| Background:Hepatocellular carcinoma(HCC)currently ranks the fourth highest incidence of malignant cancers with death rate of the third in China.There are 466000 newly diagnosed cases with HCC and 422000 cases end up with death every year in China,both of which account for more than 50%of that in worldwide.Owing to characteristics of insidious onset difficulty in early diagnosis,tendency to recurrence and metastasis after hepatectomy,and poor prognosis in advanced stage,HCC is regarded as a huge threaten for human in worldwide.Accordingly it is appealing for more studies focusing on the molecular pathological features and therapeutic targets in hepatocellular carcinoma.Objectives:1.To evaluate expression levels of HDAC1,HDAC2 and HDAC4 in HCC and their adjacent tissue thus to analyse the relationship between expression levels of HDAC1,HDAC2,HDAC4 and clinical pathological characteristics and outcomes of HCC patients,indicating the potential role of HDAC1,HDAC2 and HDAC4 as promising novel biomarkers in HCC.2.To measure effects of JNJ-26481585 on proliferation in HCC both in vitro and vivo and identify related mechanism via a series of cellular and molecular techniques thus to explore potential mechanism of cell cycle arrest and apoptosis.3.To confirm the synergistic manner induced by combination treatment of JNJ-26481585 and sorafenib and identify the mechanism.4.To evaluate toxic effects of combination treatment of JNJ-26481585 and sorafenib.Methods:1.Western Blot and immunohistochemistry assay were used to analyze expression levels of HDAC1,HDAC2 and HDAC4 in liver cancers and their adjacent tissue to compare the differences in HDAC1,HDAC2 and HDAC4 between these tissues.The immunohistochemical results of 111 HCC tissues were used to analyze the relationship between expression levels of HDAC1,HDAC2,HDAC4 and clinical pathological characteristics and outcomes of HCC patients.2.The mRNA expression levels of HDAC1,HDAC2 and HDAC4 were measured by Real-time quantitative PCR in HCC cell lines and QSG-7701 immortalized cell as normal hepatocytes group.3.Cell Counting Kit-8(CCK 8)assay,clone formation assay and EDU assay were employed to detect potential role of JNJ-26481585 on proliferation in HCC in vitro.In addition flow cytometry and western blot assay were performed to explore the potential effects of JNJ-26481585 on cell apoptosis,cell cycle and expression levels of related proteins in HCC.4.The flow cytometry assay was performed to analyse the effect of MK22062HCL on cell cycle distribution of HCC treated with JNJ-26481585 and western blot assay was employed to investigate mechanism of cell cycle arrest induced by JNJ-26481585.5.The flow cytometry assay was performed to analyse the effect of Z-VAD-FMK on apoptosis of HCC treated with JNJ-26481585 and western blot assay was employed to investigate mechanism of apoptosis induced by JNJ-26481585.6.The flow cytometry assay was performed to analyse the effect of SP600125 on apoptosis of HCC treated with JNJ-26481585 and western blot assay was employed to investigate mechanism of apoptosis induced by JNJ-26481585.7.The CompuSyn software was employed to calculate the Combination Index of the combination of JNJ-26481585 and sorafenib thus to comfirm the synergistic manner between JNJ-26481585 and sorafenib,indicating optimal contentration of JNJ-26481585 and sorafenib.8.The flow cytometry assay was performed to analyse cell apoptosis of HCC treated with combination of JNJ-26481585 and sorafenib and western blot assay was employed to identify effect on expression levels of proteins relating apoptosis.9.The flow cytometry assay was performed to explore the effect of SP600125 on apoptosis of HCC treated with JNJ-26481585 and sorafenib and western blot assay was employed to investigate mechanism of apoptosis induced by JNJ-26481585 and sorafenib.10.To evaluate the effect of JNJ-26481585 or combination treatment of JNJ-26481585 and sorafenib on cell proliferation in the HCCLM3 xenograft model.11.HE staining assay was performed to evaluate the major organs in the HCCLM3 xenograft model thus to evaluate toxic effects of combination treatment of JNJ-26481585 and sorafenib.12.The immunohistochemical assay was employed to measure expression levels of phosphorylated JNK,Cleaved-caspase-3,Cleaved-PARP and Ki67 and Tunel assay was used to detect cell apoptosis,exploring potential mechanism of JNJ-26481585 and sorafenib in vivo.Results:1.The protein expression levels of HDAC 1,HDAC2 and HDAC4 in liver cancers tissues were higher than those of adjacent tissues.2.According to immunohistochemical results of 111 HCC tissues,we found that expression levels of HD AC 1,HDAC2 and HDAC4 were closely related with clinical pathological characteristics.High expression level of HDAC 1 was related with large sizes of tumors,portal vein invasion,TNM with high degree and poor histological differentiating degree.High expression level of HDAC2 was related with large sizes of tumors and poor histological differentiating degree.High expression level of HDAC4 was related with large sizes of tumors,TNM with high degree and poor histological differentiating degree.3.Expression levels of HDAC1,HDAC2 and HDAC4 were closely related with overall postoperative survival rates of patients with hepatocellular carcinoma.The overall postoperative survival rates of patients with low expression level of HDAC1,HDAC2 and HDAC4 were longer than those with high expression level.4.The mRNA and protein expression levels of HDAC1,HDAC2 and HDAC4 in HCC cell lines were higher than those of normal hepatocytes.5.JNJ-26481585 facilitated G0/G1 cycle arrest with downregulating AKT and upregulating p21 through PI3K/AKT pathways.6.JNJ-26481585 facilitated apoptosis with activating phosphorylated JNK,upregulating Cleaved-caspase-3,Cleaved-caspase-9,Cleaved-PARP and Bax and downregulating Bcl-xl,Bcl-2 and Survivin through JNK/c-jun pathways.7.The combination treatment of JNJ-26481585 and sorafenib markedly suppressed cell proliferation and induced apoptosis in a synergistic manner both in vitro and in vivo.8.The combination treatment of JNJ-26481585 and sorafenib facilitated apoptosis with activating phosphorylated JNK,upregulating Cleaved-caspase-3,Cleaved-caspase-9,Cleaved-PARP through JNK/c-jun pathways in vitro and in vivo.9.The combination treatment of JNJ-26481585 and sorafenib did not excert toxic effects on major organs in the HCCLM3 xenograft model.Conclusion:1.The protein expression levels of HDAC1,HDAC2 and HDAC4 in liver cancers tissues were higher than those of adjacent tissues.And HDAC1,HDAC2 and HDAC4 were closely related with clinical pathological characteristics.The overall postoperative survival rates of patients with high expression level of HDAC1,HDAC2 and HDAC4 were shorter than those with low expression level.2.HDAC1,HDAC2 and HDAC4 had early-warning value for the prognosis of patients,and were expected to be novel early-warning molecular markers for postoperative patients with hepatocellular carcinoma.3.JNJ-26481585 facilitated G0/G1 cycle arrest with downregulating AKT and upregulating p21 through PI3K/AKT pathways.4.JNJ-26481585 facilitated apoptosis with activating phosphorylated JNK,upregulating Cleaved-caspase-3,Cleaved-caspase-9,Cleaved-PARP and Bax and downregulating Bcl-xl,Bcl-2 and Survivin through JNK/c-jun pathways.5.The combination treatment of JNJ-26481585 and sorafenib facilitated apoptosis with activating phosphorylated JNK,upregulating Cleaved-caspase-3,Cleaved-caspase-9,Cleaved-PARP through JNK/c-jun pathways in vitro and in vivo.6.The combination treatment of JNJ-26481585 and sorafenib did not have toxic effects on major organs in the HCCLM3 xenograft model. |
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