Ketogenic diets enhance oxidative stress and radio-chemo-therapy responses in lung cancer xenografts

Abstract

Purpose: Ketogenic diets are high in fat and low in carbohydrates as well as protein which forces cells to rely on lipid oxidation and mitochondrial respiration rather than glycolysis for energy metabolism. Cancer cells (relative to normal cells) are believed to exist in a state of chronic oxidative stress mediated by mitochondrial metabolism. The current study tests the hypothesis that ketogenic diets enhance radio-chemo-therapy responses in lung cancer xenografts by enhancing oxidative stress.

Experimental design: Mice bearing NCI-H292 and A549 lung cancer xenografts were fed a ketogenic diet (KetoCal 4:1 fats: proteins+carbohydrates) and treated with either conventionally fractionated (1.8-2 Gy) or hypofractionated (6 Gy) radiation as well as conventionally fractionated radiation combined with carboplatin. Mice weights and tumor size were monitored. Tumors were assessed for immunoreactive 4-hydroxy-2-nonenal-(4HNE)-modified proteins as a marker of oxidative stress as well as proliferating cell nuclear antigen (PCNA) and γH2AX as indices of proliferation and DNA damage, respectively.

Results: The ketogenic diets combined with radiation resulted in slower tumor growth in both NCI-H292 and A549 xenografts (P < 0.05), relative to radiation alone. The ketogenic diet also slowed tumor growth when combined with carboplatin and radiation, relative to control. Tumors from animals fed a ketogenic diet in combination with radiation showed increases in oxidative damage mediated by lipid peroxidation as determined by 4HNE-modified proteins as well as decreased proliferation as assessed by decreased immunoreactive PCNA.

Conclusions: These results show that a ketogenic diet enhances radio-chemo-therapy responses in lung cancer xenografts by a mechanism that may involve increased oxidative stress.

Figures

Figure 1. The ketogenic diet enhances chemo-radiation responses in H292 lung cancer xenografts

Nude mice (5-16 animals per group) were injected with H292 cells in the flank and tumors allowed to grow to 30 mm3. Treated mice were given 3 × 15 mg/kg carboplatin doses on three consecutive Mondays. Following each of the first two doses of carboplatin the mice were irradiated with three × 2 Gy IR fractions Monday, Wednesday and Friday for 2 weeks (12 Gy total dose) followed one final carboplatin dose on the following Monday. The KD started 2 days prior to the first chemo-radiation dose and continued until two days following the last dose for a total of 16 days. When maximum tumor diameter exceeded 1.5 cm, mice were sacrificed. (A) Tumor volume growth curve estimates using mixed linear regression analysis demonstrate that KD significantly (P<0.05) decreases tumor growth rates when combined with IR and carboplatin-IR, relative to IR and carboplatin-IR alone, respectively. (B) Pair wise group comparisons of Kaplan-Meir survival curves demonstrate that KD significantly enhances IR response (p<0.05), relative to IR alone, and approaches significance for carbo-IR sensitivity (p<0.06). (C) Pair wise group comparisons of animal weights demonstrate that all treatments were well tolerated as demonstrated by a lack of significant weight change (errors omitted for clarity).

Figure 2. A protracted KD increases radiation response in H292 lung cancer xenografts given conventional fractionated radiation

H292 xenografts were grown in nude mice as in Figure 2. Mice (6-8 per group) were treated with KD for the entire treatment time and/or a 61.2 Gy total dose of radiation in 34 × 1.8 Gy fractions three times per week (M, W , F). The experiment was terminated 81 days after the beginning of KD. (A) Tumor growth curve estimates demonstrated that the KD significantly enhances radiation response as assayed by tumor growth rates (p=0.0210). (B) Pair wise group comparisons of Kaplan-Meir survival curves also demonstrated that the KD significantly enhanced radiation response (p=0.0041).

Figure 3. The responses of H292 and A549 lung cancer xenografts to hypo-fractionated radiation were enhanced by the KD

H292 and A549 xenografts were grown in nude mice as in Figure 2. Mice (5-9 per group) were treated with KD for 2 days prior to radiation and continuing for a total of 7 days. Hypo-fractionated radiation (2 × 6 Gy fractions) was delivered on day 3 and 5 of the protocol. Tumor growth curves (A) and Kaplan-Meir survival plots (B) showed enhanced radiation responses in animals with H292 xenografts fed the KD (p

Figure 4. KD combined with radiation increases 4HNE-modified proteins in H292 mouse lung cancer xenografts

Equal protein from H292 xenograft tumor homogenates taken from animals treated as in Figure 3 (N=3 from each group) were blotted onto PVDF membranes and stained with polyclonal antibody against 4HNE-modified proteins. This analysis was repeated twice and a representative blot is shown in panel A. Positive (+) and negative (−) controls represent the immunoreativity derived from H292 cell homogenates treated with and without 100 uM genuine 4HNE for one hour at 37°C. Panel B shows quantification of dot blots by Image J analysis and samples were normalized to the background on each blot. Error bars represent ± 1SEM. One way ANOVA with Newman-Keuls Multiple Comparison Test demonstrated the K+IR was significantly greater than Control, IR, and KD alone (*p

Figure 5. KD combined with fractionated radiation treatment results in decreased immuno-reactive PCNA in tumor tissue

Mouse xenograft tumors treated as in Figure 2 were harvested 24 hours after the final 2 Gy radiation fraction. Tumor protein homogenates harvested from control, KD, IR, and KD + IR treated animals (3 from each group) were separated on denaturing gels, blotted onto membranes, and stained with anti-PCNA or anti-γH2AX antibodies (A). Quantification of dot blots was done by Image J analysis and samples were normalized to the background on each blot. Animals treated with K + IR had a significant reduction in PCNA staining, relative to IR animals (B). There was not a significant difference in γH2AX staining when IR and KD+IR were compared, suggesting that ketogenic diets do not increase DNA damage when combined with radiation (C). Error bars represent ± 1SEM. One way ANOVA with Newman-Keuls Multiple Comparison Test were used to determine which groups were significantly different from control (*p