SOX17 increases the cisplatin sensitivity of an endometrial cancer cell line

Patients with EC who have received comprehensive staging surgery and suffered disease relapse within 6 months after regular platinum containing therapy are considered to be platinum resistant [17]. We selected a group of 21 chemo-resistant and another of 21 chemo-sensitive patients. To confirm the expression levels of SOX17, we assayed SOX17 expression in paraffin-embedded tissues obtained from all 42 EC patients. SOX17 levels were lower in the chemo-resistant tissues (Fig. 1a, b; Additional file 1: Table S1). There were no significant differences between the clinical features of the patients of the two groups (Additional file 1: Table S2). To clarify the molecular mechanisms underlying CDDP resistance in EC cells, SOX17 expression levels in 29 EC patients were assayed after cisplatin-based combination chemotherapy. The results showed that SOX17 expression in blood samples from chemo-resistant patients was significantly decreased compared with that from chemo-sensitive patients (Fig. 1c).

Western blot analyses were performed to assay the expression of SOX17 in the EC cell lines. SOX17 protein was expressed in all the EC cell lines (Fig. 2a). However, only HEC-1B cells expressed high levels of p53 protein under basal conditions. To further investigate the function of SOX17 in EC cells, SOX17 overexpression and SOX17 knockdown plasmids were constructed and were used to transfect HEC-1B cells. The cells were used for further study at 72 h post transfection, and the expression levels of SOX17 were confirmed using western blot analysis (Fig. 2b, c).

Cytotoxicity tests were performed to study the influence of SOX17 on the cytotoxicity of CDDP to HEC-1B cells. After transfected for 72 h, the cells were treated with CDDP (20 μg/ml) for 24, 48, 72, 96 h. Cells overexpressing SOX17 displayed a higher sensitivity to CDDP and a lower cell viability than the control (Fig. 2d), whereas cells with a reduced expression of SOX17 had a lower sensitivity to CDDP and an enhanced cell viability (Fig. 2e). These data suggested that SOX17 increased the CDDP sensitivity of EC cells.

To further assay the effect of SOX17 on the sensitivity of HEC-1B cells to CDDP, we used flow cytometry to examine the apoptosis rate of HEC-1B cells after treatment with CDDP (20 μg/ml) for 48 h. The apoptosis rate of HEC-1B cells over-expressing SOX17 was higher than that of the control group (P < 0.05). Meanwhile, under-expression of SOX17 decreased the apoptosis rate (P < 0.05). The apoptosis rate after treatment with CDDP was 55.1 ± 2.47 and 2.36 ± 0.47 % in SOX17 up-regulated and down-regulated groups, respectively (P < 0.05) (Fig. 3a–d). These results further suggested that SOX17 increased the CDDP sensitivity of HEC-1B cells.

SOX17 overexpression increased the levels of apoptosis in EC cells. We next investigated the role of SOX17 in regulating cell apoptosis after treatment with CDDP (20 μg/ml) for 48 h. Cleaved caspase-9, the marker of mitochondrial apoptosis activation, increased with higher SOX17 expression. As a result, cleaved caspase-3 (cc-3), the executor of apoptosis, increased, whereas the level of survivin, an inhibitor of apoptosis, decreased (Fig. 4a). Similarly, pretreatment with inhibitors of caspase-9 (ZLEHD-FMK) or caspase-3 (Z-DEVD-FMK) inhibited CDDP-induced activation of mitochondrial apoptosis in HEC-1B cells (Fig. 4b).

To further evaluate the involvement of SOX17 in the mitochondrial apoptotic events after CDDP treatment, the levels of the key proteins of the p53 pathway were analyzed. After SOX17 knockdown or overexpression, HEC-1B cells were treated with CDDP (20 μg/ml) for 48 h, and then assayed for the expressions of total proteins. SOX17 overexpression increased the level of p53 protein, whereas a reduced expression of SOX17 resulted in decreased p53. We also found that the downstream product of p53, Bax pro-apoptosis protein and cc-3, the executor of apoptosis, increased with SOX17 overexpression (Fig. 4c) and decreased with SOX17 under-expression (Fig. 4d). These results suggested that SOX17 altered the levels of pro- and anti-apoptotic proteins of the Bcl-2 family and influenced the CDDP sensitivity through the p53-Bax-caspase-3 apoptotic pathway.

To further investigate the effects of SOX17 on the resistance of EC cells to CDDP, an in vivo chemosensitivity experiment was performed by the subcutaneous transplantation of transduced cells into BALB/c nude mice. Cells were suspended at a density of 10⁷ cells/ml and 100 μl was injected into the flank of each nude mouse (n = 5). Nine days post-injection, CDDP (10 mg/kg) was intraperitoneally administered every 2 days for 8 days. Three days after the first treatment with CDDP, we measured the size of the growing tumors every 5 days for 50 days using the formula (W ²  × L) (Fig. 5b, c). The mice were then euthanized and the tumors were excised from the nude mice. The tumor sizes of the SOX17 group were significantly smaller than those of the control group (Fig. 5a).

