Combination of Niraparib, Cisplatin and Twist Knockdown in Cisplatin-Resistant Ovarian Cancer Cells Potentially Enhances Synthetic Lethality through ER-Stress Mediated Mitochondrial Apoptosis Pathway

Abstract

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.

Keywords: PARPi; Twist; cisplatin; cisplatin resistance; lethality; niraparib; ovarian cancer.

Figure 1

Twist expression regulates DNA damage response (DDR) proteins, PARP1 and XRCC1 during the development of cisplatin resistance in ovarian cancer (OC) cells. (A) Schematic repre-setantion of how four intermediate sublines were generated between parental and CisR OC cells. (B) Western blot analysis of Twist and DDR proteins, PARP1 and XRCC1 in each subline of OV90-CisR and SKOV3-CisR. (C) Clonogenic cell growth assay for cisplatin, olaparib and niraparib with the doses of 20 µM, each respectively, for 72 h of treatment followed by 72 h recovery in each subline of OV90-CisR and SKOV3-CisR. (D) Measurement of 50% inhibitory concentration (IC₅₀) values of cisplatin, olaparib and niraparib on CisR OC cells compared with their respective parental cells. P: parental cell; CisR: cisplatin-resistant cells; G: sublines of each respective generation. Values were represented as mean ± SD. * p < 0.05, ** p < 0.01, compared with their respective parental cells.

Figure 2

Twist regulates DDR proteins to sensitize cisplatin and PARPi in CisR OC cells. (A) Western blot analysis of Twist-knockdown with PARP1 and XRRRC1 proteins expression at day 1, 2 and 3 in OV90-CisR and SKOV3-CisR compared with the siNC group. (B,C) The treatment effect expressed as cell survival and IC₅₀ of cisplatin and PARPi (niraparib) on CisR OV90 and SKOV3 OC cells depending on Twist expression. The both drus were treated at a various concentrations ranging from 0 to 160 µM for 72 h of incubation. CisR: cisplatin-resistant; siRNA: small interfering RNA; siNC: non-targeting negative control siRNA; siTwist: Twist siRNA. Values were represented as mean ± SD. * p < 0.05, ** p < 0.01 compared with the siNC group.

Figure 3

Twist knockdown sensitizes CisR OC cells to Cis and niraparib (Nira) therapy. (A) The dose response curve of Cis and Nira at 1:1, 2:1 and 1:2 combination ratio on parental, siNC CisR and siTwist CisR OV90 and SKOV3 cells. The cells were co-treated with increase dose of Cis and Nira (0–160 µM) for 72 h and percentage of cell survival was determined by EZ-cytox cell viability kit. (B) Measurement of combination index (CI) using compusyn software. P: parental CisR: cisplatin-resistant; siRNA: small interfering RNA; siNC: non-targeting negative control, siRNA; siTwist: Twist siRNA; DMSO: dimethyl sulfoxide, Cis: cisplatin; Nira; niraparib; Fa: fraction of affected cells.

Figure 4

The effect of Cis and Nira on DDR proteins, PARP1 and XRCC1 in parental and non-silenced CisR OC cells. (A) Measurement of cell growth rate using clonogenic assay in parental cells administered with DMSO, Cis, Nira, and Cis+Nira. The cells were cultured in absence or presence of Cis (18 µm, OV90—P; 21 µm, SKOV3-P) and Nira (17.5 µm, OV90—P; 15 µm, SKOV3-P) for 72 h followed by 72 h of recovery period. (B) Western blot analysis of DDR proteins, PARP1 and XRCC1 expression levels to determine the effect on parental and siNC cells treated with DMSO, Cis, Nira and Cis+Nira. The transfected cells were cultured in absence or presence of Cis (18 µm, OV90—P; 21 µm, SKOV3-P) and Nira (17.5 µm, OV90—P; 15 µm, SKOV3-P) for 72 h. (C) Measurement of cell growth rate using clonogenic assay in siNC transfected CisR cells administered with DMSO, Cis, Nira, and Cis+Nira. The cells were cultured in absence or presence of Cis (18 µm, OV90—CisR; 21 µm, SKOV3-CisR) and Nira (17.5 µm, OV90—CisR; 15 µm, SKOV3-CisR) for 72 h followed by 72 h of recovery period. (D) Western blot analysis of DDR proteins, PARP1 and XRCC1 expression levels to determine the effect on parental and siNC cells treated with DMSO, Cis, Nira and Cis+Nira. The transfected cells were cultured in absence or presence of Cis (18 µm, OV90—CisR; 21 µm, SKOV3-CisR) and Nira (17.5 µm, OV90—CisR; 15 µm, SKOV3-CisR) for 72 h. P: parental; CisR: cisplatin-resistant; siRNA: small interfering RNA; siNC: non-targeting negative control siRNA; siTwist: Twist siRNA. Values were represented as mean ± SD. * p < 0.05, compared with the DMSO group and ^ns p > 0.05, compared with the DMSO group.

