Could fasting while receiving chemotherapy be beneficial? 18 July 2014 Michaela Onstad MD, MPH Kristina Stemler PhD

Starvation-dependent differential stress resistance protects normal but not cancer cells against high-dose chemotherapy Lizzia Raffaghello, Changhan Lee, Fernando M. Safdie, Min Wei, Federica Madia, Giovanna Bianchi, and Valter D. Longo PNAS 2008

Quick Summary Aim – Constitutively active oncogenes prevent effective differential stress resistance Methods Stress yeast, primary cancer cell lines with H2O2 and chemo after STS Stress cancer-bearing mice and controls with chemo after STS Results (to come) Where is the field now?

Differential stress resistance against oxidants and genotoxins in yeast Deletion mutants in part mimic CR but also undergoing STS (in water media/absence of glucose prior to oxidizing agent treatment) Menadione – superoxide generating agent In A see: Activation of Oncogenic Ras (prevents the protection of CR) (A) Survival of nondividing (day 3) STS-treated yeast cells deficient in Sch9 and/or Ras2 (sch9 and sch9 ras2), or expressing constitutively active RAS2 val19 after treatment with H2O2 (30 min) or menadione (60 min). Serial dilutions (10-, 102-, and 103-fold dilutions, respectively, in the spots from left to right) of the treated cultures was spotted onto YPD plates and incubated for 2–3 days at 30°C. (D) Differential stress resistance (DSR) to chronic Cyclophosphamide treatments in mixed yeast cultures: sch9 and sch9 RAS2val19. To model the mixture of normal and tumor cells in mammalian cancer, sch9 and sch9 RAS2val19 were mixed in the same flask and incubated for 2 h at 30°C with shaking. The initial sch9:sch9 RAS2val19 ratio, measured by growth on selective media, was 25:1. Mixed cultures were then treated with either CP (0.1 M) or MMS (0.01%). Viability was measured after 24–48 h by plating onto appropriate selective media that allows the distinction of the two strains. Data from three independent experiments are shown as means SD.

In vitro DSR to H2O2 treatment Rat glioma cell line Human glioma cell line Human neuroblastoma cell line The reduced glucose is similar to how to mimic STS in cell culture (and in low serum) Cell lines were tested for glucose restriction-induced DSR. Cells were incubated in low glucose (0.5 g/liter, STS), normal glucose (1.0 g/liter), or high glucose (3.0g/liter), supplemented with 1% serum, for 24 h. Viability (MTT assay) was determined after a 24-h treatment with 0–1,000M H2O2. All data are presented as means SD. P values were calculated with Student’s t test (*, P 0.05; **, P 0.01; ***, P 0.001; of 0.5 and 1.0 g/liter vs. 3.0 g/liter glucose).

In vitro DSR to cyclophosphamide treatment Human glioma cell line Human neuroblastoma cell line (A) Glucose restriction induced DSR. Cells were incubated in either low glucose (0.5 g/liter, STS) or normal glucose media (1.0 g/liter), supplemented with 1% serum, for 24 h. Cells were then treated with CP (6–12 mg/ml) for 10 h, and viability was determined (MTT assay) (n 9). (B) Serum restriction-induced DSR. Cells were incubated in medium containing either 1% (STS) or 10% serum for 24 h, followed by a single CP treatment (15 mg/ml) for 10 h. Cytotoxicity was determined by the LDH assay (n = 12) it’s a colorimetric assay. All data are presented as means SD. P values were calculated with Student’s t test (*, P0.05; **, P0.01; ***, P 0.001). CP treatment (15 mg/ml)

Short-term starvation protects against high dose etoposide, in vivo (C) CD-1 mice were treated (i.v.) with 110 mg/kg etoposide with (STS/Eto, n 5) or without (Eto, n 5) a 60-h prior starvation. (D) Percent weight loss after etoposide treatment in STS-treated (n 5) or untreated (n 5) CD-1 mice. Asterisks indicate the day at which all mice died of toxicity. (G) Comparison of survival of all of the mice that were either prestarved (STS/Eto) or not (Eto) before etoposide injection. The survival of all STS-treated (n28) and untreated (n 37) mice from all genetic backgrounds above (A/J, CD1, and Nude-nu) has been averaged (***, P 0.05).

