The Role Of HSF1 In Drug Resistant HEPG2 Cell Which Induced By Adriamycin

BackgroundLiver cancer is the second malignant tumors in the world.There are about 780,000 new cases of human hepatocellular carcinoma(HCC)each year around the world,cases of liver cancer deaths is about 746,000.In all of deaths and new cases,about 50% occur in China.Hepatoblastoma is a type of primary hepatocellular carcinoma.This disease is mainly seen in children but can also be seen in adults.When patients go to see their doctor,half of them have already missed the best surgical treatment period.Combination use of cisplatin and adriamycin chemotherapy is an important treatment to improve their prognosis.However,the pervasive problem of anti-cancer drug resistance is its failure to chemotherapy.The most common mechanism of drug resistance is the emergence of multidrug resistance(MDR).On the surface of tumor cells,P-glycoprotein(P-gp),multidrug resistance protein(MRP),lung resistance protein(LRP)or breast cancer resistance protein(BCRP)etc.are always highly expressed to transport the drug from the intracellular to the extracellular resulting in tumor cross resistant to drugs with multiple structures,functions and targets.Nevertheless,to reverse tumor cross resisitant,there is no good reversal strategy by far.Other common tumor drug resistance mechanisms include the loss or mutation of antitumor drug targets;overexpression or abnormal activation of oncogenes(such as Ras,c-myc,Bcl-2 etc.)or inactivation of anti-oncogene(P53,Rb etc.)directly or indirectly leads to the tumor proliferation improve and anti-apoptotic signals enhance,cancer stem cells enrich,the cell cycle of cancer cell arrest,cellular self-renewal capacity enhance,cell's drugs sensitive reduce,antitumor drug targets lost or change and the alternative survival signaling pathways activate.Due to the existence of tumor heterogeneity,the same tumor cells may have different drug sensitivity and the sensitivity of different tumor cells to the same drug may also different,so that the resistance mechanisms of different tumors may be significantly different.And now the study of chemotherapy resistance of hepatocyte carcinoma is still insufficient and lack corresponding countermeasures in clinical practice.Therefore,it is of great significance to improve the patient's prognosis to explore the mechanism of chemotherapy resistance in hepatocellular carcinoma in order to seek an effective target for reverse transformation therapy.Heat shock factor 1(HSF1)is the most important heat shock transcription factor which plays a central regulatory role in the heat shock response.When cells are stimulated by high temperature,drugs,hypoxia,virus infection,etc.,HSF1 activates and up-regulates the expression of HSPs through HSR,helps proteins to maintain normal structure,reduces the pressure of protein toxicity,and participates in many basic physiological processes in organisms by role of molecular chaperones.Recent studies have shown that HSF1 is overexpressed in a variety of tumors and can regulate the expression of a wide range of genes associated with the malignant progression of tumors through HSR/HSP-dependent or independent manner,promoting tumorigenesis,proliferation,and metastasis.It has been reported that HSF1 activition can regulate the expression of MDR-1 and promote tumor resistance.Targeting its downstream protein HSP90 or HSP70 can increase the chemosensitivity of non-small cell lung cancer(NSCLC)or other cancers.HSF1 activition can up-regulate the expression of HSP70 and regulate the expression of oncogenes c-myc,c-fos,src,Ras and tumor suppressor gene P53,Rb result in cancer cell excess multiplication and anti-apoptotic.Our previous study found that HSF1 was overexpressed in Hep G2,PLC/PRF/5 and other hepatocellular carcinoma cells.And it is higher expressed in in poorly differentiated primary liver cancer tissues than in paracancerous tissues.HSF1 knockdown inhibited the