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

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.

1.2 % poly-HEMA gel was prepared in 95% ethanol and coated on the bottom of 12, 24, 48 and 96-well culture plates by 500, 300, 150 and 75 µ l, respectively. Then the culture plates were kept under laminar flow cabinet for air drying. The cells were seeded at 2 × 10 6 cells/ml in the form of 20 µl drops on the inverted lid of the 60 mm petri dish, while the dish contained 4 to 5 ml of PBS. The generated cell aggregate sheets were transferred to Poly-HEMA coated plate containing complete medium after 48 h. After 24 h, the spheroids derived from siNC and siTwist cells were cultured in the absence or presence of Cis, Nira or Cis+Nira for 120 h with routine checking under light microscopy. The cultures were placed at 37˚C in a humidified incubator under 5% CO2 atmosphere throughout the culture period.

Mitochondrial apoptosis staining
After the treatment period, the 3D organoid cell suspension were collected to a centrifuge tube and centrifugation for 5 min at room temperature at 400 ×g, then remove supernatant, followed by addition of mitostain solution and incubate at 37˚C incubator for 15 min. After incubation, centrifuged for 5 min at 400 ×g, remove supernatant, then washed with PBS. Subsequently, PBS was used to mount the chamber for imaging by fluorescence microscopy and resuspended for fluorescence ratio detection by microplate reader. In fluorescence microscopy, mitostain was imaged using an Ex/Em: 540/570 nm filter, whereas the green signal was measured at Ex/Em: 490/520 nm. The fluorescence ratio was detected by measuring red fluorescence (Ex/Em: 550/600 nm) and green fluorescence (Ex/Em: 485/535) using a fluorescence microplate reader.

Mitochondrial and cytosolic separation
After treatment period, cells were washed twice with cold PBS and treated with lysis buffer containing protease inhibitors. Then they were homogenized by gentle douncing (100 strokes) and keep on ice for 30 min. Then collected the buffer and centrifuged for 10 min at 750 ×g at 4°C, and the sediment containing the nuclei and unbroken cells was discarded. The supernatant was then centrifuged at 10,000 ×g for 10 min. The resulting supernatant was removed and used as the cytosolic fraction. The sediment containing the mitochondria was further incubated with PBS containing 0.5% Triton X-100 for 10 min at 4°C. After centrifugation at 10,000 ×g for 30 min, the supernatant was collected as the mitochondrial fraction.

Western blotting
Total protein for western blotting was extracted from cells cultured in 2D and 3D environment using

Histology and immunohistochemistry
The After the treatment of the 3D spheroids with DMSO, Cis alone, Nira alone and Cis+Nira combination, aspirated off the medium from each well and wash the spheroid by adding PBS for 2×5 mins, fixed them by adding 10 % formalin. Following paraffin embedding and casting into a block, 3 µm thick sections were cut and mounted onto slides. All slides were kept at 58°C for 60 min in a The slides were dehydrated in 95 % and 100% ethanol and followed by the clear xylene. Finally, the slides were air dried and added the coverslip with permanent mounting medium (#E01-18, Golden bridge international labs, Bothell, WA, USA).    Values were represented as mean ± SD. # p < 0.05, compared with siNC group; *p < 0.05, compared with the siTwist (DMSO) group.