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Ovarian carcinoma is a common malignant disease worldwide with a poor therapeutic response. The present study investigated the effects of Na7CrCuW11O39.16H2O (CrCuW11) on ovarian cancer cell growth and investigated the mechanisms underlying its actions. The effects of CrCuW11 on cell viability and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, acridine orange/ethidium bromide staining and electron microscopy in human ovarian cancer SKOV3 cells. The expression of bcl-2-like protein 4 (Bax), B-cell lymphoma 2 (Bcl-2), cytochrome c, phosphorylated (p)-p38 and p38 was determined by western blot analysis. Caspase-3 activity was measured by caspase-3 activity kit. CrCuW11 concentrations of 1.87×10−3 mol. l−1 at 12 h reduced viability induced apoptosis in SKOV3 cells in a concentration-and time-dependent manner. Forced expression of CrCuW11 upregulated the expression of certain proteins (Bax, cytochrome c, and p-p38), and downregulated Bcl-2 protein expression. Furthermore, CrCuW11 also enhanced caspase-3 activity. The p38 inhibitor SB203580 was able to inhibit the activity of CrCuW11. Caspase-3 and p38 signaling pathways were associated with CrCuW11-regulated multiple targets involved in SKOV3 cell proliferation. Therefore, the results of the present study indicated that CrCuW11 may be used as a novel clinical drug for the treatment of ovarian cancer.
Ovarian cancer is the leading cause of mortality from gynecological malignancies in women worldwide, causing >140,000 deaths annually (1). Each year, it is estimated that ~22,000 women will be newly diagnosed and 14,200 will succumb to disease in the USA (2). The poor overall survival is due to late-stage diagnosis and resistance to chemotherapy (3). The disease course for ovarian cancer may be complicated. Initial treatments often include surgery and chemotherapy, which may cause significant physical, psychological and social side effects (4). Ovarian cancer has a higher mortality rate in females than all other gynecological cancers combined (5). Therefore, there is an urgent requirement for a novel drug that is able to enhance the efficiency of therapy for the treatment of ovarian cancer patients.
Since 1970, polyoxometalate has been widely used for analytical chemistry, medicine, catalysis and material science (6). A number of polyoxometalates were observed to be potential antitumor, antiviral and antibacterial drugs (7–9). Initially, ‘silicotungstic acid supernatants’ were reported to be potent inhibitors of murine leukemia and sarcoma viruses (10). Since then, many polyoxometalates have been observed to possess potential antitumor, antiviral and antibacterial ability (8). They were observed to exhibit inhibition affects on tumor cells, including B16 melanoma, HeLa, HepG-2 and PAMC 82 cells (11). Furthermore, polyoxometalate may induce apoptosis at a low concentration in HEK293 cells (9). Therefore, it is worthy to consider the introduction of transition metal elements to polyoxometalate in order to improve its antitumor activity.
In a preliminary experiment by the present authors, CrCuW11 was a polyoxometalate containing the transition metal chromium. However, CrCuW11 has not been reported to induce apoptosis in any human cancer cells to the best of our knowledge. In the present study, it was demonstrated that CrCuW11 was able to induce the apoptosis of SKOV3 cells. Treatment with CrCuW11 significantly decreased viability and induced apoptosis in SKOV3 cells, due to significant activation of caspase-3 and p38-dependent signaling pathways.
Antibodies for bcl-2-like protein 4 (Bax; catalog no., D120073) and B-cell lymphoma 2 (Bcl-2; catalog no., D160117) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Cytochrome c (catalog no., 11940), p38 (catalog no., 8690) and phosphorylated (p)-p38 (catalog no., 4511) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). p38 inhibitor SB203580 was purchased from Beyotime Institute of Biotechnology (Haimen, China). Other chemicals [including Na2WO4•2H2O, Cu(NO3)2, Cr(NO3)3 and glacial acetic acid] were purchased from Sigma-Aldrich (EMD Millipore, Billerica, MA, USA).
