to be effective.

Electronic supplementary material

The online version of this article (doi:10.1186/s12885-016-2367-1) contains supplementary material, which is available to authorized users.

Keywords: Methionine, Diet, P21, Breast cancer, Cell cycle, Inhibitors, CDKN1a

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Background

One of the most common cancers worldwide is breast cancer, and it is the second leading cause of mortality in women from the United States [1, 2]. Although conventional therapies and surgical approaches have been developed, none are completely effective in removing and annihilating the cancer.

Cancer cells alter their metabolic machinery to maintain a high level of metabolism and prevent the depletion of a host of substrates, such as glucose and amino acids that are used for energy. A significant aspect of this reprogramming involves changes in the glycolytic pathway, referred to as the Warburg effect [3, 4]. These changes include an increase in pyruvate that is generated via the glycolytic pathway. Pyruvate is converted to lactic acid instead of acetyl-CoA which enters the TCA cycle and ultimately forms citrate. In addition to metabolic changes from Warburg effect, some cancers depend on glutamate metabolism for fixing ammonia to acquire the nitrogen required for cellular growth [5]. Further, glutamine synthetase is a target for activated β-catenin and is regulated by the oncogene Myc [5], which connects the metabolic regulation of cancer cells to several important growth and developmental signaling pathways.

Dietary nutritional control may be a feasible option for supplementing cancer treatment. The use of caloric restriction (CR) is effective in inhibiting cancer development in non-human primates and rodents [6, 7] and the onset of age-related diseases [6]. However, recent evidence suggests that CR can reduce function of the immune system [8, 9], which may not be ideal for people already fighting cancer. Further, the general population is not in favor of reducing food consumption as required by CR. A viable alternative to CR may be to restrict the intake of dietary methionine.

Methionine is an essential amino acid with a multitude of functions. It is prominent in protein translation, since it is the N-terminal amino acid of most mammalian proteins. Methionine is a precursor of glutathione, a tripeptide that reduces reactive oxygen species (ROS) and protects cells from oxidative stress. Methionine is needed for polyamine synthesis, in which polyamines function during nuclear and cell division. Moreover, methionine is a precursor of S-adenosyl methionine the major source of methyl groups needed for methylation of DNA, proteins and low Mr biomolecules/metabolites. In rodents, a diet low in methionine (20-35 % of regular chow) reduced adiposity in the fat depots and reduced blood levels of lipids, glucose, IGF-1, and leptin, while elevating levels of FGF21 and adiponectin [10–15]. Reduction in mitochondrial free radical production and oxidative stress also occurs during MR in organs, such as, liver, heart, and brain [16–18]. Finally, MR in rodents promotes longevity and delays onset of age-related impairments and chronic diseases [10, 19–21].

In intestinal, colon, and prostate cancer models, methionine levels were restricted or excluded from diets to examine the effects on tumor progression. In an intestinal tumorigenesis model with Apc(Min/+) mice, folate deficiency in combination with depletion of choline, methionine, and vitamin B12 resulted in reduced tumor size in mice treated by 5 weeks of age, but produced no difference when started at 10 weeks of age [22]. Azoxymethane-treated rats when fed MR diet had a significant reduction in formation of aberrant crypt foci in colon, suggesting an inhibition of cell proliferation [21]. More recently, dietary MR in TRAMP mouse model demonstrated a decrease in prostatic intraepithelial neoplasia with a concomitant drop in plasma IGF-1 levels and reduced proliferation in prostate lobe-specific manner [23]. In human prostate cancer cell lines grown in the absence of methionine, the expression of cell cycle inhibitors P21 and P27 increased and were identified as a possible mechanism for halting the cell cycle and increasing apoptosis [24]. These studies suggest that methionine depletion in human prostate cells can inhibit proliferation either by halting the cell cycle at the G1/S checkpoint or by directing cells to go through apoptosis. However, little is known about how MR alters the cell cycle in other cancers, such as breast cancer. The present study uses a xenograft model for breast cancer, an immortalized human breast cell line, and an invasive breast cancer cell line to examine whether MR alters cell cycle inhibitors that could inhibit tumor progression.

