Contributed equally
Resistance to chemotherapy poses a serious problem for the treatment of advanced epithelial ovarian cancer patients. The mechanisms of chemoresistance are complex and studies have implicated signal transducer and activator of transcription 3 (STAT3) signaling in the chemoresistance of cancer cells. The present study investigated whether corosolic acid (CA), which has been previously reported to be a STAT3 inhibitor, was able to increase the sensitivity to chemotherapeutic drugs in epithelial ovarian cancer cells. CA also markedly enhanced the anticancer effect of paclitaxel, cisplatin and doxorubicin. In addition, CA abrogated the cell-cell interactions between macrophages and epithelial ovarian cancer cells and inhibited the macrophage-induced activation of epithelial ovarian cancer cells. These data indicated that CA was able to reverse the chemoresistance of epithelial ovarian cancer cells and suppress the cell-cell interaction with tumorigenic macrophages. Thus, CA may be useful as an adjuvant treatment to patients with advanced ovarian and other types of cancer due to the multiple anticancer effects.
Epithelial ovarian cancer is the fourth most common cause of cancer-related mortality in females worldwide (
Signal transducer and activator of transcription 3 (STAT3) is a well-known signaling molecule that is associated with cell proliferation, survival, angiogenesis and immunosuppression. The activation of STAT3 is considered to be significant for cancer progression (
Corosolic acid (CA), a natural compound derived from apple pomace, is a potent STAT3 inhibitor and inhibits the proliferation of glioblastoma and osteosarcoma cells (
The present study examined whether CA has a synergistic effect with chemotherapy on epithelial ovarian cancer
The human ovarian carcinoma SKOV3, RMG-1, and ES-2 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA) and were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS). Peripheral blood mononuclear cells were obtained from healthy volunteer donors, who gave written informed consent for participation in this study. The study was approved by the ethics committee of Kumamoto University (Kumamoto, Japan). CD14+ monocytes were purified from the peripheral blood mononuclear cells by positive selection using magnetic-activated cell sorting technology (Miltenyi Biotec., Bergisch Gladbach, Germany) as described previously (
CA was isolated from the apple pomace as described previously (
STAT3 activation was determined by measuring the increased expression of phosphorylated STAT3 by western blot analysis. The protein (10 μg) was run on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidine fluoride transfer membranes (Millipore, Bedford, MA, USA). To detect the phosphorylated (phospho)-STAT3, the membranes were exposed to an anti-phospho-STAT3 antibody (D3A7, Cell Signaling, Danvers, MA, USA) and visualized by horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) with an enhanced chemiluminescence western blotting detection reagent (GE Healthcare, Tokyo, Japan). The molecular size of phospho-STAT3 that was detected by the immunoblotting procedure was ~80 kDa. To detect the STAT3 protein, the membranes were exposed to an anti-STAT3 antibody (sc-8019; Santa Cruz Biotech, Dallas, TX, USA) and visualized by horseradish peroxidase-conjugated anti-mouse IgG antibody with an ECL western blotting detection reagent. The molecular size of STAT3 that was detected by the immunoblotting procedure was ~80 kDa. These membranes were re-blotted with an anti-β-actin antibody as an internal calibration control.
Briefly, 1×104 SKOV3, RMG-1 or ES-2 cells were cultured in 96-well plates in quadruplicate as previously described. Anticancer drugs, including CA, paclitaxel (PTX), cisplatin (CDDP) or doxorubicin (DOX) (Wako), were then added to the cells. The cell viability was determined using a WST assay (WST-8 cell counting kit; Dojin Chemical, Kumamoto, Japan) according to the manufacturer’s instructions. In order to analyze the cytotoxic activity, the amount of lactate dehydrogenase (LDH) that was released into the culture supernatants was calculated using an LDH release assay (LDH-cytotoxic test kit; Wako).
The apoptotic cells in the sections were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) technique using an ApopTag Peroxidase In Situ Apoptosis Detection kit (Intergen Co., Purchase, NY, USA). To visualize the reaction, anti-digoxigenin-peroxidase was applied for 30 min at room temperature. For the negative controls, distilled water was used instead of the TdT enzyme.
The co-culture cells were fixed in 10% neutral buffered formalin and embedded in paraffin wax. Deparaffinized sections were immersed in 0.3% hydrogen peroxide solution and treated with anti-BrdU (Abbiotec, San Diego, CA, USA) and anti-pSTAT3 (Cell Signaling Technology, Tokyo, Japan) antibodies. The sections were subsequently treated with a HRP-conjugated secondary antibody (Nichieri Bioscience, Tokyo, Japan). Reactions were visualized with diaminobenzidine. The number of BrdU-positive cells were counted among 200 randomly selected tumor cells under a microscope.