Na4CrCuW11O39.16H2O plasmid was synthesized by the Department of Inorganic Chemistry and Physical Chemistry, Harbin Medical University (Harbin, China). To a solution of Na2WO4•2H2O (36.3 g) in water, 200 ml glacial acetic acid was added and heated to boiling. Then a solution of Cu(NO3)2•3H2O (2.4 g, 0.01 M) in water (50 ml) was added dropwise to the stirred solution above. The reaction mixture was stirred for 30 min. Then Cr(NO3)3 (4.15 g, 0.01 mol) in 50 ml of water was added dropwise. The solution pH of 5 was adjusted by adding glacial acetic acid. The mixture was stirred for a further 1.5 h, then cooled and filtered. The resulting mixture was added to dehydrated alcohol to produce a dark green oil. The oily crude product was frozen and purified by a dissolved-frozen method performed 2–3 times. The product was then dried in an vacuum dryer to produce dark green crystals.
SKOV3, a human ovarian cancer cell line, was provided by the Key Laboratory for Reproductive Medicine of Guangdong Province (Guangzhou, China) and cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in an incubator containing humidified air with 5% (v/v) CO2.
Cells were seeded onto 96-well plates at a density of 8×103 cells per well 24 h prior to treatment. The cells were treated with CrCuW11 at the following concentrations: 1.17×10−4, 3.69×10−4, 1.02×10−3, 1.87×10−3 or 3.52×10−3 mol. l−1. Following 12 h of incubation at 22–26°C, 15 µl (5 mg/ml) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich; EMD Millipore) was added to each well, and incubated at 37°C for 4 h. Subsequently, the MTT solution was removed and 150 µl of dimethyl sulfoxide was added to dissolve the crystals. The mixtures were shaken for 10 min to fully dissolve the crystals. A microplate reader (Tecan, Männedorf, Switzerland) was used to measure the absorbance at a wavelength of 570 nm. Cell viability was expressed as the percentage change of the absorbance values in the treatment group over the control group.
SKOV3 cells were cultured in 60 mm plates, suspended in PBS solution and fixed with 2% (v/v) paraformaldehyde containing 2.5% (w/v) glutaraldehyde (Paesel & Lorei GmbH & Co. KG, Duisberg, Germany) buffered in Hank's modified salt solution at 4°C for 4 h. The cells were further fixed in 1% (w/v) OsO4 solution buffered by 0.1 M cacodylate (pH 7.2) at 4°C for 2 h, and dehydrated in ethanol. Dehydration was performed in propylene oxide. The specimens were embedded in Araldite® (SERVA Electrophoresis GmbH, Heidelberg, Germany). Ultrathin sections (2–6 µm) were produced on an FCR Reichert Ultracut ultramicrotome (Leica Microsystems, Inc., Buffalo Grove, IL, USA), mounted on pioloform-coated copper grids and contrasted with lead citrate. Specimens were analyzed and documented with a 10A transmission electron microscope (Zeiss AG, Oberkochen, Germany).
SKOV3 cells in the exponential growth phase were cultivated on sterile coverslips for 24 h at room temperature, and were subsequently treated with CrCuW11 at concentrations of 1.17×10−4, 3.69×10−4, 1.02×10−3, 1.87×10−3 or 3.52×10−3 mol. l−1 for 12 h at 37°C. The cells were washed twice with PBS, and then mixed with 1 ml of dye mixture containing 100 mg/ml AO and 100 mg/ml EB in PBS (12). Cellular morphological changes were examined using fluorescence microscopy (magnification, ×200). The percentage of apoptotic cells was calculated by the following formula: Apoptotic rate (%)=number of apoptotic cells/total number of cells counted (13,14).
Total protein samples were extracted from SKOV3 cells. Cells cultured on 25 mm dishes or 6-well plates were lysed in lysis buffer (protease and phosphatase inhibitors; Beyotime Institute of Biotechnology). Following centrifugation at 13,500 × g for 15 min at 4°C, the lysates were collected. Protein concentration was assessed using the bicinchoninic acid protein assay. The aliquots were then mixed with Laemmli sample buffer and boiled at 100°C for 5 min. The protein samples (80 µg) were resolved by 10–12% SDS-PAGE, followed by transferal to nitrocellulose membranes and blocking with 5% (w/v) non-fat dried skimmed milk powder in PBST. For visualization, blots were probed with antibodies against Bcl-2 (1:500 dilution), Bax (1:500 dilution), cytochrome c (1:500 dilution), p38 (1:1,000 dilution), p-p38 (1:1,000 dilution) and GAPDH (1:1,000 dilution) at room temperature (21–23°C) for 1 h. Membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. 5257, 5151; 1:10,000 dilution; Cell Signaling Technology, Inc.). The obtained digital images of western blotting results were then used for densitometry measurements (Gel-Pro analyzer version 4.0; Media Cybernetics, Inc., Rockville, MD, USA). Western blotting bands were quantified using Odyssey version 3.0 software (LI-COR Biosciences, Lincoln, NE, USA) by measuring the band intensity (area × absorbance) for each group, and normalized to GAPDH bands as an internal control.
