Mechanism Of Er Stress-induced Therapeutic Resistance To Adrimycin In Hepatocellular Carcinoma

Background and AimsHepatocellular carcinoma (HCC) is a common malignant tumor in digestive tract, which threats people's life and health greatly. In China, most patients are diagnosed at mid-late stage and lose the opportunity of radical surgery. Chemotherapy is the common treatment for mid-late HCC patients. Adrimycin is frequently chosen to treat patients with HCC. But the prognosis is not ideal due to chemoresistance in HCC. Therefore, it is urgent to further study on the mechanism of therapeutic resistance to adrimycin in HCC. Currently, many scholars are investigating the mechanisms of chemoresistance in HCC from different perspectives. But little is known on the mechanism of chemoresistance induced by ER stress in HCC.Our study explored the mechanism of ER stress-induced therapeutic resistance to adimycin using both HCC tissue and HCC cell lines, which might provide a new insight on the mechanisms of chemoresistance in HCC.PartⅠThe relationship between ER stress and PI3K/Akt pathway in HCC tissueMethods30 cases of HCC tissue samples and 28 cases of normal hepatic tissue samples were collected. Expression of Bip and p-Akt were detected using Immunohistochemistry. Western blot was employed to examine the expression of Bip and p-Akt in 5 cases of HCC tissue samples and 5 cases of normal hepatic tissue samples.ResultsData from Immunohistochemistry and Western blot showed that the expression of Bip and p-Akt in HCC tissue samples were much higher than those in normal hepatic tissue samples (P<0.05).The expression of Bip was positively associated with that of p-Akt in HCC tissue samples (P<0.05)PartⅡ:The relationship of ER stress and therapeutic resistance to adrimycin in HCC cellsPhaseⅠEstablishing ER stress model in HCC cellsMethodsSMMC7721 and HepG2 HCC cells were treated with 5μg/ml TM for 0h,2h,12h and 24h, respectively. Expression of Bip at protein level at each time point was detected using Western blot. SMMC7721 and HepG2 HCC cells were treated with 5μg/ml TM for 0h and 2h, respectively. Both the expression of Bip and the splicing of XBP1 at mRNA level were detected using RT-PCR at each time point.Results1 Compared with the Bip protein expression in group 0h, that in SMMC7721 and HepG2 HCC cells treated with TM for 2h,12h and 24h was markedly increased, respectively (P<0.05)2 Compared with the Bip mRNA expression in group 0h, that in SMMC7721 and HepG2 HCC cells treated with TM for 2h was markedly increased (P<0.05)3 Compared with the mRNA expression of XBP1 splicing product XBP1s in group 0h, that in SMMC7721 and HepG2 HCC cells treated with TM for 2h was markedly increased (P<0.05) SMMC7721 and HepG2 cells were divided into the following groups:untreated control group, TM-treated group, adrimycin-treated group and TM+adrimycin-treated group. Growth inhibition rate of each group was detected Using MTT method. Apoptotic rate of each group was detected using Flow Cytometry.Results1. The growth inhibition rate of SMMC7721 cells in TM-treated group, adrimycin-treated group and TM+adrimycin-treated group was 15.1%±0.7%, 57.3%±1.5% and 36.7%±2.1%, respectively. Compared with the growth inhibition rate in control group, that in each treatment group was significantly increased (P<0.05). Compared with the growth inhibition rate in adrimycin-treated group, that in TM+adrimycin-treated group was markedly decreased (P<0.05)2. The apoptotic rate of SMMC7721 cells in control group, TM-treated group, adrimycin-treated group and TM+adrimycin-treated group was 2.1%±0.5%,13.2%±1.6%,47.6%±2.4% and 29.8%±2.0%, respectively. Compared with the apoptotic rate in control group, that in each treatment group was significantly increased (P<0.05). Compared with the apoptotic rate in adrimycin-treated group, that in TM+adrimycin-treated group was markedly decreased (P<0.05)3. The growth inhibition rate of HepG2 cells in TM-treated group, adrimycin-treated group and TM+adrimycin-treated group was 16.2%±0.4%, 54.9%±2.1% and 30.8%±1.4%, respectively. Compared with the growth inhibition rate in control group, that in each treatment group was significantly increased (P<0.05). Compared with the growth inhibition rate in adrimycin-treated group, that in TM+adrimycin-treated group was markedly decreased (P<0.05)4. The apoptotic rate of HepG2 cells in control group, TM-treated group, adrimycin-treated group and TM+adrimycin-treated group was 1.9%±0.8%,11.7%±1.1%,42.1%±2.9% and 26.9%±1.8%, respectively. Compared with the apoptotic rate in control group, that in each treatment group was significantly increased (P<0.05). Compared with the apoptotic rate in adrimycin-treated group, that in TM+adrimycin-treated group was markedly decreased (P<0.05)PartⅢ:The role of PI3K/Akt signaling pathway in ER stress induced therapeutic resistance to adrimycin in HCC cellsPhaseⅠExploring the relationship of ER stress and PI3K/Akt