| Objective The bcl-2 gene is a critical regulator of the cell death process, and it encodes a 26DK Bcl-2 protein that promotes cell survival by blocking programmed cell death(apoptosis). In small cell lung cancer, up to 90% of tumors and cell lines overexpress bcl-2.The interleukin-6 (IL-6), a cytokine, plays a important role by combining to interleukin-6 receptor (IL-6R). The different studies have showed that IL-6 expression is related to occurrence, progression and metastasis of lung cancer. Antisense technique is the highlight with its effectiveness, specificity and convenience. This subject aimed to investigate separate as well as combination effect of three antisense phosphorothioate oligodeoxynucleotides (AS-PS-ODNs), i.e. bcl-2 AS-PS-ODN, IL-6 AS-PS-ODN and IL-6R AS-PS-ODN, on the proliferation and apoptosis of small cell lung cancer cell line NCI-H446, and on mRNA expression level of bcl-2,IL-6,IL-6R and Mcl-1 in NCI-H446 cell line .Methods Proliferations of cell line NCI-H446 treated with different group of AS-PS-ODNs were evaluated by growth curve and cell clone formation rate test. DNA content assay and cell apoptosis detection kit were used to assessed the apoptosis by flow cytometry. Semi-quantitative reverse transcription-PCR was performed to detect mRNA expression level of bcl-2, IL-6, IL-6R and Mcl-1 in NCI-H446 cell line. Results 1, All groups of AS-PS-ODNs could inhibit proliferation of NCI-H446 cell line. After treatment with AS-PS-ODNs in 5umol/L for 48 hours , inhibition rate of cell proliferation is 15.46% in group of bcl-2 AS-PS-ODN, 22.79% in group of IL-6 AS-PS-ODN 22.79% , 19.85% in group of IL-6R AS-PS-ODN, 41.91 % in combined group of bcl-2 AS-PS-ODN and IL-6 AS-PS-ODN respectively, significantly higher than that in control or (N)S-PS-ODN (P<0. 05). While NCI-H446 cell line being treated with AS-PS-ODNs in 10mol/L for 7 days ,the cell clone formation rate was 13.25% inbcl-2 AS-PS-ODN, 7.00% in IL-6 AS-PS-ODN 22.79%, 1.63% in combined group of bcl-2 AS-PS-ODN and IL-6 AS-PS-ODN and 11.88% in IL-6R AS-PS-ODN respectively, also significantly lower than that in control or (N)S-PS-ODN (P<0. 01). All groups of AS-PS-ODN could induced apoptosis . The result detected by cell apoptosis detection kit demonstrated that the apoptosis rate is less than 32.0% in group of single AS-PS-ODN, and 64.7% in combined group of bcl-2 AS-PS-ODN and IL-6 AS-PS-ODN. The difference is also significant as comparing to control or (N)S-PS-ODN (P< 0. 01). Consistently, DNA content assay displayed that the apoptosis rate is less than 23.5% in group of single AS-PS-ODN, and 36.4% in combined group of bcl-2 AS-PS-ODN and IL-6 AS-PS-ODN.2, Semi-quantitative reverse transcription-PCR analysis revealed that expressions of bcl-2,IL-6 , IL-6R and Mcl-1 are quite high in NCI-H446 cell line. Three AS-PS-ODNs could obviously down-regulated expression.of itself mRNA, 62.58% in bcl-2 AS-PS-ODN, 84.07% in IL-6 AS-PS-ODN 22.79% and 60.31% in IL-6R AS-PS-ODN. Moreover, bcl-2 AS-PS-ODN could still up-regulate IL-6 expression by 74.3 % .while IL-6 AS-PS-ODN could down-regulate bcl-2 expression by 32.2 % accompanying with increased IL-6R expression by 65.8 % . However, IL-6R AS-PS-ODN could regulate a little to other gene and Mcl-1 expression could not be found in three AS-PS-ODNs group.Conclusion 1. bcl-2,IL-6 and IL-6R AS-PS-ODNs inhibit proliferation and viability of NCI-H446 and induce apoptosis by down-regulating expression of anti-apoptosis genes. 2. Combination effect of AS-PS-ODNs was better than single AS-PS-ODN. 3. IL-6 can regulate bcl-2 expression because it is upstream, and bcl-2 can also regulateIL-6 expression by feedback.. |
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