Fasting cycles retard growth of tumors and sensitize a range of cancer cell types to chemotherapy

Abstract

Short-term starvation (or fasting) protects normal cells, mice, and potentially humans from the harmful side effects of a variety of chemotherapy drugs. Here, we show that treatment with starvation conditions sensitized yeast cells (Saccharomyces cerevisiae) expressing the oncogene-like RAS2(val19) to oxidative stress and 15 of 17 mammalian cancer cell lines to chemotherapeutic agents. Cycles of starvation were as effective as chemotherapeutic agents in delaying progression of different tumors and increased the effectiveness of these drugs against melanoma, glioma, and breast cancer cells. In mouse models of neuroblastoma, fasting cycles plus chemotherapy drugs--but not either treatment alone--resulted in long-term cancer-free survival. In 4T1 breast cancer cells, short-term starvation resulted in increased phosphorylation of the stress-sensitizing Akt and S6 kinases, increased oxidative stress, caspase-3 cleavage, DNA damage, and apoptosis. These studies suggest that multiple cycles of fasting promote differential stress sensitization in a wide range of tumors and could potentially replace or augment the efficacy of certain chemotherapy drugs in the treatment of various cancers.

Figures

Fig. 1

Effect of short-term starvation on stress resistance and DXR sensitivity of cancer cell lines. (A) Effect of 24 hours of starvation before treatment on the survival of wild-type (WT) (DBY746) and yeast cells expressing constitutively active Ras (RAS2val19). Starvation was modeled by culturing nondividing yeast cells in water for 24 hours as described (19, 20). Tenfold serial dilutions of cells (from left to right) were spotted on culture plates and incubated at 30°C for 2 to 3 days. For heat shock resistance, cells were incubated at 55°C for 40 min. For oxidative stress resistance assays, cells were diluted to an absorbance at 600 nm of 1 in K-phosphate buffer (pH 6.0) and treated with 100 to 200 mM hydrogen peroxide (H2O2) for 60 min, or cells were treated with 250 mM menadione for 30 min in K-phosphate buffer (pH 7.4). (B) Effect of serum from fasted and ad lib-fed mice on survival of DXR- and CP-treated breast cancer cells (4T1) (n = 3). (C) Effect of starvation (0.5 g/liter, 1% FBS) on cellular proliferation. (D) Effect of starvation on DXR sensitivity of 17 different cancer cells in vitro (n = 3 to 6). Starvation was applied to cells 24 hours before and 24 hours during DXR treatment. Control groups were cultured in glucose (1.0 and 2.0 g/liter, for human and murine cells, respectively), supplemented with 10% FBS. Starved groups were cultured in glucose (0.5 g/liter) supplemented with 1% FBS. Survival was determined by MTT reduction. See the Supplementary Materials for additional data, including the effects of CP. (E) Effect of IGF-1 on starvation-dependent sensitization of cancer cells to DXR. Cells were treated with recombinant human IGF-1 (200 μM) during glucose restriction (0.5 g/liter versus 2.0 g/liter, under 1% FBS), followed by DXR (16 μM) treatment (n = 3). Data are from at least three independent experiments and shown as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test. Murine cells: 4T1, breast cancer; B16, melanoma; GL26, glioma; NXS2 and Neuro-2a, neuroblastoma. Human cells: PC3 and 22Rv1, prostate cancer; MCF-7 and C42B, breast cancer; U87-MG, glioblastoma; HeLa, cervical cancer; LOVO, colon cancer; ACN and SH-SY5Y, neuroblastoma; A431, epidermoid carcinoma; MZ2-MEL, melanoma; OVCAR3, ovarian cancer. See also figs. S1 to S4.

