| Objective: Ovarian cancer is one of the three gynecological malignancy. Epithelial ovarian cancer (EOC) is the most common form of ovarian cancer accounting for 90% of it. Due to the lack of effective screening strategies and the absence of symptoms in early-stage of disease, 75% of cases have progressed to an advanced stage at the time of primary diagnosis. The most effective treatment for EOC is the surgery ( cytoreduction) plus the platinum- based chemotherapy, such as cisplatin. However, the majority(70%)of patients with advanced ovarian cancer ultimately experience chemoresistance, and recurrence of their disease and the 5-year survival rate of patients is less than 30%. ovarian cancer is the most common life-threatening malignant tumors among women and the first leading cause of death from gynecological disorder. Thus,The effective anti-tumor and reversing drug resistance of platinum product need to be developed,the effective therapeutic strategies and the investigation of the molecular mechanism responsible for the effects of anti-tumor and reversing anti-resistance are exigently demanded.Recently,the natural active substances attracted the attention of resear- cher at home and abroad with the characteristics of high activity and small adverse reactions. It has become a main Approaches to obtain effective anti-cancer agents and drug-resistant reversal agents by antitumor composition extraction from Chinese herbal medicine and natural plant. Ganoderma lucidum wall-broken spores,a natural plant product , have been widely used to treat various human diseases, including cancers). significant amount of research had shown that Ganoderma lucidum exerts multiple anti-tumor activity on cancer cells. There were some reporters at home and abroad in recent years that Ganoderma wall-broken spores suppressed growth and invasive behavior of cells of breast cancer,lung cancer, nasopharyngeal darcinoma,carcinoma of stomach,colorectal cancer, prostate cancer, leucoc- ythemia, hepatoma,melanoma lymphomas and enhanced cisplatin anti-tumor effect .however,the effect of Ganoderma Lucidum on epithelial ovarian cancer (EOC) has not been reported at home and at abroad. In this study, we determined if Ganoderma Lucidum regulates EOC cell activity . Using several cell lines derived from EOC, We observed the effects of ganoderma lucidum on proliferation and apoptosis in the human ovarian cell line (ov2008 cells) , and study its possible mechanism ; investigated the inhibitory effects of Ganoderma lucidum on invasion and metastasis of human ovarian cancer cells; investigated the reversal effect of Ganoderma lucidum on multidrug resistance (MDR) in the Cisplantin-resistant ovarian cancer cell line by WST-1 assay, microscope, cell counting, Hoechst-33258 staining, flow cytometry, colony formation assay, hanging drop culture, migration assay, invasion assay and western blot . It will provide the theoretical basis for further development and clinical application of ganoderma lucidum spore in the epithelial ovarian cancer.Objective 1: To observe the effects of ganoderma lucidum on prolifer- ation and apoptosis in the human ovarian cell line, and study its possible mechanism .Objective 2: To investigate the inhibitory effects and molecular mech- anism of Ganoderma lucidum on invasion and metastasis of human ovarian cancer cells in vitro .Objective: 3 To investigate the reversal effect of Ganoderma lucidum on multidrug resistance (MDR) in the Cisplantin-resistant ovarian cancer cell line A2780CP.Method:1 Cell culture:epithelial ovarian cancer cell lines OV2008, C13*, A2780s, A2780-cp and SKOV3 were obtained and cultured. Cells were grown at 37°C in an atmosphere with 5% CO2。