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Methods

Xenograft model for testing efficacy of methionine restriction

MCF10AT1, human transformed breast cells, were a gift obtained from Steven Santner at Barbara Ann Karmanos Cancer Institute, Detroit, MI. The cells were maintained in DMEM/F12 (Invitrogen, Carlsbad, CA) with 5 % horse serum, 0.029 M sodium bicarbonate, and 10 mM HEPES, and supplemented with insulin (10 μg/ml), EGF (20 ng/ml), hydrocortisone (0.5 μg/ml), cholera toxin (100 ng/ml), and 1 % penicillin-streptomycin solution. Cells were routinely passaged weekly and maintained in 5 % CO2 at 37 °C.

The procedures and treatments used on the athymic nude mice were reviewed and approved by Penn State College of Medicine Institutional Animal Care and Use Committee (IACUC#2012-082), and followed standard procedures described in Guide for the Care and Use of Laboratory Animals, 8th edition from the National Research Council. A completed ARRIVE guidelines checklist is included in Additional file 1. A total of ten million MCF10AT1 cells with matrigel (1:1 (v/v), BD Biosciences, San Jose, CA) were injected subcutaneously into the left flank region near the mammary fat pad of each of 40 female athymic nude mice (Strain-088 homozygous; 8 weeks of age; Charles River, Wilmington, MA). The mice were fed sterile standard rodent diet for 1 week after cell implantation. These mice were divided into 2 feeding groups: (a) 20 mice on a sterile, a chemically-defined AIN-76-based diet containing 0.86 % methionine (control-fed diet; CF); and (b) 20 mice on a sterile chemically-defined AIN-76-based diet containing 0.12 % methionine (methionine-restricted diet; MR). The mice were housed in groups of five and maintained on a 12 h light-dark cycle and fed these diets ad libitum for 12 weeks. Body weights and average diet intake for each group were measured weekly.

At the end of the study, unfasted mice were euthanized using CO2; whole blood was collected by cardiac puncture, centrifuged, and plasma was collected and stored at −80 °C for analysis. Plasma samples were analyzed for amino acid content, insulin, IGF-1, FGF21, leptin, adiponectin, cholesterol, triglycerides, and glucose.

Tumors were excised, weighed, and measured prior to fixing in 10 % neutral buffered formalin (NBF). Tumor volumes were determined using the formula: 0.523 x a2 x b, where a is the smallest diameter and b is the largest. The fixed tumors were paraffin-embedded, sectioned and stained for hematoxylin & eosin. Histopathology analysis was performed by Dr Timothy Cooper (Penn State College of Medicine), and the percent of lesions morphologically resembling mild, moderate, or florid intraductal papillomas (IDPs); ductal carcinoma in situ (DCIS); or invasive carcinoma were calculated. The tumor sections also were used for immunohistochemical analysis of proliferation and apoptosis for all groups. Additionally, the mammary glands from 10 mice of each group were fixed in NBF for histology. At sacrifice, organ weights for liver, kidneys, spleen, mammary fat pads, and left gastrocnemius muscle were recorded.

Tumor cell proliferation and apoptosis

The percentages of Ki67 and cleaved caspase-3 positive cells counted in triplicate under a high power field (400X) were used as a measure of proliferation and apoptosis, respectively. Briefly, formalin-fixed paraffin-embedded mammary and MCF10AT1 tumor sections were hydrated, subjected to antigen retrieval, and incubated at room temperature with 1:100 anti-Ki67 antibody (M7240, Dako North America Inc, Carpinteria, CA) for 30 min and 1:100 cleaved-caspase-3 antibody (9661, Cell Signaling, Danvers, MA) for 1 h. Slides were developed using the Dako Envision™ +/HRP Polymer detection system (K4001, Dako North America Inc, Carpinteria, CA) and visualized with 3,3'-Diaminobenzidine (DAB) chromagen followed by hematoxylin counterstain. Staining was completed using a Dako Autostainer Plus.

Amino acid analysis

Plasma amino acid concentrations were measured using the Acquity UPLC (Waters Corporation, Milford, MA) with the AccQ.Tag Ultra derivatization Kit (Waters Corporation, Milford, MA). Plasma samples were deproteinized with a solution of 10 % sulfosalicylic acid and 250 pmol/μl of norvaline was used as the internal standard.

