近紫外UVA辅助4-硫-5-(2-噻吩基)尿苷体外对人黑色素瘤A375细胞增殖作用的影响

The effect of UVA assisted 4-thio-5-(2-thienyl) uridine on human melanoma A375 cells proliferation in vitro

目的研究新化合物4-硫-5-(2-噻吩基)尿嘧啶核苷酸在近紫外UVA辅助下,体外抑制人黑色素瘤A375细胞增殖的作用机制。方法利用MTT法筛选4-硫-5-(2-噻吩基)尿苷和近紫外UVA的协同作用剂量;采用Annexin V-FITC/PI形态学染色法和流式细胞术对协同作用引起的细胞死亡类型做以定性判断;通过蛋白免疫印迹法,探讨协同作用在细胞内的信号传递途径。结果无毒剂量的4-硫-5-(2-噻吩基)尿苷(100μmol·L^(-1))在无害剂量的UVA(15 k J·m-2)辅助下,通过降低p38蛋白和Akt蛋白的表达及磷酸化,下调Bcl-2、pro-caspase-9和pro-caspase-3蛋白的表达量,促进Bad蛋白表达及claved-PARP蛋白的活化,诱导细胞凋亡抑制人黑色素瘤A375细胞增殖。结论 4-硫-5-(2-噻吩基)尿苷在近紫外UVA的辅助下,通过诱导细胞凋亡,抑制人黑色素瘤A375细胞增殖。

Objective To research the synergism on the mechanism of UVA assisted 4-thio-5-（ 2-thienyl） uridine to inhibit human melanoma A375 cells proliferstion in vitro. Methods MTT assay to select the combined does of 4-thio-5-（ 2-thiophen） uridine and UVA. Annexin V-FITC/PI staining and flowcytometry test to identify the type of cell death for synergy effects. Western blot to explore the signaling pathway. Results Non-toxic dose of 4-thio-5-（ 2-thienyl） uridine（ 100 μmol·L^-1） could induce extensive death of A375 cells with the assist of harmless dose of UVA（ 15 kJ·m^-2）. The results of morphological analysis and flowcytometry showed that 4-thio-5-（ 2-thienyl） uridine and UVA could induce apoptosis of A375 cells by synergistic effect. Western Blot showed that the synergistic effect of 4-thio-5-（ 2-thienyl） uridine and UVA could inhibit the expression and phosphorylation of p38 protein,thus inhibiting the proliferation of cells. At the same time,the synergy could reduce the expression and phosphorylation of Akt and the expression of anti-apoptotic protein Bcl-2,weakening the inhibition of pro-apoptotic protein Bad. Further amplified the apoptosis signal,promoting the activation and cleavage of pro-caspase-9 and pro-caspase-3. Finally activated clavedPARP and performed apoptosis. Conclusion The non-toxic dose of 4-thio-5-（ 2-thienyl） uridine（ 100 μmol·L^-1） can inhibit human melanoma A375 cells proliferation by inducing apoptosis with lowdoes UVA（ 15 k J·m^-2） assist.