I observe that a certain drug treatment induces autophagy in cancer cells. I am trying to distinguish the role of autophagy, to find out whether it aids in cell survival or autophagy activation leads to cell death (Type II programmed cell death). I am looking for short term experiments which could aid in this distinction. Any help in this regard would be greatly appreciated.
I recommend that you use autophagy inhibitors and activators to help elucidate the role of autophagy in the specific context of your study.
1. Over Expression of Oncogenic mutated RAS increases autophagy level and leads to cell death that is dependent on autophagy.
2. Autophagy can inhibit TRIAL-induced apoptosis by selective degradation of active caspase-8
João Agostinho Machado-Neto , I have used the autophagy inhibitors, there is considerable decrease in survival which leads to a conclusion that it resists cell death. I haven't tried using autophagy activators though. Would there be any other way, apart from using activators and inhibitors, to distinguish this, any experimental modality? Live-cell imaging where I follow autophagic cells would be one of them. what are your thoughts?
Ganesh Pai B, If you want to follow autophagic cells individually to determine their fate, there is the option to transfect cells with vectors with Autophagy Tandem Sensor RFP-GFP-LC3B to monitor autophagy in real time. Personally, I have not experienced using these vectors, but it has been used extensively and there are commercial kits. However, if you just want to know if the autophagy induced by your compound participates in cell death or survival, the use of inhibitors and activators can be a good start. I recommend using more than one inhibitor and activator to make the evidence more robust.
João Agostinho Machado-Neto thank you for the suggestions. I will try it with the autophagy inhibitors-3MA and CQ. Also, I am going to use rapamycin for the activation part.
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