藥學(xué)專業(yè)英語文章及翻譯

1、Expression of livin in gastric cancer and induction of apoptosis in SGC-7901 cells by shRNA-mediatedsilencing of livin geneBackground-Because of increased resistance to apoptosis in tumor cells, inhibition of specific antiapoptotic factors may provide a rational approach for the development of novel

2、 therapeutic strategies.Livin, a novel inhibitor of apoptosis protein family, has been found to be expressed in various malignancies and is suggested to have poorly prognostic significance. However, no data are available concerning the significance of livin in gastric cancer. In this study, we detec

3、ted the expression of livin in human gastric carcinoma and investigated the apoptotic susceptibility of SGC-7901 cell by shRNAmediated silencing of the livin gene.Methods-The mRNA and protein expression of livin were analyzed by RT-PCR and western blot assay.The relationship between livin expression

4、 and clinical pathologic parameters was investigated. The small interfering RNA eukaryotic expression vector specific to livin was constructed by gene recombination, and the nucleic acid was sequenced. Then it was transfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR and Western blot assay we

5、re used to validate gene-silencing efficiency of livin in SGC-7901 cells. Stable clones were obtained by G418 screening. The cell apoptosis was assessedby flow cytometry (FCM). Cell growth state and 50 % inhibition concentration (IC50) of 5-FU and cisplatin was determined by MTT method.Results-The e

6、xpression of livin mRNA and protein were detectedin 19 of 40 gastric carcinoma cases (47.5%) and SGC-7901 cells. No expression of livin was detected in tumor adjacent tissues and benign gastric lesion. The positive correlation was found between livin expression and poor differentiation of tumors as

7、well as lymph node metastases (P 0.05). Four small interfering RNA eukaryotic expression vector specific to livin were constructed by gene recombination. And one of them can efficiently decrease the expression of livin, the inhibition of the gene was not less than 70% (P 0.01). The recombinated plas

8、mids were extracted and transfected gastric cancer cells. The stable clones were obtained by G418 screening, and were amplified and cultured. When livin gene was silenced, the reproductive activity of the gastric cancer cells was significantly lower than the control groups(P 0.05). The study also sh

9、owed that IC50 of 5-Fu and cisplatin on gastric cancer cells treated by shRNA was decreased and the cells were more susceptible to proapoptotic stimuli (5-Fu and cisplatin) (P 104 cells/well) and 12-well plates (1.5 105 cells/well) for 24 h before transfectionThe cells were transfected with 4 mg/wel

10、l of empty pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control plasmid using Li-pofectAMINE 2000 (Life Technologies, Inc., Grand Island,NY) according to the manufacturer?盡 instructions. Forty-eight hours after transfection, the cells were passaged at 1:15 (v/v) and cultured in mediumsupp

11、lemented with Geneticin (G418) at 1000 g/ml for 4 weeks. Stably transfected clones were picked and maintained in medium containing 400 g/ml G418 for additional studies.2.7. Assay of anchorage-dependent cell growthParent cells and cells stably expressing empty pGPU/GFP/Neo vector, pGPU/GFP/Neo/livin

12、or pGPU/GFP/Neo/Control were seeded into 6-well plates. Cells from triplicate wells were collected every other day. Cell numbers were determined using a Coulter counter (Coulter Electronics, Miami, FL). The number of cells per well is reported as the average SD at the indicated number of days after

13、plating.2.8. MTT assayCytotoxicity was measured by MTT assay. Cells growing exponentially were plated onto 96-well plates at a density of 10000 cells/well for 24 h. The cells were then treated with different concentrations of drugs for 48 h. One hundred microliters of MTT stock solution (1 mg/ml) we

14、re added to each well, and the cells were further incubated at 37 for 4 h. The supernatant was replaced with isopropyl alcohol to dissolve formazan production. The absorbanceat wavelength 595 nm was measured with a micro-ELISA reader (ClinBio-128, SLT, Austria). The ratio of the absorbance of treate

15、d cells relative to that of the control cells was calculated and expressed as a percentage of cell death.2.9. Flow cytometryCells were collected and fixed with ice-cold 70% ethanol in PBS and stored at -4 until use. After resuspension,cells were incubated with 100 ml of RNase I (1 mg/ml) and 100 ml

16、of PI (400 mg/ml) at 37 and analyzed by flow cytometry (BD, USA).2.10. Statistical analysisData were expressed as the means of at least three different experiments SD. The results were analyzed by Student -test, a ndsPt 0.05 was considered statistically significant.3. Results3.1. Expression of livin

17、 in gastric carcinomasIn the present study, for the first time, we evaluated by RTCPCR and westen blot the presenceof livin expression in 40 gastric cancinomas, 13 para-canceroustissues and 13 benign lesions of gastric mucosa. In para-cancerous tissues and benign lesions of gastric mucosa, no detect

18、able levels of either mRNA isoforms were revealed, while among tumor tissues, 19/40(47.5%) showed mRNA and protein expression of livina and livinb (Figs. 1 and 2). Livin expression correlated with some of the known prognostic variables, such as histologic grade and lymph node metastasis, but not wit

19、h age, sexuality, stage and tumor infiltration extent (Table 2).3.2. Characterization of stable transfectants expressing pGPU/GFP/Neo/livin and pGPU/GFP/Neo/ControlWe established SGC-7901 stable transfectantswith either pGPU/GFP/Neo/livin, pGPU/GFP/Neo/Control plasmid, or empty pGPU/GFP/Neo/vector (

