Human ovarian cancer cell lines SKOV3 and OCAVAR8 were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM culture solution containing 12% fetal bovine serum, respectively, and placed in an incubator at 5% CO2, 37°C. Lentivirus contain DCTPP1 shRNA (target:GCCCTTCAAGAGGAGCTTA) and negative controls (5′-GTT​CTC​CGA​ACG​TGT​CAC​GT-3′) were purchased from Shanghai Gima Pharmaceutical Co., Ltd. SKOV3 and OCAVAR8 cells in logarithmic growth phase were planted in 24-well plates with a planting density of 5 × 10⁴/well. When the cell growth density reached 50%, infection was performed for 24 h according to the instructions and then transected cells are selected by puromycin to establish DCTPP1 knockdown cell. The cells of each group were expanded cultured for follow-up work.

Ovarian cancer cells were lysed by Trizol method, then total RNA was extracted and synthesized into cDNA.qPCR assay was performed follow the instruction manual of SYBR premix Ex Taq Kit. qPCR reaction program was set to two steps: (a) pre-incubation at 95°C for 30 s; (b) 95°C for 5 s and 55 °C for 30 s, 72 °C for 34 s for 40 cycles. Samples were assayed in triplicate using the ABI Prism 7500 detection system (Applied Biosystems, Foster City, CA, United States). The relative quantization value was then calculated by subtracting the average CT from the corresponding average CT for 18SRNA.

Cells were harvested and lysed in ice-cold lysis buffer (10 mM Hepes, pH 7.9; 150 mM NaCl; 1 mM EDTA); The protein concentration of cell Lysate was determined by BCA protein assay kit. Samples were separated by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore,United States), which was then blocked in 5% BSA blocking buffer at 4°C overnight. All antibodies (dilution, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States) were incubated on shaking bed overnight at 4°C respectively. Secondary antibody was incubated at room temperature for 30 min.Densitometric quantification of protein bands were visualized using ECL-Plus detection reagents (Santa Cruz Biotechnology, Santa Cruz, CA, United States) and performed with GAPDH as an internal control using ImageJ.

The genomic DNA was routinely extracted according to the instructions of the genome extraction kit. Two different promoter sequences of DCTPP1 (P1: 2000 -0 bp upstream of DCTPP1 coding sequence; P2:674-0 bp upstream of DCTPP1 coding sequence) were cloned and inserted into the luciferase vector. The luciferase reporter plasmids named luc-2000 and luc-674 respectively, were transfected into cells with empty vector as a control. The Dual luciferase reporter assay kit (promega, Madison, Wisconsin, United States) was used to detected the luciferase signal.

Cells (1 × 10³/100 μL) were seeded in 96-well plates (Corning, New York, United States) and cell viability was analyzed by using CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. The absorbance of 450 nm was measured on a microplate reader (BioRad, CA, United States) and the cell growth curves were calculated.

Cells (1 × 10⁵) were Collected for cell apoptosis assay using propidium iodide (PI, Sigma, Shanghai, China) and apoptosis assay using FITC-Annexin V Apoptosis Detection Kit (Biolegend, San Diego, CA, United States) according to the manufacturer’s instructions. Stained Cells (10,000 events for each sample) were acquired by FACS (BD Pharmingen, San Diego, CA, United States) and the results were analyzed with MLFT32 software.

ROS levels were detected using dihydroethidium (DHE) (Sigma-Aldrich, CA, United States). Cells were harvested and incubated with DHE (10 µ M) for 10 min at 37°C in the dark. Relative red fluorescence was measured using a flow cytometer (BD Pharmingen,CA, United States).

Briefly, type I collagen (Cellmatrix Type CD; Nitta Gelatin, Inc., Osaka, Japan), 10× F-12 medium, and reconstitution buffer were mixed together at a ratio of 8:1:1. The prepared tumor cells and collagen solution are inoculated at a volume ratio of 10:1, so that the final density of cells in the collagen droplets is 2 × 10⁵–5 × 10⁵ pieces/mL. The collagen-cell mixture (30 μL/drop) was transferred to the 6-well multiplate and cultured at 37°C in a CO₂ incubator. After culture for 16 days, Each collagen droplet was stained with neutral red, fixed with 10% neutral formalin buffer, washed with water, and quantified by culture cell analysis system. Image analysis at 540 nm would quantify the number of DCTPP1 knockdown cells and control cells.

Ovarian cancer specimens are surgical resection specimens, the specimens are fixed with 10% neutral formaldehyde after removal from the body. Immunohistochemistry was done using 8 μm serial sections placed onto glass slides using a single-staining procedure.