DSR in cancer-bearing mice (A and B) Survival of neuroblastoma (NXS2)-bearing mice. All mice were inoculated (i.v.) with 200,000 NXS2 cells per mouse. The different groups were treated as follows: NXS2 (control group, 16 mice), i.v. inoculation with NSX2 tumor cells on time 0; NXS2/STS (STS, 8 mice), i.v. inoculation with NSX2 tumor cells at time 0 followed by a 48-h starvation; NXS2/STS/Eto (STS/Eto, 16 mice), i.v. inoculation with NSX2 tumor cells at time 0, followed by a 48-h starvation, followed by an i.v. injection with 80 mg/kg etoposide and feeding at 48 h; NXS2/Eto (Eto, 6 mice, two deaths caused by the injection procedure), i.v. inoculation with NSX2 tumor cells at time 0, followed by an i.v. injection of 80 mg/kg etoposide at 48 h. The survival period of the NXS2 (control) and NXS2/STS/Eto groups was significantly different (P0.001), whereas that of the NXS2 (control) and Eto groups was not (P0.20). In addition, the survival periods of the NXS2/STS/Eto and NXS2/Eto groups were not significantly different (P 0.12). (C) Procedure for the in vivo experiment.

Model for DSR in response to STS In normal cells, downstream elements of the IGF1 and other growth factor pathways, including the Akt, Ras, and other proto-oncogenes, are down-regulated in response to the reduction in growth factors caused by starvation. This down-regulation blocks/reduces growth and promotes protection to chemotherapy. By contrast, oncogenic mutations render tumor cells less responsive to STS because of their independence from growth signals. Therefore, cancer cells fail to or only partially respond to starvation conditions and continue to promote growth instead of protection against oxidative stress and high-dose chemotherapy.

Effect of diet on serum IGF-1 in murine models All of the diets in D will see reduction in glucose levels too D) Serum IGF-1 levels for mice once 80% bodyweight was reached. Red line represents mean; *** p b 0.001, ANOVA, Tukey's multiple comparison. E) Serum IGF-1 levels after 9 days of ad lib feeding. Lines represent mean; *p b 0.05,*** p b 0.001, ANOVA, Tukey's multiple comparison compared to AIN93G control. two different low protein diets (20% P-1 and 20% P-2), a diet low in carbohydrates but high in protein (LCHP), a protein deficient diet (0% P) and a high fat diet (60% HF) and ketogenic diet (90% HF) Other areas of research then: Protective effects of DR on chemo side effects These lead to implications for human research therefore, what has been done in human research? Brandhorst et al., Exp Gerontology 2013

Experiments on DR and genetic or chemical alteration of nutrient sensing pways. Aging and stress resistance are related Long term effects in humans are not really known (nutrient restriction is a form of stress resistance) Blanket DR is bad because it decreases functional immunity and wound healing! Experimental DR leads to Increased stress resistance (ROS, DNA damage, hypoxia, ER stress) However little evidence for a role of increased OX stress resistance alone leading to increased longevity… Fontana et al., Science 2010

Conserved nutrient sensing pathways regulating longevity The TxnFs are related to anti-aging and are activated by DR and regulate metabolic activities that increase life span Nutrient presence sequesters the TxnFs in the cytoplasm to keep them inactive Major focus on the GH/IGF pathway and dietary interventions, show work in murine model (plug for Energy Balance Center here?) Fontana et al., Science 2010

Fasting and cancer treatment in humans: A case series report Fernando M. Safdie, Tanya Dorff, David Quinn, Luigi Fontana, Min Wei, Changan Lee, Pinchas Cohen, and Valter D. Longo Aging 2009

