Establishment of Acquired Cisplatin Resistance in Ovarian Cancer Cell Lines Characterized by Enriched Metastatic Properties with Increased Twist Expression

Ovarian cancer (OC) is the most lethal of the gynecologic cancers, and platinum-based treatment is a part of the standard first-line chemotherapy regimen. However, rapid development of acquired cisplatin resistance remains the main cause of treatment failure, and the underlying mechanism of resistance in OC treatment remains poorly understood. Faced with this problem, our aim in this study was to generate cisplatin-resistant (CisR) OC cell models in vitro and investigate the role of epithelial–mesenchymal transition (EMT) transcription factor Twist on acquired cisplatin resistance in OC cell models. To achieve this aim, OC cell lines OV-90 and SKOV-3 were exposed to cisplatin using pulse dosing and stepwise dose escalation methods for a duration of eight months, and a total of four CisR sublines were generated, two for each cell line. The acquired cisplatin resistance was confirmed by determination of 50% inhibitory concentration (IC50) and clonogenic survival assay. Furthermore, the CisR cells were studied to assess their respective characteristics of metastasis, EMT phenotype, DNA repair and endoplasmic reticulum stress-mediated cell death. We found the IC50 of CisR cells to cisplatin was 3–5 times higher than parental cells. The expression of Twist and metastatic ability of CisR cells were significantly greater than those of sensitive cells. The CisR cells displayed an EMT phenotype with decreased epithelial cell marker E-cadherin and increased mesenchymal proteins N-cadherin and vimentin. We observed that CisR cells showed significantly higher expression of DNA repair proteins, X-ray repair cross-complementing protein 1 (XRCC1) and poly (ADP-ribose) polymerases 1 (PARP1), with significantly reduced endoplasmic reticulum (ER) stress-mediated cell death. Moreover, Twist knockdown reduced metastatic ability of CisR cells by suppressing EMT, DNA repair and inducing ER stress-induced cell death. In conclusion, we highlighted the utilization of an acquired cisplatin resistance model to identify the potential role of Twist as a therapeutic target to reverse acquired cisplatin resistance in OC.