proliferation and colony formation of PLC/PRF/5 cell.However,it is not clear whether direct targeting of HSF1 can effectively reverse the chemotherapy resistance of liver cancer.This study intends to use the acquired Adriamycin resistance Hep G2 cell,Hep G2/ADR/62.5 n M as a chemotherapeutic drug resistance cell model for analyzing the role and mechanism of HSF1 in chemotherapy resistance of hepatoblastoma to provide potential targets for reversing the resistance of liver cancer.Objective To establish an adriamycin-induced human hepatoma resistant cell line Hep G2/ADR/62.5 n M,and use this drug-resistant cell line as a research model to reveal the role and mechanism of HSF1 in its chemotherapy resistance to further reverse the resistance of liver cancer chemotherapy.Drug research provides cell models and experimental evidence.Methods The cell line Hep G2 was cultured by gradually increasing dose of adriamycin in vitro to generate the resistance cell line Hep G2/ADR/62.5 n M.The morphological changes of ADR-resistant cell were observed.The drug-resistance level and cell proliferation of this cell line was analyzed by MTS assay.Flow cytometry was used to detect cell cycle changes and drug efflux ability.Immunoblotting,Real-time PCR,and immunofluorescence were used to analyze gene and protein expression levels of MDR genes.Western-blot,Real-time PCR,and transplantation experiment were used to analyze the CSCs(tumor stem cells)phenotypes of this drug-resistant cell.The cell's survival signal(MAPK,PI3K/ AKT,Jak/STAT3)changes were detected by Western-blot and so do the expression of HSF1,p-HSF1 and HSPs.Lentivirus infection method was used to construct Hep G2-TAP-HSF1 cell which can stably overexpress HSF1 and its control cell which called Hep G2-TAP then analysis the express of MDR1 in Hep G2-TAP-HSF1 by immunoblotting and the effect of overexpressing HSF1 on drug-resistance level of Hep G2 cell by MTS assay.The effect of HSF1 activation on the transcriptional activity of ABCB1 promoter was analyzed by dual-luciferase reporter system and so does the transcriptional activity of HSF1 promoter in Hep G2 and Hep G2/ADR/62.5 n M cell.Stable transfection HSF1/sh RNA in resistant cells to observe the knockdown of HSF1 and the effect of underexpressing HSF1 on the expression of drug-resistant related proteins(MDR1,HSP27),survival signal pathway(MAPK,JAK/STAT3);FACS is used to detect the change of cell cycle;MTS method is used to detect the effect on adriamycin resistance of the cell that had HSF1 deficient;and Q-PCR is used to detect the effect on CSCs phenotype.Results1.Established adriamycin-resistant hepatoma resistant Hep G2/ADR/62.5 n M cell lines;2.Cell morphology of Hep G2/ADR/62.5 n M cell obviously changed and the cell cycle of G0/G1 phase arrested;3.Hep G2/ADR/62.5 n M cell acquired very apparent multidrug resistance phenotype;4.HepG2/ADR/62.5 nM cell acquired very apparent CSCs phenotype;5.Too actived Erk1/2 and Stat3 signal is related to the drug resistance of Hep G2/ADR/62.5 n M cell;6.HSF1 activation enhanced in Hep G2/ADR/62.5 n M cell and so that upregulated the expression of HSP27;7.Activation of HSF1 in Hep G2 cell promotes adriamycin resistance;8.Activation of HSF1 in Hep G2 cell can upregulate MDR1 expression;9.Downregulated HSF1 can't reverse the cell cycle arrest of G1 phase of Hep G2/ADR/62.5 n M cell;10.Downregulated HSF1 can inhibit the activity of ERK1/2 but it can't inhibit the activation of Stat3 signal;11.Downregulated HSF1 can't reverse Hep G2/ADR/62.5 n M cell resist to Adriamycin;12.Downregulated HSF1 can't inhibit the expression of MDR1 in Hep G2/ADR/62.5 n M cell;13.Downregulated HSF1 can't effectively inhibit the molecular marker of CSCs Hep G2/ADR/62.5 n M cell resist to AdriamycinConclusions HSF1 participate in Hep G2/ADR/62.5 n M cell acquired Adriamycin resistance by upregulated MDR1,HSP27 and the activity of ERK1/2,nevertheless,it isn't the master regulator of this cell's drug resistance.