Figure 5

The Cis+Nira therapy induces cell death with blocking DDR capacity on Twist-knockdown CisR OC cells. (A) Measurement of cell growth rate using clonogenic assay in siTwist transfected cells administered with DMSO, Cis, Nira, and Cis+Nira. The siTwist transfected cells were cultured in absence or presence of Cis (18 µm, OV90—CisR; 21 µm, SKOV3-CisR) and Nira (17.5 µm, OV90—CisR; 15 µm, SKOV3-CisR) for 72 h followed by 72 h of recovery period. (B) Western blot analysis of DDR proteins, PARP1 and XRCC1 expression levels to determine the effect of siNC and siTwist transfected cells treated with DMSO, Cis, Nira and Cis+Nira. The transfected cells were cultured in absence or presence of Cis (18 µm, OV90—CisR; 21 µm, SKOV3-CisR) and Nira (17.5 µm, OV90—CisR; 15 µm, SKOV3-CisR) for 72 h. (C) Measurement of relative cell viability in siNC and siTwist transfected cells administered with DMSO, Cis, Nira, and Cis+Nira in OV90-CisR (18 µm of Cis and 17.5 µm of Nira) and SKOV3-CisR (21 µm of Cis and 15 µm of Nira) OC cells for 4 days. (D) Western blot analysis of cell death proteins, including Bax, cleaved caspase-9 and cleaved caspase-3 and Bcl-2 proteins and their expression levels to determine the effect of siNC and siTwist transfected cells treated with DMSO, Cis, Nira and Cis+Nira. The transfected cells were cultured in absence or presence of Cis (18 µm, OV90—CisR; 21 µm, SKOV3-CisR) and Nira (17.5 µm, OV90—CisR; 15 µm, SKOV3-CisR) for 72 h. CisR: cisplatin-resistant; siRNA: small interfering RNA; siNC: non-targeting negative control siRNA; siTwist: Twist siRNA. Values were represented as mean ± SD. # p < 0.05, compared with siNC group; * p < 0.05, compared with the siTwist (DMSO) group.

Figure 6

The Cis+Nira therapy is effective against three-dimensional (3D) cultures of Twist-knockdown CisR OC cells. (A) The schematic diagram showing the experimental protocol for the formation 3D spheroid and treatment plan of DMSO, Cis, and Nira. (B) The images representing 3D spheroids formation of siNC and siTwist transfected cells at 0, 1, 3 and 5 days on OV90-CisR and SKOV3-CisR (Magnification, 10×, scale bar 100 µm). The cells were cultured in absence or presence of Cis (18 µm, OV90—CisR; 21 µm, SKOV3-CisR) and Nira (17.5 µm, OV90—CisR; 15 µm, SKOV3-CisR) for 120 h. (Magnification, 10×, scale bar 100 µm) (C) Relative cell viability of siNC and siTwist transfected 3D culture cells administered with DMSO, Cis, Nira, and Cis+Nira in OV90-CisR (18 µm of Cis and 17.5 µm of Nira) and SKOV3-CisR (21 µm of Cis and 15 µm of Nira) OC cells. CisR: cisplatin-resistant; siRNA: small interfering RNA; siNC: non-targeting negative control, siRNA; siTwist: Twist siRNA; DMSO: dimethyl sulfoxide, Cis: cisplatin; Nira: niraparib. Values were represented as mean ± SD. # p < 0.05, compared with siNC group; * p < 0.05, compared with the siTwist (DMSO) group.

Figure 7

The Cis+Nira therapy enhances ER stress in 3D cultures of Twist-knockdown CisR OC cells. Western blot analysis of ER stress proteins, GRP78, calnexin, cleaved ATF6 and CHOP in 3D cultures of Twist-knockdown (A) OV90-CisR and (B) SKOV3-CisR OC cells. CisR: cisplatin-resistant; siRNA: small interfering RNA; siNC: non-targeting negative control, siRNA; siTwist: Twist siRNA; DMSO: dimethyl sulfoxide, Cis: cisplatin; Nira; niraparib. Values were represented as mean ± SD. # p < 0.05, compared with siNC group; * p < 0.05, compared with the siTwist (DMSO) group.

Figure 8

The Cis+Nira therapy significantly reduced mitochondrial membrane potential in Twist-knockdown CisR OC cells. Microscopic visualization and quantification of mitochondrial membrane potential in 3D cultures of Twist-knockdown (A) OV90-CisR and (B) SKOV3-CisR OC cells. Magnification, 10×, scale bar 100 µm. CisR: cisplatin-resistant; siRNA: small interfering RNA; siNC: non-targeting negative control, siRNA; siTwist: Twist siRNA; DMSO: dimethyl sulfoxide, Cis: cisplatin; Nira; niraparib. Values were represented as mean ± SD. # p < 0.05, compared with siNC group; * p < 0.05, compared with the siTwist (DMSO) group.

Figure 9

The combination therapy efficiently activated mitochondrial-dependent apoptotic pathway in Twist-knockdown CisR OC cells. (A) Western blot analysis of Bax, Bcl-2 and cytochrome C in 3D cultured of Twist knockdown OC cells. (B) The ratio of cytochrome C in cytosolic fraction to that in mitochondria fraction. (C) H&E staining for morphology evaluation and immunohistochemistry of the cell proliferation marker, ki-67 and apoptosis marker, cleaved caspase-3 immunoreactivity prediction (Magnification, 40×, scale bar 20 µm). CisR: cisplatin-resistant; siRNA: small interfering RNA; siNC: non-targeting negative control, siRNA; siTwist: Twist siRNA; DMSO: dimethyl sulfoxide, Cis: cisplatin; Nira; Niraparib. Values were represented as mean ± SD. # p < 0.05, compared with siNC group; * p < 0.05, compared with the siTwist (DMSO) group.

Figure 10

The pathway involved in the synthetic lethality of PARPi with combination of cisplatin on Twist knockdown CisR OC cells.

Figure 11

The proposed therapeutic strategy to overcome cisplatin resistance in OC.