Introduction
The most lethal gynecological malignancy, ovarian cancer (OC) is the third most prevalent after cervical and uterine cancers and is the fifth leading cause of cancer-associated death in women worldwide [1,2]. Platinum-based chemotherapy has been the standard treatment for advanced OC  20, 40, 80 to 100 µM) of cisplatin for intermittent incremental treatment methods to generate CisR OC cells. A total of four sublines were generated, two from each cell line, including OV-90/CisR1, OV-90/CisR2, SKOV-3/CisR1 and SKOV-3/CisR2 ( Figure 1). On morphological evaluation, the parental cells displayed a polygonal shape with more regular shapes and sizes and were attached to the culture dish in discrete clusters, where CisR cells had variation in cell sizes, occasional enlarged multinucleated "giant" cells, prominent macronucleoli, and increased number of cellular processes (dendrites). The CisR cells also demonstrated stronger adhesion to the culture dish than parental cells ( Figure 2A). Furthermore, the parental and CisR OC cells were analyzed for spheroid formation capacity in Poly-HEMA coated 12-well plates by utilizing hanging drop method. CisR cells exhibited more cancer stem cell (CSC)-like characteristics than their parental OC cells. The spheroids in CisR cells were more round, solid and tightly compact compared to their parental cells (Figure 2A).
Inhibitory concentration (IC50) values were evaluated for parental and CisR cells by measuring the percentage of inhibition of cisplatin at 24, 48 and 72 h. It was observed that a significant increase in the dose of cisplatin was required to inhibit 50% of cell growth in both CisR cells compared to their corresponding parental cells ( Figure 2B). The IC50 values of cisplatin in the OV-90/parental cell line were 57. 55 16.75 ± 0.83 µM) at 72 h, which showed a 3.53-fold (OV-90/CisR1) and 4.19-fold (OV-90/CisR2) increase in the concentration of cisplatin required to obtain a 50% inhibition in cell growth ( Figure S1A). In SKOV-3 cells, the IC50 values of CisR cells, SKOV-3/CisR1 and SKOV-3/CisR2, were determined as 91.59 ± 8.47 On morphological evaluation, the parental cells displayed a polygonal shape with more regular shapes and sizes and were attached to the culture dish in discrete clusters, where CisR cells had variation in cell sizes, occasional enlarged multinucleated "giant" cells, prominent macronucleoli, and increased number of cellular processes (dendrites). The CisR cells also demonstrated stronger adhesion to the culture dish than parental cells ( Figure 2A). Furthermore, the parental and CisR OC cells were analyzed for spheroid formation capacity in Poly-HEMA coated 12-well plates by utilizing hanging drop method. CisR cells exhibited more cancer stem cell (CSC)-like characteristics than their parental OC cells. The spheroids in CisR cells were more round, solid and tightly compact compared to their parental cells (Figure 2A).
Taken together, these initial data revealed that a cisplatin-resistant phenotype in four OC sublines induced metastatic signals of increasing cell migration ability following chronic in vitro exposure to cisplatin.
Our results demonstrated that the extracellular matrix (ECM)-like fibronectin was associated with a decreased sensitivity to cisplatin-based drug treatment, as CisR cells displayed higher cell-ECM adhesion compared to the parental OC cells ( Figure S5). In OV-90 cells, the cell-ECM adhesion rate was higher in CisR sublines, OV-90/CisR1 (133.25 ± 3.7% vs.% of parental) and OV-90/CisR2 (141.4 ± 3.5% vs.% of parental). In SKOV-3 cells, the cell-ECM adhesion rate was also higher in CisR sublines, SKOV-3/CisR1 (148.2 ± 3.5% vs.% of parental) and SKOV-3/CisR2 (154.6 ± 3.4% vs.% of parental). For further confirmation, we examined the most common epithelial (E-cadherin) and mesenchymal (N-cadherin and vimentin) markers. Our Western blot data demonstrated that CisR OC cells displayed EMT phenotype ( Figure 4B). The expression level of the epithelial marker E-cadherin was lower in CisR cells compared to parental cell. However, the CisR cells showed significantly higher expression of mesenchymal marker proteins, N-cadherin and vimentin. Taken together, these results demonstrated that chronic exposure to cisplatin induced metastatic signals leading to EMT phenotypes.     [43]. Thus, we investigated whether acquired cisplatin resistance was associated with a significant change in DNA repair proteins. We examined the clonogenic growth rate of both cisplatin resistance and parental OC cell lines to confirm survival capability. The CisR cells had increased clonogenic activity as early as 24 h of cisplatin incubation followed by the ninth day of recovery period compared to parental OC cells ( Figure 5A). Furthermore, our Western blot analysis revealed that CisR cells significantly altered DNA repair protein (XRCC1 and PARP1) expression compared to parental OC cells ( Figure 5B), which could enhance survival by repairing the cisplatin-damaged DNA and rescuing the cell from apoptosis.  [43]. Thus, we investigated whether acquired cisplatin resistance was associated with a significant change in DNA repair proteins. We examined the clonogenic growth rate of both cisplatin resistance and parental OC cell lines to confirm survival capability. The CisR cells had increased clonogenic activity as early as 24 h of cisplatin incubation followed by the ninth day of recovery period compared to parental OC cells ( Figure 5A). Furthermore, our Western blot analysis revealed that CisR cells significantly altered DNA repair protein (XRCC1 and PARP1) expression compared to parental OC cells ( Figure 5B), which could enhance survival by repairing the cisplatin-damaged DNA and rescuing the cell from apoptosis. Since many chemotherapeutic drugs, including cisplatin, cause cell death by inducing endoplasmic reticulum (ER)-stress-mediated apoptosis, an altered ER stress-dependent apoptotic response diminishes the efficacy of these drugs in resistant cells [18,[44][45][46][47]. Studies on colon cancer, breast cancer and osteosarcoma have demonstrated that acquired chemotherapy resistant cancer cells have resistance to ER stress-triggered cell death [18,48,49]. Thus, we evaluated whether ER stressmediated apoptosis is involved in acquired cisplatin resistance in OC cells. To accomplish this, both Since many chemotherapeutic drugs, including cisplatin, cause cell death by inducing endoplasmic reticulum (ER)-stress-mediated apoptosis, an altered ER stress-dependent apoptotic response diminishes the efficacy of these drugs in resistant cells [18,[44][45][46][47]. Studies on colon cancer, breast cancer and osteosarcoma have demonstrated that acquired chemotherapy resistant cancer cells have resistance to ER stress-triggered cell death [18,48,49]. Thus, we evaluated whether ER stress-mediated apoptosis is involved in acquired cisplatin resistance in OC cells. To accomplish this, both the parental and the CisR OC cells were treated with 50 µM of cisplatin for 24 h. Our results revealed that CisR cells had a poor apoptotic signature profile with a reduced level of apoptotic proteins (Bax, cleaved caspase-9 and cleaved caspase-3) and an increased level of anti-apoptotic protein (Bcl-2) compared to parental OC cells ( Figure 5C,D) [50]. Subsequently, cisplatin significantly increased the expression of molecular markers of ER stress, such as GRP78, CHOP in parental OC cells compared to CisR OC cells [51,52]. Taken together, these findings demonstrate that cisplatin-induced ER stress-mediated apoptosis was significantly diminished in the CisR cells compared to the parental OC cells, which may point to an important underlying mechanism of CisR OC to avoid cellular death by cisplatin.

