Acid Ceramidase Expression Modulates the Sensitivity of A375 Melanoma Cells to Dacarbazine

Abstract

Dacarbazine (DTIC) is the treatment of choice for metastatic melanoma, but its response in patients remains very poor. Ceramide has been shown to be a death effector and to play an important role in regulating cancer cell growth upon chemotherapy. Among ceramidases, the enzymes that catabolize ceramide, acid ceramidase (aCDase) has been implicated in cancer progression. Here we show that DTIC elicits a time-and dosedependent decrease of aCDase activity and an increase of intracellular ceramide levels in human A375 melanoma cells. The loss of enzyme activity occurred as a consequence of reactive oxygen species-dependent activation of cathepsin B-mediated degradation of aCDase. These events preceded autophagic features and loss of cell viability. Down-regulation of acid but not neutral or alkaline ceramidase 2 resulted in elevated levels of ceramide and sensitization to the toxic effects of DTIC. Conversely, inducible overexpression of acid but not neutral ceramidase reduced ceramide levels and conferred resistance to DTIC. In conclusion, we report that increased levels of ceramide, due to enhanced degradation of aCDase, are in part responsible for the cell death effects of DTIC. These results suggest that downregulation of aCDase alone or in combination with DTIC may represent a useful tool in the treatment of metastatic melanoma.

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells

“…Notably, whilst DTIC treatment (40 µM) did not induce apoptosis, combined pitavastatin-DTIC treatment resulted in significant levels of apoptosis as evidenced by the increased level of apoptosis markers, cleaved PARP and active caspase 3, as well as annexin V staining. The results of the present study are in agreement with a number of studies which demonstrate that high concentrations of DTIC are necessary to induce apoptosis in melanoma cells (19,32,36). Whilst there has been insufficient research to determine which type of apoptosis is induced by DTIC, sensitization to the intrinsic apoptotic pathway has been revealed to augment DTIC-induced melanoma tumour cell death (37).…”

“…Chemical inhibition of autophagy resulted in enhancement of cell viability, suggesting that pitavastatin-DTIC-induced autophagy occurs as a cell death mechanism. In support of this, previous studies have suggested that DTIC and pitavastatin may serve a function in the induction of autophagy and specifically as a mode of cell death (15,19,38).…”

“…Whilst DTIC chemotherapy is the standard treatment, <20% of patients respond at all to the treatment and <5% continue to respond to long-term treatment (28,29). Previous studies have demonstrated that, in the case of melanoma, resistance to DTIC is associated with low levels of apoptosis and increasing levels of anti-apoptotic proteins (19,29,30).…”

“…The results revealed that 4 µM pitavastatin treatment resulted in the death of >50% of cells, suggesting that it exerts potent cytotoxic effects against melanoma cells specifically at high concentrations. DTIC has previously been demonstrated to inhibit A375 cell survival but only at a high concentrations of 25-100 µM (19). Therefore, the present study aimed to determine whether the combined treatment of pitavastatin and DTIC may have a greater anticancer effect on A375 and WM115 cells.…”

Development of nanostructured lipid carrier for dacarbazine delivery

“…It is metabolized in the liver to an alkylating agent (diazomethane) that destroys cancer cells [8,9], although different mechanism of Dac-toxicity was also suggested [10]. The only available formulation for clinical use is delivered through intravenous injection.…”