Knockdown of Survivin Expression by siRNA Induces Apoptosis of Hepatocellular Carcinoma Cells

Survivin, a newly identified member of lAP family, is a powerful apoptosis-inhibiting factor. It is expressed in embryonic tissues as well as in the majority of human cancers, but not in most normal adult tissues. The cancer-specific expression of survivin makes it a potential target for cancer treatment. A survivin-specific small inhibitory RNA （siRNA） was introduced into hepatocellular carcinoma cells to investigate its effect on cancer cell apoptosis, growth and sensitivity to chemotherapeutic drugs. It was found that expressions of survivin protein and proliferation index （PI） in siRNA groups were significantly decreased, the apoptosis index （AI） of siRNA groups was significantly higher than those of others groups, and the growth inhibition rate （GIR） of chemotherapeutic drugs in siRNA groups were significantly higher than those of other groups. Our study suggests that the expression of survivin may be significantly decreased in hepG2 cell after siRNA transfection. siRNA targeting survivin could induce cell apoptosis, inhibit cell proliferation and sensitize hepatocarcinoma cells to chemotherapy. Our findings provide preliminary evidence for the therapeutic use of survivin-targeted RNA interference for human tumors that express high levels of this molecule.

Cyclin L2, a novel RNA polymerase Ⅱ-associated cyclin, is involved in pre-mRNA splicing and induces apoptosis of human hepatocellular carcinoma cells

We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

Essential Oil from Siegesbeckia pubescens Induces Apoptosis through the Mitochondrial Pathway in Human HepG2 Cells

Siegesbeckia pubescens（SP） has been used as a traditional medicine for the treatment of and inflammatory diseases. However, the activities of SP against hepatocellular carcinoma and the related mechanisms remain unclear. The present study aimed to examine the effects of the essential oil of SP（SPEO） on the proliferation of hepatocellular carcinoma cells and the possible mechanisms. The growth inhibition of Hep G2 cells was analyzed by MTT assay. Hoechst 33258 and fluorescence microscopy were utilized to observe the nuclear morphological changes of apoptotic cells. Flow cytometry was used to detect cell apoptosis and cell cycle. The expressions of the target proteins were detected by Western blotting. The results showed that SPEO obviously inhibited the proliferation of Hep G2 cells in a dose-dependent manner. SPEO activated a series of apoptotic proteins in Hep G2 cells, increasing expression levels of Bax, caspase-3 and caspase-9, and decreasing the bcl-2 expression level. SPEO displayed promising anti-hepatocellular carcinoma activities in vitro, partly by inducing apoptosis in Hep G2 cells through activating the mitochondrial pathway.

APOPTOSIS IN GASTRIC CANCER INDUCED BY TRICHOSANTHIN AND RELATIONSHIP OF THIS APOPTOSIS WITH EXPRESSION OF C-MYC

Objective To investigate the apoptosis in gastric cancer induced by trichosanthin (TCS) and the relationship between this apoptosis and expression of c-myc. Methods In in vitro experiment, morphologic test and TUNEL staining method were used to detect quantitatively and qualitatively the apoptosis status of gastric adenocarcinoma cell line SGC-7901 before and after the treatment of 0.1μg/ml TCS. Immunohistochemical staining method and Northern Blot hybridization were used to detect the expression status of apoptosis-related genes c-myc before and after TCS treatment. Results Some typical apoptotic morphologic changes appeared after 36h treated by TCS. The apoptotic indexes increased significantly in the TCS treated group than those of the untreated group after 36h,42h and 48h (P<0.01). After 24h treated by TCS, an increased expression of c-myc protein product occurred, the staining density was increased from + or ++ to +++ (P< 0.01), and the expression of c-myc RNA increased significantly (P<0.05). Conclusion TCS is able to induce the apoptosis in gastric cancer. This apoptosis may be mediated by up-expression of apoptosis-regulated gene c-myc.

CO-EXPRESSIONS OF SURVIVIN GENE, BCL-2 AND BAX PROTEINS IN OVARIAN CARCINOMA

Objective To characterize the cellular properties of ovarian cancer, we examined the correlation between the expression of apoptosis-related gene survivin and those of Bcl-2 and Bax proteins. Methods Expressions of survivin mRNA, and Bcl-2 and Bax proteins in 35 cases of ovarian carcinoma, 10 cases of borderline carcinoma, 10 cases of benign tumors and 10 cases of normal tissue were evaluated by reverse transcription polymer-ase chain reaction (RT-PCR) and immunohistochemistry SABC method, respectively. Results Expression of survivin gene was detected in a significantly greater proportion in ovarian carcinoma and borderline carcinoma than those in benign tumors and normal tissues. Although there was no relationship between expression of survivin gene and FIGO stage, histologic grade, pathological type and lymphatic metastasis, expressions of Bcl-2 and Bax proteins were positively and negatively correlated with that of survivin gene, respectively. Conclusion Survivin may play an important role in pathogenesis of ovarian carcinoma, with a synergistic role of apoptosis-related gene Bcl-2 protein and an antagonistic role of Bax protein in formation and progression of ovarian carcinoma.

Heat shock protein 70 expression in relation to apoptosis in primary bladder transitional cell carcinoma

Bladder carcinoma is the most common tumor in the urinary system. In 1996, a sampleinvestigation showed that bladder carcinoma, in which more than 90% was mainly primary bladder transitional cell carcinoma （BTCC）, was one of the ten highest mortality malignant tumors in China. Bladder carcinoma represented 2% of all malignant tumors and has the fifth most common malignancy in men in Europe and North America.

Expression of Bcl-2 and Survivin in uterine cervical carcinogenesis and correlation between the expression and infection of HPV_(16/18)

To investigate the expression of Bcl-2 and Survivin and HPV16/18 infection in uterine cervical carcinogenesis, formalin-fixed, paraffin-embedded specimens from cervical carcinomas, cervical intraepithelial neoplasia （C1N） and normal cervical tissues were studied. Using streptavidin-biotin peroxidase （S- P） immunohistochemical technique, the authors examined the expression of Bcl-2 and Survivin in these specimens. The number of apoptosis cells was assessed in situ by TdT-mediated dUTP-biotin end labeling （TUNEL） method. The infection of HPV type 16, 18 DNA were determined by PCR. It was found that there were significant differences in Bcl-2, Survivin and apoptotic index （AI） between cervical carcinomas, CIN and normal cervical tissues, respectively. Expression of Bcl-2 and AI were correlated with tu- mor grades, clinical stages and lymph node metastasis and expression of Survivin was associated with tu- mor grades and lymph node metastasis. There were different positive rate of HPV^s between cervical car- cinomas, C1N and normal tissues and were not associated with tumor grades, clinical stages and lymph node metastasis. The infection of HPV16/18 was associated with the expression of Bcl-2, Survivin and AI, respectively. It was concluded that the abnormal expression of Bcl-2, Survivin and infection of HPV16/18 were associated with cervical carcinomas. They possibly can be useful indexes for the primary screening and prognosis of cervical carcinomas.