Twist Knockdown Can Affect the Metastasis Potential of Acquired CisR OC Cells
To investigate the role of Twist in cisplatin resistance, we generated the Twist knockdown (siTwist) OV-90/CisR1, OV-90/CisR2, SKOV-3/CisR1 and SKOV-3/CisR2 OC cells and siRNA negative control (siNC) cells ( Figure 6A). In order to justify re-sanitization capacity of CisR cells by Twist knockdown, we included non-transfected parental cell in our experiment. The siTwist cells significantly changed 3D spheroid formation capacity than siNC. The siTwist showed reduced spheroid roundness and solidity than siNC ( Figure 6B). The siTwist cells displayed the reduced wound healing capacity then siNC ( Figure 6C).

Twist Knockdown Attenuated Cell Metastasis Properties via Suppression of Cell Invasion and EMT Phenotype in Acquired CisR OC Cells
To investigate the molecular mechanism by which Twist mediated cisplatin resistance, we investigated cell metastasis capacity by invasion and the expression level of EMT-related proteins by Western blot. The siTwist cells showed lower cell invasion ( Figure 7A) ability than siNC cells. The expression of epithelial cell marker protein, E-cadherin was significantly increased in siTwist cells than siNC cells, while N-cadherin and vimentin, mesenchymal cell marker significantly downregulated in siTwist than siNC ( Figure 7B).

Twist Knockdown Reduces Cell Survival Potential via Downregulation of DNA Repair Pathway and Activation of ER-Stress-Mediated Cell Death in CisR OC Cells
We observed that the expression of DNA repair proteins-PARP1 and XRCC1 ( Figure 8A and Figure S7) and the cell survival capacity ( Figure 8B) were lower in siTwist cells than siNC cells. Twist knockdown increased the ER stress response in CisR cells, as indicated by significant increase in the expression of GRP78, cleaved ATF-6 and CHOP ( Figure 8C and Figure S7).
The result also showed that knockdown of Twist in CisR cells significantly reduced relative cell viability ( Figure 9A) and induced the magnitude of cell death by downregulation of regulation anti-apoptotic protein Bcl-2 and upregulation of apoptotic proteins Bax, cleaved caspase-9 and cleaved caspase-3 ( Figure 9B and Figure S8).
Under ER stress, cellular dysfunction and cell death often occurred. As expected, the CisR cells were treated with tunicamycin (5 µg/mL) for 48 h to induce ER stress. The upregulation of GRP78, cleaved ATF-6 and CHOP were observed at 24 and 48 h, as indicated elevation of ER stress ( Figure 9C).

