All-trans retinoic acid enhances temozolomide-induced autophagy in human glioma cells U251 via targeting Keap1/Nrf2/ARE signaling pathway

The present study evaluated the retinoic acid (RA) enhancement of temozolomide (TMZ) effects on the human glioma cells U251 and explored its underlying molecular mechanism. The cell growth was detected using the MTT assay and the cell cycle was assessed by flow cytometry. Cell apoptosis was analyzed by Annexin V/propidium iodide staining, and the cell morphology was evaluated using transmission electron microscopy (TEM). Additionally, reverse transcription-PCR and western blot analysis were applied to detect the mRNA and protein levels. The RA treatment in combination with TMZ in the human U251 cells further inhibited cells growth, arresting cell cycle progression at the G0/G1 phase, and significantly induced apoptosis of U251 cells. After the RA+TMZ treatment of U251 cells, autophagy associated proteins Beclin 1 and LC3B were significantly increased, and the TEM analysis were consistent with autophagy protein levels. Moreover, Keap1/Nrf2/ARE expression was downregulated significantly, indicating the involvement in the mechanisms that RA treatment could enhance the TMZ effects on U251 cells. RA treatment in combination with TMZ may provide some experimental evidence for the possible effect of RA+TMZ against the growth and the proliferation of glioma cells. Therefore, the RA+TMZ administration may have potential utility for glioblastoma treatment.