Assays on plasma parameters

Enzyme-linked immunosorbent assay (ELISA) kits were used to measure insulin (ALPCO Diagnostics, Salem, NH), leptin, IGF-1, adiponectin (R&D Systems, Minneapolis, MN), and FGF21 (Millipore Corp., Billerica, MA). Colorimetric assays were used to determine plasma triglycerides (TG) and total cholesterol (TC) (Thermo Electron Corp. Beverly, MA). Blood glucose was measured using an AbbottH Freestyle glucometer and glucose test strips.

Methionine restriction in cell culture

MDA-MB-231 and MCF10A cells were obtained from American Type Culture Collection (Manassas, VA). MDA-MB-231 cells were maintained in DMEM/F12 media with 10 % fetal bovine serum, and MCF10A cells were maintained in DMEM/F12 media containing 5 % horse serum with the addition of EGF (20 ng/ml), hydrocortisone (0.5 μg/ml), cholera toxin (100 ng/ml), and insulin (10 μg/ml). Cells were routinely passaged weekly and maintained in 5 % CO2 at 37 °C. Methionine restricted media were specially formulated without methionine, cysteine, and cystine (Life Technologies, Grand Island, NY), and the FBS or horse serum was dialyzed in PBS pH 7.2 three times over a period of 24 h at 4 °C using a Slide-A-Lyzer™ dialysis flask with a 3.5 K MWCO (Thermo Fisher Scientific, Waltham, MA) to remove all amino acids. Media were specially formulated to be 80 % reduced in methionine from the regular DMEM/F12 media (17.24 mg Met/L), with no cysteine or cystine, but with all amino acids and non-essential amino acids (methionine-restricted, cysteine-depleted (MRCD) media). Cells were plated and grown to 80 % confluence, washed once with PBS, and then given either MRCD or regular DMEM/F12 media. Twenty-four hours later, RNA was harvested from cells using Tri-Reagent (Molecular Research Center, Cincinnati, OH), according to the manufacturer’s instructions.

In vitro proliferation assay

Cells (MCF10A and MDA-MB-231) were plated with 40 μl of 1X105 cells/ml per well in a 96 well cell-culture treated dish. The next day cells were washed with PBS, and then treated with either MRCD or regular DMEM/F12 media. At 0 and 24 h cells were assayed for changes in proliferation by treating with the Cell Titer 96® Aqueous One Solution Cell Proliferation assay reagent, according to the manufacturer’s instructions (Promega, Madison, WI). Four experiments with 6 replicates per treatment were conducted. Results were analyzed using a 2-way ANOVA with Sidak’s comparison test.

Real-time PCR

RNA isolated from frozen tissue or cells from culture was converted to cDNA using the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Primers were generated for HPRT, P21, P27, and cyclin D1 (Additional file 1: Table S1). Real-time PCR was performed using a SYBR-green GoTaq qPCR system (Promega, Madison WI), with 40 cycles at 95 °C for 15 s, and then 60 °C for 1 min using Applied Biosystems StepOne Plus Real-Time PCR System. Ct values were analyzed using the ΔΔ − Ct method [25].

RNA isolation and RT-PCR from paraffin sections

Ten micron sections were obtained from the formalin-fixed, paraffin-embedded MCF10AT1 tumors and mammary fat pads of the athymic nude mice. Five sections were collected in a microcentrifuge tube, and RNA was isolated using miRCURY RNA isolation kit (Exiqon, Woburn, MA) according to the manufacturer’s instructions. RNA (250 ng) was converted into cDNA for real-time PCR analysis and analyzed as described above.

The Cancer Genome Atlas data analysis

The cBioPortal for Cancer Genomics (http://www.cbioportal.org) was used to assess the changes in P21 and P27 expression in breast cancer patients from the Cancer Genome Atlas (TCGA) invasive breast carcinoma cancer study (TCGA, Nature 2012) [26, 27]. This data set was screened for those patients containing data on mRNA levels from Agilent microarrays. Data retrieved from the TCGA controlled-access database was collected using tumors from patients who provided informed consent according to guidelines laid out by the TCGA Ethics, Law and Policy Group which is in compliance with the Helsinki Declaration (http://www.wma.net/en/30publications/10policies/b3/index.html).