ER Stress Inhibition Reversed the Twist Knockdown-Induced Cell Death
To investigate the implication of ER stress, we used 2.5 mM of 4-phenylbutyric acid (4-PBA) to diminish ER stress. The knockdown of Twist reduced cell growth and induced cell death ( Figures 8B and 9A,B). Interestingly, 4-PBA treatment reversed the Twist knockdown-induced cell growth ( Figure 10A), and also rescued the CisR cells from Twist knockdown-induced cell death by attenuating apoptotic protein cleaved caspase-3 expression ( Figure 10B). These results represented how Twist knockdown acted via ER stress to induce cell death in CisR OC cells.   To investigate the molecular mechanism by which Twist mediated cisplatin resistance, we investigated cell metastasis capacity by invasion and the expression level of EMT-related proteins by Western blot. The siTwist cells showed lower cell invasion ( Figure 7A) ability than siNC cells. The expression of epithelial cell marker protein, E-cadherin was significantly increased in siTwist cells than siNC cells, while N-cadherin and vimentin, mesenchymal cell marker significantly downregulated in siTwist than siNC ( Figure 7B).

ER Stress Inhibition Reversed the Twist Knockdown-Induced Cell Death
To investigate the implication of ER stress, we used 2.5 mM of 4-phenylbutyric acid (4-PBA) to diminish ER stress. The knockdown of Twist reduced cell growth and induced cell death ( Figures 8B  and 9A,B). Interestingly, 4-PBA treatment reversed the Twist knockdown-induced cell growth ( Figure 10A), and also rescued the CisR cells from Twist knockdown-induced cell death by attenuating apoptotic protein cleaved caspase-3 expression ( Figure 10B). These results represented how Twist knockdown acted via ER stress to induce cell death in CisR OC cells.

Discussion
In this study, we established CisR OC models using high dose pulse treatment and a stepwise increasing dose of cisplatin to characterize the evolution of acquired resistance during cisplatin-based anti-cancer therapy. The pulse dosing is identical to that used in hospital treatment with a pulse therapy regimen, using a high dose of the chemotherapy drug followed by a rest period calculated to allow the patient to recover from adverse effects [53,54]. The treatment method using stepwise drug dose escalation can be clinically effective for an oral drug given daily or twice daily, as a relatively constant amount of the drug is present within the body [24,55]. When establishing resistant

