Cancer Biology Division, School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Deemed to be University, Campus-11, Patia, Bhubaneswar, 751024, Odisha, India.
Cancer Biology Division, School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Deemed to be University, Campus-11, Patia, Bhubaneswar, 751024, Odisha, India.
Exp Cell Res. 2022 Nov 1;420(1):113338. doi: 10.1016/j.yexcr.2022.113338. Epub 2022 Sep 6.
Although sensitization of BRCA-mutated, homologous recombination (HR)-deficient breast cancer cells through PARP inhibitor is widely studied, not much is known about the treatment of BRCA-wild-type, HR-proficient breast cancer. Here, we aim to investigate whether a bioactive compound, Resveratrol (RES), can induce DNA double-strand breaks in HR-proficient breast cancer cells and Olaparib (OLA), a PARP inhibitor, can enhance the RES-mediated apoptosis by deregulating the HR repair pathway. The detailed mechanism of anti-cancer action of RES + OLA combination in breast cancer has been evaluated using in vitro, ex vivo, and in vivo preclinical model systems. OLA increased RES-mediated DNA damage, downregulated the HR pathway proteins, caused a late S/G2 cell cycle arrest, enhanced apoptosis and cell death in RES pre-treated breast cancer cells at much lower concentrations than their individual treatments. Direct measurement of HR pathway activity using a GFP plasmid-based assay demonstrated reduced HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. Moreover, RES + OLA treatment also caused significant reduction in PARP1-mediated PARylation and efficiently trapped PARP1 at the DNA damage site. Upon RES treatment, PARylated PARP1 was found to interact with BRCA1, which then activated other HR pathway proteins. But after addition of OLA in RES pre-treated cells, PARP1 could not interact with BRCA1 due to inhibition of PARylation. This resulted in deregulation of HR pathway. To further confirm the role of BRCA1 in PARP1-mediated HR pathway activation, BRCA1 was knocked down that caused complete inhibition of HR pathway activity, and further enhanced apoptosis after RES + OLA treatment in BRCA1-silenced cells. In agreement with in vitro data, similar experimental results were obtained in ex vivo patient-derived breast cancer cells and in vivo xenograft mice. Thus, RES + OLA combination treatment enhanced breast cancer cell death by causing excessive DNA damage and also simultaneously inhibiting the HR pathway.
虽然通过 PARP 抑制剂使 BRCA 突变、同源重组 (HR) 缺陷的乳腺癌细胞敏化已得到广泛研究，但对于 BRCA 野生型、HR 功能正常的乳腺癌的治疗知之甚少。在这里，我们旨在研究生物活性化合物白藜芦醇 (RES) 是否可以在 HR 功能正常的乳腺癌细胞中诱导 DNA 双链断裂，以及 PARP 抑制剂奥拉帕利 (OLA) 是否可以通过下调 HR 修复途径来增强 RES 介导的细胞凋亡。使用体外、离体和体内临床前模型系统评估了 RES+OLA 联合在乳腺癌中的抗癌作用的详细机制。OLA 增加了 RES 介导的 DNA 损伤，下调了 HR 途径蛋白，导致晚期 S/G2 细胞周期停滞，在 RES 预处理的乳腺癌细胞中以比单独治疗低得多的浓度增强了细胞凋亡和细胞死亡。使用 GFP 质粒为基础的测定法直接测量 HR 途径活性表明，在用 OLA 处理的 I-SceI 内切酶转染的细胞中，HR 效率降低。此外，RES+OLA 处理还导致 PARP1 介导的 PAR 化明显减少，并有效地将 PARP1 捕获在 DNA 损伤部位。在用 RES 处理后，发现 PAR 化的 PARP1 与 BRCA1 相互作用，然后激活其他 HR 途径蛋白。但是在用 RES 预处理的细胞中加入 OLA 后，由于 PAR 化抑制，PARP1 无法与 BRCA1 相互作用。这导致 HR 途径失调。为了进一步证实 BRCA1 在 PARP1 介导的 HR 途径激活中的作用，敲低 BRCA1 导致 HR 途径活性完全抑制，并且在用 RES+OLA 处理 BRCA1 沉默的细胞后进一步增强了细胞凋亡。与体外数据一致，在离体患者来源的乳腺癌细胞和体内异种移植小鼠中也获得了类似的实验结果。因此，RES+OLA 联合治疗通过引起过度的 DNA 损伤并同时抑制 HR 途径来增强乳腺癌细胞死亡。
