摘要
为了探讨醛酮还原酶AKR1C3 在乳腺癌阿霉素耐药中的作用,成功建立了阿霉素耐药的人乳腺癌细胞株MCF-7/DOX以及AKR1C3稳定过表达和敲低的乳腺癌细胞株。Western blot发现MCF-7/DOX细胞内AKR1C3的表达水平明显高于敏感株MCF-7细胞。CCK-8细胞药物敏感实验和 DAPI 染色实验发现在AKR1C3高表达的细胞中,阿霉素的细胞毒性明显降低(IC50增加了6倍);集落形成实验也证实AKR1C3过表达细胞中,集落形成能力增高。进一步实验证实,AKR1C3介导的β-catenin入核增多是导致乳腺癌细胞阿霉素耐药的原因之一。应用β-catenin 抑制剂XAV939,能够逆转AKR1C3诱导的阿霉素耐药。上述结果提示,AKR1C3可以作为逆转阿霉素耐药的潜在治疗靶标。
关键词
乳腺癌(breast cancer, BC)是女性中最常见的肿
AKR1C3是醛酮还原酶家族(aldo-keto reductases, AKRs)的一员,是体内类固醇代谢的关键酶,AKR1C3 异常表达会导致类固醇代谢紊乱,从而引起各种疾病。AKR1C3参与了体内性激素的合成,在性激素依赖性的疾病中起着举足轻重的作用。它可以作用于雄激素受体(AR)、雌激素受体(ER)和孕激素受体(PR)来调节雄激素、雌激素和孕激素的含
阿霉素(上海阿拉丁生化科技股份有限公司);DMEM培养基,RPMI 1640培养基,胎牛血清(FBS)及0.25%胰蛋白酶溶液(美国Gibco公司);β-catenin特异性抑制剂XAV939(上海基屹生物科技有限公司);嘌呤霉素(无锡纳奥生物医药有限公司);AKR1C3过表达质粒OE(北京安诺伦生物科技有限公司);AKR1C3敲低质粒shRNA-AKR1C3(苏州吉玛基因股份有限公司);质粒提取试剂盒(北京康为世纪生物科技有限公司);慢病毒转染试剂盒(上海翊圣生物技术股份有限公司);DAPI染色试剂盒(上海碧云天生物技术股份有限公司);核浆分离试剂盒(南京凯基生物技术股份有限公司);Bcl-2,Bax一抗(武汉Abclonal公司);AKR1C3(英国Abcam公司);Wnt3a,p-GSK-3β,β-catenin,LaminA/C,β-actin一抗(武汉proteintech公司);HRP山羊抗兔IgG和HRP山羊抗鼠IgG(武汉Abclonal公司)。其余试剂均为市售分析纯。
将MCF-7细胞接种于含10% FBS,1%青霉 素-链霉素溶液的DMEM高糖培养基中,置于37 ℃、5% CO2 恒温细胞培养箱中培养,每2~3天传代1次,取对数生长期细胞进行实验。
采用浓度梯度递增
将AKR1C3过表达质粒OE和AKR1C3敲低质粒shRNA-AKR1C3用慢病毒转染试剂盒进行转染,收集病毒上清液,过滤分装于-80 ℃保存。用病毒上清液侵染MCF-7和MCF-7/DOX细胞,然后用2 μmol/L嘌呤霉素进行抗性筛选两周后,选出一个稳定生长的细胞进行单克隆培养,最终得到稳定高表达AKR1C3的MCF-7细胞株(MCF-7/AKR1C3)和稳定低表达AKR1C3的MCF-7/DOX的细胞株(MCF-7/DOX-KD)。
取对数期生长状态良好的MCF-7和MCF-7/DOX细胞,消化后以每孔1 × 1
取对数生长期状态良好的MCF-7和MCF-7/DOX细胞,消化后以每孔1 × 1
将对数生长期状态良好的MCF-7和MCF-7/DOX细胞置于6孔板中,每孔1 000个细胞,用阿霉素处理后培养12~14 d。存活细胞克隆用4%甲醛固定20 min后,用0.1%结晶紫染色20 min,冲洗干净晾干后进行拍照观察细胞增殖情况。
将对数生长期状态良好的MCF-7、MCF-7/AKR1C3、MCF-7/DOX以及MCF-7/DOX-KD的细胞,消化后以每孔6 × 1
将对数生长期状态良好的MCF-7、MCF-7/AKR1C3、MCF-7/DOX以及MCF-7/DOX-KD的细胞均匀接种在6孔板上,培养至细胞覆盖率为80%以上。收集细胞,使用Trizol法提取RNA,逆转录得到cDNA,加入SYBR Green,使用实时PCR系统进行实时扩增。扩增条件为:95 ℃ 15 s,60 ℃ 1 min,95 ℃ 1 min,65 ℃ 10 s,一共35个循环。采用GAPDH作为内参,所用引物序列如
将对数生长期状态良好的MCF-7、MCF-7/AKR1C3、MCF-7/DOX以及MCF-7/DOX-KD细胞,消化后以每孔6 × 1
取对数生长期状态良好的MCF-7、MCF-7/AKR1C3、MCF-7/DOX以及MCF-7/DOX-KD细胞,接种于提前放置好玻片的6 孔板上,加入培养基培养24 h后取出6孔板。用PBS洗3次,每次5 min。4%多聚甲醛固定5 min后,加入0.3% Triton反应5 ~ 15 min,PBS洗3次,每次5 min。然后,加入3% BSA封闭2 h;PBS洗3次,每次5 min。加入β-catenin一抗80 ~ 100 μL(1∶100)后,置于37 ℃,1 h后,4 ℃过夜。然后,PBST洗3次,每次5 min。避光,加入HRP山羊抗兔IgG二抗溶液,常温反应30 ~ 40 min,PBST洗4次,每次5 min。最后,加入含抗荧光淬灭剂的DAPI封片液进行封片;封片后,在荧光显微镜下观察并拍照。
本研究采用浓度梯度递增法,成功建立了对阿霉素耐药的乳腺癌细胞株MCF-7/DOX。用CCK-8细胞药物敏感实验,检测到阿霉素对MCF-7细胞的IC50为(2.865 ± 0.042 13)μmol/L,而对MCF-7/DOX细胞的IC50为(171.0 ± 0.027 23)μmol/L(

