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目的研究三氧化二砷(As_2O_3)联合γ-谷氨酰半胱氨酸合成酶抑制剂丁硫氨酸亚砜胺(BSO)对肿瘤多药耐药细胞株 K562/ADM 细胞的诱导凋亡效应,及对 P 糖蛋白(P-gp)和 mdrlmRNA表达的抑制作用,探讨谷胱甘肽(GSH)的含量变化与 As_2O_3作用效果的关系。方法 As_2O_3(0.5、2.0、5.0 μmol/L)单独及联合100 μmol/L BSO 作用于 K562/ADM 细胞,应用 MTT 比色法检测 K562/ADM细胞的增殖活性;膜联蛋白 V/碘化丙锭(Annexin V/PI)标记法观察 K562/ADM 细胞的凋亡效应;分光光度法检测 K562/ADM 细胞内 GSH 含量变化;流式细胞术(FCM)检测 P-gp 水平变化;RT-PCR 方法检测 mdr1mRNA 的表达变化。结果 K562/ADM 细胞内的 GSH 含量为(81.13±3.91)mg/g 蛋白,在BSO 降低 GSH 含量后,临床剂量(0.5、2.0 μmol/L)As_2O_3联合 BSO(100 μmol/L)24 h内即可抑制K562/ADM 细胞的增殖活性,诱导 K562/ADM 细胞发生凋亡,处理48 h凋亡率分别为(59.29±6.01)%,(65.06±8.29)%;72 h凋亡率分别为(82.15±9.28)%,(92.72±9.41)%;其诱导凋亡效果均明显强于单用 As_2O_3临床剂量和高剂量(5.0 μmol/L)组。K562/ADM 细胞 P-gp 表达阳性率为98.1%,mdr1mRNA 的相对表达水平为1.85±0.13,临床剂量 As_2O_3联合 BSO 处理48 h,其抑制 mdr1mRNA 表达的作用效果以及处理72 h对 P-gp 的抑制作用均明显强于单用高剂量 As_2O_3组。结论GSH 的含量变化与 As_2O_3的作用效果密切相关,As_2O_3联合 BSO 可有效诱导 K562/ADM 细胞发生凋亡,有效抑制 P-gp 及 mdr1mRNA 的表达。
Objective To investigate the apoptosis-inducing effects of arsenic trioxide (As_2O_3) combined with γ-glutamylcysteine synthase inhibitor butylthionine sulfoxide (BSO) on multidrug resistant K562 / ADM cell line P-glycoprotein (P-gp) and mdrlmRNA, and to explore the relationship between the content of glutathione (GSH) and the effect of As 2 O 3. Methods K562 / ADM cells were treated with As2O3 (0.5,2.0,5.0 μmol / L) alone or in combination with 100 μmol / L BSO. The proliferation of K562 / ADM cells was detected by MTT assay. Annexin V / propidium iodide The apoptotic effect of K562 / ADM cells was observed by Annexin V / PI staining; the content of GSH in K562 / ADM cells was detected by spectrophotometry; the level of P-gp was detected by flow cytometry (FCM) mdr1 mRNA expression changes. Results The GSH content in K562 / ADM cells was (81.13 ± 3.91) mg / g protein. After BSO decreased GSH content, the clinical dose (0.5,2.0 μmol / L) of As_2O_3 and BSO (100 μmol / L) (59.29 ± 6.01)% and (65.06 ± 8.29)%, respectively. The apoptosis rates at 72 h after treatment were (82.15 ± 6.01)%, and the apoptotic rates of K562 / ADM cells were ± 9.28)% and (92.72 ± 9.41)%, respectively. The induction effect of apoptosis was significantly stronger than that of As2O3 alone and high dose (5.0 μmol / L). The positive rate of P-gp expression in K562 / ADM cells was 98.1%, the relative expression level of mdr1 mRNA was 1.85 ± 0.13, and the effect of inhibiting the expression of mdr1 mRNA by As 2 O 3 and BSO for 48 h as well as the inhibition of P-gp at 72 h The effect was significantly stronger than single high-dose As_2O_3 group. Conclusion The change of GSH content is closely related to the effect of As 2 O 3. As 2 O 3 combined with BSO can effectively induce the apoptosis of K562 / ADM cells and effectively inhibit the expression of P-gp and mdr1 mRNA.