丁硫氨酸硫酸亚胺逆转人结肠癌细胞多药耐药的机制

The mechanism of buthionine sulfoximine reverses multidrug resistance in human colon cancer cells

目的探讨丁硫氨酸硫酸亚胺（BSO）逆转人结肠癌LoVo／Adr细胞多药耐药性的可行性，并初步探讨其逆转机制。方法MTT法检测BS0对多药耐药LoVo／Adr细胞抗癌药物敏感性的影响；RT-PCR法检测细胞中多药耐药1（mdr1）基因及谷胱甘肽巯基转移酶（GST）-π mRNA水平；免疫组化染色检测GST-π蛋白水平；1氯-2，4-二硝基苯法测定GST活性。结果BS0作用后亲本LoVo细胞中阿霉素、丝裂霉素的IC50无显著变化，而在LoVo／Adr细胞中，阿霉素的IC50由（3．08±0．84）mg／L降至（1．31±0．53）mg／L（t=3．09，P〈0．05），丝裂霉素的ICs0由（1．24±0．45）mg／L降至（0．54±0．32）mg／L（t=165．50，P〈0．01）；BS0对mdr1基因mRNA水平无影响，BS0作用前后GST-π mRNA表达量分别为1．75±0．24和0．84±0．19（t=5．11，P〈0．05），BSO作用后LoVo／Adr细胞GST-π蛋白表达显著低于处理前；BSO处理前后LoVo／Adr细胞GST活性分别为（144．26±50．13）和（129．58±41．36）U／mg（t=0．57，P〉0．05）。结论BSO可有效增强人结肠癌耐药细胞LoVo／Adr对阿霉素、丝裂霉素的敏感性，具有逆转作用，其机制与mdrl基因无关，但与GST-π基因下调有关，可能是通过降低细胞内GST-π含量，增强对化疗药物的敏感性。

Objective To determine the feasibility of reversing multidrug resistant （MDR） in human colon carcinoma LoVo/Adr cells by buthionine sulfoximine（BSO） and investigate the mechanism of BSO reversing MDR. Methods The effect of BSO on sensibility of anti-cancer drug in MDR LoVo/Adr cell was detected by MTT assay. The level of mdrl and glutathione S-transferase （GST）-π mRNA were measured by RT-PCR. The expression of GST-π was detected by immunohistochemistry and the activity of GST was measured by 1-C1-2, 4-2 nitrobenzene method. Results The level of IC50 of adriamycin and mitomycin in LoVo cells had no significant change after treated with BSO. The level of IC50 of adriamycin was decreased from （3.08 ± 0.84） mg/L to（1.31 ± 0.53） mg/L（t= 3.09, P〈0.05）, and the level of IC50 of mitomycin was decreased from（1.24 ±0.45）mg/L to（0. 54 ± 0. 32）mg/L（t = 165.50, P〈0.01） by BSO in LoVo/Adr cells. The level of MDR1 mRNA was not changed by BSO. The level of GST-π mRNA in LoVo/Adr cells was 1.75 ± 0.24 before treated with BSO and was 0.84 ± 0.19（t= 5.11, P 0. 05） after treated with BSO. The level of GST-π protein was decreased significantly after treated with BSO in LoVo/Adr cells. The activity of GST in LoVo/Adr cells was（ 144.26± 50. 13）U/mg, （129.58 ± 41.36）U/mg before and after treated with BSO, respectively. Conclusions BSO increased the sensitivity of human colon carcinoma LoVo/Adr cells to adriamycin and mitomycin with reversal of MDR. The mechanism of reverse was not associated with MDR1 gene, but was associated with decreased expression of GST-π. It is possible to increase the sensitivity of drug through decreasing GST-π in cells.