Figure 1 Effects of different concentrations of doxorubicin(DOX) on cell viability and apoptosis in MCF-7 and doxorubicin-resistant cell line MCF-7/DOX()
A: CCK-8 assay of DOX on cell viability of MCF-7 cells; B: CCK-8 assay of DOX on cell viability of MCF-7/DOX cells; C: Cells images after 24 h treatment of DOX(red arrow head points at apoptotic cells) (400 ×,DAPI)
为了进一步验证耐药细胞株MCF-7/DOX对阿霉素的耐药性,用DAPI染色检测凋亡细胞情况。如
已有报道表明,在乳腺癌细胞中AKR1C3的表达水平较正常乳腺细胞中

Figure 2 Expression of AKR1C3 in MCF-7 and MCF-7/DOX cells()
A: Expression of AKR1C3 detected by Western blot; B: AKR1C3 mRNA detected by QRT-PCR
为了进一步研究AKR1C3在乳腺癌阿霉素耐药中的作用,本研究在MCF-7细胞中稳定过表达AKR1C3蛋白,构建过表达细胞株MCF-7/AKR1C3。实验结果表明,过表达细胞株MCF-7/AKR1C3中AKR1C3蛋白表达明显上调(

Figure 3 Effect of AKR1C3 on the sensitivity of DOX in MCF-7 cells()
A: AKR1C3 expression in MCF-7 and MCF-7/AKR1C3 cells detected by Western blot; B: AKR1C3 mRNA in MCF-7 and MCF-7/AKR1C3 cells detected by QRT-PCR; C: Cell viability of MCF-7 and MCF-7/AKR1C3 cells detected by CCK-8 assay; D: Colony formation of MCF-7 and MCF-7/AKR1C3 cells after treatment of DOX; E: DAPI staining of MCF-7 and MCF-7/AKR1C3 cells after treatment of DOX(red arrow head points at apoptotic cells); F: Western blot verified that expression of AKR1C3 in MCF-7/DOX-KD cells is knocked down compared with MCF-7/DOX cells; G: QRT-PCR verified that AKR1C3 mRNA in MCF-7/DOX-KD cells is knocked down compared with MCF-7/DOX cells; H: CCK-8 analysis showed MCF-7/DOX sensitivity to DOX was enhanced after AKR1C3 knockdown
本研究通过CCK-8实验验证MCF-7/AKR1C3细胞对阿霉素的敏感性。结果显示,阿霉素对MCF-7/AKR1C3和MCF-7细胞的IC50分别为(14.460 ± 0.043) μmol/L和(2.425 ± 0.034) μmol/L。MCF-7/AKR1C3细胞对阿霉素的耐药指数(RI)约为6(
为了进一步确证AKR1C3介导乳腺癌细胞耐药的作用,本研究构建了敲低MCF-7/DOX的AKR1C3的细胞株MCF-7/DOX-KD,并检测了其对阿霉素的敏感性(
以上结果表明,过表达AKR1C3可增加乳腺癌细胞对阿霉素的耐药性,低表达AKR1C3会增加细胞对阿霉素的敏感性,证实了AKR1C3在乳腺癌细胞阿霉素耐药中具有介导作用。
有报道称β-catenin与许多肿瘤的耐药性密切相