signaling pathway in HCC cellsMethodsSMMC7721and HepG2 HCC cells were treated with 5μg/ml TM for 0h,2h,12h and 24h, respectively. Phosphorylation level of Akt protein at each time point was detected using Western blot.ResultsCompared with the phosphorylation level of Akt protein in group 0h, that in SMMC7721 and HepG2 HCC cells treated with TM for 2h,12h and 24h was markedly increased, respectively (P<0.05)PhaseⅡInvestigating the role of PI3K/Akt signaling pathway in ER stress induced therapeutic resistance to adrimycin in HCC cells MethodsSMMC7721 cells were divided into the following groups:untreated control group, TM-treated group and and LY294002 (Wortmannin)+TM-treated group. Phosphorylation level of Akt protein in each group was detected using Western blot.SMMC7721 cells were divided into the following groups:untreated control group, adrimycin-treated group, TM+adrimycin-treated group and LY294002 (Wortmannin)+TM+adrimycin-treated group. Growth inhibition rate of each group was detected Using MTT method. Apoptotic rate of each group was detected using Flow Cytometry.Results1 Phosphorylation level of Akt protein in TM-treated group was much higher than that in both control group and LY294002 (Wortmannin)+TM group (P<0.05) There was no difference in the phosphorylation level of Akt protein between control group and LY294002 (Wortmannin)+TM group (P>0.05)2 The growth inhibition rate of SMMC7721 cells in control group, adrimycin-treated group, TM+adrimycin-treated group and LY294002+TM+adrimycin-treated group was 58.1%±1.6%,36.8%±1.4% and 46.0%±1.6%, respectively. Compared with the growth inhibition rate in control group, that in each treatment group was significantly increased (P<0.05). Compared with the growth inhibition rate in adrimycin-treated group, that in TM+adrimycin-treated group was markedly decreased (P<0.05). Compared with the growth inhibition rate in TM+adrimycin-treated group, that in LY294002+TM+adrimycin-treated group was significantly increased (P<0.05). Compared with the growth inhibition rate in adrimycin-treated group, that in LY294002+TM+adrimycin-treated group was also significantly decreased (P<0.05) 3 The apoptotic rate of SMMC7721 cells in control group, adrimycin-treated group, TM+adrimycin-treated group and LY294002+TM+adrimycin-treated group was 2.7%±0.7%,50.3%±3.0%,28.5%±1.9% and 35.4%±2.3%, respectively. Compared with the apoptotic rate in control group, that in each treatment group was significantly increased (P<0.05). Compared with the apoptotic rate in adrimycin-treated group, that in TM+adrimycin-treated group was markedly decreased (P<0.05). Compared with the apoptotic rate in TM+adrimycin-treated group, that in LY294002+TM+adrimycin-treated group was significantly increased (P<0.05). Compared with the apoptotic rate in adrimycin-treated group, that in LY294002+TM+adrimycin-treated group was also significantly decreased (P<0.05)4 The growth inhibition rate of SMMC7721 cells in control group, adrimycin-treated group, TM+adrimycin-treated group and Wortmannin+TM+ adrimycin-treated group was 58.5%±1.6%,37.2%±0.6% and 46.4%±0.7%, respectively. Compared with the growth inhibition rate in control group, that in each treatment group was significantly increased (P<0.05). Compared with the growth inhibition rate in adrimycin-treated group, that in TM+adrimycin-treated group was markedly decreased (P<0.05). Compared with the growth inhibition rate in TM+adrimycin-treated group, that in Wortmannin+TM+adrimycin-treated group was significantly increased (P<0.05). Compared with the growth inhibition rate in adrimycin-treated group, that in Wortmannin+TM+adrimycin-treated group was also significantly decreased (P<0.05)5 The apoptotic rate of SMMC7721 cells in control group, adrimycin-treated group, TM+adrimycin-treated group and Wortmannin+TM+adrimycin-treated group was 2.2%±0.6%,48.1%±2.7%,27.3%±1.8% and 37.6%±1.9%, respectively. Compared with the apoptotic rate in control group, that in each treatment group was significantly increased (P<0.05). Compared with the apoptotic rate in adrimycin-treated group, that in TM+adrimycin-treated group was markedly decreased (P<0.05). Compared with the apoptotic rate in TM+adrimycin-treated group, that in Wortmannin+TM+adrimycin-treated group was significantly increased (P<0.05). Compared with the apoptotic rate in adrimycin-treated group, that in Wormannin+TM+adrimycin-treated group was also significantly decreased (P<0.05)Conclusions1 In HCC tissue, ER stress is present and PI3K/Akt pathway is activated. The presence of ER stress is closely associated with the activation of PI3K/Akt pathway in HCC tissue.2 ER stress induces therapeutic resistance to adrimycin in HCC cells.3 The activation of PI3K/Akt pathway is involved in ER stress-induced therapeutic resistance to adrimycin in HCC cells. Blocking the PI3K/Akt pathway partially abrogated the ER stress-induced therapeutic resistance to adrimycin in HCC cells...