Caspase-3 activity was analyzed using a caspase-3 activity assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol, using substrate peptides Ac-DEVD-pNA (p-nitroanilide), Ac-IETD-pNA and Ac-LEHD-pNA. Briefly, the supernatant of cell lysate was mixed with buffer containing the substrate peptides for caspase attached to peptide nucleic acid (pNA). The release of pNA was quantified by determining the absorbance with an ELISA reader at 405 nm. The caspase activities were expressed as a percentage, in terms of the treatment group over the control group.
Data are presented as the mean ± standard deviation of 3–6 independent experiments and were evaluated by unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using GraphPad Prisim 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA).
The antiproliferative effect of CrCuW11 on SKOV3 cells was examined by exposing the cells to various concentrations (1.17×10−4, 3.69×10−4, 1.02×10−3, 1.87×10−3 or 3.52×10−3 mol. l−1) of CrCuW11 for 6, 12 and 24 h. Cell growth was inhibited in a dose- and time-dependent manner (Fig. 1). In the presence of 1.87×10−3 mol. l−1 CrCuW11, SKOV3 cells exhibited ~50% inhibition of proliferation following treatment for 12 h. Therefore, 1.87×10−3 mol. l−1 and 12 h were used in the subsequent experiments.
Cell viability is a balance between proliferation and apoptosis (15). To investigate whether CrCuW11 is able to regulate apoptosis of ovarian cancer cells, AO/EB staining was performed to detect apoptotic cells. The results from fluorescence microscopic analysis are presented in Fig. 2. A total of 3 types of cell were recognized under a fluorescence microscope: Live cells (green), apoptotic cells (yellow) and necrotic cells (red). Forced expression of CrCuW11 induced substantial apoptosis (P<0.05).
To investigate the mechanisms by which CrCuW11 induces apoptosis in SKOV3 cells, the present study measured the levels of the downstream proteins in the CrCuW11 apoptotic signaling pathway, including Bax, Bcl-2 and cytochrome c, demonstrating that treatment with CrCuW11 may upregulate Bax and cytochrome c expression, and downregulate Bcl-2 expression, thereby inducing apoptosis (Fig. 3A and B). In addition, caspase-3 activity, a marker of apoptosis, was significantly increased 2.1-fold by CrCuW11 treatment (Fig. 3C).
To investigate the apoptotic mechanism of CrCuW11, p38 was inhibited by SB203580 treatment, followed by treatment of cells with CrCuW11. The expression of p38 was detected by western blotting. Cell surface microvilli reduction, nuclear chromatin condensation, margination and membrane blistering were detected by electron microscopy. As shown in Fig. 4A, without SB203580, CrCuW11 was able to upregulate p-p38 expression, whereas total p38 expression exhibited no change. Following treatment with SB203580, neither p-p38 nor t-p38 expression was altered compared with the control. Under an electron microscope, the cells treated with CrCuW11 also exhibited robust changes in microstructure, including cell surface microvilli reduction, nuclear chromatin condensation, margination and membrane blistering (Fig. 4B), but these changes were not observed following treatment with SB203580.
In the present study, it was proven that CrCuW11, containing the transition metal element chromium, exerted potent anti-proliferative effects on SKOV3 cells by causing apoptosis. Activation of the caspase-3-dependent signaling pathway and p38 overload were identified as important mechanisms for CrCuW11-induced apoptosis. Therefore, the current study may provide a novel insight into the clinical role of CrCuW11 in ovarian cancer.
Ovarian cancer is a highly metastatic disease and >70% of patients are diagnosed at an advanced stage, with widespread intraperitoneal invasion and metastasis (16). It has been considered that invasion and metastasis largely contribute to the high mortality in ovarian cancer patients (17).
