Rottlerin induce early autophagy and late apoptosis via PKC-delta independent pathway.

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Rottlerin, a widely selective protein kinase C delta inhibitor, is isolated from Mallotus philippinensis. Recently, many reports have shown the possible apoptotic inducing effect of rottlerin in lung cancer, breast cancer, chronic lymphocytic leukemia and multiple myeloma cells. However, the mechanisms of cytotoxic effects are not fully understood. In present study, we investigated the mechanisms of cell death induced by rottlerin in human fibrosarcoma HT1080 cells.Upon treatment of rottlerin, the cell growth was inhibited in dose and time dependent manner. Rottlerin markedly induced cytoplasmic vacuolation of HT1080 cells. We showed that these vacuoles are acidic autolysosomes by electron microscopy, acidic vesicular organelle staining and transfection of green fluorescent protein-LC3. Rottlerin also caused DNA fragmentation, accumulation of sub-G₁ population with the loss of mitochondrial membrane potential and translocation of AIF from mitochondria to nucleus. However, the activation of caspase-3,-8,-9 and cleavage of PARP were not detected. Interestingly, autophagosome was first detected within 12hr of treatment with 10 μM rottlerin. But DNA fragmentation was first detected at 48hr. These results indicate that rottlerin induce early autophagy and late apoptosis. Knockdown of protein kinase C-delta by RNA interference did not affected growth inhibition and cytoplasmic vacuolation. From the above results, we suggest that rottlerin induces cell death by early autophagy and late apoptosis via PKC-delta independent pathway in HT1080 cells