In addition, tumor growth curves indicated that after 10 days treatment, the tumors of the SOX17 group were smaller than those of the control. TUNEL assays on the tumor samples confirmed the hypothesis that the inhibition of tumor growth in the SOX17 group was due to increased cell apoptosis (Fig. 5d, e). To further explore the mechanism underlying this phenomenon, western blot analysis was performed to measure the expression levels of p53, Bax, and cc-3 (Fig. 5f). The tumors from the SOX17 group contained a significantly higher level of these proteins compared with those of the control group.

The protocols used in the study were approved by the Hospital’s Protection of Human Subjects Committee. Samples were acquired with written informed consent from the Shanghai First People’s Hospital Affiliated to Shanghai Jiaotong University.

Paraffin-embedded tissues of 42 EC patients were included in the study, and all the patients received cisplatin-based combination chemotherapy between 12/2013 and 08/2014. The average age of the patients with EC was 53.9 ± 3.8 years (mean ± SD; median, 55 years; range, 41–63 years). The difference between the trial and the control was not significant (P = 0.317). The stages (II–III) and histological grades (G2–G3) of these tumors were established according to the criteria of the International Federation of Gynecology and Obstetrics (FIGO) surgical staging system (2009) [28]. None of the patients had undergone hormone therapy or radiotherapy before surgery.

A total of 29 EC patients were included in the study, 17 being cisplatin-sensitive and 12 cisplatin-resistant. Blood specimens of these patients were acquired, and all the patients received cisplatin-based combination chemotherapy between 12/2013 and 08/2014 (median age 54.3 years, range 43–58 years).

Human EC cell lines Ishikawa, HEC-1B, AN3CA and SPEC-2 were obtained from the China Center for Type Culture Collection (CCTCC) and maintained as recommended by the American Type Culture Collection (ATCC, Manassas, VA).

Immunohistochemical staining was performed using the 2-Step Plus Poly-HRP Anti IgG Detection System (ZSGB-Bio, Beijing, China) according to the manufacturer’s recommendations. Mouse polyclonal anti-SOX17 (diluted 1:100–10 μg/ml; Abcam, Cambridge, UK) was used as the primary antibody. Scoring was performed by two independent pathologists who had not seen the clinical and pathological data. Protein staining was evaluated as described [29].

The extraction of total RNA and reverse transcription (RT) reactions were performed according to the manufacturer’s protocol. Real-time PCR was performed using a standard protocol from the SYBR Green PCR kit (Toyobo, Osaka, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a reference for mRNAs. DCt values were normalized to GAPDH mRNA levels. The 2^−△△Ct method was used to determine the relative gene expression levels. The primer pairs used were human SOX17 forward: 5′-ATCCTCAGACTCCTGGGTTT-3′, reverse: 5′-ACTGTTCAAGTGGCAGACAAA-3′. GAPDH forward: 5′-GGCTCCCTTGGGTATATGGT-3′, reverse: 5′-TTGATTTTGGAGGGATCTCG-3′.

Western blot analysis to assess protein expression was performed as previously described [30]. Primary antibodies included mouse anti-SOX17 (Abcam, Cambridge, UK) and mouse anti-GAPDH (Sigma, ST Louis MO, USA). All the other antibodies were purchased from CST (Boston, USA).

Transient transfection of HEC-1B cells was performed with lipofectamine 2000 following the manufacturer’s recommendations. Tumor cell clone overexpressing SOX17 was successfully established as previously described [31]. Cells were cultured in 10 % Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10 % fetal bovine serum (Wisent, Quebec, Canada) at 37 °C with 5 % CO₂.

The percentage of cells undergoing apoptosis was evaluated using simultaneous Annexin V-FITC and propidium iodide (PI) staining. Cells were treated with CDDP (20 μg/ml) for 48 h. The apoptotic cells were analyzed by flow cytometry on a FACScan (Beckton-Dickson, New Jersey, USA).

Cells were plated for 24 h, then treated with CDDP for an additional 48 h. The number of viable cells was determined by using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described [32].

Cells were incubated with CDDP for 48 h. The subsequent steps were performed according to the manufacturer’s protocol. DNA content was measured on a FACSCalibur-HTS flow cytometer (BD Biosciences, New Jersey, USA).

We designed an oligonucleotide as the target genes of SOX17 mRNA in accordance with the law of multiple biological information (GeneBank number NM 022454). The RNA interference target sequence: 5′-GGTATATTACTGCAACTAT-3′. A BLAST search confirmed the non-silencing siRNA had no matches with the complete human genome (http://​www.​ncbi.​nlm.​nih.​gov). The sequence of NC target gene is 5′-TTCTCCGAACGTGTCACGT-3′. ShRNA was cloned into pCDNA3.1 vector. The positive clones were identified by PCR and sequenced. SOX17cDNA plasmid was purchased from Origene (Cat no. RC220888, USA).

All tumors were collected by sacrificing the mice 50 days after treatment. In situ Death Detection Kits (Roche, Mannheim, Germany) were used to detect apoptotic cells by specific staining. TUNEL assays for determining cell apoptosis were performed according to the manufacturer’s protocol. The apoptotic rate of cancer cells was calculated as apoptotic cells/total cells ×100 %.

All experiments were repeated in triplicate. Data were expressed as the mean ± SD. The statistical significance between two groups was determined by Student’s t test. The association between SOX17 expression and clinicopathological parameters was examined by the Chi square test. P < 0.05 were considered statistically significant.