Kinease 3D?F show that PRIMA-1 inhibited the binding of p53 to its binding online sites on MAP4K4 gene in all breast cell lines irrespective in the standing of its p53 function. The data clearly indicate the JNK signaling pathway may not be concerned in PRIMA- 1-induced apoptosis in breast cancer cells as attested from the lack of p53 binding to its promoter on MAP4K4 gene. Even though the prior report by Li et al. showed the involvement of JNK signaling in PRIMA-1-induced apoptosis in colorectal carcinoma cells, the authors didn’t perform in vivo research of p53 binding to the promoter web-sites on MAP4K4 gene in these cell lines. On top of that to our in vivo p53 binding research to your promoter sites of Bax, PUMA, and MAP4K4, we also investigated the binding of p53 to the promoter web sites of other p53 proapoptotic genes such as Noxa, which encodes a BH3-only protein and therefore is likely to contribute to p53-mediated apoptosis inside a equivalent manner to PUMA and Bax.
Our ChIP evaluation failed to help the involvement of Noxa in PRIMA-1-induced apoptosis in breast cancer cells . Therefore, it appears that, in response to PRIMA-1 restoration of p53 these details transcriptional transactivation function, p53 activates the ??intrinsic?? mitochondrial apoptotic pathway by inducing the expression of at the least two Bcl-2 proapoptotic family members?PUMA and Bax shifting the stability in the direction of proapoptotic impact. Our recent expression proteomics research has supported these findings and showed the activation of mitochondrial intrinsic pathway on PRIMA-1 treatment method of breast cancer cells .
Determined by these data, we concluded that the involvement of JNK signaling in PRIMA-1-induced apoptosis in breast cancer cells seems unlikely. p53-dependent induction of Bax and PUMA by the treatment of PRIMA-1 To verify our ChIP data that in vivo p53 binding to its promoter on Bax and PUMA resulted within the activation of Bax and PUMA proteins, we utilized siRNA precise to hop over to this site p53 to downregulate the expression of p53 gene. Incubation of MDA-231 and GI-101A cells with p53siRNA for 48 h resulted in the reduction of p53 expression by approximately 80% compared to cells treated with siRNA control . Induction of PRIMA-1-mediated expression of Bax and PUMA in MDA-231 and GI-101A breast cancer cells was completely inhibited by p53siRNA when in contrast to control siRNA-treated cells . These information obviously indicate that PRIMA-1-mediated activation of Bax and PUMA is p53 dependent and therefore confirmed our ChIP data .
Collectively these data also indicate that the activation of proapoptotic targets, Bax and PUMA, plays a major function while in the induction of PRIMA-1- induced apoptosis . Downregulation of JNK signaling pathway by PRIMA-1 c-Jun NH2-terminal kinase , a member from the mitogen-activated kinase family members, may be a primary regulator of apoptosis .