5-Aza-dC对恶性黑色素瘤细胞的抑制作用及机制研究

Inhibitory effect of 5-Aza-dC on malignant melanoma in vitro and its mechanism

目的研究5-氮杂-2’-脱氧胞苷(5-Aza-dC)对小鼠黑色素瘤B16细胞增殖、凋亡及体外迁移、侵袭等恶性生物学行为的影响及其作用机制。方法2022年1月至6月,通过CCK-8法、细胞划痕实验、Transwell侵袭实验及流式细胞术检测5-Aza-dC对小鼠恶性黑色素瘤B16细胞体外增殖、迁移、侵袭及凋亡等恶性生物学行为的影响;蛋白免疫印迹法检测凋亡相关蛋白Bcl-2、Bax及缝隙连接蛋白Cx43的表达水平。采用Mann-Whitney U检验、Welch检验和单因素方差分析。结果CCK-8实验结果显示:与空白对照组及DMSO对照组相比,5-Aza-dC给药组细胞存活率均明显降低(均P<0.01);同一药物浓度作用下不同给药时间细胞存活率对比显示,细胞存活率随着作用时间的增加而降低(P<0.01)。细胞划痕实验结果显示:5-Aza-dC给药组细胞划痕愈合率均明显低于空白对照组[(74.67±11.91)%比(100.00±0.00)%,P<0.01]。Transwell侵袭实验结果显示:空白对照组侵袭细胞数量明显多于5-Aza-dC给药组[(444±65)个比(2±1)个],差异有统计学意义(P<0.01)。流式细胞术(Annexin V/PI双染法)结果显示:5-Aza-dC给药组细胞凋亡率高于空白对照组[(11.35±0.66)%比(2.51±0.04)%],差异有统计学意义(P<0.01)。Western blot结果显示:与空白对照组比较,5-Aza-dC给药组Bcl-2表达下调,Bax表达上调,Cx43蛋白表达增强。结论5-Aza-dC可以促进小鼠恶性黑色素瘤B16细胞凋亡,抑制其增殖活力及迁移、侵袭能力,从而发挥抗肿瘤作用,其抑制作用可能与上调Cx43蛋白表达相关。

Objective To study the effect of 5-Aza-2'-deoxycytidine(5-Aza-dC)on the malignant biological behaviors of mouse melanoma B16 cells,such as proliferation,apoptosis,migration,and invasion in vitro,and its mechanism.Methods This study was from January to June 2022.The effects of 5-Aza-dC on the proliferation,migration,invasion,and apoptosis of murine malignant melanoma B16 cells were examined by CCK-8 assay, cell scratch test, Transwell invasiontest, and flow cytometry test. The expression levels of apoptosis-related proteins, Bcl-2 and Bax, andconnexin 43 were detected by Western blotting. Mann-Whitney U and Welch tests and one-wayANOVA were applied. Results The results of CCK-8 assay showed that the cell survival rates of the5-Aza-dC groups were significantly lower than those of the blank control group and the DMSO group(all P<0.01). The comparison of the cell survival rates at different times of drug administration underthe same drug concentration showed that the cell survival rate decreased as the time of drugadministration prolonged (P<0.01). The results of cell scratch test showed that the cell scratchhealing rate of the 5-Aza-dC group was significantly lower than that of the blank control group[(74.67±11.91)% vs. (100.00±0.00)%;P<0.05]. The Transwell invasion test results showed that thenumber of invasive cells in the blank control group was significantly higher than that in the 5-Aza-dCgroup [(444±65) vs. (2±1);P<0.01]. The flow cytometry test (Annexin V/PI double-staining) resultsshowed that the apoptosis rate of the 5-Aza-dC group was significantly higher than that of the blankcontrol group [(11.35±0.66)% vs. (2.51±0.04)%;P<0.01]. The Western blot results showed thatcompared with those in the blank control group, the expression of BCL-2 was down-regulated andthe expressions of Bax and CX43 were up-regulated in the 5-Aza-dC group. Conclusions 5-Aza-dCcan promote the apoptosis and inhibit the proliferation, migration, and invasion of murine malignantmelanoma B16 cells. The anti-tumor effect of 5-Aza-dC may be related to the up-regulation of CX43protein expression.