Study On The Role And Mechanisms Of A Novel Marine Agent SD118-2in Human Hepatocellular Carcinoma Cell

Because keeping on exploring marine resources, researchers gradually found a new typeof anticancer drugs with clinical values in this decade. Such as Eribulin, a drug extractedfrom a marine microbial, is a compound inhibiting microtubule dynamics, it plays ananticancer effect by combining to microtubule proteins to suppress cell mitosis andtriggering the apoptosis. In our study, the compound we used, SD118-2(structure formulaC18H12N2O2, also is extracted from deep-sea sediments, besides the activity of resistanceto common Penicillium, it also can inhibit a variety of cancer cell proliferation in vitro.The first part: effects of SD118-2on cytotoxic activity, cell cycle and apoptosisPurpose: To detect the effect of compound SD118-2on proliferation, cell cycle andapoptosis of human hematomas HepG2cells. Method: detect the L02of normal humanliver cells HepG2to determine the concentration of half inhibitory. The result ofcalculating of IC50was the reference concentration to deal with liver cancer cells, and thendetect the cell cycle and apoptosis by flow cytometers. Result: MTT assay showed that theSD118-2play a role in HepG2cells’ proliferation after48h later, its role depends on itsconcentration; however it shows low toxicity to normal human liver cells. The result ofIC50is24.3μM, this is the guidance concentration of the compound used to HepG2cellsto continue the subsequent experiments. The result of flow cytometer detection showedthat SD118-2had no significant effect on cell cycle of HepG2, the number of apoptosisrate of cells are less than7%, then we used the transmission electron microscopy toobserve the morphological feathers of HepG2cells and found a very small number ofapoptosis cells, but we found autophagosome in cytoplasm, in which there are remnants oforganelles. Conclusion: The compound SD118-2significantly inhibits normal liver cells’proliferation, but has no significant effect on HepG2cells’ cycle and apoptosis. SD118-2influences cell proliferation in other ways.The second part: The signaling pathways of SD118-2induced autophagy on HepG2Purpose: To study the signaling pathway of SD118-2inducing HepG2cells to producingautophagy further. Method: we use transmission electron microscopy to observe thepresence of autophygosome in morphology; In biochemmistry we detected the expressionlevel of macrophage-related genes by RT-PCR, and verified them at the protein level bywestern blot; determine the role of SD118-2on the signaling pathway of autophagy. Results: after SD118-2affected on HepG2we observed autophagy lysosomes. At themRNA levels we detected the autophagy gene Beclin1and LC3are up regulated, the effectis time dependent, at protein level we also found the autophagy protein beclin1is upregulated and contribute to autophagy-specific protein LC3-I convers to LC3-II; withrespect to the autophagy signaling pathway, we found the total level of mTOR has nosignificant change between medication group and control group; but phosphorylatedmTOR, p-ERK1/2and Bcl-2are reduced after treated by SD118-2. Conclusion: SD118-2induced HepG2cells’ autophagy by inhibiting the MEK/ERK-mTOR signaling pathwayand enhanced type III PI3K/Beclin1signaling pathway.The Third part:3-MA partially attenuate the autophagic effect of SD118-2Purpose: by using the specific autophagy inhibitor3-MA reducing antophage,we observedthe effect of SD118-2on HepG2. Method: we detect the macrophage proteins changesafter treated with3-MA by western blotting, analysis the effect of3-MA using flowcytometer, detect the proliferation level of cells using MTT assay. Results: after treatedwith autophagy inhibitor3-MA, the apoptosis rate is instable, and there is no statisticsignificant, but at protein level the autophagy-specific protein Beclin1is significantlydown-regulated, and the rate of LC3-I converse to LC3-II is also down-regulated; Theresult of MTT assay, after added3-MA, the survival rate of cells is significantly increased.Conclusion: After using3-MA, the ratio of cell apoptosis changes is unstable, throughstatistical analysis, there is no reference value, which indicates that, SD118-2inducedHepG2cells’ autophage has no direct relation with apoptosis. We verified3-MA reducedthe expression of autophagy proteins. The result of MTT assay shows that SD118-2cancause HepG2cells’ autophagy and autophagy death.In summary, A compound named SD118-2(C18H12N2O2), isolated from Penicilliumcommune in a deep-sea sediment sample, has been shown to inhibit the growth of severalcancer cell lines in vitro (data unpublished). In present study, we employed a growthinhibition assay and apoptotic analysis to identify the biological effect and detailedmechanism of SD118-2in human hepatocellular carcinoma (HepG2) cells. SD118-2demonstrated a concentration-dependent inhibitory effect on the growth of HepG2cellsand caused slight cellular apoptosis and significantly induced autophagy. Autophagy wasdetected as early as12h by the conversion of microtubule-associated protein1light chain3(LC3-I) to LC3-II, following cleavage and lipid addition to LC3-I. The pharmacologicalautophagy inhibitor3-methyladenine largely attenuates the growth inhibition and autophagic effect of SD118-2in HepG2cells. Our data also indicated that the autophagiceffect of SD118-2occurs via the down-regulation of the MEK/ERK signaling pathway andthe up-regulated of the class III PI3K/Beclin1signaling pathway.