2 Preparation of Ganoderma lucidum:The Ganoderma lucidum used in this study was purchased from Energene Naturals Inc (Toronto, On) which contains wall-broken spores and polysaccharides. A stock solution of 50mg/ml was prepared by adding water to the power and then boiling for 5 min. After a brief centrifugation, the supernatant was collected and passed through a 0.2 m filter. The stock solution was stored at 4C.3 Cell proliferation and viability assays: Cell proliferation and viability were determined using manual counting and WST-1 assays. For manual counting, cells were seeded in 24 well plates. After treatment with Ganoderma lucidum, the cell morphology was observed under microscope, and then viability cells number were determined by manual counting . For WST-1 assay, cells were seeded on 96-well culture plates at a density of 10,000 cells per well. After treatment with different concentrations of Ganoderma for 48h, WST-1 reagent was added to the cells and absorbance was measured at 450 nm using an ELISA plate reader. The inhibition rate of the cells was calculated as follows: inhibition rate (%)=1-the absorbance of drug-treated group / the absorbance of control group×100%.4 Cell cycle analysis: To determine the cell cycle phase distribution, flow cytometry was performed. 48h after treatment with different concentration Ganoderma lucidum 0,0.5,1,2mg/ml , 2X106 cells were collected and washed twice with PBS, and fixed with 3 ml ice cold 70% ethanol. The cells were then washed twice with cold PBS, and resuspended with 1ml staining buffer containing 50ug/ml RNAse A and 50ug/ml propipdium iodide. Stain cells for 1 hour at room temperature in the dark. Finally, samples were analyzed by flow cytometry machine .5 Assessment of apoptosis by Hoechst-33258 : The changes of apoptotic morphological changes in ovarian cancer cells after treated with ganoderma lucidum were detected by Hoechst 33258 DNA staining and and examined by fluorescence microscopy.Control and treated cells were fixed in 4% paraf- ormaldehyde in 1xPBS for 20 min, washed with 1XPBS, stained with Hoechst 33258 at 1μg/ml in PBS for 15 min. Stained cells were washed 2 times with 1xPBS. The changes in nuclei were observed with a fluorescent microscope through UV-filter. 6 Ganoderma Lucidum inhibited Epithelial ovarian cancer cell growth: Epithelial ovarian cancer cell lines were treated with different concentration of Ganoderma Lucidum . The cell growth were determined by Colony formation assay and Hanging drop culture for spheroid formation. Formation of cell Colony and Multicell Spheroids were examined.7 Ganoderma Lucidum inhibited Epithelial ovarian cancer cell adhesion, migration and invasion: Epithelial ovarian cancer cell lines were treated with different dose of Ganoderma Lucidum .The ability of cell adhesion,migration and invasion were detected by adhesion assay,Wound healing assay (migr- ation assay), and invasion assay.For Wound healing assay, Ten points were marked randomly down the wound and pictures were taken at the same points at regular time intervals(0, 24,48h). Area migrated by the cells was measured by computer software Simple PCI.8 Reversal effect of Ganoderma lucidum on cisplantin resistance in ovarian cancer cell line:Cells cultured in 24 well plate at a density of 6X104 cells per well. Group 1: Cells were treated with different concentrations of cisplatin. Group 2: Cells were treated with different concentrations of Gano- derma lucidum. Group 3: Cells were treated with both different concentrations of cisplatin and 0.5mg/ml Ganoderma lucidum. Live cell numbers were determined using manual counting and WST-1 assays. The cell morphology was observed under microscope. The cell inhibition rate, drug resistance factor and reverse factor were calculated using result of WST-1 assay. The reversing factor (RF) was calculated as follows: RF=IC50(control group)/IC50 (drug- treated group). resistance factor=IC50 (cisplantin treated group)/IC50 (cispla- tin and Ganoderma lucidum treated group)9 Western blot analysis: Epithelial ovarian cancer cells were seeded into 6-well plates. Five experimental groups were divided. Group 1: To investigate the molecular mechanism of ganoderma lucidum on proliferation and apoptosis in the human ovarian cell line. Epithelial ovarian cancer cells were seeded into 6-well plates. The cells were treated with different dose (0,1,2mg/ml)of ganoderma lucidum. 