The protocols used with DCTPP1 and Ki67 antibody (dilution, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, United States) are carried out according to the instructions of Immunohistochemistry kit, and negative and positive controls were seted. Double-blind reading method was used to judge the staining results: two experienced doctors read the pictures separately, 10 representative fields were randomly observed in each slice, and comprehensive analysis and scoring was performed according to the respective staining degree and number of stained cells.

Alkaline unwinding and electrophoresis conditions was carried out according to instructions for the comet assay kit (Phgene, KM,China). A minimum of 100 individual cells per sample were scored in duplicate from three independent experiments. According to the DNA tail lengths, cell damage is divided into three levels: level 1, none (no damage); level 2, medium (moderate damage); level 3, long (severe damage);

Graphpad prism 5.0 and Excel 2016 was used to generate the graphs and process the data. The significance was analyzed with the unpaired t-test and one-way ANOVA, p values < 0.05 were considered significant. Data were expressed as the mean ± SD from at least three independent experiments.

Immunohistochemistry was used to measure DCTPP1 protein expression, represented by immunoreactivity score (IRS) in OC tissues and adjacent non-cancerous fallopian tubes tissues. As shown in Figure 1A, Expression of DCTPP1 was weakly or almost no stained in non-cancerous fallopian tubes tissues, positive signals of DCTPP1 was detectable mainly in the nucleus and cytoplasm of cancerous tissues. Statistical analyses indicated that DCTPP1 expressions in OC tissues of Ⅲ and Ⅳ stage are significantly higher than that inadjacent non-cancerous fallopian tubes tissues with p < 0.05 (Figure 1B).

Correlation between DCTPP1 expression and clinicopathological parameters was further analyzed, the expression of DCTPP1 was higher in older patients (>56 years old), no evident correlations were observed between DCTPP1 expression with the FIGO stage, lymph node and omentum metastasis, and CA125 (Table 1). The correlation between DCTPP1 expression and disease prognosis was analysis using the Kaplan–Meier assay. As shown in Figure 1C, 887 patients in the DCTPP1-high group exhibited a poorer prognosis when compared with 768 patients in the DCTPP1-low group (p = 0.022, Figure 1C). These results indicate that DCTPP1 is strongly associated with ovarian cancer prognosis and might play some role in ovarian cancer progression. And under the condition of cisplatin treatment, there was significant difference in the overall survival (p = 0.0034) between the patients in DCTPP1-high group and the patients in DCTPP1-low group. This results suggested that patients with high expression of DCTPP1 had a poor response to cisplatin treatment (Figure 1D).

In order to determine the role of ROS in cisplatin-induced cytotoxicity, we tested the level of ROS generation in the two ovarian cancer cells after cisplatin treatment. The results showed that cisplatin increased ROS levels and correspondingly increased apoptosis ratio in SKOV3 and OVCAR8 cells (Figures 2A,B), while cisplatin-induced apoptosis decreased when N-acetyl-l-cysteine (NAC) was added (Figure 2C). These results suggest cisplatin-induced generation of reactive oxygen species (ROS) has been correlated to its cytotoxic effects, which makes specific ROS scavengers being great significance to cisplatin resistance.

As shown in Figure 2D, induction effect of cisplatin on DCTPP1 was observed in both SKOV3 and OVCAR8 cells at the protein level after cisplatin treatment (p < 0.05). These results suggest that DCTPP1 might be a response factor for cisplatin. And then, we investigated our hypothesis that DCTPP1 expression could be induced by cisplatin through ROS generation. The dual luciferase reporter gene system is used to efficiently detect the transcriptional activation of DCTPP1. As shown in Figure 2E, the DCTPP1 promoter can be activated by the ROS inducer H₂O₂ (500uM), suggesting that DCTPP1 is a response factor of oxidative stress. At the same time, the activation of the DCTPP1 promoter by cisplatin can be reversed by N-acetyl-l-cysteine (NAC), the oxidative stress inhibitor (p < 0.05, Figure 2E). These results show that intracellular ROS accumulation induced by cisplatin up-regulated the transcription of DCTPP1. In summary, DCTPP1 expression could be induced by cisplatin through ROS generation.

Figure 3A shows cell lines with stable DCTPP1 knockdown were constructed, and the knockdown efficiency was assessed by Western blot respectively (p < 0.05). The OVCAR8 and SKOV3 cell lines with stable knockdown of endogenous DCTPP1 by short hairpin RNA (shRNA) were named as S-SH and O-SH respectively, the control cell lines were named S-NA and O-NA respectively. These four cell lines were selected for use in subsequent experiments.