Statistical analysis and data presentation

Histological analyses of mammary and tumor sections from the nude mice were analyzed using an unpaired Mann-Whitney one-tailed t-test to determine significance. Ct values from RT-PCR results were analyzed unpaired one-tailed t-test to determine significance. In vitro proliferation assay data were analyzed using a 2-way ANOVA with Sidak’s comparison test. All analyses were performed using the statistical software package GraphPad PRISM (GraphPad Software Inc, La Jolla, CA).

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Results

Physiological changes

Physiological parameters of mice on CF and MR were examined. Similar to previous studies [11], MR reduced the body weight of mice, whereas percent weight of liver, spleen, kidney, and mammary fat pad (MFP) were not changed (Table 1). Muscle mass decreased in the mice on MR diet. Future studies may provide a more in depth understanding of the changes that were observed in the muscle.

Table 1

Physiological parameters of mice on MR and CF diets. A 2-tailed t-test was conducted

One of the hallmarks of cancer is increased metabolic activity of the cells. Decreased plasma amino acid levels in cancer patients supports the concept that the tumor is parasitizing the organism and altering protein metabolism that would then affect the amino acid concentrations [28]. In this study, increased levels of alanine, glutamine, histidine, ornithine, and serine occurred in the plasma, which may suggest that MR is maintaining or improving the metabolic condition of the animals (Table 2). Equally important is the finding that the sulfur-containing amino acids (methionine, cysteine, and taurine) were reduced significantly in plasma, providing clear evidence that the MR diet reduces sulfur-containing amino acids throughout the animal.

Table 2

Plasma amino acid concentrations from mice on MR and CF diets. Levels of methionine, cysteine, and taurine in the plasma from mice on the MR diet are significantly lower than those on the control diet. The Kruskal-Wallis test was used to analyze the differences ...

As predicted from previous studies of mice on a MR diet [11, 29], reductions in plasma triglycerides, IGF1, and glucose were observed (Fig. 1). Plasma levels of adiponectin and FGF21 increased significantly in the MR mice compared to the CF mice (Fig. 1), similar to what was reported previously to occur in rodents on MR [11, 13, 30]. Cholesterol levels were slightly elevated in the MR mice. Leptin was not significantly changed (Fig. 1), whereas leptin levels of C57BL/6 J mice on a MR diet were reported to be lower [11]. These differences in leptin levels may be due to strain differences between the two studies.

Fig. 1

Selected analytes measured in the plasma from nude mice on MR or CF diet. A 2-tailed t-test with Welch’s correction for unequal variance was conducted. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, ...

Methionine restriction inhibits tumor progression

The MCF10AT1 cell-derived tumors from mice on MR diet were reduced in size when compared to the mice on CF diet (Representative animals from MR and CF groups in Fig. 2). Tumor weights averaged 20.2 ± 6.1 mg in mice on the CF diet. The mice on the MR diet had a notable decrease in tumor weight to 11.4 ± 4.0 mg that corresponds to a reduction of 55 % (p ≤ 0.0001) in MR mice when compared to the CF mice (Table 1). Descriptive characteristics of the tumors indicated that the mice on the MR diet trended to have fewer florid IDPs (Table 3). There were no significant differences in mild or moderate IDPs, but there was a significant difference in the number of DCIS lesions between the two groups (Table 3). This suggests that lesions formed prior to the initiation of the MR diet were not eliminated by MR, but that MR slowed tumor progression and reduced the size of tumors formed by the implanted MCF10AT1 cells. The fact that there was a population of mice that acquired an invasive carcinoma phenotype suggests that some cells may be able to escape the MR inhibition through an unknown mechanism.

Fig. 2

Representative MCF10AT1 breast tumors (black circles left-flank) in CF and MR fed nude mice at termination. Left mammary gland (#4) is indicated with arrows

Table 3

Characteristics of tumor lesions formed from MCF10AT1 cell implants in the nude mice. Classifications in order of tumor progression included mild intraductal papilloma (IDP), moderate IDP, florid IDP, ductal carcinoma in situ (DCIS), and invasive carcinoma ...