Previous studies have revealed that polyoxometalates are able to exert anticancer activities via regulation of invasion, proliferation and cell migration in a variety of malignancies, including breast (18), kidney (19), lung (19), ovary (20), pancreas (21) and prostate cancer (22). However, CrCuW11 has not been reported to induce apoptosis in any human cancer cells. It appears that whether CrCuW11 inhibits cell proliferation may depend upon the cell type; specific cell types have various predominant signaling pathways that lead to apoptosis (23).
Apoptosis (programmed cell death) is an important homeostatic mechanism that balances cell division and cell death, thus maintaining the appropriate cell number in the body (24). Therefore, searching for novel drugs that trigger the apoptosis of tumor cells has become an attractive strategy in anticancer medication research (25). The Bcl-2 family members constitute essential intracellular players in the apoptotic mechanism (26). Several studies have reported that Bax, Bcl-2 and caspase-3 are the key molecules participating in apoptosis in ovarian cancer cells (27,28). Cactus pear extract triggers apoptosis by increasing the ratio of Bax/Bcl-2 in SKOV3 cells (29). Cactus pear extract increased caspase-3 and Bax protein levels, and decreased Bcl-2 levels in ovarian cancer cell line SKOV3 (30). Epidermal growth factor induces ovarian cancer cell line apoptosis by activating caspase-3 via upregulating Bax and downregulating Bcl-2 (31). In the present study, it was observed that the expression of Bcl-2 protein in SKOV3 cells was significantly reduced compared with the control group (P<0.05), whereas the expression of Bax protein was significantly increased compared with the control group (P<0.05). The ratio of Bax/Bcl-2 significantly increased, indicating that SKOV3 cell apoptosis occurred following CrCuW11 treatment. These data indicated that the caspase-3-dependent apoptotic signaling pathway has a significant role in the anticancer activity of CrCuW11.
The caspase family has a significant role in apoptosis, with the cysteine aspartic acid protease caspase-3 acting as a key molecule that is able to transmit apoptotic signals in a number of signaling pathways (32). The results of the present study revealed that the caspase-3 activity in cells was increased following treatment with CrCuW11. Cytochrome c, which is also involved in apoptosis, is able to stimulate unlimited cell proliferation and promote cell division, and is closely associated with the occurrence and development of a wide variety of tumors (33). The results of the present study revealed that treatment of SKOV3 cells with CrCuW11 for 12 h significantly induced the expression of cytochrome c protein.
p38 is an important member of the MAPK signaling pathway, and the p38 MAPK pathway was found to increase the sensitivity of human colon cancer cells to drug treatment (34). As a kinase activated by oxidative stress, p38 primarily participates in apoptosis (35). Under normal circumstances, p38 is located in the cytoplasm; however, once activated it will be rapidly translocated into the nucleus to activate mitogen-activated protein kinase-activated protein kinase-2 and 3 and caspase family members (36). These findings suggest that activation of the p38 signaling pathway, may increase the effects of the antitumor signaling pathway in SKOV3 cells.
In the present study, it was observed that the expression of p38 protein in the SKOV3 cells was unaltered compared with the control group (P<0.05), whereas the expression of p-p38 protein was significantly increased compared with the control group (P<0.05). When 10 µl SB203580 (a selective p38 inhibitor) was used to treat the cells for 4 h prior to treatment with 1.87×10−3 mol. l−1 of CrCuW11 for 12 h, it was observed that the protein level of the total p38 did not change. When electron microscopy was used to examine the cells, it was observed that the cells did not alter compared with the control group in cell surface form and other parameters, including apoptotic bodies.
In conclusion, the present study demonstrated, for the first time to the best of our knowledge, that CrCuW11 markedly inhibits cell proliferation by inducing apoptosis in ovarian cancer cells. This is likely to be associated with CrCuW11-regulated multiple targets in SKOV3 cell proliferation. CrCuW11 increased the expression of Bax and decreased the expression of Bcl-2, and subsequently mediated an increase in the ratio of Bax over Bcl-2, leading to activation of caspase-3, and subsequent activation of the p38 signaling pathway. The results of the present study suggest that CrCuW11 may be a potential drug for the clinical treatment of ovarian cancer.
The present study was supported by the Medical Science Foundation of Guangdong Province (grant no., A2014316).