| Ovarian cancer is one of the highest mortality of gynecological malignant tumors in clinical, most of them are treated with surgery combination with chemotherapy. However, due to special anatomy location of ovary, most patients with ovarian cancer are already in the last stage and missed the best time for surgery when ovarian cancer symptoms are present. In a word, ovarian cancer often leads to death. Patients in intermediate stage and advanced stage were mainly treated with chemotherapy for remission in clinical. At present, although undergoing standardized full courses of chemotherapy, the five-year survival rate is still low in the vast majority of patients. Drug resistance was generated in ovarian cancer cells after long-term treatment of first-line chemotherapy drugs, for example cisplatin. Thus, failure of chemotherapy eventually leads to death. Some measures have been taken in response to multidrug resistance such as combined chemotherapy drugs and increased the dosage, but the accompanying side effects also increased. Finally, body organism functions failed, and immune regulation effects declined, ultimately resulting in tumor proliferation and spread. These have seriously affected the clinical effect of chemotherapy. Drug-resistant mechanisms of ovarian cancer are as follows:drug efflux mediated by p-glycoprotein, enhancement on the repair capacity of DNA damage, increase anti-apoptotic capacity and so on. In view of this, based on the specific mechanism for drug resistance of ovarian cancer, new drugs which can reverse drug resistance and improve the treatment of first-line anti-cancer drugs, will become an important way of anti-tumor adjuvant therapy.Ganoderma lucidum polysaccharides (GLP) is a kind of active polysaccharide and an important component of ganoderma lucidum. The related studies on GLP are mainly focused on its contributions to anti-tumor, anti-oxidation, anti-aging, antibacterial, antiviral and immune-modulation. GLP has been used clinically in the adjuvant treatment of some diseases such as cancer, chronic diseases, elder diseases and also used to improve immunity. The effect of GLP on the anti-tumor has been studied for many years. As a variety of biological activity and good bio-security, GLP could be used as an adjuvant drug to treat the resistant tumor combined with the first-line chemotherapy drugs. Such useful attempts may improve the drug resistance, enhance the efficacy of chemotherapy drugs and increase the satisfaction rate of chemotherapy in patients with drug resistance ovarian cancer. So far, GLP which may reverse the chemotherapy drug resistance in ovarian cancer cells has not been reported.In this study, we try to discuss the effect of GLP on DDP resistance. And we investigated the reversal effect of GLP combination with DDP on the DDP resistance in ovarian cancer cell line SKOV-3/DDP, and to explore its possible mechanism of action. We hope for the clinical application of resistant tumor treatment and provide the theoretical support and the experimental basis.Firstly, the stable DDP resistant human ovarian cancer cells of SKOV-3had been established successfully in vitro, named as SKOV-3/DDP, with small dose and increasing concentrations of DDP gradient way by repeating intermittent administration. Through in-vitro model, we studied the expression of GSTs of SKOV-3/DDP and discussed its mechanism of GLP on proliferation, differentiation, apoptosis, migration, invasion ability and drug resistance to DDP. We also induced SKOV-3/DDP tumor ball by ultra-low adsorption so as to imitate the survival and migration patterns in cancer cells in vivo. Through establishing ovarian cancer animal model of orthotopic transplantation tumor, GLP group and GLP combination with DDP group had studied the treatment of DDP resistant ovarian cancer in mice and further discussed the influence and mechanism of GLP on DDPresistant ovarian cancer. Specific research as follows:In vitro, the stable DDP drug-resistant ovarian cancer cell lines SKOV-3/DDP were successfully established. Inhibition rate of GLP and DDP on SKOV and SKOV-3/DDP cell proliferation was tested by MTT methods which included the effects of different concentrations of the GLP and DDP on SKOV and SKOV-3/DDP cells on different times. The results showed GLP and DDP suppressed the proliferation of SKOV-3cells than DDP or GLP. GLP could suppress SKOV-3/DDP cell proliferation. In addition, increasing the dose and prolonging the effect time could enhance the role of the inhibition. In the combination of two drugs, the effect of suppression on SKOV-3and SKOV-3/DDP cells proliferation was significantly increased. This suggested combination of GLP and DDP had a synergistic anti-tumor effect and GLP improved DDP resistance on SKOV-3/DDP cells. We designed primers according to the gene sequences of GSTM1, GSTTland GSTP1in GenBank. We amplified GSTM1, GSTT1and GSTP1gene fragments via Real Time-PCR and detected their different expressions on SKOV-3/DDP cells treated with GLP and DDP. We also explored Western-blotting to check the differences of expressions of GSTM1, GSTT1and GSTP1related resistance protein. The results showed both the expressions of GSTM1, GSTT1and GSTP1gene and its protein used GLP were significantly decreased, compared with DDP and the control group (P<0.05). Experimental results suggested that GLP had great effect of inhibition on SKOV-3/DDP cells and this could be proved by the inhibition of resistance GSTM1, GSTT1and GSTP1gene expression. Therefore, DDP resistance of SKOV-3/DDP cells could be reversed. In the study, we discussed the intracellular peroxides levels after observing intracellular ROS by fluorescence microscopy and detecting differences of GSH-Px and CAT gene expression by Real-time PCR. The results showed: GSH-Px and CAT gene expression was significantly decreased with the GLP concentration increasing. This confirmed that GLP could increase ROS levels in tumor cells by reducing GSH-PX and CAT activity of oxygen free radical scavenger. Another study, we used different dose of GLP to affect the SKOV-3/DDP cells and observed the morphological changes. We found cell proliferation became slower after using GLP and the cell division phase also decreased. We detected apoptosis via acridine orange fluorescent microscopy and flow cytometry and found that apoptosis increased after using GLP which affected the SKOV-3/DDP cells. The results suggested GLP could induce the apoptosis and finally exerted its anti-tumor effects.In vivo, The mice were immunosuppressed with cyclophosphamide. Mouse ovarian cancer peritoneal implantation tumor model was established, using SKOV-3/DDP cell lines and SKOV-3/DDP cell balls. The results showed SKOV-3/DDP cell balls had higher infection rate in mice (P<0.05). Murine ovarian cancer animal model of orthotopic transplantation tumor was established after immune suppression using implanted tumor tissue block, there was pathological changes in ovarian cancer by HE staining observation; ovarian cancer orthotopic transplantation tumor infection was successfully confirmed. Using GLP, DDP alone and combined effects on ovarian cancer orthotopic xenograft animal testing the expression of GSTM1, GSTT1, GSTP1. Our results showed that the level of GSTM1and GSTT1was higher in cells treated with DDP and GLP than in cells treated with DDP or GLP. But the level of GSTP1was not different in cells treated with DDP and GLP than in cells treated with DDP or GLP. In vivo assay, we als believed that GLP accelerated SKOV-3/DDP apoptosis by promoting lipid peroxidation damage of tumor cells.In summary, this study confirms GLP reverses cisplatin resistance in ovarian cancer cells. the mechanism of drug resistance are inhibiting resistance gene expression, promoting damage of lipid peroxidation and apoptosis of tumor cells. Research for the development of GLP in the clinical wider application provides experimental foundation and theoretical basis.
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