Small Molecule Oxidizing-reducing Compounds Inhibit Glycolysis Of Ovarian Cancer

Background and purpose:Tempo is small molecule nitroxide radical,because its molecular structure contains a single electron,it has oxidability and reducibility.Also,it has many features,such as penetrating cell membranes,low toxicity and non-carcinogenicity.At low concentrations,Tempo presents oxidation resistance.When concentrations are higher than 1mM,it presents the property of promoting oxidation.Cisplatin(DDP)is preferably used for chemotherapy of ovarian cancer in clinical practice,while its use is often limited because of its dose-limiting toxicities and the development of drug resistance.Exploiting a method to reduce DDP toxicity,at the same time maintaining its efficacy is significantly important for the chemotherapy on ovarian cancer.Silybin is one kind of flavonoids.Because its molecular structure contains conjugated double bonds and alcoholic groups,it has oxidability and reducibility as well.In clinical,Silybin is mainly used for the prevention and treatment of diabetes,chronic hepatitis and atherosclerosis.It also plays an important role in anti-tumor.The principle way of tumor metabolism is glycolysis,which is one of the characteristics of tumors.nNOS in intracellular catalyzes L-arginine to react oxidation reaction,producing nitric oxide(NO).NO is one of the factors that positively regulate cancer glycolysis.Our team's preliminary experiments also prove that nNOS promotes the glycolysis of ovarian cancer.Therefore,we have interests on whether Tempo and Silybin affect the function of nNOS on cancer glycolysis and increases cisplatin sensitivity of ovarian cancer.Results:Chapter One.Tempo enhanced Cisplatin-induced apoptosis in ovarian cancer cellsThe results of MTT assay showed that Tempo inhibited the proliferation of SKOV3 and OVCAR3 cells and enhanced DDP-induced proliferation inhibition of OVCAR3 cells.The results of cell apoptosis assay showed that Tempo enhanced DDP-induced apoptosis of OVCAR3 cells.The results of Western Blot analysis demonstated that Tempo decreased DDP-induced the expression ratio of Bcl-2/Bax in OVCAR3 cells.The results of ROS level assay showed that Tempo enhanced the DDP-induced apoptosis through ROS generation in OVCAR3 cells.This part of the experiments suggested that Tempo enhanced Cisplatin-induced apoptosis in ovarian cancer cells.Chapter Two.Tempo inhibited the glycolysis of ovarian cancer cells through interfering the function of nNOSThe results of Griess assay and Western Blot analysis showed that Tempo inhibited the process of producing NO by nNOS and the expression of nNOS protein level.The results of glucose consumption and lactic acid generation assay,ATP and NADPH generation assay showed that Tempo inhibited the glycolysis of OVCAR3 cells.Our previous experiments showed that nNOS promoted the glycolysis of ovarian cancer through promoting the levels of S-nitrosation of two key glycolytic enzymes PFK1 and PKM2.The results of Nitrosylation assay showed Tempo decreased the levels of S-nitrosation PFK1 and PKM2.This part of the experiments suggested that Tempo decreased the levels of S-nitrosation PFK1 and PKM2 through inhibiting the process of producing NO by nNOS.Chapter Three.Silybin inhibited the glycolysis of ovarian cancer cells through interfering the combination of HSP90 and nNOS.The results of cell apoptosis assay showed that Silybin enhanced the apoptosis of ovarian cancer cells.The results of MTT assay showed that Silybin enhanced DDP-induced proliferation inhibition of ovarian cancer cells.The results of Griess assay and Western Blot analysis showed that Silybin inhibited the process of producing NO by nNOS and the expression of nNOS protein level.The results of glucose consumption and lactic acid generation assay demonstated that Silybin inhibited the glycolysis of ovarian cancer cells.The results of Co-IP assay suggested that HSP90 could combina with nNOS and Silybin inhibited the function of nNOS through interfering the combination of HSP90 and nNOS.