Proteomic Study On SMMC-7721 Hepatocellular Carcinoma Cell Line Induced By Camptothecin

ObjectiveCamptothecin(CPT) was originally isolated from the bark of the Chinese tree, Camptotheca acuminate(Nyssaceae.family),and is a DNA Topoisomerase I inhibitor that reversibly binds to Top 1 cleavage complexes(Top lccs).Several CPT derivatives such as Irinotecan and Topotecan are now commonly used in the treatment of certain human cancers,especially hepatocellular carcinoma.The conversion of Top lccs into irreversible Top 1 covalent complexes results in DNA damage,the induction of DNA repair mechanisms,and changes in expression for cell-cycle proteins related to growth arrest and apoptosis,and defects in key factors of these pathways are essential to the oncogenic process,and are related to the effects of Top 1 inhibitors.For example,CPT can activate Poly(ADP-ribose) polymerase(PARP) which is involved in DNA-repair mechanisms and plays a crucial role in maintaining gene stability.PARP-deficient and knockout cells have been shown to be hypersensitive to CPT and more susceptible to oncogenic transformation. Furthermore,administration of a PARP inhibitor increased the anti-tumor activity of CPT derivatives in a mouse model.Inhibitors of CHK1,CHK2,and NF-κB,which are involved in CPT-induced cellular pathways,have also shown enhanced cytotoxicity when combined with CPT treatment.It is therefore essential to discover new regulation factor induced by CPT in the model system,in order to fulfill the specific regulation network induced by cytotoxic drugs in cancer cell and build theoretical foundation for cancer therapy.The goal of the present study was to use proteomics to identify proteins that change expression following treatment with CPT in the hepatocellular carcinoma cell line,SMMC-7721.Galectin-1 is a member of the lectin family which has many receptors and is involved in mediating cell adhesion and proliferation,as well as tumor metastasis, apoptosis,immune escape,and angiogenesis.In normal liver tissue,Galectin-1 expression is barely detectable,however,it was found to be overexpressed in primary hepatocellular carcinoma cells.Moreover,administration of siRNA targeted to galectin-1 gene increased the sensitivity of siRNA-expressing cells to the cytotoxic effects of SN-38,a CPT derivative,in the glioma cell line,U87 MG.Expression of recombinant Galectin-1 partially abrogated the increase in sensitivity observed with siRNA expression.The proximal promoter region of the Galectin-1 gene is extremely GC-rich.Therefore,we hypothesized that DNA methyltransferases(DNMTs) may be involved in CPT-induced signaling and affect cell proliferation inhibition and apoptosis induced by CPT.MethodsSMMC-7721 cells were treated with 55μg/ml CPT for 6 h,then total protein of CPT treated group and control group were collected and separated by two dimensional electrophoresis(2-D).Several spots differentially expressed in two groups were analyzed by a mass spectrometer MALDI-TOF-MS/MS(ABI 4700).Then Quantitative real-time PCR(RT-PCR) and Western blot analysis confirmed mRNA and protein expression changes in Galectin-1 respectively.DNMTs activity of SMMC-7721 cell line after CPT treatment was analyzed by ELISA assay.To evaluate whether regulation of DNMTs is associated with CPT-induced cell death,SMMC-7721 cells were pre-treated with the DNMT inhibitor,5-aza-2'deoxycytidine(DAC),and cell proliferation inhibition and apoptosis were evaluated after CPT treatment.Finally cell cycle distributions were analyzed after DAC treatment.ResultsIsolation and Identification of Galectin-1Addition of CPT to SMMC-7221 cells resulted in concentration dependent growth inhibition.CPT treatment(55μg/ml) was used to generate samples for two-dimensional electrophoresis.The silver-stained gel provided a comprehensive view of the major proteins expressed in the hepatocellular carcinoma cell line,SMMC-7721. Although some horizontal streaks persisted,protein spots were generally well resolved. 2D-gels for both treated samples and control gels consistently provided good sensitivity and high reproducibility with detection of more than 700 silver stained proteins.High efficiency matching was achieved in each group.A specific protein that showed decreased expression following treatment with CPT,was subsequently identified as Galectin-1 using MALDI-TOF-MS/MS analysis.Confirmation of Galectin-1 IdentificationTo confirm the changes in Galectin-1 protein expression observed by 2D-gel electrophoresis,both mRNA and protein expression levels were evaluated following CPT treatment using Quantitative Real-Time RT-PCR and Western blot analysis,respectively. Based on data from real-time RT-PCR analysis,6 h after treatment with CPT(55μg/ml) mRNA expression of Galectin-1 was significantly down regulated.However,24 h after treatment with CPT,Galectin-1 mRNA expression returned to its baseline level.Using the same time points,Western blot analysis of protein expression levels of Galectin-1 in SMMC-7721 cells following CPT treatment were consistent with the data from real-time RT-PCR analysis.Staining forβ-actin was used as a control.CPT induces down-regulation of DNMTsThe proximal promoter region of the Galectin-1 gene is extremely GC-rich. Therefore,we hypothesized that DNA methyltransferases(DNMTs) may be involved in CPT-induced signaling.The activity of DNMTs decreased with incubation of SMMC-7721 cells with CPT.Significant differences in the activity of DNMTs were observed between: control samples and samples collected at the 3rd h timpoint,between the 3rd h and 9th h timepoint samples,and between the 9th h and 24th h timepoint samples.These results confirmed our hypothesis that DNMTs were involved in CPT-induced cellular responses.Enhanced cell cytotoxicity after DAC and CPT co-treatmentTo evaluate whether regulation of DNMTs is associated with CPT-induced cell death,SMMC-7721 cells were pre-treated with the DNMT inhibitor,5-aza-2'deoxycytidine (DAC),and cell viability and apoptosis were evaluated after CPT treatment.MTT and Flow Cytometry assays were used to detect differences in cell proliferation and apoptosis in SMMC-7721 cells with and without DAC pre-treatment followed by incubation with CPT for 48 h.DAC treatment alone could not affect cell proliferation and apoptosis in SMMC-7721 compared to blank control group,but inhibition of DNMTs by DAC followed by administration of CPT resulted in a significant inhibition of proliferation and enhancement of apoptosis in SMMC-7721 cells compared to CPT treatment alone.DAC and cell cycle distributionsCPT is a cell cycle specific drug,mainly worked in S phase.DAC could lead cell cycle arrest in S phase and G2/M phase in some kinds of cell type.Therefore changes in cell cycle may play a role in enhanced cytotoxic effects of CPT by pretreatment of DAC. However DAC did not affect cell cycle distribution of SMMC-7721 cell line.Conclusion A 2-D method with high resolution and good reproductivity was built in this study, and was proved to be an effective and viable way to discover new regulation proteins induced by CPT.This study provided 2D map of SMMC-7721 cell line,and compared the differences in protein expressions between CPT treated group and control group.Our study reported on the discovery of a novel downstream protein,DNA methyltransferase(DNMT),linking Topoisomerase I inhibition by CPT leading to the decrease expression of Galectin-1 and growth inhibition in hepatocellular carcinomas SMMC-7721 cells.Moreover,in combination with the DNMT inhibitor DAC,CPT markedly enhanced its antitumor activity.We also provided new evidence to indicate that CPT can modulate Galectin-1 gene and protein expression in the human hepatocellular carcinoma cell line,SMMC-7721.Our results provided new insight into the molecular mechanisms of CPT and the potential to optimize therapies for treatment of hepatocellular carcinomas.