P-glycoprotein-independent cytotoxic effects of 5-aminopyrazole in L1210 leukemia cells

We previously investigated the cytotoxic effects of 5-aminopyrazoles (5-APs) on leukemia cells both negative and positive for P-glycoprotein (P-gp) expression, a common contributor to multidrug resistance. Five derivatives (A1-A5), each containing perfluorinated methylbenzene at the N3 nitrogen of the pyrazole ring, were tested on murine lymphoblastic cell line variants: parental S cells (P-gp positive), R cells (vincristine-resistant and P-gp positive), and T cells (human P-gp transfected). Among these, A1 and A4 exhibited the strongest effects, especially in R cells, with lesser but similar effects observed in S and T cells. Cell death assays revealed both apoptosis and necrosis, with apoptosis confirmed by DNA fragmentation and activation of caspases 3/7, 8, and, to a lesser extent, 9, suggesting a predominance of extrinsic apoptosis. The compounds also induced autophagy, identified by LysoTracker Green and monodansylcadaverine staining. All derivatives, except A5, suppressed P-gp activity, though they did not alter P-gp expression at the mRNA or protein level. Cell cycle analysis showed changes in the G0/G1 and S phases. The heightened sensitivity of R cells to 5-AP, despite P-gp expression, likely results from an altered phenotype due to vincristine-induced stress rather than from P-gp inhibition alone. This conclusion is supported by the fact that T cells expressing P-gp are as sensitive to 5-APs as S cells.