An active medicinal component of plant origin with an ability to overcome autophagy by inducing apoptosis should be considered a therapeutically active lead pharmacophore to control malignancies. In this report, we studied the effect of concentration-dependent 3-AWA (3-azido withaferin A) sensitization to androgen-independent prostate cancer (CaP) cells which resulted in a distinct switching of 2 interrelated conserved biological processes, i.e. autophagy and apoptosis. We have observed 3 distinct parameters which are hallmarks of autophagy in our studies. First, a subtoxic concentration of 3-AWA resulted in an autophagic phenotype with an elevation of autophagy markers in prostate cancer cells. This led to a massive accumulation of MAP1LC3B and EGFP-LC3B puncta coupled with gradual degradation of SQSTM1. Second, higher toxic concentrations of 3-AWA stimulated ER stress in CaP cells to turn on apoptosis within 12 h by elevating the expression of the proapoptotic protein PAWR, which in turn suppressed the autophagy-related proteins BCL2 and BECN1. This inhibition of BECN1 in CaP cells, leading to the disruption of the BCL2-BECN1 interaction by overexpressed PAWR has not been reported so far. Third, we provide evidence that pawr-KO MEFs exhibited abundant autophagy signs even at toxic concentrations of 3-AWA underscoring the relevance of PAWR in switching of autophagy to apoptosis. Last but not least, overexpression of EGFP-LC3B and DS-Red-BECN1 revealed a delayed apoptosis turnover at a higher concentration of 3-AWA in CaP cells. In summary, this study provides evidence that 3-AWA is a strong anticancer candidate to abrogate protective autophagy. It also enhanced chemosensitivity by sensitizing prostate cancer cells to apoptosis through induction of PAWR endorsing its therapeutic potential.
Keywords: 3-AWA, 3-azido withaferin A; 3-azido withaferin A; AO, acridine orange; ATG, autophagy-related; AVOs, acidic vesicular organelles; BAD, BCL2-associated agonist of cell death; BAF A1, bafilomycin A1; BCL2; BCL2, B-cell CLL/lymphoma 2; BECN1; BECN1, Beclin 1, autophagy-related; CASP3, caspase 3; CASP9, caspase 9; CQ, chloroquine; CYCS, cytochrome c, somatic; CaP, prostate cancer cells; DAPI, 4’6-diamidino-2-phenylindole; DCF, dichlorofluorescein; DDIT3/CHOP, DNA-damage-inducible transcript 3; EIF2AK3/PERK, eukaryotic initiation translation factor 2-α kinase 3; ER, endoplasmic reticulum; HSPA5/GRP78, heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa); MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 β; MDC, monodansylcadaverine; MEFs, mouse embryonic fibroblasts; MMPψ, mitochondrial membrane potential; MTOR, mechanistic target of rapamycin; NAC, N-acetyl-L-cysteine; PARP1, poly (ADP-ribose) polymerase 1; PAWR; PAWR/Par-4, PRKC, apoptosis, WT1, regulator; PRKCZ/PKCζ, protein kinase C, zeta; SQSTM1/p62, sequestosome 1; WT1, Wilms tumor 1; apoptosis; autophagy; myrAKT1, myristoylated v-akt murine thymoma viral oncogene homolog 1.