TRAIL-resistant glioma cells resist Withania somnifera mediated apoptosis via USP5 upregulation.

The tumor necrosis factor related apoptosis inducing ligand (TRAIL) shows a potential therapeutic by inducing apoptosis in glioma cells and sparing normal cells, but TRAIL resistance and chemotherapeutic resistance still prevails in glioma and hinder its apoptotic effect. To address TRAIL resistance, we investigated the potential of Withania somnifera, a medicinal herb with recognized anti-cancer properties. The response against the therapeutic approaches led glioma to get regress but relapse is a major challenge. The study aims to evaluate the biological effect of the W. somnifera fruit extract in TRAIL resistant glioma cell lines (U87MG, LN229) and TRAIL sensitive (T98G) glioma cell lines. MTT cell viability assay were performed to assess the effect of W. somnifera fruit extract. Fluorophore conjugated Annexin V APC -PI staining was performed to study the apoptotic effect of an extract obtained from W. somnifera. The underlying mechanism comprises of USP5 mediated DR5 regulation in cell survival and its subsequent knock down led to apoptosis enhancement. The apoptotic relevant proteins SMAC, and semi quantitative PCR analyzed abundance of DR4 & DR5 receptor was quantitated. Therefore, the expression of USP5 was analyzed across 592 glioma tumor data and was correlated with the abundance of EGFR, the data was accessed from cBioportal (https://www.cbioportal.org/). Our study findings proposed the leading upregulation of deubiquitinating enzyme USP5 in TRAIL resistant U87MG and LN229 glioma cells upon treatment with Withania somnifera fruit extract promotes cell survival. Furthermore, depletion of USP5 followed with Withania somnifera fruit extract-treatment, upregulates TRAIL receptor (DR5) and SMAC protein led to apoptosis activation, reveals a survival promoting characteristic of Withania somnifera fruit extract in TRAIL resistant U87 MG and LN229 glioma cells. Furthermore, being USP5 a key promoter in tumorigenesis but is found overexpressed/mutated in 3% of glioma tumor. The leading upregulation of USP5 found interesting in our treatment set to correlate with the EGFR abundance across the tumor samples. Since, EGFR is overexpressed in 47% tumor and acquiring the potential chemo-resistant pathway led to USP5 upregulation may be detrimental causing recurrent tumor. Therefore, from our studies USP5 deubiquitinating enzyme plays a key role in latent survival. Deubiquitinating enzyme family protein USP5 upregulation and stability of apoptotic protein SMAC are found important indicator in balancing the TRAIL resistance glioma to opt unidirectional pathway which is not aberrant but rewired on W. somnifera FE treatment. Therefore, knockdown of USP5 make cells sensitive to undergo direct apoptosis.