Discussion
In this study, we established CisR OC models using high dose pulse treatment and a stepwise increasing dose of cisplatin to characterize the evolution of acquired resistance during cisplatin-based anti-cancer therapy. The pulse dosing is identical to that used in hospital treatment with a pulse therapy regimen, using a high dose of the chemotherapy drug followed by a rest period calculated to allow the patient to recover from adverse effects [53,54]. The treatment method using stepwise drug dose escalation can be clinically effective for an oral drug given daily or twice daily, as a relatively constant amount of the drug is present within the body [24,55]. When establishing resistant cell lines, the pulse dosing method has generally been considered as inferior compared to the intermittent incremental method due to relatively lesser stability and strength of drug resistance [24,56,57]. In our study, we established a genetically stable and clinically relevant CisR OC cell by modifying the conventional pulse dosing method with gradually increasing duration of drug incubation period with constant high dose [58].
In ovarian cancer research, in vitro cell line models become effective tools to understand the molecular mechanisms underlying acquired chemo-resistance development in ovarian cancer [59][60][61][62]. We derived and confirmed the acquired cisplatin resistance cell model through both pulse and stepwise dose increasing methods, which offer a useful tool for describing the molecular mechanisms of acquired cisplatin resistance. Morphologically, the CisR cells were different from the parental OC cell. CisR cell lines exhibited, multinucleated "giant" cells, greater variability in cell size, and prominent macronucleoli compared to the parental OC cell. These CisR cells were confirmed with cisplatin IC 50 values at least 3-5 times greater than those of the corresponding parental cell.
Chemotherapy resistance with metastasis is a major obstacle to successful cancer treatment [63,64]. Epithelial OC patients receiving chemotherapy usually develop acquired drug resistance within one year, which leads to tumor recurrence and uncontrolled metastases [65]. Metastasis, one of the most important hallmarks of malignancy, is a complex process that involves migration and invasion of cancer cells [66][67][68]. Several studies have suggested that chemotherapy resistance is acquired by the metastatic growth of tumor cells that may closely parallel each other [69][70][71]. It has been demonstrated that metastasis-related genes play a vital role in cisplatin chemo-resistance [72]. Our results suggested that CisR in OC cells altered their characteristics and retained the majority of metastatic properties by increasing the cell proliferation, cancer stem cell (CSC)-like characteristics, migration and invasion abilities of cancer cells [73].
DNA nucleotide excision repair (NER) and base excision repair (BER) are the most common DNA repair mechanisms that arise to repair DNA damage caused by cisplatin [74][75][76][77][78]. Poly (ADP-ribose) polymerases 1 (PARP1) interacts with X-ray repair cross-complementing protein 1 (XRCC1) to trigger the BER DNA repair process [79][80][81][82]. NER and BER are the mechanisms in repairing the DNA crosslink induced by cisplatin [83,84]. Therefore, DNA repair proteins, XRCC1 and PARP1, have been associated with significantly aggressive clinical outcome of ovarian cancer patients and decreased cisplatin sensitivity in OC cells. XRCC1 is a scaffolding protein that interacts with BER factors, including Ligase III, DNA polymerase β and PARP1, to recruit them to the DNA breaks, thus vital to BER [85]. PARP1 is an important protein that is recruited to the site of DNA damage to trigger poly (ADP-ribosylation) of multiple substrates, which leads to the activation of DNA repair [81]. On such basis, PARP inhibitors have been clinically effective for their anti-cancer effects [86,87]. The results of our study are also consistent with previous studies because the significantly increased level of XRCC1 and PARP1 correlated with cisplatin resistance in both SKOV-3 and OV-90. Taken together, these results support our findings that XRCC1 and PARP1 are important to the development of cisplatin resistance in OC.
We observed that in CisR OC cells had significantly decreased apoptotic proteins of the ER stress pathway and mitochondrial pathway compared to parental OC cells. Anti-apoptotic protein Bcl2 in CisR OC cells were significantly increased compared to parental cells, in agreement with other previous in vitro studies involving CisR cell lines [88]. However, our observations of significantly decreased GRP78 protein level conflict with previous reports in other malignancies showing that GRP78 exerts pro-survival and chemo-resistant effects. GRP78 is an important chaperone protein of the ER which has been implicated in cancer resistance against chemotherapy involving apoptotic pathways. This has been demonstrated through increased sensitivity against therapeutic drugs by knockdown of GRP78 in glioblastoma. In addition, GRP78 has been associated with poor survival in breast, liver, prostate, colon and gastric cancers with the exception of lung cancer [89][90][91][92][93]. These conflicting data regarding the role of GRP78 may be related to the differences in organs and cell types, as well as the inadequacy of using a single model to explain the complex process of cisplatin resistance.
EMT, a hallmark of aggressive and highly invasive cancers, contributes to CisR in OC cells by suppressing the epithelial marker (E-cadherin) and enhancing the expression of mesenchymal marker proteins (N-cadherin and vimentin) [94]. The EMT of cancer cells has been considered to be an important mechanism for cancer metastasis and chemotherapy resistance [65,95]. EMT-associated proteins are highly expressed in chemotherapy resistance and have been associated with enhanced migration and metastasis of tumor cells [96][97][98]. Many studies have demonstrated that chemotherapy resistance and metastasis are controlled by reversible phenotypic transitions between epithelial and mesenchymal phenotypes (EMT and MET), which is referred to as epithelial plasticity [99][100][101][102][103]. The tumor cells can also reach a state of partial or intermediate EMT during transitioning between EMT and MET, called hybrid epithelial/mesenchymal phenotype [95,104,105].
One of the important events contributing to EMT is the activation of EMT-transcription factors (TFs), such as Twist that act as repressors for epithelial genes and as activators for mesenchymal genes [106][107][108][109]. We demonstrated significant increased levels of Twist, N-cadherin and vimentin and significantly decreased levels of E-cadherin in CisR cells compared to parental cells. This was in agreement with studies that noted that EMT-TFs that regulated EMT, such as Twist, have been demonstrated to mediate the development of cancer cell resistance to platinum-based anti-cancer drugs in various cancers [33][34][35]110]. As CisR cells exhibited EMT phenotypes, we speculate that EMT contributes to the cisplatin resistance mechanism of OC cells.
Twist has been reported to be involved in the development of acquired chemoresistance, leading to a poorer progression in various human cancer [111][112][113][114][115]. Some study suggested that Twist promotes platinum resistance in ovarian cancer via activation of collagen type XI alpha 1 (COL11A1), GAS6, L1CAM, and Akt signaling [35,116]. Recently, acquisition of therapeutic resistance in ovarian cancer correlated with Twist, EMT phenotype and micro-RNA [117][118][119][120]. Various approaches were applied to overcome the clinical challenges of metastasis and chemoresistance in OC including nanoparticle delivery of siRNA against Twist [121]. In agreement with previous studies, the present study revealed that Twist knockdown reduced metastasis properties by suppressing CSC-like characteristics, migratory ability and invasiveness in CisR OC cells [28,113]. Twist-deficient CisR OC cells exhibited EMT phenotypic characteristics by increasing E-cadherin and reducing N-cadherin and vimentin expression [122]. Twist knockdown CisR OC cells exhibited reduced DNA repair capacity and increased ER stress mediated cell death. In addition, our studies suggested that Twist knockdown could promote cell death via ER stress pathway. The ER stress markers GRP78, cleaved ATF-6 and CHOP were markedly increased in Twist-deficient CisR cell. Intriguingly, ER stress inhibition markedly rescued cell growth and reversed Twist knockdown-induced cell death. Finally, we limited our experimental analysis of OC cell lines to two types in vitro, which may not reflect the in vivo patient environment. Therefore, we cannot conclude that the majority of OC cancers will be correlated with Twist and ERstress-mediated cell death.