20、Fig. 3). Some clones from each transfection were selected and analyzed by RT-PCR and Western blot to determine the livin mRNA and protein expression, and others were selected for expansion and additional studies. As shown in (Figs. 4 and 5), the level of livin mRNA and protein in SGC-7901 pGPU/GFP/N

21、eo/livin2 transfectants was reduced by more than 90%. The suppression of livin expression was not observed in pGPU/GFP/Neo/livin1 transfectants and negative control. So SGC-7901 pGPU/GFP/Neo/livin2 transfectants was chosen for subsequent experiment.3.3. Inhibition of cell growth in stable transfecta

22、ntsThe growth rate of SGC-7901 pGPU/GFP/Neo/livin2 transfectants was significantly inhibited. As shown in Fig. 6, SGC-7901 pGPU/GFP/Neo/livin2 transfectant cells number had significant decreases at 72 h and 96 h after plating (P 0.01) compared with negative control and parent cells.3.4. Stable trans

23、fectants were more susceptible to proapoptotic stimuliWe treated SGC-7901 pGPU/GFP/Neo/livin2 transfectants and negative control cells with cytotoxic drugs (5-fluorouracil and cisplatin). MTT assay showed that SGC-7901 pGPU/GFP/Neo/livin2 transfectants were more sensitive to cisplatin and 5-fluorour

24、acil than negative control and parent cells (Figs. 7A, 6B). The number of apoptotic cells induced by cisplatin and 5-fluorouracil increased to about 2.5 C3-fold in pGPU/GFP/Neo/livin2 transfectants compared with their control cells (P 0.001; Fig. 7C). Furthermore, stable transfectants underwent spon

25、taneous apoptosis more readily without proapoptotic stimuli than the control cells (P 0.05; Fig. 7C).4. DiscussionIn this study, we show that livin, a new member of the IAP family, was found to be not expressed in any of the NOT cancerous gastric tissues, and expressed only in a proportion of gastri

26、c cancer patients (47.5%), and also show that suppressinglivin expression or function causes spontaneous apoptosis and inhibition of SGC- 7901 cells growth and make cells more susceptible to proapoptotic stimuli. It was thought that livin has two isoforms, a and b. Although both isoforms are involve

27、d in blocking apoptosis induced by TNF-a and anti-CD95 in vitro, they show some different antiapoptotic properties. livin b seemsto be more effective than livin a in blocking apoptosis induced by DNA damaging agents13.Some study on tissue distribution of livin has recently shown that elevated levels

28、 of both livin isoforms a and b have been detected in heart, placenta, lung, spleen and ovary, while livin balone has been detected specifically in fetal tissues and dult kidney and livin a alone has been detected in brain, skeletal muscle and peripheral blood lymphocytes 11-14. Furthermore, while l

29、ivin expression was detected in a variety of cancerous cell lines and some tumor tissues 14-18 and anti-livin antibody was recognized in sera of gastric cancer and lung cancer patients 19,20, no data were available concerning the expression of livin isoforms in gastric tumor tissues. Our study for t

30、he first time demonstrates that livin isoforms a and b were almost both expressed in a proportion of gastric cancer tissues (47.5%) and livin expression correlate with some of the known prognostic variables, such as grade and lymphonode metastasis.Data from the literature have demonstrated that both

31、 livin isoforms are involved in blocking apoptosis and may give cells with livin overexpression a strong resistance to chemotherapy-induced apoptosis. Gastric cancer in general is highly resistant to chemoradiotherapy and moderately resistant to apoptosis 21. These result suggested thatoverexpressio

32、n of livin may effect the responsibility of chemotherapy on some gastric cancer patients and prognosis of patients.The specific interference with factors contributing to the apoptosis resistance of tumor cells may provide a novel basis for the development of rational intervention strategies in cance

33、r therapy 22,23. Since the expression of livin could contribute to the apoptosis-resistant phenotype of cancer cells and its specific expression in tumors could make livin an interesting therapeutic target for tumor-specific intervention strategies, we chose the livin gene as a molecular target. The

34、 shRNA technology representiong an extremely powerful tool to inhibit endogenous gene expression 24,25 be made to inhibit livin gene and attempt to correct the apoptosis deficiency of gastric tumor cells. The efficacy of shRNAs to silence expression of a tageted gene is different, relation with the

35、half-life and abundance of the gene product as well as with accessibility of target mRNA 24-27. In this study, we observed that si-livin1 was regularly more strongly silence the livin gene than si-livin2. Our study results also shown that silencing livin gene expression may strongly increase apoptot

36、ic response of SGC-7901 cells in the presence or absence of several proapoptotic agents and inhibit the cells growth, which indicate that the interference with livin leads to a sensitization to proapoptotic stimuli. The similar result on hela cell was reported by Crnkovic-Mertens 18.In summary, our

37、results showed that inhibition of livin expression and function resulted in spontaneousapoptosis and inhibitor cell growth enhanced sensitivity to cytotoxic drugs in vitro. Because of the preferential expression of livin in gastric cancer but not in normal tissues, these data suggest that targeting

38、the livin pathway alone or with cytotoxic drugs may be useful in the treatment of gastric cancer. Despite their therapeutic potential, major technical hurdles still have to be overcome, in order to apply shRNAs as drugs. Under therapeutic aspects,will have to meet the general challenges of gene therapy approaches, such as efficient delivery into the target cells or the circumvention of immune responses. Notably, recent in vivo studies showed that shRNAs could be directly