Bax/Bim upregulation was followed by Bcl-2 downregulation beginning from 8 hours post PMC-A exposure and culminating at 24 hours post-exposure. This dramatic downregulation Nilotinib Leukemia was preceded by a transient slight increase in cellular Bcl-2 in the early hours of exposure in both wt and p53?/? cell lines. Truncation of Bid which may mark secondary involvement of the extrinsic apoptotic pathway took place as a late response to PMC-A in both cell lines. However, it should be noted that Bid cleavage was relatively delayed in p53?/? cells as cleavage product was not visible until 24 hours post-exposure. Figure 6 Bcl-2 downregulation and Bid processing follows Bax/Bim elevation in a p53-independent fashion in response to PMC-A.

The relatively late decrease in Bcl-2 expression was dose-dependent and confirmed to take place at the transcriptional level by quantitative real-time polymerase chain reaction (qRT-PCR) analysis (Fig. 7A, B). Bcl-2 expression was decreased in PMC-A-treated cells to almost one fifth of the expression in untreated control cells. Moreover, interrogation of HCT116 cells that were transiently transfected with the human Bcl-2 gene revealed that this regulation was critical in response to PMC-A. Annexin-V binding analysis shows that cells that ectopically express Bcl-2 were significantly more resistant to PMC-A induced apoptosis (Fig. 7C). Figure 7 Transcriptional downregulation of Bcl-2 is critical to mediate PMC-A induced apoptosis. Finally, we forced HCT116 wt cells to express BH3-only proapoptotic Bim which displayed early cellular increase in response to PMC-A (Fig.

6). Use of BimL expression vector was exploited to interrogate whether this upregulation is a contributing factor to apototic effects of PMC-A (Fig. 8). Flow cytometry analysis showed that it is possible to amplify the apoptotic response to PMC-A by ectopic Bim expression. Figure 8 Early upregulation of Bim is important to mediate PMC-A induced apoptosis. Discussion An elaborate network of pathways is required to tightly control apoptotic events in mammalian cells. Failure of surveillance/maintanence over cell fate decision could result in malignant transformation. Evasion from apoptosis as a hallmark of cancerous cells may involve dysregulation of Bcl-2 family proteins as evidence indicates that a wide variety of solid and hematopoietic tumors rely on elevated antiapoptotic Bcl-2 protein expression for survival [33]�C[36]. In this view, inspection of cellular levels and activation statuses of pro- and antiapoptotic Bcl-2 AV-951 proteins may provide valuable clues about anticancer drug action.