Generation of Acquired CisR Ovarian Cancer (OC) Cell Lines
The acquired CisR OC cell lines were generated by following the previously described method with slight modifications [71,123].
The two resistant sub-clones were established over a period of 8 months. All the resistant cells were maintained in a medium containing 2 µM of cisplatin supplemented with 10% FBS, 1% penicillin and streptomycin. Cells were kept at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air. These cell lines grew in monolayers and were passaged when cultures were 70-80% confluent. No experiments were performed until all the cells had been maintained in drug-free medium for 1 month.

siRNA Transfection
To create a knockdown of Twist, we transfected cells using Twist siRNA (#sc38604, Santa Cruz Biotechnology, Dallas, TX, USA) and control siRNA (#sc37007). The lyophilized siRNA duplex was reconstituted in RNase-free water to create 10 µM stock solutions. Lipofectamine 2000 RNAiMAX (#13778030, Invitrogen, Waltham, MA, USA) was used to transfect the siRNA into cells according to the manufacturer's instructions. The transfected cells were incubated for 48 h before experiments.

Colony Formation Assay
Colony formation assay was determined using a clonogenic assay [125]. In brief, the parental and CisR cells were cultured in 6-well plates at low density (~1000 cells per well) for 24 h and then treated with 50 µM cisplatin for 24 h followed by 9 days of recovery. The plates were then washed with PBS and stained with 0.1% crystal violet solution. The cells were washed until no stain was visible, air dried, and photographed. The dye was extracted using 1% sodium dodecyl sulfate (SDS) solution by continuous shaking and then quantified using a spectrophotometer at 570 nm.

