Autophagy Induced By Cardamonin Is Involved In Glycolysis Inhibition Via MTOR In SKOV3 Cells

ObjectiveOvarian cancer is one of the common cancers in women, with lower rate of 5 years survival. Although chemotherapy works in ovarian cancer, significant toxicity and the rapid onset of drug resistance limit their clinical application. It was demonstrated that cardamonin (CAR) had an effect of mTOR inhibition, and it might be a novel drug with high efficiency and safety on ovarian cancer therapy. Studies showed that autophagy was closely related with the occurrence, development, treatment and prognosis of ovarian cancer. Therefore the regulation of tumor cell autophagy will be a new direction for the treatment of ovarian cancer. It has been known that cancer development depend on activation of aerobic glycolysis. However, when nutrition deprivation, autophagy will be induced by AMPK. Therefore, the project is designed to study the effect of autophagy induction by CAR and to elucidate its mechanism on glycolysis suppression in ovarian cancer cell.MethodsHuman ovarian cancer cells lines SKOV3 were grown at 37 ℃ in McCoy’s 5A supplemented with 10% fetal bovine serum,100 U·mL-1 penicillin and 100 μgmL-1 streptomycin in a humidified 5% CO2 atmosphere. Cells were incubated in medium containing CAR (5,10,20 μM), Rapamycin (RAP,0.1 μM),2-deoxy-D-glucose (2-DG,10 mM), Compound C (10 μM) and Compound C+CAR (10 μM+20 μM), respectively. Colorimetric method was used to analyze lactate secretion, ATP content, glucose content and the activity of HK and LDH. Western Blot was performed to analyze the protein expression of P-mTOR, mTOR, P-S6K1, S6K1, P-AMPK, AMPK, HK2, LC3, and LAMP1. The level of autophagy was detected by MDC staining method.Results1. SKOV3 cells were treated with CAR (5,10,20 μM), RAP (0.1 μM),2-DG (10 mM) at 24, 36,48,60,72 h, respectively. The lactate secretion was significantly inhibited in a dose-dependent and time-dependent way. The inhibition rate of CAR was higher at 48 h than that at any other time.2. ATP content and glucose uptake were decreased by CAR in a dose-dependent way when cells treated with different drugs at 48 h.3. The activity of HK and LDH was decreased by CAR and RAP, while the activity of LDH decreased by CAR was in a dose-dependent manner when cells treated with different drugs at 48 h.4. The expression of LC3 and LAMP1 was significantly increased. Fluorescent spots with MDC staining were strengthened when cells treated with different drugs at 48 h.5. The expression of P-mTOR, P-S6K1 and HK2 was decreased, while expression of P-AMPK was increased when cells treated with different drugs at 48 h.6. The expression of LC3 and P-AMPK were lower by CAR combined with compound C than that CAR used alone.Conclusions1. CAR inhibited glycolysis in SKOV3 cells.2. CAR induced autophagy in SKOV3 cells.3. Autophagy induced by CAR was involved in glycolysis inhibition via mTOR on SKOV3 cells.