Methionine restriction decreases proliferation while increasing apoptosis

To understand a possible mechanism for the decreased tumor size in the mice on the MR diet, proliferation and apoptosis were examined. Histological sections of the both tumor and nearby mammary tissue were stained for Ki67, a marker for proliferation. A total of 20 animals per treatment were examined for levels of Ki67 staining. Representative images demonstrate that the number of Ki67-positive stained cells was reduced in the tissue from mice on the MR diet (Fig. 3a and andc).c). The number of proliferating cells in the tumor tissue was 13.8 ± 0.8 % cells in CF mice, while 11.6 ± 1.1 % cells in MR mice (Fig. 3e; p < 0.05). Consecutive sections of tumor tissue were stained for activated caspase-3, as an indicator of apoptosis (Fig. 3b and andd);d); MR mice had significantly elevated levels of activated caspase-3 (MR: 3.82 ± 0.66 % cells, p < 0.05), compared with CF mice (CF: 2.44 ± 0.30 % cells) (Fig. 3f).

Fig. 3

Increases in apoptosis and decreases in proliferation occur in mice on the MR diet when compared to mice on the CF diet. a, c are representative images of Ki67 positive staining in mammary tissue near the tumor. b, d represent images of cleaved caspase-3 ...

Changes in cell cycle regulators by methionine restriction

The decrease in proliferation combined with the increase in apoptosis suggests that MR may perturb the cell cycle. To investigate this possibility, RNA was harvested from frozen mammary tissue obtained from CF- and MR-treated nude mice. Expression of murine cell cycle inhibitors P21 and P27 and the cell cycle regulator cyclin D1 were measured by real-time PCR in the nude mice. Although there was a slight decrease in cyclin D1, this effect was not significant (Fig. 3a). P21 expression was significantly increased (Fig. 4b, p ≤ 0.01) in the mammary gland of MR mice, while P27 was not affected (Fig. 4c). To determine whether the human cell-derived tumors were affected similarly by MR, paraffin sections of embedded tumors from CF or MR mice were collected, and RNA was isolated. Real-time PCR using primers specific to human P21 and P27 were examined. There was a trend indicating that human P21 was increased in the tumors from MR fed mice (Fig. 4d, p = 0.09), and a slight decrease in P27 (Fig. 4e, p ≤ 0.05).

Fig. 4

Methionine restriction increases cell cycle inhibitors P21 and P27, while decreasing cyclin D1. a Murine cyclin D1 expression from mammary tissue near the MCF10AT1 tumor in CF and MR mice. N = 5 CF, =3 MR. b Murine P21 expression from ...

To determine whether the changes in cell cycle control could occur in other breast cancer cells, breast cancer cell line MDA-MB-231 and MCF10A immortal cells were examined under conditions of MR. Cells were grown in regular media and plated at 70 % confluency. The next day, media was changed to either regular media (17.24 mg/L of methionine), or MRCD (3.45 mg/L of methionine) media. Cells were harvested for RNA extraction after 24 h. Real time PCR revealed in both MCF10A and MDA-MB-231 cells that P21 (p ≤ 0.001) and P27 (MCF10A p ≤ 0.01, MDA-MB-231 p ≤ 0.05) were significantly increased by MR (Fig. 4f-i). This suggests that the MR effect on the cell cycle inhibitors may be a similar response to MR in both breast cancer cells and immortalized breast cells. Both MCF10A and MDA-MB-231 cells were examined for changes in proliferation over 24 h (Fig. 5). At 24 h, MDA-MB-231 had a significant reduction of proliferation (p ≤ 0.05) by diet, and was further affected by the interaction of diet and time (p ≤ 0.001). Proliferation in MCF10A cells was not initially affected by MR, but there were differences in proliferation over time (p = 0.0013). The differences in proliferation may indicate that MR may be more effective at hindering invasive breast cancer cells.

Fig. 5

Reduced levels of proliferation were seen by 24 h in MDA-MB-231 and to a lesser degree in MCF10A cells. In both cell types, time had significant affect on proliferation. In MDA-MB-231 cells, time, diet, and the interaction of time and diet on ...

