Suppression of autophagy sensitizes multidrug resistant cells towards Src tyrosine kinase specific inhibitor PP2

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Upregulated Src activity has been implicated in a variety of cancers. Thus, Src family tyrosine kinase (SFK) inhibitors are often effective cancer treatments. Here, we employed 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2), a selective SFK inhibitor, to determine the possible involvement of tyrosine phosphorylation in the modulation of autophagy, for overcoming multidrug resistance. We found that multidrug-resistant v-Ha- ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) were more susceptible to PP2 treatment than were their parental cells (Ras-NIH 3T3). The antiproliferative activity of PP2 appeared to be due to cell-cycle arrest at G 1/S without induction of apoptosis. Interestingly, PP2 preferentially induced autophagy in Ras-NIH 3T3 cells but not in Ras-NIH 3T3/Mdr cells, which implies that a high level of autophagy may protect PP2-treated cells from undergoing cell death. PP2-induced autophagy in Ras-NIH 3T3 cells is accompanied by an inhibition of the mTOR signaling pathway. However, we found that in Ras-NIH 3T3/Mdr cells, PP2-induced mTOR inhibition was uncoupled from the induction of autophagy—likely due to the hyperactivation of AMPK by delayed Raf activation. We also found that PP2-induced dissociation of Beclin 1 from Bcl-2 leads to autophagy in Ras-NIH 3T3 cells. Taken together, these results suggest that functional autophagy in response to PP2 may lead to cell survival in Ras-NIH 3T3 cells, while defective autophagy may contribute to inhibition of growth in Ras-NIH 3T3/Mdr cells. Thus, modulators of autophagy may be used beneficially as adjunctive therapeutic agents during the treatment of cancers with SFK inhibitors.