Institute of Parasitology, Macdonald Campus, McGill University, Ste. Anne de Bellevue, Québec, H9X-3V9, Canada.
Department of Cardiac Surgery, Quebec Heart & Lung Institute, Université Laval, Québec, Canada.
BMC Cancer. 2023 Jan 6;23(1):24. doi: 10.1186/s12885-022-10474-x.
P-glycoprotein (P-gp), a member of the ATP Binding Cassette B1 subfamily (ABCB1), confers resistance to clinically relevant anticancer drugs and targeted chemotherapeutics. However, paradoxically P-glycoprotein overexpressing drug resistant cells are "collaterally sensitive" to non-toxic drugs that stimulate its ATPase activity.
Cell viability assays were used to determine the effect of low concentrations of tamoxifen on the proliferation of multidrug resistant cells (CHOC5 and MDA-Doxo), expressing P-gp, their parental cell lines (AuxB1 and MDA-MB-231) or P-gp-CRISPR knockout clones of AuxB1 and CHOC5 cells. Western blot analysis was used to estimate P-gp expression in different cell lines. Apoptosis of tamoxifen-induced cell death was estimated by flow cytometry using Annexin-V-FITC stained cells. Oxidative stress of tamoxifen treated cells was determined by measuring levels of reactive oxygen species and reduced thiols using cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and 5,5-dithio-bis-(2-nitrobenzoic acid) DTNB, respectively.
In this report, we show that P-gp-expressing drug resistant cells (CHOC5 and MDA-Doxo) are collaterally sensitive to the anti-estrogen tamoxifen or its metabolite (4-hydroxy-tamoxifen). Moreover, P-gp-knockout clones of CHOC5 cells display complete reversal of collateral sensitivity to tamoxifen. Drug resistant cells exposed to low concentrations of tamoxifen show significant rise in reactive oxygen species, drop of reduced cellular thiols and increased apoptosis. Consistent with the latter, CHOC5 cells expressing high levels of human Bcl-2 (CHOC5-Bcl-2) show significant resistance to tamoxifen. In addition, the presence of the antioxidant N-acetylcysteine or P-gp ATPase inhibitor, PSC-833, reverse the collateral sensitivity of resistant cells to tamoxifen. By contrast, the presence of rotenone (specific inhibitor of mitochondria complex I) synergizes with tamoxifen.
This study demonstrates the use of tamoxifen as collateral sensitivity drug that can preferentially target multidrug resistant cells expressing P-gp at clinically achievable concentrations. Given the widespread use of tamoxifen in the treatment of estrogen receptor-positive breast cancers, this property of tamoxifen may have clinical applications in treatment of P-gp-positive drug resistant breast tumors.
P-糖蛋白（P-gp）是 ABCB1 亚家族的一员，它使癌细胞对临床相关的抗癌药物和靶向化疗药物产生耐药性。然而，具有讽刺意味的是，P-糖蛋白过表达的耐药细胞对刺激其 ATP 酶活性的非毒性药物具有“旁系敏感性”。
细胞活力测定用于确定低浓度他莫昔芬对表达 P-糖蛋白的多药耐药细胞（CHOC5 和 MDA-Doxo）及其亲本细胞系（AuxB1 和 MDA-MB-231）或 AuxB1 和 CHOC5 细胞的 P-gp-CRISPR 敲除克隆的增殖的影响。Western blot 分析用于估计不同细胞系中 P-gp 的表达。用 Annexin-V-FITC 染色的细胞通过流式细胞术估计他莫昔芬诱导的细胞死亡的细胞凋亡。用细胞通透性 2',7'-二氯二氢荧光素二乙酸酯（H2DCFDA）和 5,5-二硫代双（2-硝基苯甲酸）DTNB 分别测量反应性氧物种和还原型硫醇的水平来确定他莫昔芬处理细胞的氧化应激。
在本报告中，我们表明表达 P-糖蛋白的耐药细胞（CHOC5 和 MDA-Doxo）对雌激素他莫昔芬或其代谢物（4-羟基他莫昔芬）具有旁系敏感性。此外，CHOC5 细胞的 P-gp 敲除克隆显示对他莫昔芬的旁系敏感性完全逆转。暴露于低浓度他莫昔芬的耐药细胞显示出活性氧显著增加，细胞内还原型硫醇减少，细胞凋亡增加。与后者一致的是，表达高水平人 Bcl-2 的 CHOC5 细胞（CHOC5-Bcl-2）对他莫昔芬表现出显著的耐药性。此外，抗氧化剂 N-乙酰半胱氨酸或 P-gp ATP 酶抑制剂 PSC-833 的存在可逆转耐药细胞对他莫昔芬的旁系敏感性。相比之下，鱼藤酮（线粒体复合物 I 的特异性抑制剂）与他莫昔芬协同作用。
本研究表明，他莫昔芬可用作旁系敏感性药物，可在临床可达到的浓度下优先靶向表达 P-糖蛋白的多药耐药细胞。鉴于他莫昔芬在治疗雌激素受体阳性乳腺癌中的广泛应用，他莫昔芬的这种特性可能在治疗 P-糖蛋白阳性耐药性乳腺癌肿瘤的临床应用中具有重要意义。
