The Tumor Inhibitory Effect And Synergistic Effect With Radiotherapy Of Ketogenic Diet With Calorie Restriction On Lung Cancer And Its Mechanisms

Lung cancer is a serious threat to people’s life and health because of its high incidence and mortality.The main treatment methods for non-small cell lung cancer(NSCLC)include surgery,radiotherapy,chemotherapy,molecular targeting therapy,and immunotherapy,but its overall efficacy is still unsatisfactory.Therefore,it is very necessary and urgent to explore the relevant factors that affect the prognosis of NSCLC for accurately evaluating the prognosis,and take targeted measures to improve the therapeutic effect.Increasing evidence suggests that abnormal lipid metabolism is significantly associated with the risk and prognosis of malignant tumors.Dyslipidemias(i.e.abnormal blood lipid levels)is one of the main manifestations of abnormal lipid metabolism in cancer patients,and is closely related to the prognosis of various malignancies.Although previous studies have reported abnormalities in blood lipids of lung cancer patients and their correlation with prognosis,they are all single-center studies,with a small number of patients included,and lack of long-term follow-up data.In view of this,we conducted a prospective,multicenter clinical study that analyzed the serum lipid levels of 2524 patients with lung cancer in 21 centers nationwide.It was found that the incidences of abnormalities in serum triglycerides(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C)were 30.7%,16.4%,52.7%,and 25.9%,respectively.Through long-term follow-up over 5 years and univariable and multivariate Cox regression analysis,it was found that serum TG,TC,HDL-C,and LDL-C were independent prognostic factors for lung cancer patients.According to the Yoden index screening method,we also determined the cutoff values(TG,2.04 mmol/L;TC,5.98 mmol/L;HDL-C,1.34 mmol/L;LDL-C,3.80 mmol/L)for survival prediction of various lipid indicators,and constructed a new combined serum lipids index(CSLI)model in predicting prognosis.CSLI=(0.28894 × HDL-C)-(0.03277 × TG)-(0.03867 ×TC)-(0.05566 × LDL-C).Time dependent AUC analysis confirmed that CSLI has a higher accuracy and stability in predicting survival of lung cancer than any single lipid index.In the subgroup analysis of 300 NSCLC patients undergoing radical radiotherapy,various blood lipid indicators and CSLI also showed good prognostic predictive efficacy,suggesting that blood lipid levels also have a significant impact on the radiation effect of lung cancer.Based on the high incidence of dyslipidemia and its adverse effects on prognosis and radiotherapy in lung cancer patients discovered in the first part of the multicenter clinical study,targeted modulation of dyslipidemia may become a new strategy to improve the therapeutic efficacy of lung cancer and the synergistic effect of radiotherapy,but further research and validation are needed.As we all know,dietary regulation is one of the means to regulate tumor lipid metabolism and blood lipid levels,but it is unclear which dietary pattern has an advantageous regulatory effect.Therefore,we conducted a comparative study on the effects of four common dietary patterns,namely,ad libitum(AL),ketogenic diets(KD),caloric restriction(CR),and letogenic diet with calorie restriction(KR),on blood lipid levels and tumor growth in tumor-bearing mice.The results showed that the KR diet had better blood lipid regulation effects(significantly increasing HDL-C and CSLI,but not TG,TC,and LDL-C)and the best anti-tumor effect.When combined with radiotherapy,the KR diet significantly reduced the tumor growth rate and tumor weight after radiotherapy compared to the AL diet,and prolonged the survival time of tumor bearing mice,indicating that the KR diet has a synergistic effect on radiotherapy.This part of research has confirmed the feasibility of achieving synergistic effects of lipid regulation,tumor suppression,and radiation therapy on lung cancer through dietary regulation.However,little is known about the mechanism of how the KR diet exerts these effects.Exploring the metabolic effects of macro dietary regulation on the micro tumor microenvironment may help us find the answer.Therefore,we further utilized metabolomics and proteomics to screen 84 differential metabolites and 477 differential proteins in tumor tissues of mice fed the AL and KR diets.Differential protein enrichment pathway analysis found that KR diet significantly inhibited cholesterol biosynthesis,binding,and transport,as well as LDL-C and very low density lipoprotein cholesterol(VLDL-C)receptor expression,which may be related to the role of KR diet in lipid regulation.Differential metabolite analysis found that the KR diet significantly increased 5α-cholestanol levels in tumor tissue,combined with metabolite databases and previous relevant literature reports,we speculate that 5α-cholestanol may be one of the key metabolic molecules for KR diet to play a synergistic role in tumor suppression and radiotherapy,but further research is needed to verify it.In order to verify the antitumor and radiosensitization effects of KR dietary metabolites 5α-cholestanol selected in the second part of animal model research,we first applied it alone to A549 and LLC cells and found that it can inhibit the proliferation,migration,and invasion ability of cancer cells,rather than induce G1 phase cell cycle arrest,promote apoptosis,and affect the cytoskeleton α-tubulin and β-actin.Then,we confirmed its radiosensitization effect on LLC and A549 cells through cell cloning experiments.The radiosensitization ratios calculated by the L-Q model were 1.874 and 1.916,respectively.Next,we used flow cytometry,TUNEL,and Western blot to detect that 5α-cholestanol inhibited Bcl-2 expression,and upregulated Bax expression in a dose dependent manner,and promoted radiation induced apoptosis.Immunofluorescence detection found that 5α-cholestanol increased the γH2AX foci of LLC and A549 cells 24 hours after 4Gy irradiation.Finally,Western blot analysis of the DNA damage repair protein revealed that it inhibited the homologous recombination and non homologous terminal junction repair of radiation induced DNA damage in a dose dependent manner.These results confirm that 5α-cholestanol has inhibitory effects on lung cancer cell proliferation and radiosensitization,and may be one of the key molecules for the synergistic effects of KR diet on anti-tumor and radiotherapy.In summary,this study has identified the high incidence of dyslipidemia in lung cancer patients and its adverse effects on prognosis and radiation therapy through three levels and multiple methods.It has also found the lipid regulatory effect,tumor inhibitory effect,and radiation synergistic effect of KR diet on lung cancer bearing mice.The high concentration of 5α-cholestanol in tumor tissue induced by KR diet can promote apoptosis of lung cancer cells and inhibit DNA damage repair caused by radiotherapy,which may be one of the potential mechanisms for its above effects.The results of this study provide a preliminary research basis for the KR diet and5α-cholestanol as a potential metabolic regulatory therapy and radiation sensitization method,but further clinical research and validation are still needed.