Cell Migration Assays
Cell migration assay was performed by using a culture medium-treated 6.5 mm transwell chamber with 8.0 µm pore polycarbonate membranes (#3422, Corning Life Sciences, Corning, NY, USA). In brief, the parental and CisR cells were cultured in 6-well plates and were treated with 50 µM cisplatin for 24 h. Cells were then harvested from cell culture plates by serum-free medium and 2 × 10 4 cells per 300 µL of serum-free medium plated into the transwell insert, while the bottom chamber was filled with 700 µL medium containing 10% FBS. After incubation in a humidified incubator with 5% CO 2 at 37 • C for the desired period of times (12 and 24 h), non-migratory cells were scraped off from the top of the transwell using a cotton swab. The cells attached to the bottom side of the membrane were fixed by methanol, stained with 0.1% crystal violet, dried, and photographed under a light microscope. The number of migrating cells was measured using a microplate reader at (Synergy H1; BioTek Instruments, Inc., Winooski, VT, USA) absorbance 570 nm. The calculations of percentage of cell migration, migration speed and average migration speed are given in Table 1.

Cell Invasion Assays
Cell invasion assay was performed by using a culture medium-treated Corning BioCoat Matrigel Invasion Chamber with 8.0 µm pore polycarbonate membranes (#354480, Corning Life Sciences, Corning, NY, USA). In brief, the parental and CisR cells were cultured in 6-well plates and were treated with 50 µM cisplatin for 24 h. Cells were then harvested from cell culture plates by serum-free medium and 2 × 10 4 cells per 300 µL of serum-free McCoy's 5A medium was plated into the transwell insert, while the bottom chamber was filled with 700 µL medium containing 10% FBS. After incubation in a humidified incubator with 5% CO 2 at 37 • C for the desired period of time (12 and 24 h), non-invasive cells were scraped off from the top of the transwell with a cotton swab. The cells attached to the bottom side of the membrane were fixed by methanol, stained with 0.1% crystal violet, dried, and photographed under a light microscope. The number of invading cells was measured by the microplate reader (Synergy H1; BioTek Instruments, Inc., Winooski, VT, USA) at absorbance 570 nm. The equations for calculating the percentage of cell invasion and the invasion speed are given in Table 2.

Wound Healing Assay
Cell mobility was assessed using a scratch wound healing assay. In brief, the parental and CisR cells were cultured in 6-well plates for 24 h and then treated with 50 µM cisplatin for another 24 h.
Cells were re-suspended and again 2 × 10 5 cells were seeded into six-well plates and cultured to monolayers, which were then wounded using sterile 1 mL pipette tips. Cells were washed with PBS to remove any debris. Photos were captured at 0, 12 and 24 h after wounding. The gap distance can be quantitatively evaluated using software such as ImageJ (National Institutes of Health, Bethesda, MD, USA). The equations for calculation of percentage of wound closure, wound healing speed and relative wound area are given in Table 3. Table 3. Calculations for measuring wound healing.

Adhesion Assay
The adhesion assay was performed with the CytoSelect cell adhesion kits, which utilizes a Fibronectin-coated 48-well plate (#CBA-050, Cell Biolabs, Inc., San Diego, CA, USA). In brief, the parental and CisR cells were cultured in 6-well plates for 24 h and then treated with 50 µM cisplatin for another 24 h. Then, under sterile conditions the Fibronectin adhesion plate was allowed to warm up at room temperature for 10 min (min) and a cell suspension containing 1 × 10 6 cells/mL was prepared in serum-free medium. Then, 150 µL of cell suspension was added to the inside of each well (BSA-coated wells act as a negative control) and incubated for 90 min in cell culture incubator (37 • C, 5% CO 2 atmosphere). After that, the researchers carefully discarded or aspirated media from the wells and gently washed each well 4-5 times with 250 µL of PBS. Then, the researchers added 200 µL of cell stain solution and incubated for 10 min at room temperature. The cell staining solution was discarded or aspirated from the wells, and each well was gently washed 4-5 times with 500 µL of deionized water and allowed to air dry. After taking photographs of each group, 200 µL of extraction solution was added per well, followed by incubating for 10 min on shaking machine. Next, 150 µL was transferred from each extracted sample to a 96-well microtiter plate, and the OD 570 nm was measured in the plate reader (Synergy H1; BioTek Instruments, Inc., Winooski, VT, USA).

