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Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor is known to inhibit the growth of various cancer cells including melanoma. Here in this study, we have found that PTX induces autophagy in human melanoma cell lines (A375 and MeWo). Induction of autophagy is associated with the increase in Atg5 expression as knockdown of Atg5 effectively inhibited PTX mediated autophagy. A decrease in mTOR activation was also observed after PTX treatment. We observed that autophagy was activated as a downstream effector mechanism of ER stress induced by PTX. ER stress response was confirmed by upregulation of IRE-1α, GRP78 and CHOP expression. PTX treatment also resulted in an increase in intracellular calcium (Ca(2+)) level. Ca(2+) is the central player as blocking Ca(2+) by intracellular calcium chelator (BAPTA-AM) effectively inhibited the PTX induced ER stress response as well as autophagy. Moreover, silencing of CHOP also resulted in autophagy inhibition with a decrease in Atg5 expression. Collectively, PTX triggers ER stress response followed by induction of autophagy via involvement of Ca(2+)→CHOP→Atg5 signalling cascade. Interestingly, inhibition of intracellular calcium level by BAPTA-AM significantly increased PTX mediated cell death by augmenting intrinsic apoptotic pathway. Inhibition of autophagy by the ATG5 siRNA and pharmacological inhibitor, chloroquine also enhances PTX induced cell death. Taken together, our results clearly indicate that activation of ER stress response and autophagy provides resistance to PTX mediated apoptosis, and thus, interferes with the anticancer activity of PTX in human melanoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising anticancer agent but cutaneous T lymphoma cells (CTCL) are less sensitive to TRAIL-induced apoptosis. Here, we report that pentoxifylline (PTX), a phosphodiesterase inhibitor, augments TRAIL-mediated apoptosis in HuT-78 and MyLa cells through modulating extrinsic death receptors and intrinsic mitochondria dependent pathways. Our results clearly show that PTX augments TRAIL-mediated activation of caspase-8 and induces cleavage of Bid, although PTX alone cannot activate caspase-8. This is followed by cytochrome c release and subsequent, activation of caspase-9 and caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). Combined treatment downregulates the expression of various antiapoptotic proteins including c-FLIP, Bcl-xl, cIAP-1, cIAP-2 and XIAP. PTX induces the expression of death receptors DR4 and DR5 on cell surface of both the cell types where c-Jun NH2-terminal kinase (JNK) pathway plays an important role. Moreover, combined silencing of DR4 and DR5 by small interfering RNA abrogates the ability of PTX to induce TRAIL-mediated apoptosis. Thus, this is the first demonstration that PTX can potentiate TRAIL-mediated apoptosis through downregulation of cell survival gene products and upregulation of death receptors.