Quick Summary Aim- Determine feasibility and effect of fasting in cancer patients undergoing chemotherapy Methods Descriptive case series 10 patients with different malignancies Voluntary fasting during chemotherapy Reported side-effects Effects on tumor volume, serum tumor markers Results Fasting was well tolerated Fasting was associated with reduced fatigue, weakness and GI side effects No adverse effects on tumor volume or serum tumor markers

Cases 7 women and 3 men, ages 44-78 were reported on in this case series. 4 suffered from breast cancer, 2 patients had prostate cancer, 1 esophageal, 1 lung, 1 uterine, 1 ovarian cancer.

Individuals fasted for a total of 48-140 hours prior to chemotherapy and/or 5-56 hours following chemotherapy 4 of the patients fasted during all cycles of chemotherapy received. 6 of the patients fasted for some of the cycles, but did not fast for the remaining cycles. The chemotherapy regimens were varied. Most contained a taxane or a platinum, or both.

Fasting Regimens Varied in length 48-140 hours prior to chemotherapy 5-56 hours following chemotherapy What was consumed during fasting was not well defined for most patients Diet during non-fasting chemotherapy cycles was not described

Measurement of Side Effects Survey was mailed to participants. They were instructed to complete it 7 days after each treatment cycle

Measurement of Side Effects Compares self-reported side effects from the closest 2 cycles compared to one another. Report lower fatigue and weakness in the fasting cycles compared to non-fasting cycles ? Is this the best way to compare

Other Reported Effects Complete blood count Absolute neutrophil count Hemoglobin Platelets Tumor markers Tumor size on imaging studies For each case, a graph of variables from CBC is given. For some cases, it is suggested that bone marrow suppression is mitigated during fasting cycles. Tumor markers overall declined for all of the patients receiving chemotherapy On imaging, tumor size tended to improve or remain stable. One patient had progression of disease during chemotherapy.

Adverse Effects of Fasting No specific survey described to detect adverse side effects of fasting “Minor complaints that arose during fasting included dizziness, hunger, and headaches at a level that did not interfere with daily activities” Weight loss “Weight lost during fasting was rapidly recovered in most of the patients and did not lead to any detectable harm” Other measures of nutritional status?

Conclusions Fasting was well tolerated Self-reported reduction in chemotherapy-induced side effects Fasting did not prevent chemotherapy-induced reduction of tumor volume or tumor markers

Current trials Mayo Clinic (NCI) All cancer types, goal recruitment 12 patients Fast 24 hours before day 1 of course 2 of chemo. If well tolerated, escalate fasting by 12 hours for each subsequent course for up to 3 courses in the absence of unacceptable toxicity Evaluate weight changes Get a preliminary estimate of the longest fewasible fasting perod prior to chemo Evaluate toxicity profile of chemo during fasting Evaluate changes in glucose, insulin, IGF1

Current Trials University of Southern California Patients with advanced urothelial and pulmonary malignancies (on gemcitabine, cisplatin) Goal 70 patients Groups Group I- patients fast for 24 hours prior to chemo Group II- Fast for 48 hours prior to chemo Group III- Fast for 72 hours prior to chemo Group IV- Modified 48 hour fast with minimal caloric intake Evaluate toxicity, change in plasma markers

Current Trials Charite University, Berlin 30 women with breast or ovarian cancer Randomized controlled cross-over trial Fast for 60-72 hours during the first half of a 4-6 cycle regimen of chemo or fast for the same time period during the second half of chemo Evaluate QOL using FACT-O Evaluate fatigue, intensity of adverse affects, and laboratory assessments

Further discussion What is the real world application, would patients be amenable? Can we utilize fasting induced chemoprotection in the clinic? Weight loss goal or length of time? Current standard of care for diet during chemo? Risks related to fasting that should be considered? Endpoint of patient impact? Controversy of findings?