The Resistant Mechanisms Of Bcl-2Inhibitors In Killing Hepatocellular Carcinoma Cells And The Associated Sensitizing Methods

Background and objective:Hepatocellular carcinoma (HCC) is one of the tumors that severely threaten the health ofmankind, which ranks the fifths of morbidity and third of mortality in all tumors, especially inChina, HBV-associated HCC has a very high morbidity. The current therapeutics for HCC aremainly surgical treatment, accompanied by radiotherapy and chemotherapy. However, anumber of patients can not take the surgery because the inconfirmty of the surgical indicationsin clinical diagnosis. Therefore, the efficacy of HCC therapies is not quite satisfactory with avery low five year survival rate and it is urgent to find safe and effective therapies. There arereports indicate that the overexpression of anti-apoptotic Bcl-2family members is associatedwith the grade, prognosis and drug resistance in HCC patients, which are crucial targets forHCC treatment.Bcl-2family members are proteins that regulate the cell survival and programmed celldeath, which consist of three categories: anti-apoptotic family members (Bcl-2, Bcl-xL, Mcl-1,Bcl-w, and A1), pro-apoptotic BH3-only proteins (Bim, Noxa, Puma, Bid, Bad, etc) andexecutors of apoptosis (Bak and Bax). The proper interaction of these proteins is important forhomeostasis and survival for both normal and tumor cells. Nowadays, according to thefunctions and structures of anti-apoptotic proteins, several natural or synthesized chemicalinhibitors have been developed for both basical and clinical experiments. These chemicalsinteract with anti-apoptotic proteins and release the pro-apoptotic proteins, such as Bak, Baxor BH3-like proteins, inducing the occurrence of apoptosis. Several Bcl-2inhibitors havebeen applied in Phase I~III clinical trials, with promising efficacy in both single therapy orcombined usage in multiple kinds of tumors.There are reports indicating that some kinds of tumors are relatively resistant to Bcl-2inhibitors and have trends of drug resistance, among which HCC is one. However, theresistant mechanisms of Bcl-2inhibitors in HCC are not quite clear, the possible reasons may be Mcl-1upregulation, induction of protective autophagy, deficiency of apoptotic-relatedgenes and etc. In our study, we mainly concentrate on the molecular mechanisms of Bcl-2inhibitors resistance in HCC cells and try to identify the sensitizing methods in Bcl-2inhibitors treatment, aiming to improve the efficacy of chemotherapies in HCC.Main contents:Part One The molecular mechanisms of Bcl-2/xL inhibitor ABT-263-inducedMcl-1upregulation induced in HCC cells1. To investigate whether Mcl-1upregulation is one of the mechanisms of ABT-263resistance in HCC cells;2. Detection of Mcl-1mRNA and protein levels upon ABT-263treatment;3. Identification of whether ABT-263activates Mcl-1transcription using luciferasereporter assay, whether ABT-263increases Mcl-1mRNA stability using RNA stability assay;4. Detection whether ABT-263influences the phosphorylation status of Mcl-1proteins,whether these phosphorylations influence Mcl-1expression levels in combination ofinhibitors of associated pathways;5. Detection of whether the inhibitors of corresponding pathways sensitize the anti-HCCeffect of ABT-263, using CCK-8, trypan blue exclusion assay, flow cytometry and etc.Part Two The molecular mechanisms of Bcl-2/xL inhibitor ABT-737-inducedprotective autophagy in HCC cells1. Identificaiton of whether ABT-737sensitivity is correlated with Bcl-2expressionlevels in HCC cells;2. The molecular mechanisms of protective autophagy induced by ABT-737in high Bcl-2HCC cells.Part Three The sensitizing methods and associated mechanisms of Bcl-2inhibitor(-)-gossypol in killing HCC cells1. Identification of whether Hsp90inhibitor17-AAG sensitizes (-)-gossypol in killingHCC cells;2. Exploration of mechanisms of17-AAG sensitizing (-)-gossypol, whether this effect ismediated by inhibiting protective autophagy;3. Whether the inhibition of protective autophagy by17-AAG is mediated by influencingthe stability of autophagy-related proteins; 4. Whether the inhibition of protective autophagy and Mcl-1accumulation by17-AAG ismediated by ERK inactivation;5. The mechanisms of PI3K/mTOR dual inhibitor BEZ-235downregulating Mcl-1, thussensitizing the anti-HCC effect of (-)-gossypol.Methods:1. In vitro culture and passage of HCC cell lines PLC/PRF/5, Hep3B, HepG2and Huh7;2. Detection of mRNA and protein levels using qPCR and Western blot;3. Knockdown or overexpression of associated genes by transient transfections ofsiRNAs or expression plasmids;4. Detection of the mechanisms of ABT-263upregualting Mcl-1mRNA levels byluciferase reporter assay and RNA stability assay;5. Detection of the mechanisms of ABT-263upregualting Mcl-1protein levels by proteinsynthesis inhibition assay and Western blot;6. Identification of growth inhibition, cell death and cell apoptosis using CCK-8, trypanblue exclusion assay and flow cytometry;7. Observation of GFP-LC3dots upon different treatments to reflect the autophagiclevels in the cells using fluorescence microscope;8. Co-immunoprecipitation tests to investigate the possible interations of differentproteins.Results:1. ABT-263-induced Mcl-1upregulation is an important mechanism of HCC resistance;2. ABT-263can increase the mRNA stability of Mcl-1, meanwhile, ABT-263enhancesMcl-1protein stability by two pathways: activation of ERK and JNK-induced Mcl-1Thr163phosphorylation thus increasing Mcl-1protein stability; activation of Akt and inhibition ofGSK-3β to decrease the protein degradation of Mcl-1;3. Inhibition of ERK, JNK or Akt pathways can sensitize the anti-tumor effect ofABT-263in HCC cells;4. ABT-737induces protective autophagy by enhancing ROS generation, which results indrug resistance;5.17-AAG and BEZ-235can sensitize (-)-gossypol in killing HCC cells;6.17-AAG inhibits (-)-gossypol-induced protective autophagy by decreasing ERK activation, instead of influencing autophagy-related protein stabilities;7.17-AAG and BEZ-235partially reverse (-)-gossypol-induced Mcl-1upregulation byinhibiting ERK and mTOR phosphorylation, respectively.Conclusions:HCC cells can resist the anti-tumor effect of Bcl-2inhibitors by upregulation of Mcl-1and induction of protective autophagy. Inhibition of ERK, JNK, Akt or Hsp90can sensitizeBcl-2inhibitors in killing HCC cells.