Distinct Roles Of Autophagy In Initiation And Progression Stage Of Hepatocellular Carcinoma

【Background and Object】Hepatocellular carcinoma(HCC) is the sixth most frequently diagnosed cancer and thethird leading cause of cancer mortality worldwide. China is a high incidence area of HCC,and HCC mortality accounts for2nd of cancer cause of death. Many researches suggestthat hepatocarcinogenesis is a long and complicated process, and has association withmany factors. The major risk factor for HCC is chronic liver inflammation due to manypathogenic factors. However, the mechanism of the relationship between HCC risk factorsand tumorigenesis has not been fully clarified. Among the numerous known factors,macroautophagy (autophagy hereafter) obtains increasing attention.Autophagy is an evolutionarily conserved self-digestive mechanism, which involvesaggregation of double-membrane, formation of autophagosome which engulfs the targetedregion, fusion with lysosomes and digestion in autolysosomes. Under starvation or otherstresses, autophagy is required for cell survival to eliminate damaged cellular componentsand maintain nutrition and energy homeostasis. Autophagy has close relationship withmany biologic or pathologic phenomena including various cancers. Many researchessuggest that autophagy has tumor-suppressive function. Autophagy inhibition couldpromotes various spontaneous cancers. However, some researches exhibited thatautophagy is essential to many oncogene-mediated tumorigenesis. Moreover, autophagycould aid tumor cells to accommodate their harsh microenvironment. These raises aparadox that autophagy inhibits tumorigenesis while promoting tumor development.Actually, cancer development is a multiple-step process, which may be the reason for theparadoxical role of autophagy in cancer.In the process of cancer development, the genetic and epigenetic alteration, whichresult from many damaged factors, initiate and trigger the transformation from normal cellto cancer cell. This process could be divided between initiation and progression stage. Inthe initiation stage, normal cells gradually evolve to incipient cancer cells, which couldslow but consistent proliferation. In the progression stage, incipient cancer cells further evolve to maligant cancer cells, which ultimately lead to the formation of maligant tumor.As a protect mechanism, autophagy could affect many tumorigenesis-associated factors. Inthe different stage of tumorigenesis, autophagy may plays a dynamic role accompanied bythe transformation of cells and the alteration of tumorigenesis-associated factors.In our study, SD rats were used chloroquine(CQ) to inhibit autophagy in the initiationand progression stage of N-diethylnitrosamine(DEN)-induced hepatocarcinogenesis,respectively. We attempted to explore the different role of auophagy in initiation andprogression stage of HCC, and further investigated the underlying mechanism. Thisresearch could offer a new insight into our understanding of how autophagy affecttumorigenesis, and will contribute to the appropriate application of anti-autophagy cancertherapy.【Methods】1,Establishing the rat model of DEN-induced hepatocarcinogenesis, and divide its processbetween initiation and progression stage.2,Using western blot(WB) and electron microscope(EM) to explore the influnence ofDEN on the autophagy of rat liver cell, and examine the effect of CQ combination.3,Using CQ to inhibit autophagy in the initiation and progression of HCC, respectively.Distinguish the different influence of two kinds of treatment on hepatocarcinogenesis.4. Using immunohistochemistry(IHC), Tunel staining, WB and qPCR to explore theinfluence of autophagy inhibition on apoptosis, cell proliferation, DNA damage andinflammatory cytokins expression of liver cells in the DEN-treated rats.5. Using direct and indirect reactive oxygen species(ROS) indicator to exam the influnenceof autophagy inhibition on ROS accumulation in the DEN-treated rat livers.6. Using antioxidant BHA to testing the impact of ROS accumulation on thetumor-promotive effect of autophagy inhibiton in the initiation stage of HCC.7. Using WB, EM to varify the inhibited effect of CQ on autophagy level of tumor cells,and explore the influence of autophagy inhibition on cell proliferation and apoptosis oftumor cells by IHC and WB.8. Testing the impact of autophagy inhibition on ROS accumulation of tumor cells, andusing BHA to testing the influence of ROS accumulation on the tumor-suppressive effectof autophagy inhibition in the progression stage of HCC.9. Testing the important intermediates of Tricarboxylic acid cycle, including citrate,isocitrate and acetyl-CoA, and ATP level of tumor cells to analysis the influence of autophagy inhibition on tumor cell metabolism.【Results】1. Initiaion stage of HCC is the period from0to9weeks, and progression stage is theperiod from10to17weeks. Autophagy inhitibiton exerts tumor-promotive effect ininitiation stage and tumor-suppressive effect in progression stage.2. DENcould induce autophagy in the liver of rats, and CQ inhibits this auophagy flux.3. Autophagy inhibition has no impact on DEN-induced cell death in the rat liver, butsignificantly promotes cell proliferation, DNA damage and the expression of IL-1β, TNFαand IL-6.4. Autophagy inhibition enhances DEN-induced ROS accumulation in the rat livers.5. Antioxidant BHA could restrain the tumor-promotive effect of autophagy inhibition ininitiation stage of HCC.6. Autophagy inhibition by CQ could decrease cell proliferation and increase cell apoptosisin the tumor during the progression stage of HCC.7. Autophagy inhibition also enhances ROS accumulation in the tumor. However, ROSaccumulation only involves autophagy inhibition induced cell apoptosis, which also hasROS-independent factors resulting from autophagy inhibition.8. Autophagy inhibition significantly suppressed tumor cell metabolism.【Conclusion】Our research demonstrates that autophagy inhibition promotes tumorigenesis in theinitiation stage of HCC, and suppresses tumorigenesis in the progression stage. In theinitiation stage, autophagy could suppress tumorigenesis by inhibiting cell proliferation,decreasing DNA damage and expression of inflammtory cytokins. Inhibition of ROSaccumulation is the key factor of tumor-suppressive effect of autophagy in the initiationstage of HCC. However, in the progression stage of HCC, autophagy could support cellproliferation and inhibit apoptosis in the tumor and exerts tumor-promotive effect.Autophagy protect liver cells in the initiation stage and tumor cells in the progression stage.This lead to the phenomenon that autophagy plays distict roles in the initiation andprogression stage of HCC.