5-氮杂-2'-脱氧胞苷联合曲古菌素A对乳腺癌SKBR-3细胞中性激素受体基因表达的影响

Transcription regulation of gene expression of human sex hormone receptors in breast cancer cells induced by 5-aza-dC and trichostatin A

目的:探讨5-氮杂-2'-脱氧胞苷(5-aza-2'-deoxycytidine,5-aza-dC)联合曲古菌素A(trichostatin A,TSA)能否诱导性激素受体阴性的乳腺癌细胞系SKBR-3同时重新表达功能性雌激素受体α(estrogen receptoralpha,ERα)和雄激素受体(androgen receptor,AR),并进一步研究诱导前后SKBR-3细胞对乳腺癌内分泌治疗敏感性的变化。方法:分别采用RT-PCR法和免疫细胞化学法检测5-aza-dC联合TSA诱导前后SKBR-3细胞和阳性对照MCF-7细胞中ERα、AR、孕激素受体(progestone receptor,PR)、雌激素调节蛋白pS2与前列腺特异性抗原(prostate specific antigen,PSA)mRNA和蛋白的表达水平;采用CCK-8法检测各内分泌治疗药物对5-aza-dC联合TSA诱导后重新表达ERα和AR的乳腺癌SKBR-3细胞增殖能力的影响。结果:5-aza-dC联合TSA能够诱导激素受体阴性的乳腺癌SKBR-3细胞同时重新表达ERα和AR,它们的下游产物PR、pS2以及PSA也相应表达;诱导后SKBR-3细胞中ERα和AR的表达量明显低于在MCF-7细胞中的表达量(P<0.05)。5-aza-dC联合TSA能明显抑制SKBR-3细胞的增殖能力(P<0.05),加入雌激素类药物17β-雌二醇(17β-estradiol,E2)后细胞的增殖能力略有上升,但差异无统计学意义(P>0.05)。在加入E2的基础上,分别再加入雌激素拮抗剂他莫昔芬(tamoxifen,TAM)或孕激素醋酸甲地孕酮(megestrol acetate,MA)后,二者都能使肿瘤细胞的增殖能力明显下降(P<0.05);联合加入TAM和MA后,肿瘤细胞的增殖能力进一步明显下降(P<0.05)。结论:5-aza-dC联合TSA能够诱导乳腺癌细胞系SKBR-3同时恢复表达功能性的ERα和AR,抑制肿瘤细胞的生长,同时使SKBR-3细胞恢复了对激素的依赖性和内分泌治疗的敏感性。

Objective： To investigate whether breast cancer SKBR-3 cells with negative expressions of ERα （estrogen receptor alpha） and AR （androgen receptor） can re-express these two sex hormone receptors after combined treatment with 5-aza-dC （5-aza-2’-deoxycytidine） and TSA （trichostatin A）, and to study the change of sensitivity of SKBR-3 cells against endocrine therapy after introduction of 5-aza-dC and TSA. Methods： The expression levels of ERα, AR, PR （progestone receptor）, estrogen regulated protein pS2 and PSA （prostate specific antigen） mRNAs and proteins in SKBR-3 cells （before and after combined treatment with 5-aza-dC and TSA） and MCF-7 cells （as positive control cells） were detected by RT-PCR （reverse transcription-PCR） and ICC （immunocytochemistry）, respectively. The effects of different endocrine medications on the proliferation of SKBR-3 cells re-expressing ERα and AR after treatment with 5-aza-dC and TSA were detected by CCK-8 （cell counting kit-8）. Results： ERα and AR and their downstream products including PR, pS2 and PSA were reexpressed in SKBR-3 cells after cotreatment with 5-aza-dC and TSA, and the expression levels of ERα and AR were lower in SKBR-3 cells than in MCF-7 cells （P〈0.05）. The proliferation of SKBR-3 cells was suppressed significantly by 5-aza-dC and TSA intervention （P〈0.05）, and this inhibition effect could be slightly reversed by E2 （17β-estrodiol） intervention （P〉0.05）. The proliferation of SKBR-3 cells could be significantly suppressed by treatment with E2 followed by TAM （tamoxifen） or MA （megestrol acetate） intervention （P〈0.05）, and it could be further suppressed after cotreatment with TAM and MA （P〈0.05）. Conclusion： The proliferation of breast cancer SKBR-3 cells re-expressing ERα and AR after cotreatment with 5-aza-dC and TSA can be inhibited; meanwhile, the sensitivity against endocrine therapy and the hormone dependence are reobtained in SKBR-3 cells.