Does the phosphorylation of MAPK proteins alter over time during drug treatment in cancer models? For instance, if I treat melanoma cells with current line of therapy, then do the pERK and pMEK changes at early time like 2hrs, 6hrs, 12hrs, 24hrs, 48hrs upon treatment ? Does it conclude anything?
Certainly you will see the change from 6 hrs, I am not sure about the 2 hrs time period. In melanoma BRAF-MEK-ERK pathway is a major one, so in general, if you treat with some BRAF inhibitor eg. PLX-4032 / PLX-4720, you should see the inhibition of MEK/ERK phosphorylation. It is conclusive only, about the activity of these 2 proteins and nothing else. Hope it helps, Gud luck !!!
Chronic inhibition of MAPK survival/ proliferative signal finally results in the paradoxical enhanced signal activation due to the secretion of ligands in autocrine or paracrine manner, internalization of receptor tyrosine kinases, and the alternative signal activation which leads to cross-talk with MAPK signal transduction. This is not only the case in the long-termed anti-tumor therapy in the clinical settings or in the animal experiments, but you would often encounter in the cancer cell lines. I strongly recommend that the optimal time-course of the kind of experiment you mentioned above is up to 12 hours.
Dr. Yoshida made a valid point. I have tried different time point experiment for MAPK and AMPK pathway. The phosphorylation changes with time and sometimes you can see compensatory change that totally opposite than what we all believe. I always tell m y students and colleagues that as long as the cells or animals can survive the insults with treatment then some compensatory mechanisms will fire up.
Performing a time course for activation may give some indication of what is happening in the cell, regarding a specific signaling pathway, but it does not provide any major conclusions due to possible intrinsic compensatory mechanisms, just as Ru-Jeng suggests. I have done this kind of work in primary human melanocytes, where I looked at ERK, JNK and p38 and downstream targets. If you want to understand if MAPK activation is playing a role, you need to work on elucidating the signal mechanism involved over the time points you detect, including inhibitors and/or knockout technology.
Thank you so much for your comment guys. I will consider this for my future experiments :)
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