All data are representative of two or three independent experiments that were performed in quadruplicate. The data are expressed as the mean ± standard deviation. The Mann-Whitney U test was used for the two-group comparison. P<0.05 was considered to indicate a statistically significant difference.
The effect of CA on the proliferation of epithelial ovarian cancer cells was measured. CA was observed to inhibit the proliferation of the SKOV3, RMG-1 and ES-2 cells at a concentration of at least 30 μM (
To elucidate whether CA enhances the anticancer activity of the anticancer drugs in the epithelial ovarian cancer cells, the combinational effects of CA and the anticancer drugs, including PTX, CDDP and DOX, was examined. As CA demonstrated no anticancer effects at a concentration of 20 μM, three epithelial ovarian cancer cell lines (SKOV3, RMG-1 and ES-2) were incubated with 20 μM CA for 24 h, concurrently with an incubation of 10 μM anticancer drugs. As shown in
Direct cell-cell interactions between M2 macrophages and epithelial ovarian cancer cells have previously been reported to induce STAT3 activation and a tumorigenic microenvironment in the ascites fluid of advanced epithelial ovarian cancer patients (
The present study demonstrated that the anticancer effects of CA on epithelial ovarian cancer cells are due to its suppressive effect on STAT3. Furthermore, CA enhanced the anticancer effect of chemotherapeutic agents. STAT3 activation is a well-known signal that is associated with cell survival or resistance to apoptosis and this effect is considered to be induced by the upregulation of anti-apoptotic genes, including Bcl-X, MCL-1 and survivin (
Numerous tumor-associated macrophages (TAMs) are detected in the cancer tissues (
In conclusion, the
The authors would like to thank Mr. Takenobu Nakagawa, Mrs. Emi Kiyota and Miss. Yui Hayashida for their technical assistance. This study was supported in part by the Kanazawa Medical Research Foundation and Grants-in-Aid for Scientific Research (nos. 23790407, 23790747 and 20390113) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Effect of CA on the proliferation of ovarian carcinoma cells. The ovarian carcinoma cells (SKOV3, RMG-1, and ES-2) were incubated with the indicated concentrations of CA for 48 h, followed by (A) determination of cell viability and (B) cell cytotoxicity, by WST-8 assay and LDH assay, respectively (as described in Materials and methods). (C) The ES-2 cells were incubated with the indicated concentrations of CA for 5 h, followed by the determination of cell apoptosis by TUNEL staining (as described in Materials and methods). Data are presented as the mean ± SD. *P<0.01 and **P<0.001 vs. the control. CA, corosolic acid; DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling; LDH, lactate dehydrogenase.
Effect of CA on STAT3 activation of ovarian carcinoma cells. Ovarian carcinoma cells, (A) SKOV3, (B) RMG-1 and (C) ES-2, were incubated with the indicated concentrations of CA for 24 h, followed by determination of pSTAT3, STAT3 and β-actin expression by western blot analysis (as described in Materials and methods). CA, corosolic acid; STAT3, signal transducer and activator of transcription 3; pSTAT3, phosphorylated signal transducer and activator of transcription 3.
Combined effect of CA and anticancer drugs on the proliferation of ovarian carcinoma cells. Ovarian carcinoma cells, (A) SKOV3, (B) RMG-1 and (C) ES-2, were incubated with 10 μM anticancer drugs, PTX, CDDP and DOX, concurrently with or without 20 μM CA for 24 h. Cell viability was then determined using a WST-8 assay (as described in Materials and methods). Data are presented as the mean ± SD. *P<0.01 vs. the control. CA, corosolic acid; PTX, paclitaxel; CDDP, cisplatin; DOX: doxorubicin.
Combinational effect of CA and anticancer drugs on STAT3 activation of ovarian carcinoma cells. The ovarian carcinoma cells, (A) SKOV3, (B) RMG-1 and (C) ES-2, were incubated with 20 μM CA and/or 10 μM paclitaxel for 24 h, followed by determination of pSTAT3, STAT3 and β-actin expression by western blot analysis (as described in Materials and methods). CA, corosolic acid; STAT3, signal transducer and activator of transcription 3; pSTAT3, phosphorylated signal transducer and activator of transcription 3.
Effect of CA-treated macrophages on STAT3 activation and cell proliferation in epithelial ovarian carcinoma. (A) HMDM were treated with 20 μM CA for 24 h and the SKOV3 cells were incubated with CA-treated macrophages for 24 h, followed by determination of BrdU-positive cells by immunohistochemistry (as described in Materials and methods). (B) HMDM were treated with 20 μM CA for 24 h, while the SKOV3 cells were incubated with CA-treated macrophages for 24 h, followed by determination of STAT3 activation by immunohistochemistry (left) and western blot analysis (right) (as described in Materials and methods). CA, corosolic acid; STAT3, signal transducer and activator of transcription 3; HMDM, human monocyte-derived macrophages; Mø, macrophage.