The reduction in population growth induced by nontargeting siRNA is likely due to non-specific toxicity as reported by others ; importantly, it was not associated with an increase in INCB18424 molecular weight apoptosis marker expression. The results are consistent with reports of a critical role for BIRC6 in the survival of a variety of cancer cells. Cell cycle analysis showed that BIRC6 reduction did not result in significant change in cell cycle distribution, suggesting that the reduction in cell viability was attributable to apoptosis. In this study, a decrease in cell viability induced by BIRC6 reduction did not confine to cells expressing wild-type p53, contrary to previous reports suggesting that apoptosis resulting from BIRC6 knockdown in H460 cells and breast cancer cells requires functional p53. We showed that both wild type p53 and p53 null cells were sensitive to BIRC6 siRNA induced growth inhibition. This variation reflects that apoptosis induction by loss of BIRC6 may be facilitated by different mechanisms in different models. Further investigation is necessary to understand the underlying mechanism leading to apoptosis after BIRC6 reduction in p53 null cells and that will provide further insight in the possibility of targeting BIRC6 in cancer cells lacking functional p53. Elevated levels of BIRC6 have been linked to apoptosis resistance, for instance in the SNB-78 U0126 glioma cell line and over-expression of BIRC6 in human fibrosarcoma cells supports resistance to anti-cancer drugs and death receptor ligation. Furthermore, down-regulation of BIRC6 expression in SNB-78 cells was shown to sensitize the cells to apoptosis induced by cisplatin and camptothecin. It is therefore conceivable that the elevated expression of BIRC6 observed in castration-resistant prostate cancers may be responsible for the treatment resistance of refractory disease. The specific reduction of BIRC6 expression in LNCaP cells leading to a decrease in the expression of LC3B-II and Beclin-1 and decline in autophagosome accumulation, suggest that there is a novel role for BIRC6 in the regulation of autophagy. The reduced expression of LC3B-II indicates that loss of BIRC6 expression results in a lower number of autophagosomes. However, based on LC3B-II levels alone, it is not possible to determine whether the reduced number of autophagosomes is due to a decrease in autophagosome formation or to an increase in autophagosome degradation. To provide further insight into regulation of autophagy by BIRC6, BIRC6-depleted LNCaP cells were also examined for changes in the levels of Beclin-1. In mammalian cells enhanced expression of Beclin-1 has been shown to increase their autophagic response and the suppression of Beclin-1 has been shown to impair autophagy and sensitize cells to starvation-induced apoptosis. The reduced expression of Beclin-1 in the BIRC6-depleted LNCaP cells suggests that the lower numbers of autophagosomes in these cells is likely due to inhibition of autophagy initiation and autophagosome formation. In addition, in the LC3-GFP puncta formation assay where LNCaP cells were treated with chloroquine, a potent inhibitor of autophagosome degradation, BIRC6-depleted cells also showed less punctate structures than non-targeting controls. This evidence highlights the relationship between BIRC6 expression and autophagy initiation in particular. Taken together, these data demonstrate that loss of BIRC6 expression in LNCaP prostate cancer cells.