To further examine the connection of P21 and P27 expression in breast cancer, patients from the Cancer Genome Atlas (TCGA) invasive breast carcinoma cancer study were examined to determine whether expressions of Cdkn1a (P21) and Cdkn1b (P27) genes were inhibited in breast cancer tumors (TCGA, Nature 2012) [26, 27]. This data set contains 825 patients, of which data are available for 526 patients regarding mRNA expression levels from Agilent microarrays. Alterations in P21 or P27 expression occurred in 10 % of the 526 patients. Of those 52 patients, 56 % had decreases in P21, P27, or both. Four patients had decreases in both P21 and P27, 11 patients had decreases in P21, and 14 patients had decreases in P27. Of the 52 patients, information was available regarding human epidermal growth factor receptor 2 (Her2), estrogen receptor (ER), and progesterone receptor (PR) status in 32 patients. Interestingly, 45 % of patients with decreased expression of P21 were Her2, ER, and PR negative (Fig. 6a), and patients with lower levels of P21 or P27 had reduced survival times following diagnosis (Fig. 6b). This may suggest that cancer patients following an MR diet may benefit, since increased levels in P21 and P27 can inhibit the cancer cell proliferation, providing a longer period of time for other established cancer therapies to be effective.

Fig. 6

Patients from the TCGA Invasive Breast Carcinoma study [, ] with altered P21 and P27 had a tendency to be Her2 negative and had decreased survival. a. Estrogen receptor (ER), progesterone receptor (PR), and ErbB2 receptor (Her2) status in the 11 patients ...

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Discussion

Dietary MR has been identified as a strategy for disease prevention and increased lifespan in experimental animals [10, 19, 31, 32]. We hypothesize that MR may be used as a potential strategy for inhibiting carcinogenesis, and to test this hypothesis, we used the xenograft model for breast cancer by injecting MCF10AT1 cells into nude mice and examined the development tumors in these mice for 12 weeks. Our findings support this hypothesis and indicate that MR inhibits the growth of breast cancer tumors and induces apoptosis.

High levels of IGF1 and insulin in humans have been linked to increased risk of breast cancer [33]. In carcinogenesis, growth factors, such as IGF1 and insulin, stimulate growth and progression of cancer [34]. Therefore, the reduction in plasma IGF1 and insulin by MR suggest that the reduced levels in the insulin/IGF1 axis may inhibit tumor development in this xenograft model.

Methionine functions as the donor for the C2-C4 and amine nitrogen during the synthesis of the polyamines spermidine and spermine. Ornithine, the precursor of putrescine (which in turn is the precursor of spermidine and spermine), is increased in mice on the MR diet. Polyamines are involved in the regulation of proliferation, growth, and survival of cells [35]. High levels of polyamines have been identified in several cancers [36], and the inhibition of polyamine synthesis has been shown to have antitumor effects on skin, colon, and prostate cancers [37, 38]. The increase in ornithine suggests an inhibition of polyamine production. The metabolism of polyamines is strictly controlled and contains two rate-limiting enzymes: ornithine decarboxylase and S-adenosyl methionine decarboxylase [39]. Ornithine, spermidine, and spermine were previously reported to be increased in the liver of MR rats [29]. In the Perrone et al. 2012 study [29], gene expression of the two rate-limiting enzymes of polyamine synthesis in the liver were not inhibited. The conflicting findings regarding polyamine synthesis suggest that either MR effects on the tumor are independent of polyamine synthesis, or alternatively, polyamine function and synthesis are regulated differently in liver and mammary gland.

