The Mechanism Of Autophagy And Apoptosis Induced By Reduction Stress In Hepatocellular Carcinoma Cells Was Studied Based On Optical Probe

Liver cancer is one of the most common malignant tumors in the world.China is the worst-hit area of suffering liver cancer,accounting for more than 50%of the cases in the world.Its morbidity and mortality rate are the first in Asia and even the world,and they are increasing year by year,which seriously endangers people's health.Liver cancer belongs to solid malignancy,and hypoxia is one of the important characteristics of solid malignancy.Hypoxia plays an important role in the occurrence,development and recurrence of HCC after chemotherapy by promoting the proliferation/invasion and angiogenesis of HCC cells.Currently,the treatment methods of liver cancer mainly focus on surgery,ablation,liver transplantation,arterial interventional therapy,chemotherapy and combination therapy.Due to the symptoms of liver cancer in the early stage are relatively hidden,early diagnosis is difficult,most patients have been in the middle or late stage when the clinical diagnosis is clear.For patients with advanced liver cancer,chemotherapy is the main treatment method.However,the hypoxic microenvironment of the tumor will reduce the sensitivity of the tumor to chemotherapy and produce drug resistance.Therefore,the study on the anti-tumor mechanism of reductive anti-tumor drugs under hypoxic conditions can provide references for exploring methods that can improve the chemotherapy effect of hypoxic tumor and seeking drugs that can utilize the tumor hypoxic microenvironment.It is an urgent problem to be solved in the treatment of hypoxic tumors.Selenium has anticancer effects,but its anticancer mechanism has not been clearly studied,especially in the low oxygen microenvironment of tumors.Hydrogen selenide?H₂Se?,as a common intermediate metabolite of dietary selenium compounds,is a kind of highly reduced selenide with high volatility and reactivity,which is difficult to be directly detected in cells and animal models.In the past,its role has not been clarified due to the lack of detection methods.In this study,a new mechanism of selenium against liver cancer was revealed with the help of a fluorescent probe that could specifically detect H₂Se.Under the simulated tumor microenvironment?hypoxia?conditions,the intracellular H₂Se level was significantly increased after the HCC cells were treated by pharmacological concentration of sodium selenite,and the increase of H₂Se level occurred before the cell death.The increase of H₂Se level was also accompanied by the increase of NAD?P?H and GSH contents which are two reductive stress markers.The test results of reactive oxygen species showed that the content of H₂O₂ did not change significantly in this process.Therefore,under hypoxic conditions,the intracellular accumulation of H₂Se caused reductive stress rather than oxidative stress.However,under the normoxic conditions,the intracellular H₂Se level did not increase after the pharmacological concentration of sodium selenite acted on liver cancer cells,but H₂O₂ content increased which resulting in the oxidative stress in cells.Further studies found that,under hypoxic conditions,the H₂Se metabolized by sodium selenite could break the disulfide bond in HMGB1 protein to make HMGB1 protein in a reduced state.The reduced HMGB1 was secreted to play a signal molecule role outside the cell and induced autophagy.Autophagy was significantly weakened after H₂Se was cleared.Here,autophagy played a dual role:mild autophagy inhibited apoptosis,while excessive autophagy eventually led to autophagy-related cell death.Differently,under normoxic conditions,H₂Se generated by sodium selenite metabolism was rapidly oxidized to reactive oxygen species,which led to oxidative stress in cells;and oxidized HMGB1 protein was secreted into the extracellular environment and induced cell apoptosis.These results indicate that H₂Se plays a key role in the process of hepatocarcinoma cell death induced by sodium selenite and plays different roles at different oxygen concentrations.Therefore,H₂Se can be used as the research target of selenium treatment for cancer to explore new methods to improve the therapeutic effect of selenium.Natural antioxidants?such as resveratrol,curcumin,celastrol,etc.?are self-protective substances produced by organisms in the long-term evolution process to resist environmental factors,chemical factors and reactive oxygen species generated by their own metabolism,which have anti-inflammatory,antioxidant and free radical scavenging effects.Recent studies have found that resveratrol,curcumin and celastrol have anti-tumor effects in addition to anti-inflammatory and antioxidant effects,but the anti-tumor mechanism is not clear,and the research results are controversial.In this study,with the help of a small molecule fluorescence probe for specifically imaging the NAD?P?H which is a reductive stress marker,we found that the NAD?P?H level was increased after pharmacological doses of resveratrol,curcumin or celastrol treatment of liver cancer cells under the simulated hypoxic microenvironment conditions,and the level of NAD?P?H increased was appeared before cell death.The content of reactive oxygen species had no obvious change.The increase of NAD?P?H led to reductive stress rather than oxidative stress.However,there was no significant change in NAD?P?H level during cell death induced by resveratrol,curcumin or celastrol under normoxic conditions.We speculated that natural antioxidants may induce tumor cell death through reductive stress in tumor hypoxic microenvironment.In order to further study the role of reductive stress,we selected resveratrol as an example and analyzed the anti-liver cancer mechanism of natural antioxidants under hypoxic conditions by means of transcriptome research.Analysis of redox-related genes showed that 94.2%of the redox-related genes were up-regulated after resveratrol treatment of liver cancer cells under hypoxic conditions.GO functional enrichment analysis showed that these genes were mainly involved in the oxidation-reduction process,the cellular redox homeostasis and oxidoreductase activity.Further analysis of the oxidoreductase activity showed that oxidoreductase catalyzed NAD?P?⁺to NAD?P?H after resveratrol treatment of cells under hypoxic conditions,while catalyzed NAD?P?H to NAD?P?⁺under normoxic conditions.This was consistent with the results detected by NAD?P?H fluorescence probe?the level of NAD?P?H increased after the treatment with resveratrol under hypoxia,but not significantly changed under normoxia?.The results of antioxidant enzyme gene analysis showed that all the antioxidant genes with significant differences were up-regulated after resveratrol treated cells under hypoxic conditions.The above results suggested that resveratrol did not induce oxidative stress but induce reductive stress after acting on liver cancer cells under hypoxia.The enrichment results of KEGG signaling pathway showed that resveratrol mainly activated TNF signaling pathway and autophagy related signaling pathway after acting on HCC cells under hypoxic conditions.The analysis also found that after resveratrol treatment of liver cancer cells,there were some genes whose expression was up-regulated under hypoxic conditions but down-regulated under normoxic conditions or genes whose expression was down-regulated under hypoxic conditions but up-regulated under normoxic conditions.These genes with opposite expression in different oxygen conditions could be used as research targets for the reductive stress.In conclusion,this study revealed a new anti-cancer mechanism of selenium and natural antioxidants in the treatment of hypoxic tumors,clarified the role of reductive stress in the treatment of hypoxic tumors,and provided a new research direction for the study of selenium and natural antioxidants in the treatment of hypoxic tumors.It is suggested that the study of reductant/antioxidant antitumor drugs should be carried out under the corresponding hypoxic conditions of tumors,because different oxygen conditions may lead to different research results,which may also be one of the reasons that many theoretical research results are inconsistent with clinical tests.