Apoptosis Assay
Hoechst 33,342 staining was conducted to distinguish apoptotic cells from normal cells. In brief, cells were seeded in 6-well plates with 2 × 10 5 cells per well in culture media and were allowed to attach overnight. The cells were treated with cisplatin at doses of 50 µM and incubated at 37 • C for 24 h. After incubation, the seeded cells were washed on the 6-well plate PBS once, then incubated with 5 µg/mL Hoechst 33,342 for 15 min. Finally, cells were washed twice with PBS and observed using inverted fluorescence microscopy (Axioskop 2 plus microscope, Carl Zeiss, Oberkochen, Germany). The apoptotic nuclei were counted from five non-overlapping fields and expressed as a percentage of the total number of nuclei counted.

Conclusions
The generation and characterization of OC cell lines in this study provide an important scientific resource for studying the molecular mechanism of acquired chemotherapy resistance in ovarian cancer therapy. The individual signature ( Figure 11) exhibited by the CisR cell model offers a framework for the development of new molecular targets to treat ovarian cancer disease. In particular, the role of Twist and ER-stress-mediated cell death could allow us to understand the molecular mechanism of complex acquired cisplatin resistance, and develop a new strategy to overcome CisR OC.

Conclusions
The generation and characterization of OC cell lines in this study provide an important scientific resource for studying the molecular mechanism of acquired chemotherapy resistance in ovarian cancer therapy. The individual signature ( Figure 11) exhibited by the CisR cell model offers a framework for the development of new molecular targets to treat ovarian cancer disease. In particular, the role of Twist and ER-stress-mediated cell death could allow us to understand the molecular mechanism of complex acquired cisplatin resistance, and develop a new strategy to overcome CisR OC. Figure 11. The possible molecular mechanism of acquired cisplatin resistance in ovarian cancer (OC) cells. Prolong or high dose of cisplatin lead to increase expression EMT transcription factor Twist followed by enriched metastasis and acquired chemotherapy resistance. The CisR cells reserved the metastasis properties, including cell proliferation, migration, invasion and cell adhesion. The CisR Figure 11. The possible molecular mechanism of acquired cisplatin resistance in ovarian cancer (OC) cells. Prolong or high dose of cisplatin lead to increase expression EMT transcription factor Twist followed by enriched metastasis and acquired chemotherapy resistance. The CisR cells reserved the metastasis properties, including cell proliferation, migration, invasion and cell adhesion. The CisR cells acquire the ability to repair DNA and reduce ER-stress-mediated cell death. The EMT-related phenotypes are strongly expressed in the CisR, which could lead to enhanced metastasis and acquired cisplatin resistance.

Supplementary Materials:
The following are available online at http://www.mdpi.com/1422-0067/21/20/7613/s1: Figure S1: The 50% inhibitory concentration (IC 50 ) value of cisplatin in parental and CisR OC cells; Figure S2: The migration capability of parental and CisR OC cells assessed by transwell migration assay; Figure S3: The migration capability of parental and CisR OC cells assessed by scratch wound healing assay; Figure S4: The invasion capability of parental and CisR OC cells assessed by transwell migration assay; Figure S5: The adhesion capability of parental and CisR OC cells assessed by fibronectin-adhesion assay; Figure S6: The expression of DNA repair proteins, PARP1 and XRCC1 to relative beta actin in parental, siTwist and siNC cells; Figure S7: Twist knockdown induces the expressions of ER stress proteins, GRP78, cleaved ATF-6 and CHOP relative to beta actin in parental, siTwist and siNC cells; Figure S8: Twist knockdown induces cell death protein expression in CisR OC cells.