Figure 4 Effect of AKR1C3 on the Wnt/β-catenin pathway in MCF-7 and MCF-7/DOX cells()
A: Western blot showed that the expression of AKR1C3,Wnt3a and p-GSK-3β in MCF-7/AKR1C3 cells were elevated compared with MCF-7 cells; B: Expression of β-catenin in the cytoplasm and nucleus of MCF-7 and MCF-7/AKR1C3 cells; C: Nuclear localization of β-catenin in MCF-7/AKR1C3 cells detected by immunofluorescence; D: Immunofluorescence showed that nuclear β-catenin is decreased after AKR1C3 knocked down; E-F: Western blot showed that expression of AKR1C3,Wnt3a and p-GSK-3β proteins were decreased and nuclear β-catenin decreased after AKR1C3 knockdown
在敲低AKR1C3之后, 免疫荧光结果表明胞质β-catenin含量增多,核内表达减少(
为了进一步确证β-catenin在AKR1C3 诱导的阿霉素耐药中的作用,本研究使用β-catenin特异性抑制剂XAV939来抑制胞内β-catenin的表达水平。结果表明,XAV939不仅会抑制β-catenin总蛋白水平,也会其降低核内的表达水平(

Figure 5 Effects of β-catenin inhibitor XAV939 combined with DOX on the cell viability and apoptosis in MCF-7 and MCF-7/AKR1C3 cells ()
A: Western blot showed XAV939 inhibited the expression of β-catenin and its nuclear translocation; B: CCK-8 analysis found β-catenin inhibitor enhanced the sensitivity of DOX to MCF-7/AKR1C3 cells; C: Western blot showed β-catenin inhibitor increased the pro-apoptotic Bax expression and decreased the level of anti-apoptotic protein Bcl-2
本研究探讨了AKR1C3/β-catenin轴在乳腺癌阿霉素耐药中的作用,证明了AKR1C3可能通过Wnt/β-catenin介导了MCF-7细胞的阿霉素耐药。关于AKR1C3参与乳腺癌阿霉素耐药虽然已有报道,但大多数报道认为,AKR1C3是作为氧化还原酶,将阿霉素还原成低毒性的阿霉素醇,从而降低了阿霉素的毒性作用。本研究主要探讨AKR1C3在生物调控网络中的作用,旨在阐明其酶活之外的阿霉素耐药的介导作用。
本研究发现AKR1C3在阿霉素耐药的乳腺癌细胞中高表达。AKR1C3基因过表达或敲低可增强或抑制乳腺癌细胞的阿霉素耐药性,这反映在细胞活力、集落形成能力和凋亡的结果上。深入研究发现,AKR1C3诱导的阿霉素耐药性可能与β-catenin信号通路相关。过表达的AKR1C3促进β-catenin的入核,增强肿瘤细胞集落形成能力,增强乳腺癌细胞的阿霉素耐药性;敲低AKR1C3减少β-catenin的核转位,降低乳腺癌细胞活力,减弱癌细胞的耐药性。然而,相比于敏感株细胞MCF-7,即使敲低AKR1C3的细胞株MCF-7/DOX-KD对阿霉素的敏感性仍然较低,提示在阿霉素耐药过程中,除AKR1C3之外,还有其他通路影响耐药细胞的敏感性,具体机制有待进一步探讨。XAV939是一种有效的端锚聚合酶(TNKS)1和2小分子抑制剂,通过抑制TNKS活性,增加肿瘤细胞中Axin-GSK3β复合物的蛋白水平,并促进β-catenin的降
综上所述,在阿霉素耐药乳腺癌MCF-7细胞模型中,AKR1C3/β-catenin是阿霉素耐药的原因之一,靶向AKR1C3/β-catenin在乳腺癌阿霉素耐药的治疗中可能具有良好的应用前景。
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