Bufalin Induces The Interplay Between Apoptosis And Autophagy In Glioma Cells Through Endoplasmic Reticulum Stress

Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Bufalin is the major component of Chan-Su (a traditional Chinese medicine) extracts from the venom of Bufo gargarizan. At present, the studies about anti-tumor mechanisms of bufalin focus on various liver cell types. It has not been reported about the anti-tumor effect of bufalin on gliomas. In this study, we evaluated the growth inhibitory effect of bufalin on glioma cells and explored the underlying molecular mechanisms.U87MG cells were treated with bufalin at various concentrations combined with other treatments for 24 or 48 h. The MTT assay was used to assess cytostatic activity induced by different treatments. Flow cytometry was adopted to evaluate the level of ROS and apoptosis after various treatments in U87MG cells. Formation of autophagic vacuoles was observed by staining with acridine orange or MDC under fluorescence microscope. Si-RNAs were transfected into U87MG cells with Lipofect2000 reagent to knockdown certain genes. Western blot was used to demonstrate the expression of proteins involved apoptosis, autophagy and ER stress following diverse treatments.The results of MTT assay indicated that bufalin significantly repressed the proliferation of U87MG cells. Proof that bufalin induced apoptosis through mitochondrial apoptotic pathway was supported by the upregulation of Bax/Bcl-2 and release of cytochrome c as well as the increase of Cleaved Caspase-3 and Cleaved PARP.Evidence of bufalin-induced autophagy included formation of the acidic vesicular organelles, increase of autophagolysosomes and LC3-Ⅱ accumulation. Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1.Inhibition of autophagy with 3-MA enhanced the apoptosis induced by bufalin and promoted the expression of cleaved caspase-3 and cleaved PARP. Knockout of autophagy-related genes Beclin 1 or Atg5 in U87MG cells attenuated bufalin-induced LC3-Ⅰ to LC3-Ⅱ conversion and upregulated the level of cleaved PARP. These results suggested that autophagy played a protective role in bufalin-induced apoptosis.Evidence of bufalin-induced ER stress involved augmented phosphorylation of UPR sensors, PERK, IRE 1α and eIF2α as well as an increase of ER stress markers, GRP78 and GRP94. Activation of CHOP and caspase 4 indicated that bufalin induced ER stress-mediated apoptosis in U87MG cells. Downregulation of CHOP using siCHOP RNA attenuated bufalin-induced apoptosis, further confirming the important role of ER stress response in mediating bufalin-induced apoptosis. In addition, TUDC and silencing of eIF2α or CHOP partially blocked bufalin-induced accumulation of LC3-Ⅱ, which indicated that ER stress preceded bufalin-induced autophagy and PERK/eIF2α/CHOP signaling pathway played a major part in the process.All together, the main conclusions in this study are as follows:Bufalin suppresses the proliferation of glioma cells and induces both mitochondria- and ER stress mediated apoptosis. In addition to apoptosis, bufalin also induces autophagy by activating AMPK/mTOR pathway. ER stress especially PERK/eIF2α/CHOP pathway participates in the autophagy process induced by bufalin. Inhibition of autophagy potentiates the proapoptotic effect of bufalin, suggesting appropriate regulation of autophagy is necessary for sensitizing tumor cells to anticancer therapy.