48h after treatment, the total protein were extracted. The protein lysates were separated in 12% SDS polyacrylamide gels, transferred to Immobilon PVDF Membranes and probed with apoptosis-associated proteins such as anti-bcl-2(1:1000),bax(1:4000),cleaved- caspase-3(1:500),P27(1:1000), ECL kit was used for chemiluminescent detection of immobilized proteins.Group 2: To study the molecular mechanism of ganoderma lucidum on invision and metastasis in the human ovarian cell line. Epithelial ovarian cancer cells were treated with different dose(0,0.5,1,2mg/ml)of ganoderma lucidum. The E-cadherin(1:500), N-cadherin(1:500),vimentin(1:1000)MMP-9 and TIMP-1 were detected by western blot after treated with Ganoderma lucidum as described above.Group 3: To determine the expression of bcl-2, Akt, P-Akt , P53 in chemosensitive cell line A2780s and chemoresistant cell line A2780cp. The cells both A2780s and A2780cp were seeded into 6-well plate and cultured for 48h. The total proteins were extracted. The bcl-2,Akt,P-Akt,P53 was detected by western blot .Group 4: Epithelial ovarian cancer cells were treated 48h with different dose(0,0.5,1,2mg/ml)of ganoderma lucidum. The expression of protein bcl-2, Akt , P-Akt and P53 were detected by western blot respectively.Group 5: To Study the molecular mechanism of the reversal effect of Ganoderma lucidum on multidrug resistance (MDR) in the Cisplantin-resistant ovarian cancer cell line A2780CP. Cells cultured in 24-well plate at a density of 6X104 cells per well and treated with both 0.5 mg/ml Ganoderma lucidum and different concentrations of cisplatin for 48 hours. At the end of the experiment, the total proteins were extracted. The expression of protein bcl-2, Akt, P-Akt and P53 were detected by western blot. Protein loading was evaluated using mouse anti-β-actin.Result:1 Ganoderma Lucidum inhibited cell growth and viability in Epithelial ovarian cancer cells: To determine the effect of Ganoderma Lucidum on ovarian cancer cells, we first treated cells with 0.1, 0.5 and 1 mg/ml of Ganoderm Lucidum. Ovarian cancer cell lines had a decrease number of cells after treatment with higher doses of Ganoderma. A significant effect on cell growth was observed at 1mg/ml Ganoderma. To confirm these results, WST-1 assays were performed in dose-dependent and time-course experiments. Again, we observed that OV2008 cells treated with 2mg/ml of Ganoderma exhibited very significant decrease in cell viability at 48h after treatment. The results of WST-1 assays showed that Ganoderma lucidum could inhibit the A2780S and A2780CP cells growth as well. WST-1 assays showed that 0.5 mg/ml of ganoderma lucidum spore can be used to do reverse resistance experiment as lower toxicity dose (cell growth rate more than 80%).2 Ganoderma induced apoptosis and blocked cell cycle progression at G1: We determined if Ganoderma regulates apoptosis. Hoechst 33258 staining showed that ganoderma lucidum was capable of inducing apoptosis in OV2008 cells, which was characterized by features of apoptotic nuclei such as nuclear shrinkage, chromatin condensation. To examine how Ganoderma inhibited cell growth and viability, we used flow cytometry to determine the effect of Ganoderma on cell cycle progression. Analysis of cell cycle profile in control and Ganoderma-treated cells revealed that Ganoderma increased the population of cells in G1 phase induced G1 phase arrest of OV2008 cells and decreased the number of cells in G1 phase. It did not change the percentage of cells in S phase.3 Ganoderma lucidum inhibited epithelial ovarian cancer cell colony formation: colony formation assays were used to test the effect of Ganoderma lucidum on cell colony growth. Skov3 cells were treated without or with Ganoderma and plated. At 7 days after plating, colonies were stained and photographed. The result showed Ganoderma lucidum strongly inhibited colony formation in a dose-dependent manner. Cloning formation rate reduced gradually. Moreover, the size and the number of single cell colony decreased in a dose-dependent manner under inverted microscope.4 Ganoderma lucidum inhibited epithelial ovarian cancer cell adhesion ability: Cell adhesion is an important step in the initiation of tumor formation and matastasis. We tested the effects of Ganoderma lucidum spores on ?epithelial ovarian cancer cell adhesion. Ovarian cancer skov3 cells were incubated with Ganoderma lucidum spores for 3h and 6h, unattached cells were removed and the plates were washed. The attached cells were pictured and counted. Our experiments indicated that Ganoderma lucidum spores inhibited epithelial ovarian cancer cell adhesion ability. The number of adhesion cells decreased in a dose-dependent manner under inverted microscope and cell counting.5 Ganoderma lucidum inhibited epithelial ovarian cancer cell multicell spheroid