CCK-8 assay were conducted to determine the cell growth. As shown in Figure 3B, the growth curves indicated that the growth rate was significantly lower in DCTPP1 knockdown cells than the control cells. A xenograft tumor mouse model was established to identify the impact of DCTPP1 on tumour growth in vivo. As shown in Figure 3C, loss of DCTPP1 expression resulted in a significant delay in tumor growth, where the difference in tumor volume was statistically significant after 6 weeks (p < 0.05). Ki67, a marker of tumour proliferation, was also observed to be decreased in DCTPP1 knockdown groups by immunohistochemistry (Figure 3D). As mounting evidence indicates that PIK3/Akt pathway always plays a crucial role in tumour growth, DCTPP1 deletion in both SKOV3 and OVCAR8 cells resulted in an elevated level of p21 and, at the same time, a decreased level of pAkt/Akt (Figure 3E). These results might predict the DCTPP1 knockdown would sensitize the sensitivity of cisplatin of ovarian cancer cells. Correspondingly, we have also observed an increase in the expression of apoptosis-related proteins PARP1 and c-PARP1, which suggested that the knockdown of DCTPP1 may stimulate cell apoptosis by down-regulating the activation of the Akt pathway.

Cisplatin is a well-known inducer of ROS and our previous researches have demonstrated that the cytotoxic effect of cisplatin was related to the increasing intracellular ROS level. We hypothesized that DCTPP1 promotes resistance of ovarian cancer cells to cisplatin-induced intracellular ROS production. To reveal the effect of DCTPP1 knockdown on intracellular ROS generation, we detected the total ROS levels in ovarian cancer cells using DHE staining. Although the effect of DCTPP1 knockdown on the level of intracellular ROS is not obvious, it has certain statistical significance (Figure 4A, p < 0.05) in OVACAR8 cells. It is worth noting that we have observed a significant downregulation of Nrf2 after DCTPP1 knockdown (Figure 4B, p < 0.05). More importantly, DCTPP1 knockdown also led to a decrease in glutamate-cysteine ligase, catalytic subunit (GCLC) and heme oxygenase 1 (HO-1), two of the major antioxidants located downstream of Nrf2 to suppress oxidative stress (Figure 4B, p < 0.05). These results suggest that overexpression of DCTPP1 in ovarian caner might activate the Nrf2-dependent antioxidative response in ovarian cancer cells, thus avoiding the oxidative stress and maintaining survival. Correspondingly, we observed changes in the total antioxidant capacity in the cells. DCTPP1 knockdown reduced the total antioxidant capacity (Figure 4C, p < 0.05), which further proved that DCTPP1 can participate in the redox homeostasis of the cell by regulating the Nrf-2-dependent antioxidant response.

To determine whether there was a causal link between cisplatin-induced upregulation of DCTPP1 and cisplatin-sensitivity, we observed the intracellar ROS in DCTPP1 knockdown cells under cisplatin treatment compare to the control. Compared to their parental cell lines, significantly higher intracellular ROS concentrations were detected after DCTPP1 knockdown, which indicated that DCTPP1 knockdown significantly increases intracellular ROS levels in ovarian caner cells cotreated with cisplatin (p < 0.05, Figure 5A). These results verified our hypothesis that the high expression of DCTPP1 may rebalance the excessive accumulation of ROS induced by cisplatin treatment. DCTPP1 knockdown might cause more oxidative stress in ovarian cancer cells under cisplatin treatment. The viability of SKOV3 and OVCAR8 cells exposed to cisplatin with or without DCTPP1 knockdown were assessed by CCK-8 assay and CD-DST assay. There is significant difference in cell viability between DCTPP1 knockdown group and the control group both in CCK-8 assay (p < 0.05, Figure 5B) and CD-DST assay (p < 0.05, Figure 5C). Both assays showed that DCTPP1 knockdown inhanced cisplatin sensitivity of ovarian cancer cells in both OVCAR8 and SKOV3 cells. Given that the main biological function of DCTPP1 is to maintain the safety of nucleic acids, comet assay was used to access the occurrence of double-strand DNA breaks (DSB), which is a natural progression of oxidative DNA damage lesions. Figure 5D shows that DCTPP1 knockdown lead to more long-tail cells in SKOV3 and OVCAR8 cells, which represent an increasing of DSB (p < 0.05). Then, we stained cells with Annexin V/PI following exposure to cisplatin with or without DCTPP1 knockdown, and Figure 5E shows that cisplatin-induced apoptosis was increased by DCTPP1 knockdown both in SKOV3 and OVCAR8 cells (p < 0.05). These results suggest that cisplatin-induced upregulation of DCTPP1 may be one of the mechanisms by which ovarian cancer resists the cytotoxicity of cisplatin. DCTPP1 knockdwon might enhence the cisplatin sensitivity of ovarian cancer cells and might be a promising therapeutic strategy.