Fig. 2

Effect of fasting on the sensitivity of allograft and xenograft tumors to chemotherapeutic agents in mice. (A to F) Effect of fasting on tumor progression as percent of initial tumor size (A, C, and E) and body weight (B, D, and F) in breast tumors (4T1; n = 12) (A and B), melanoma (B16; n = 11) (C and D), and glioma (GL26; n = 8) (E and F). Fasting in the glioma model was applied only once due to the rapid tumor growth in the control (ad lib, no chemotherapy) group. (G) Effect of fasting on human breast cancer cells (MDA-MB-231) subcutaneously xenografted into nude mice. Four cycles of fasting (48 hours) and/or DXR were performed. Tumor measurements from mice that were fed ad lib and treated with DXR were terminated at day 11 due to death of all mice from DXR toxicity (n = 5). (H) Effect of fasting on human ovarian cancer cells (OVCAR3) subcutaneously xenografted into nude mice. Two cycles of fasting (48 hours) and/or DXR were performed. Tumor measurements from mice that were fed ad lib and treated with DXR were terminated at day 9 due to death of all mice from DXR toxicity (n = 5). In both xenograft models, fasted mice treated with DXR did not experience death from chemotherapy toxicity. *P < 0.05, Student’s t test, compared to control. (I and J) Effect of fasting alone (48 hours for five cycles) in nude mice on tumor progression of a xenografted human neuroblastoma cell line (ACN; n = 7) (I) and on body weight (J). One-way ANOVA with Tukey’s post test [Student’s t test for (C) day 27)]. *P < 0.05; **P < 0.01; ***P < 0.001. All data are means ± SEM. See also figs. S5 and S6.

Fig. 3

Effect of fasting on survival and tumor load in metastatic mouse models of cancer treated with chemotherapeutic agents. (A and B) Effect of 48 hours of fasting on survival of DXR-treated mice with metastatic murine melanoma (B16; n = 9 to 10; P < 0.05) (A) and metastasis (left) and number of organs with metastases (right). (*P < 0.05 compared to control, Student’s t test; mean ± SEM) (B). (C) Effect of 48 hours of fasting on survival of CP-treated mice with metastatic murine breast cancer (4T1) (n = 16 to 18; P < 0.001). (D) Effect of 48 hours of fasting on survival of DXR-treated mice with metastatic murine neuroblastoma NXS2 (n = 5 to 12; P < 0.001). (E and F) Effect of 48 hours of fasting on survival (E) and weight loss (F) of mice with metastatic murine neuroblastoma Neuro-2a treated with a chemotherapy cocktail (n = 6 to 12; P = 0.005). Statistical significance for all survival curves was determined by log-rank test. See also fig. S7.

Fig. 4

Effect of fasting on genes involved in growth and proliferation. (A and B) Microarray analysis on the liver, heart, skeletal muscle, and subcutaneous breast tumors (4T1) from normally fed or fasted (48 hours) mice of cellular proliferation pathways (A) and translational machinery including ribosome assembly/biogenesis (B). (C) Effect of starvation on protein concentration in 4T1, B16, and GL26 cells (n = 3). (D) Effect of starvation on the rate of AHA (methionine analog) incorporation in 4T1 cells (n = 3). Data are means ± SEM [(C) and (D)]. (E and F) Effect of fasting on Akt, S6K, and eIF2α phosphorylation in murine breast cancer cells (4T1) in vivo (E) and in vitro (F). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test. See also fig. S8.

Fig. 5

Effect of fasting on chemotherapy-induced DNA damage. (A to C) Effect of starvation alone and when combined with CP in breast cancer (4T1) (A) and with DXR in melanoma (B16) (B), and with DXR in glioma (GL26) (C) cells as determined by Comet assay (n = 6). The green signal represents intact and fractured DNA. Cells in both groups were cultured in normal glucose (2.0 g/liter) or low glucose (0.5 g/liter), respectively, supplemented with 1% FBS. Drugs were selected to match those in Fig. 2, A, C, and E. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test. Data are from at least three independent experiments and shown as means ± SEM. (D) Effect of starvation on intracellular oxidative stress as estimated by a superoxide marker (DHE) in vitro. Corrected total cell fluorescence was quantified with ImageJ (NIH) and corrected for background fluorescence. *P < 0.05; ***P < 0.001, Student’s t test. (E and F) Effect of fasting on caspase-3 cleavage in allografted 4T1 breast tumors (E) and in 4T1 breast cancer cells cultured under starvation, that is, restricted glucose (0.5 g/liter) and growth factor (1% FBS) concentration (F). See also fig. S9.

Fig. 6

A model for fasting-dependent sensitization of tumor cells to chemotherapy. In response to fasting, glucose, IGF-1, and other pro-growth proteins/factors (including oncogenes) are reduced in the serum. Malignant cells respond to this reduction by activating Akt/S6K. Notably, S6K can also be activated independently of Akt via energy-sensing pathways such as AMPK-mTORC1 (34). These changes lead to an increase in oxidative stress, an increase in DNA damage, activation of caspase-3, and eventually cell death, particularly in the presence of chemotherapy.