Investigation of the autophagic effects of betulinic acid on T98 and U87 cancer cell lines

Therapeutic possibilities against Glioblastoma multiforme (GBM), which constitutes 50% of brain tumors, are limited because of blood-brain barrier (BBB). Betulinic acid (BeA), isolated from white birch (Betula sp.), is pentacyclic triterpenoid that can cross BBB and reach the brain. BeA exhibits low cytotoxicity in somatic cells and high apoptosis in tumor cells. Thus, in this study, we aimed to examine the apoptotic effects of BeA on two GBM cell lines. 3-(4,5-dimethyltriazol-2-il)-2,5 diphenyltetrazolium bromide (MTT) for IC50 and monodansylcadaverine (MDC) for finding effective doses of rapamycin and chloroquine were administered and the effects of BeA on autophagy inhibition and activation were investigated by applying doses in combination. Amount and localization of eNOS, iNOS, LC3-I-II proteins by immunofluorescence and western blotting techniques, changes in vesicles with MDC, changes in mitochondrial membrane potential (MMP) with JC-1, and healthy-apoptotic-necrotic cell ratios by ANNEXIN-V/PI flow cytometry were analyzed. It was determined that BeA application increased apoptosis in both cell lines and doubled it with autophagy inhibition and activation. It was found that BeA did not show high autophagic activity, but disrupted MMP. MMP damage was increased in the combinations and the damage was higher in BeA+rapamycin group. These findings suggest that BeA initiates apoptosis via the mitochondrial pathway by not using the autophagic pathway. eNOS and iNOS expressions in BeA group was not drastically changed for both cell lines, but changes are more prominent in combinations. In this study, it was indicated that BeA causes apoptosis in GBM cells, but apoptosis increased drastically in activator or inhibitor combination groups, and this apoptotic cell death originated from MMP disruption. Changes on eNOS and iNOS in application groups have shown that there is a connection between oxidative and mitochondrial stress. In conclusion, we report that BeA triggers apoptosis on GBM, however application of BeA with autophagy activator or inhibitor significantly increases cell death. Therefore, it can be suggested that the chemotherapeutic effect of BeA can be enhanced by autophagic mechanisms, and it can be used as a new chemotherapeutic agent on GBM in the future.