Taurine is a sulfur-containing amino acid that contributes to cell volume homeostasis and affects apoptosis mechanisms [40]. Taurine plasma levels are decreased in mice on MR. In cancer, volume-sensitive taurine correlates with cisplatin resistance [41]. Recently, taurine has been shown to induce apoptosis through PUMA (p53- up regulated modulator of apoptosis) in human colorectal, ovarian, and cervical cancer cells [41–43]. A decrease in taurine levels resulted in reduced cell volume that induced levels of activated caspase-3, which led to apoptosis in cervical adenocarcinoma cells [44]. Therefore, the MR effects on blood taurine levels (Table 2) could affect PUMA and lead to an increase in apoptosis in the mammary gland tissue and MCF10AT1 tumors of the MR mice, but this would need to be confirmed in future studies. Taurine has been examined as a novel tumor marker in female breast cancer patients. A decrease in serum taurine levels was observed in people with breast cancer or at high risk of breast cancer. Further, an inverse correlation between vascular endothelial growth factor (VEGF,marker for angiogenesis) and taurine concentrations has been demonstrated [45]. Therefore, the connection between taurine, MR, and cancer is convoluted and complicated, and further research is needed to understand the implication of reduced plasma levels of taurine in MR mice.

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Conclusion

In both the xenograft model and in breast cancer cell lines, the mechanism by which MR inhibits tumor progression appears to be a coordinated effort of inhibiting the cell cycle by stimulating the cell cycle inhibitors, P21 and P27. In both the MCF10AT1 tumors and surrounding mouse mammary tissue levels of P21 expression were elevated. Levels of P27 were not significantly changed in the xenograft model, suggesting that the effect of MR is more related directly to inhibiting P21 than P27. Both the metastatic breast cancer cell line (MDA-MB-231) and the immortalized breast cell line (MCF10A) responded to MR similarly to that seen in the xenograft model further supporting the relevance of P21 induction. In particular MDA-MB-231 cells had reduced cell proliferation after 24 h in MRCD media. Additionally, P27 also was elevated in the breast cancer cell lines, suggesting that it too may be involved in the suppression of tumor progression. Further evidence of P21 and P27 importance in patients with breast cancer was revealed in the analysis of the TCGA Breast Carcinoma Cancer study data set [26, 27]. Survival analysis revealed that patients with decreased P21 or P27 expression had reduced survival. This suggests that, if MR increases P21 and/or P27 expression, there may be an increased length of survival in breast cancer patients.

The results from the present study indicate that MR may be a protective agent against cancer progression, but does not completely inhibit cancer progression. Therefore, the application of MR in a clinical setting could be both safe and a feasible option for arresting the progression of breast cancer while providing patients with more time to be treated by conventional methods to eradicate the cancer. Further studies are needed to examine the effect of MR in combination with chemotherapeutic and immunotherapeutic treatments.

Articles from BMC Cancer are provided here courtesy of BioMed Central

Cancer Research: Intravenous Curcumin

STEP ONE

Be informed. Read information below.

STEP TWO

Schedule consultation with R. Douglas Wichman, MD.

STEP THREE

Submit "New Patient Form", current labwork, & imaging results prior to consultation.

STEP FOUR

Establish if you are a candidate for this therapy

in accordance with the Georgia

"Access to Medical Treatment Act".

Used as an Anti-Inflammatory and Anti-Oxidant by Indian doctors, compelling studies reveal that Curcumin can reduce the growth of many types of cancer.

Intravenous Curcumin Benefits:

Faster recuperation from chemotherapy.
Enhances Liver Function
Prevents blood supply to cancerous cells
Compelling evidence Curcumin can prevent breast, prostate, pancreatic, lung and colon cancer.
A natural, safe anti-inflammatory.

What is Curcumin?

Derived from turmeric, a spice that is a mainstay of Indian cooking, Curcumin has been relied upon as an anti-inflammatory to treat arthritis and muscle and joint pain. However, new studies from top cancer researchers have discovered that Curcumin reduces cancer growth and is able distinguish between cancer cells and non-affected cells.

During the last two decades, hundreds of studies from esteemed cancer research centers such as the University of Texas MD Anderson Cancer have shown that Curcumin treatments significantly reduce the rate of cancer cells in human and animal subjects. It attacks cancer at the earliest stages and prevents blood flow to cancer cells, slowing its growth and improving recovery rates.

While it’s not effective against all types of cancer, studies show it’s effective again breast, pancreatic, prostate, lung, and colon cancer.

How it Works:

Curcumin runs interference against toxins and enzymes that contribute to cancer growth. Also, it seeks out abnormal cells and induces apoptosis, a programmed death of a cell. With the cancer’s cells life cycle interrupted, cell growth is inhibited. Many cancer treatments seek to cause cancer apoptosis later in its life cycle, but Curcumin causes this at the G2 stage, a lower stage of cancer. Low-grade cancers tend to grow and spread more slowly than high-grade cancers.

