The Study Of A Novel Fusion Protein UPAg-KPI On Inhibition Of Ovarian Cancer And Protection Of Visceral Organ

Ovarian cancer is a serious threat to women’s health, the mortality rate ranks first in gynecological malignancies. Although the treatment program is clear for ovarian cancer, surgical equipments are also getting better, chemotherapy drugs are emerging, but the five year survival rate of ovarian cancer is still only 20%-30%. There are two main reasons,(1) ovarian cancer is usually accompanied by metastasis and it is difficult to carry out the ideal cytoreductive surgery;(2) chemotherapy has serious toxic and side effects, such as myelosuppression, liver and renal function injury, all these lead to the patients unable to complete the chemotherapy. So it is very important to find a new drug that can have both the ability to inhibit ovarian cancer and protective function of visceral organ.Urokinase type plasminogen activator(uPA) is a serine protease, through binding to the uPA receptor(uPAR) via uPA’s growth factor domain(GFD), Urokinase-type plasminogen activator(uPA) results in the degradation of extracellular matrix and basement membrane, activation of MAPK and SRC and FAK and PI3 K and other signaling pathway, tumor angiogenesis, cell proliferation, migration and invasion. uPA and uPAR are high expressioned in variety of solid tumors, including ovarian cancer, the expression level are closely related to the prognosis of patients.Kunitz type protease inhibitor(KPI) is a kind of serine protease inhibitor. On the one hand, KPI can inhibit the function of uPA, and then inhibit the function of uPA/uPAR system in tumor growth and metastasis. On the other hand, KPI can inhibit inflammatory medium release, inhibit the production of superoxide anion free radical, reduce lipid peroxidation injury, inhibit the degradation of lysosomal, inhibit Ca2+-activated K+ channels, delay cell apoptosis and death. The studies on KPI in the field of anti tumor metastasis and organ protection are increasing day by day.Thus, in our present study, we constructed and expressed a fusion protein “uPAg-KPI”. uPAg-KPI was fused uPA-GFD with KPI, a kunitz protease inhibitor domain that was found in amyloid beta-protein precursor(APP). Incorporation of KPI-amyloid protein precursor, the fusion protein was allowed for inhibition of serine protease hydrolysis. Our current studies provided the insightful information for the effects of uPAg-KPI on anti-ovarian cancer in vivo and vitro and the protective effects of liver and kidney injury. Object:We used the constructed fusion protein, uPAg-KPI, to evaluate the effects on anti-ovarian cancer in vivo and vitro and the protection effects on visceral organ. Methed:(1) In vitro, MTT assay, colony formation assay, flow cytometry, ‘wound’ closure assay, transwell migration and invasion assay, western blot assay were used to evaluate the effects of fusion protein on SKOV3 ovarian cancer cells.(2) In vivo, BALB/c female mices were inoculated with human SKOV3 ovarian cancer cells(5 × 106) into subcutaneous, Compared with the xenograft size, weight of xenograft and weight of nude mice, then Immunohistochemistry was used to detect the expression of P-ERK,P-AKT,PCNA,VEGF protein between uPAg-KPI group and control group to speculate the inhibition mechanism in ovarian cancer.(3) we established an acute kiney injury model, through serum BUN, CRE texting and histological observaton in kiney,we evaluated the effects of uPAg-KPI on acute kiney injury, then though SOD, CAT, GSH-PX, MDA activity texting levels to speculate the protection mechanism.(4) we established an acute liver injury model, through serum ALT,AST texting and histological observaton in liver,we evaluated the effects of uPAg-KPI on acute liver injury, then though SOD, CAT, GSH-PX, MDA activity texting levels to speculate the protection mechanism. Results :(1) In vitro, we found that uPAg-KPI had an inhibitory effect on SKOV3 ovarian cancer cells in MTT assay, the inhibition resulted in a time and concentration-dependent manner. Under 0.5μg/μl, 48 h, the inhibition rate was 50%. uPAg-KPI could decrease the colony forming ability. Through FCM(flow cytometry), we confirmed that uPAg-KPI blocked the cell cycle of SKOV3 by increasing G1 phase and decreasing G2/M phase cell. In Transwell migration and invasion assay, the uPAg-KPI group had the less penetrating cells. The ‘wound’ closure assay showed the same result. In addition, treatment of SKOV3 cells with uPAg-KPI resulted in a significant decreasing expression of P-ERK and P-AKT.(2) In vivo, the tumor size and weight of nude mice in uPAg-KPI group were less than the control group’s, the combined dosing group’s tumor volume and weight tend to be less than DDP group’s and uPAg-KPI group’s. When compared the weight of nude mice in the four groups, DDP combined uPAg-KPI group and the DDP group decreased significantly. The expression of P-ERK, P-AKT, PCNA, VEGF protein were decreased in uPAg-KPI group in immunohistochemistry.(3) uPAg-KPI could reduce the level of CRE, but this had no significance. The high-dose of uPAg-KPI could significantly reduce the level of BUN in serum. The content of SOD, CAT, GSH-PX activity in kidney were increased in uPAg-KPI group, MDA was decreased. Histological observaton showed that uPAg-KPI could reduce the kidney inflammation, protein tube type and swelling of renal tubular epithelial cells.(4) uPAg-KPI could reduce the level of ALT and AST. The content of SOD, CAT, GSH-PX activity in liver were increased in uPAg-KPI group, MDA activity was decreased. Histological odservaton showed that uPAg-KPI could reduce the liver inflammation, liver cell necrosis and degeneration. Conclusion:(1) uPAg-KPI has an inhibitory effect on t he proliferation, invasion and metastasis of ovarian cancer SKOV3 cells, which may be correlated with cell cycle blocking, P-ERK and P-AKT related signaling pathway’s interfering.(2) uPAg-KPI has an inhibitory effect on ovarian cancer in vivo, the inhibition effect was enhanced when combined with cisplatin. The inhibitory effect of uPAg-KPI may be related to the down-regulation of P-ERK, P-AKT, PCNA, and VEGF protein expression.(3) uPAg-KPI could protect the renal function that was damaged by cisplatin, reduce the renal tubular expansion, the formation of the protein tube, inflammatory cell infiltration and epithelial degeneration. The protective function was related to the increasing of antioxidant enzymes and decreasing of lipid peroxidation products.(4) uPAg-KPI could protect the liver function that was damaged by CCl4, reduce the infiltration of inflammatory cells and the degeneration and necrosis. The protective function was related to the increasing of antioxidant enzymes and decreasing of lipid peroxidation products.