ABSTRACT
Background & objectives: Erythropoietin (EPO) has cytoprotective and anti-apoptotic effects in pathological conditions, including hypoxia and ischaemia-reperfusion injury. One of the targets to protect against injury is ATP-dependent potassium (KATP ) channels. These channels could be involved in EPO induced ischaemic preconditoning like a protective effect. We evaluated the cell cytoprotective effects of EPO in relation to KATP channel activation in the renal tubular cell culture model under hypoxic/normoxic conditions. Methods: Dose and time dependent effects of EPO, KATP channel blocker glibenclamide and KATP channel opener diazoxide on cellular proliferation were evaluated by colorimetric assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] under normoxic and hypoxic conditions in human renal proximal tubular cell line (CRL-2830). Evaluation of the dose and time dependent effects of EPO, glibenclamide and diazoxide on apoptosis was done by caspase-3 activity levels. Hypoxia inducible factor-1 alpha (HIF-1 α) mRNA levels were measured by semi-quantative reverse transcription polymerase chain reaction (RT)-PCR. Kir 6.1 protein expresion was evalutaed by western blot. Results: Glibenclamide treatment decreased the number of living cells in a time and dose dependent manner, whereas EPO and diazoxide treatments increased. Glibenclamide (100 μM) treatment significantly blocked the anti-apoptotic effects of EPO (10 IU/ml) under both normoxic and hypoxic conditions. EPO (10 IU/ml) and diazoxide (100 μM) treatments significantly increased (p<0.01) whereas glibenclamide decreased (p<0.05) HIF-1 α mRNA expression. Glibenclamide significantly (p<0.01) decreased EPO induced HIF-1 α mRNA expression when compared with the EPO alone group. Interpretation & conclusions: Our results showed that the cell proliferative, cytoprotective and anti-apoptotic effects of EPO were associated with KATP channels in the renal tubular cell culture model under hypoxic/normal conditions.
ABSTRACT
OBJECTIVE: The aim of the study was to investigate whether lithium chloride and medroxyprogesterone acetate can potentiate the cytotoxicity of imatinib mesylate in human endometrial cancer in vitro and the effect of midkine in these therapies. METHODS: Imatinib mesylate (50 microM), lithium chloride (100 microM), medroxyprogesterone acetate (200 microM) and their combination were applied to monolayer and three dimensional cultures of human Ishikawa endometrial cancer for 72 hours. The cell proliferation index, apoptotic index, caspase-3 and midkine levels, cell cycle distributions in monolayer cultures and cell ultrastructure in spheroid cultures were evaluated. Results were statistically analyzed using the Student's t-test. RESULTS: All drug applications inhibited cell proliferation (p<0.05), however the combination were the effective groups for 72 hours (p<0.05). Interestingly, although the loss of efficiency was seen higly seen every 24 hours at single applications, the inhibition rates of the combination groups were almost same for 72 hours. In concordance with these results, the apoptotic index, caspase-3 levels (p<0.05), cell morphology and ultrastructure damages were much higher in the combination groups. Imatinib mesylate induced S-phase arrest, however other groups induced G0+G1-phase arrest at 24 hours and all groups induced G0+G1 arrest at 72 hours (p<0.05). Imatinib mesylate and imatinib mesylate with medroxyprogesterone acetate induced highest decrease in midkine levels, respectively (p<0.05). CONCLUSION: The present study showed that the combination of imatinib mesylate with lithium chloride and medroxyprogesterone acetate is highly active in Ishikawa endometrial carcinoma in vitro and the inhibition of midkine involved in their mechanism of action against endometrium defense.