Cloning Of Human β-smac Gene And Its Dubious Effect Of Enhancing Cisplatin-induced Apoptosis On Gastric Cancer Cell Line Sgc7901

Objective: The disregulation of apoptotic signaling pathway plays a critical role in the development of malignancies. Second mitochondria-derived activator of caspase is an established endogenious pro-apoptosis protein, which moderates the caspase inbitibion of IAPs. Mature Smac competes with caspases for the same binding domain of IAPs with its N terminal peptides"AVPI", thus rendering the release of the activated-caspases initially binded to IAPs.β-Smac is a natural transcript of Smac without the essential"AVPI"peptides in the N terminal and is incapable of binding to IAPs. However, there is a study showing thatβ-Smac still possesses desirable pro-apoptotic activities. This study is to clone humanβ-Smac gene and then investigate whether ectopic overexpression of it would enhance Cisplatin-induced apoptosis on gastric cancer cell line SGC7901, the results of which would contribute to the validation of the reported proapoptotic activity ofβ-Smac and shed more light on the role of AVPI playing in the apoptosis mediated by Smac.Methods: (1) According to the sequence registered in GeneBank, a pair of PCR primers forβ-Smac gene was designed and subsequently synthesized with lower primer containing a sequence of Flag to faciliate detection of ectopic overexpression.β-Smac fragements amplificated from SGC7901 genome were cloned into plasmid pcDNA3.1 to construct the desiredβ-Smac transfectants. The transfectants were replicated within Ecoli and then extracted and digested. The bacterial from which the transfectants showed right band after digestion was further sequenced.(2)Repeatedly replicate within Ecoli and then extract transfectants to make sure enough transfectants were obtained. Well growing cells were seeded in 6 wells plates with 1×106 cells per well. After 24 hours of incubation, equal amount of either experimental or control transfectants were transfected. Total cell RNA was extracted after 24 hours of transfecation and total cell protein extracted after 48 hours. The discrepancy inβ-Smac mRNA expression between cells transfected withβ-Smac transfectants or control vectors was investigated by RT-PCR, while the ectopic overexpression ofβ-Smac protein in cells was detected by western blot. (3) Cells were seeded in 6 wells plates with three replicates for every experimental and control group. After transfected with eitherβ-Smac transfectants or control vectors for 48h, cells were then incubated for another 24h in the absence or presence of Cisplatin at concentrations of 0,2,8,16μg/ml respectively. Cells were harvested and dyed with both PI and Annexin V. Then apoptosis was determined by flow cytometry.The differences in apoptotic rate between experimental and control group were analized with Levene's test and Student's test.Results: (1) The expression ofβ-Smac was detected in SGC7901 cells. After digested with both HindⅢand Xho I ,transfectants showed two bands at the corresponding sites on gel electrophoresis. Nucleotide sequence analysis indicated that the clonedβ-Smac sequence was 100% homology withβ-Smac gene registered in GenBank and had the exact sequence encoding Flag. (2) The concentration of extracted transfectants were determined with spectrophotometer . Compared with cells transfected with control vectors, theβ-Smac mRNA expression was significantly increased and ectopic overexpression ofβ-Smac protein was only detected in cells transfected withβ-Smac transfectants. (3) The apoptotic rates(%) of the experimental groups treated with cisplatin of 0,2,8,16μg/ml were 35.58 + 11.20,65.99 + 4.26,75.95 + 8.39,77.91 + 3.17 respectively and those of the corresponding control groups were 32.09 +2.56,51.92 + 2.75,70.37 + 3.76,80.75 + 2.39. The variance between experimental and control groups were equal according to Levene's test. While the difference of apoptotic activity between cells incubated in 2μg/ml Cisplatin with and without ectopicβ-Smac overexpression is statistically significant (P=0.009), there are no statistical differences regarding the other three groups.Conclusion: (1)SGC7901 cells endogeniously expressβ-Smac.(2)β-Smac transfectant was successfully constructed and ectopically overexpressed in SGC7901. (3) The effect is conditional and probably there is no effect of ectopicβ-Smac overexpression in enhancing Cisplatin-induced apoptosis in SGC7901,which lends supports to the theory that AVPI is essential for Smac and further studies are needed to discover any potential contributions to the proapoptotic activities of Smac other than from AVPI.