From literature, BAX is pro-apoptotic and BCL2 is pro-survival/anti-apoptotic. Our compound caused apoptosis (data from AnnexinV/PI FITC, Cleaved PARP in Western blot). However qPCR showed the upregulation of both BAX and BCL2 genes after treatment. Is it possible for BCL2 upregulation to cause apoptosis? We tested against HCT 116 and HT-29 colorectal cancer cells.
It's not about absolute levels of individual Bcl/2 proteins. The apoptotic threshold is mainly decided by the stoichiometry between the pro and anti-apoptotic members. Therefore it is still possible that the balance is tipped toward the pro-apoptotic side in your case. I would also like to bring it to your notice that recently cells undergoing necroptosis has been also shown to Annexin V positive. Therefore check for caspase9 and 3 activations. Also, increased mRNA does not necessarily mean increased protein levels so look the effect of treatment at post-transcriptional level.
Thanks
Bcl-2 inhibits Caspase-3 activity by preventing Cytochrome c release from the mitochondria and/or by binding to the apoptosis-activating factor (APAF-1).Increase in Caspase-3, increase in PARP cleavage, and decrease in Bcl-2 all indicate apoptosis stimulation. Increase in Caspase-3 levels is not always a direct indication of inducing the apoptosis due to the tight binding of cleaved Caspase-3 with X-linked inhibitor of apoptosis protein (XIAP). XIAP undergoes auto-ubiquitilation, but this process delays apoptosis until all XIAP is removed. So I think it could also increase the your Bcl-2 levels as a result of possible upstream signaling for more Bcl-2 production as it is not being inhibited by negative feedback since it is already inhibited downstream (inducing apoptosis as a result of your compound). Something like this could result in more Bcl-2 production. What I tried say is cell could ask to produce more Bcl-2 since it is not doing the job (inhibiting the Caspase-3) that it suppose to do. Other possibility is another anti-apoptotic pathway may activate as a result of this compound treatment. Hope this helps.
It's not about absolute levels of individual Bcl/2 proteins. The apoptotic threshold is mainly decided by the stoichiometry between the pro and anti-apoptotic members. Therefore it is still possible that the balance is tipped toward the pro-apoptotic side in your case. I would also like to bring it to your notice that recently cells undergoing necroptosis has been also shown to Annexin V positive. Therefore check for caspase9 and 3 activations. Also, increased mRNA does not necessarily mean increased protein levels so look the effect of treatment at post-transcriptional level.
Thanks
Hello,
At this level the role of increased Bcl-2 levels can not be predicted....gene speaks through their proteins.....so i would suggest you to go for western blotting .....so as to check the bax...bcl-2 protein levels,,,,
Cheers.....
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