formation ability: The effect of Ganoderma lucidum on spheroid formation was assessed using a hanging drop culture. When droplets of skov3 cells were plated on the inner cover of culture dish, cells proliferate, aggregate at 24h after hanging drop, and form tight spheroids at day 4 after hanging drop. When cells were treated with 1 and 2 mg/ml Ganoderma, they only formed lose spheroids and irregular spheroids. The ability of spheroid formation reduced gradually with the increasing of dose of Ganoderma lucidum spore. The result indicated that Ganoderma lucidum inhibited epithelial ovarian cancer cell multicell spheroid formation in a dose dependent manner.6 Ganoderma lucidum inhibited epithelial ovarian cancer cell migration ability: Since Ganoderma lucidum inhibits cell growth and viability in ovarian cancer cells, to determine if Ganoderma lucidum inhibits cell migration, the migration assays were performed. The skov3 cells were treated without or with different doses of Ganoderma lucidum and a wound healing assay was carried out. Briefly, cells were cultured in 6-well plates until cells reached confluence and a wound was made using a 200μl pipette tip. After scratching, the culture medium was refreshed and different doses of Ganoderma lucidum (0, 0.5, 1, 2 mg/ml) were added. Ten points were marked randomly down the wound and pictures were taken at the same points before and 24h after incubation with Ganoderma lucidum. The area migrated by the cells was measured by computer software Simple PCI. We found that wound were almost closure at 24h after hanging drop in untreated group and the migration ?ability were inhibited in treated group in a dose-dependent manner. After treated with 2mg/ml of Ganoderma lucidum, the wounds almost no difference between 24hour and at 0hour after scratching. The experiment suggested Ganoderma lucidum strongly inhibited wound closure in skov3 cell lines.7 Ganoderma lucidum inhibited epithelial ovarian cancer cell invasion ability: In cell invasion assay, matrigel poured on the surface of porous filter membrane formed a structure similar to the natural basal membrane. The ability of the cells to penetrate matrigel reflected its invasive capability. The results showed that the average number of skov3 cells that penetrated the membrane was 162.8±8.1, 132.8±6.5, 65.2±3.9 and 39.2±1.3respectively. Statistical analysis showed that there was significant difference between the cell invasion ability of control and that of Ganoderma lucidum treated cells (P>0.05), indicating that Ganoderma lucidum inhibited epithelial ovarian cancer cell invasion ability.8 Cisplantin inhibited epithelial ovarian cancer chemosensitive cell line A2780S and chemoresistant cells line A2780-cp proliferation: To determine inhibition effect of Cisplatin on epithelial ovarian cancer chemosensitive cell line A2780S and chemoresistant cells line A2780-cp, A2780S cells were treated with 0,1,2.5,5umol/L of cisplatin and A2780-cp cells were treated with 0,5,10,20umol/L of cisplatin. At 48h after treatment, Cell proliferation and viability were determined using WST-1 assays; and morphology and number of cell were determined by microscope and manual counting. The result showed that the number of A2780-cp was similar to A2780S after treated with different dose ciaplatin. A2780-cp and A2780S were treated with the same dose of cisplatin. We found that the number of A2780S less than that of A2780-cp obviously at 48h after treatment and the cisplatin inhibition rate to A2780S was significant higher than that of A2780-cp. The IC50 of A2780S and A2780-cp was 10.32 and 29.94mg/ml respectively. The result indicated that A2780CP cell line result in resistance to cisplatin, the resistance factor was 2.9.9 Ganoderma lucidum enhanced the anti-tumor effect of cisplatin and Reversal the drug resistance of cisplatin on ovarian cancer chemoresistant cells A2780-cp: Since we observed that Ganodema lucidum was effective in inhibiting growth/viability in both chemosensitive and chemoresistant cells, we examined if it will enhance the sensitivity of ovarian cancer cells to a chemo-therapeutic agent, cisplatin and Reversal their chemoresistance to cisplatin. A2780-cp cells were treated with different concentration of cisplatin in the presence or absence of a low dose of Ganoderma lucidum. The