Curcumin also prevents angiogenesis, the formation of blood vessels from existing blood vessels. For cancer to thrive, it needs a blood supply like regular, healthy cells. However, this enhances its spread to other tissues. Curcuminoids prevent angiogenesis to cancer cells, hence starving it of nutrients and slowing its growth.

What’s even more interesting is how Curcuminoids regulate cell growth. It promotes health cell growth while slowing tumor growth. According to a 2005 study in Biochemical Pharmacology, Curcuminoids shut down transcription factors, which regulate growth of tumor cells. In addition, thanks to its anti-inflammatory properties, it prevents inflammatory molecules from activating, which further cancer growth.

Our Intravenous Curcumin Treatment:

The Advanced Rejuvenation Institute specializes in Intravenous Curcumin. Under the supervision of a medical doctor or a licensed RN, an intravenous (IV) treatment of Curcumin is administered. Curcumin is readily absorbed into your system. The treatment is done in our office and will not take up much of your time.

Cancer Research: Gallium Maltolate

STEP ONE

Be informed. Read information below.

STEP TWO

Schedule consultation with R. Douglas Wichman, MD.

STEP THREE

Submit "New Patient Form", current labwork, & imaging results prior to consultation.

STEP FOUR

Establish if you are a candidate for this therapy

in accordance with the Georgia

"Access to Medical Treatment Act".

Image result for gallium maltolate Part of periodic table

Orally administered Gallium Matolate is being used as a treatment for several types of cancer. Already used as a topical analgesic for skin ailments, it’s anti-inflammatory and antiproliferative properties is showing success in clinical trials against some types of cancer including hepatocellular carcicoma, liver cancer, lymphoma and skin cancer.

While gallium does not naturally occur in the body, it’s similar to iron which is necessary for cell division and DNA production. Once it enters iron’s pathways into the cancer cell, it stops ribonucleotide reductase activity which then slows DNA production and cell division. Once the DNA production has slowed in the cancer cell, the malignant cells begin to die off.

Sources and Research:

Gallium Maltolate is a Promising Chemotherapeutic Agent for the Treatment of Hepatocellular Carcinoma

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Tumor treating fields: a novel treatment modality and its use in brain tumors

Abstract

Preclinical Data and Mechanism of Action

Clinical Experience:

Glioblastoma as a Proof of Concept Model

TTFields in Recurrent Glioblastoma

The EF-11 Trial

TTFields in Daily Practice: PriDE Dataset

TTFields in Newly Diagnosed GBM (EF-14 Trial)

Toxicity, Support, and Quality of Life with TTFields

Discussion

Conclusions & Future Perspectives

Repurposing metformin for cancer treatment: current clinical studies

Abstract

INTRODUCTION

PRECLINICAL DATA

EPIDEMIOLOGY

METHODS

CLINICAL TRIALS

DISCUSSION

CONCLUSIONS

Methionine-restricted diet inhibits growth of MCF10AT1-derived mammary tumors by increasing cell cycle inhibitors in athymic nude mice

Abstract

Background

Methods

Results

Conclusion

Electronic supplementary material

Background

Methods

Xenograft model for testing efficacy of methionine restriction

Tumor cell proliferation and apoptosis

Amino acid analysis

Assays on plasma parameters

Methionine restriction in cell culture

In vitro proliferation assay

Real-time PCR

RNA isolation and RT-PCR from paraffin sections

The Cancer Genome Atlas data analysis

Statistical analysis and data presentation

Results

Physiological changes

Methionine restriction inhibits tumor progression

Methionine restriction decreases proliferation while increasing apoptosis

Changes in cell cycle regulators by methionine restriction

Discussion

Conclusion

Used as an Anti-Inflammatory and Anti-Oxidant by Indian doctors, compelling studies reveal that Curcumin can reduce the growth of many types of cancer.

Intravenous Curcumin Benefits:

What is Curcumin?

How it Works:

Our Intravenous Curcumin Treatment:

Hyperbaric Oxygen Therapy

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