CPUK02 sensitizes U87 glioblastoma cell lines to TMZ treatment via autophagy flux inhibition.

Adjuvant chemotherapy with TMZ (Temozolomide) does not improve the survival of patients suffering from GBM (Glioblastoma). Given the importance of autophagy and UPR (Unfolding Protein Response) in chemotherapy resistance, as well as the role of Beclin-1, LC3IIβ, and P62 in the regulation of autophagy, we evaluated the effect of TMZ along with CPUK02 on U87 cells as a model of Glioblastoma cancer in this study. To achieve this goal, we treated the U87 cells with different doses of TMZ (50, 100, 200, 400, and 800 μM) and CPUK02 (1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.01, and 0.007 μM); then, cell viability was assessed by MTT assay. The gene expression of Beclin1, P62, LC3IIβ, and XBP-1s was analyzed using quantitative real-time polymerase chain reaction. The comparison of the control group with the groups treated with the TMZ drug showed that, in 48 and 72 hours, doses of TMZ more than IC₅₀ (100 μM) (p<0.001) significantly led to cell death. CPUK02 doses more than 0.125 (p<0.0001) significantly led to cell death. TMZ and CPUK02 combination therapy (100 and 0.03 μM, respectively) increased the expression of Beclin-1, LC3IIβ, and P62 and activated the IRE-1 arm of UPR by increasing the expression of XBP-1s. TMZ and CPUK02 treatment inhibits the autophagic flux (p62, LC3IIβ). Increased XBP-1s expression might contribute to the enhanced TMZ sensitivity. This combination therapy is promising for TMZ-resistant cancers, but it needs further investigation.