College of Pharmacy, Research Institute of Pharmaceutical Sciences, Gyeongsang National University, Jinju 52828, Korea.
Department of Pathology, Ilsan Paik Hospital, Inje University, Goyang 10380, Korea.
Int J Mol Sci. 2021 Apr 10;22(8):3916. doi: 10.3390/ijms22083916.
Poly (ADP-ribose) polymerase 1 inhibitors (PARPi) are used to treat recurrent ovarian cancer (OC) patients due to greater survival benefits and minimal side effects, especially in those patients with complete or partial response to platinum-based chemotherapy. However, acquired resistance of platinum-based chemotherapy leads to the limited efficacy of PARPi monotherapy in most patients. Twist is recognized as a possible oncogene and contributes to acquired cisplatin resistance in OC cells. In this study, we show how Twist knockdown cisplatin-resistant (CisR) OC cells blocked DNA damage response (DDR) to sensitize these cells to a concurrent treatment of cisplatin as a platinum-based chemotherapy agent and niraparib as a PARPi on in vitro two-dimensional (2D) and three-dimensional (3D) cell culture. To investigate the lethality of PARPi and cisplatin on Twist knockdown CisR OC cells, two CisR cell lines (OV90 and SKOV3) were established using step-wise dose escalation method. In addition, in vitro 3D spheroidal cell model was generated using modified hanging drop and hydrogel scaffolds techniques on poly-2-hydroxylethly methacrylate (poly-HEMA) coated plates. Twist expression was strongly correlated with the expression of DDR proteins, PARP1 and XRCC1 and overexpression of both proteins was associated with cisplatin resistance in OC cells. Moreover, combination of cisplatin (Cis) and niraparib (Nira) produced lethality on Twist-knockdown CisR OC cells, according to combination index (CI). We found that Cis alone, Nira alone, or a combination of Cis+Nira therapy increased cell death by suppressing DDR proteins in 2D monolayer cell culture. Notably, the combination of Nira and Cis was considerably effective against 3D-cultures of Twist knockdown CisR OC cells in which Endoplasmic reticulum (ER) stress is upregulated, leading to initiation of mitochondrial-mediated cell death. In addition, immunohistochemically, Cis alone, Nira alone or Cis+Nira showed lower ki-67 (cell proliferative marker) expression and higher cleaved caspase-3 (apoptotic marker) immuno-reactivity. Hence, lethality of PARPi with the combination of Cis on Twist knockdown CisR OC cells may provide an effective way to expand the therapeutic potential to overcome platinum-based chemotherapy resistance and PARPi cross resistance in OC.
聚（ADP-核糖）聚合酶 1 抑制剂（PARPi）因其生存获益更大且副作用更小而被用于治疗复发性卵巢癌（OC）患者，尤其是那些对铂类化疗有完全或部分缓解的患者。然而，铂类化疗的获得性耐药导致 PARPi 单药治疗在大多数患者中的疗效有限。Twist 被认为是一种可能的癌基因，并有助于 OC 细胞对顺铂的获得性耐药。在这项研究中，我们展示了 Twist 敲低的顺铂耐药（CisR）OC 细胞如何阻断 DNA 损伤反应（DDR），从而使这些细胞对顺铂作为铂类化疗药物和尼拉帕利作为 PARPi 的联合治疗敏感，这种治疗方法在体外二维（2D）和三维（3D）细胞培养中进行。为了研究 PARPi 和顺铂对 Twist 敲低的 CisR OC 细胞的致死作用，我们使用逐步剂量递增法建立了两种 CisR 细胞系（OV90 和 SKOV3）。此外，我们使用改良的悬滴和水凝胶支架技术在聚-2-羟乙基甲基丙烯酸酯（poly-HEMA）包被的平板上生成体外 3D 球体细胞模型。Twist 的表达与 DDR 蛋白 PARP1 和 XRCC1 的表达强烈相关，并且这两种蛋白的过表达与 OC 细胞的顺铂耐药有关。此外，根据组合指数（CI），顺铂（Cis）和尼拉帕利（Nira）的联合治疗对 Twist 敲低的 CisR OC 细胞产生了致死作用。我们发现 Cis 单独、Nira 单独或 Cis+Nira 治疗在 2D 单层细胞培养中通过抑制 DDR 蛋白来增加细胞死亡。值得注意的是，尼拉帕利和顺铂的联合治疗对 Twist 敲低的 CisR OC 细胞的 3D 培养非常有效，因为内质网（ER）应激上调，导致线粒体介导的细胞死亡的启动。此外，免疫组织化学染色显示 Cis 单独、Nira 单独或 Cis+Nira 显示较低的 ki-67（细胞增殖标志物）表达和较高的 cleaved caspase-3（凋亡标志物）免疫反应性。因此，PARPi 与 Cis 的联合治疗对 Twist 敲低的 CisR OC 细胞的致死作用可能为克服 OC 中的铂类化疗耐药和 PARPi 交叉耐药提供一种有效的方法来扩大治疗潜力。