Ganoderma lucidum treatment alone slightly decreased cell number. However, when added with cisplatin, it significantly enhanced the effect of cisplatin on inhibiting cell growth. The median inhibitory concentration(IC50) reduced from 29.94 to12.16umol/L. These results indicated that Ganoderma lucidum can reverse the drug resistance of chemoresistant cells A2780-cp to Cisplantin. The RF(reversing factor) was2.46. The results were consistent with of cell counting and microscope.10 Effects of Ganoderma lucidum on proteins expression on apoptosis, invasion, matastasis and drug-resistance in epithelial ovarian cancer cell line:Group1: The results of Western blot suggested that the Bax,P27 protein level was increased after treatment with ganoderma lucidum and the levels of bcl-2 decreased in a dose-dependent manner. The 35 kD proenzyme caspase-3 was cleaved to its active 19 kD form after treatment .Group2: To study the molecular mechanism of Ganoderma lucidum inhibiting effect on invasion and metastasis in epithelial ovarian cancer. The E-cadherin, N-cadherin and vimentin were detected by western blot. The expression of E-cadherin and TIMP-1 of human ovarian cancer cells were increased, that of N-cadherin, vimentin and MMP-9 was decreased in dose-dependent manner.Group3: To understand the mechanisms underlying the chemo-enhancing effect of Ganoderma lucidum, we tested if it could regulate Akt,P-Akt ,bcl-2 and p53 expression and activation. The western blot was carried out to detect Akt,P-Akt ,bcl-2 and p53 .The results of western blot suggested that The expression of Akt,P-Akt ,bcl-2 in A2780CP cell was significantly higher than that in A2780S cell, whereas the expression of P53 showed decre- ased .The P53 protein level was increased and the level of Akt,P-Akt ,bcl-2 was decreased after treatment with Ganoderma lucidum . Moreover,when A2780CP cells were exposed to Ganoderma lucidum and Cisplantin simultaneously,the level of Akt,P-Akt,bcl-2 reduced markedly and the P53 increased than that exposed to Cisplantin alone. Conclusion:To the best of our knowledge, this is the first report of Ganoderma lucidum on epithelial ovarian cancer cells. Here, we demonstrated that Ganoderma lucidum has potent anti-tumor effects on chemosensitive and chemoresistant ovarian cancer cells and reversing drug-resistance effect on chemoresistant ovarian cancer cells.Conclusion 1:Ganoderma lucidum inhibited cell proliferation and indu- ced apoptosis in human ovarian cancer cells through inducing cell cycle G1phase arrest and up-regulation of bax , p27, down-regulation of bcl-2 and activation of caspase-3.Conclusion 2:Ganoderma lucidum may inhibit invasion and metastasis in human ovarian cancer cells by inhibiting adhesion, migration, invasion and increasing the expression of E-cadherin and TIMP-1 of human ovarian cancer cells, decreasing that of N-cadherin, vimentin and MMP-9.Conclusion3:Ganoderma lucidum can enhance chemosensitivity of epi- thelial ovarian cancer cells to cisplatin and reverse the resistance of A2780- CP cells to Cisplantin by downregulating the expression of Akt,P-Akt,bcl-2 and upregulating the expression of P-53 in A2780CP cells. Ganoderma lucidum may be a promising multidrug resistance modulator.Taken together, we have demonstrated that Ganoderma lucidum exerts multiple anti-tumor effects on ovarian cancer cells such as inhibit proliferation, adhesion, migration, invasion and induce apoptosis. We have also provided evidence that Ganoderma lucidum enhances chemosensitivity of epithelial ovarian cancer cells to cisplatin and reverses resistance to cisplatin in chemoresistance cell line A2780CP. Our findings suggest that Ganoderma lucidum may be useful in treatment of chemosensitive and chemoresistant epithelial ovarian cancer. Therefore, Ganoderma lucidum spore can be as useful supplementary means of ovarian cancer conventional chemotherapy with its anti-cancer properties and can improve the curative effect, benefit to the cytoreductive surgery (surgical debulking) if combining neoadjuvant chemotherapy. Combining chemotherapeutics, Ganoderma lucidum can enhance anti-cancer effect and prevent and reverse chemoresistance after surgery. Ganoderma lucidum may be served as a possible therapeutic agent as a dietary supplement for cancer patients. |
PDF Full Text Request