Effects Of Temozolomide Combined With Pyrimethamine On Proliferation Of αT3-1Cells

Objective: To observe the effects of Tomozolomie combined with Pyrimethamineon proliferation of αT3-1cells and find a new drug therapy for non-functioning pituitaryadenoma.Methods: Proliferation/viability study: The effect of TMZ, PYR and TMZ/PYRcombination on αT3-1cells in vitro was accessed by Cell Counting Kit-8(CCK-8) assay.Flow cytometry analysis of apoptosis: αT3-1cells treated with TMZ, PYR andTMZ/PYR combination were determined using a FACS flow cytometer and wereanalyzed with CFlow Plus analysis software. Cell cycle analysis：DNA content of αT3-1cells treated with TMZ, PYR and TMZ/PYR combination was analyzed using a FACSflow cytometer. TUNEL staining was performed to detect apoptotic αT3-1cells.Caspase-3/7,8, and9activity was determined to further investigate the mechanism ofapoptosis induced by TMZ, PYR, or TMZ/PYR combination. Effects of TMZ, PYR, or(TMZ/PYR) combination on H2AX phosphorylation, Bcl-2, Bax and poly (ADP-ribose)polymerase (PARP)(intact and cleaved fragments) were analyzed by western blotting.Results: Proliferation/viability study: TMZ/PYR combination treatmentsignificantly inhibited the proliferation/viability of αT3-1cells (P<0.05). Flowcytometry analysis of apoptosis:100μmol/L TMZ induced apoptosis of αT3-1cells.And TMZ/PYR combination treatment significantly induced apoptosis of αT3-1cells(P<0.05). Cell cycle analysis：100μmol/L TMZ induced αT3-1cells accumulation in Sphase. And TMZ/PYR combination treatment notably induced αT3-1cellsaccumulation in both S phase. When PYR was combined with TMZ, the number ofTUNEL-positive cells was dramatically increased. In addition, PYR in combinationwith TMZ dramatically decreased the expression of Bcl-2. The increased expression ofcleaved PARP and Bax along with elevated Caspase-3/7、8and9activity were also observed in combination group.Conclusion: TMZ/PYR combination treatment represents an anti-proliferationeffect on pituitary adenoma cell lines—αT3-1,and provided a new drug treatment optionfor these nonfunctional pituitary adenoma which are failed in conventional treatment.