Pyrimethamine Induces Apoptosis of Melanoma Cells via a Caspase and Cathepsin Double-Edged Mechanism

Abstract

The unresponsiveness of metastatic melanoma to conventional chemotherapeutic and biological agents is largely due to the development of resistance to apoptosis. Pyrimethamine belongs to the group of antifolate drugs, and in addition to antiprotozoan effects, it exerts a strong proapoptotic activity, which we recently characterized in human T lymphocytes. However, no data regarding pyrimethamine anticancer activity are available thus far. To this end, we examined the in vitro effects of pyrimethamine on apoptosis, cell cycle distribution, and cell proliferation of human metastatic melanoma cell lines. The in vivo antitumor potential of pyrimethamine was evaluated in a severe combined immunodeficiency (SCID) mouse xenotransplantation model. Our data indicate that pyrimethamine, when used at a clinically relevant concentration, induced apoptosis in metastatic melanoma cells via the activation of the cathepsin B and the caspase cascade (i.e., caspase-8 and caspase-9) and subsequent mitochondrial depolarization. This occurred independently from CD95/Fas engagement. Moreover, pyrimethamine induced a marked inhibition of cell growth and an S-phase cell cycle arrest. Results obtained in SCID mice, injected s.c. with metastatic melanoma cells and treated with pyrimethamine, indicated a significant inhibitory effect on tumor growth. In conclusion, our results suggest that pyrimethamine-induced apoptosis may be considered as a multifaceted process, in which different inducers or regulators of apoptosis are simultaneously implicated, thus permitting death defects of melanoma cells to be bypassed or overcome. On these bases, we hypothesize that pyrimethamine could represent an interesting candidate for the treatment of metastatic melanoma.

Antifolate Activity of Pyrimethamine Enhances Temozolomide-Induced Cytotoxicity in Melanoma Cells

“…PYR has been shown to reduce melanoma growth in a severe combined immune-deficient mouse model (40). Consistent with previous reports (39,40), we observed that PYR at a concentration of 0.5 μmol/L increased the number of cells in S phase (Fig.…”

“…It has been reported that PYR can induce apoptosis and S phase accumulation in various cell lines (37)(38)(39)(40). PYR has been shown to reduce melanoma growth in a severe combined immune-deficient mouse model (40).…”

β-Actin-binding Complementarity-determining Region 2 of Variable Heavy Chain from Monoclonal Antibody C7 Induces Apoptosis in Several Human Tumor Cells and Is Protective against Metastatic Melanoma

“…As suggested, ROS are critical regulators of caspase 8-mediated apoptosis. Accordingly, in a study with pyrimethamine in metastatic melanoma, it was found that the drug, which generates ROS, induced upstream caspase activation (caspase 8), bypassing CD95/Fas engagement, similar to what had been previously observed by the same authors in activated lymphocytes from a patient with a lymphoproliferative syndrome (45).…”

Cystatin B inhibition of TRAIL-induced apoptosis is associated with the protection of FLIPL from degradation by the E3 ligase itch in human melanoma cells

“…[11][12][13]23 However, inhibition of cathepsin L did not have any effect on TRAILinduced apoptosis (Supplementary Figure 2), whereas inhibition of cathepsin B partially blocked apoptosis induced by TRAIL in only two (ME4405 and Mel-AT) of eight melanoma cell lines (Figure 2b). It should be noted that the two lines were most sensitive to TRAIL (Supplementary Figure 1A), even though there was no overall correlation between the levels of cathepsin B expression and sensitivity of melanoma cells to TRAIL-induced apoptosis (data not shown).…”

“…11,12 Moreover, it has been reported to have a role in induction of apoptosis of melanoma cells by the antifolate agent pyrimethamine. 13 The cytosol contains endogenous cysteine cathepsin inhibitors, cystatins, which function as threshold inhibitors to protect cells from detrimental consequences caused by lysosomal release of the cathepsins. 10,14 Among them, cystatin B appears to be of particular interest, in that cystatin B-deficient mice exhibit increased apoptosis of cerebellar granule cells that is associated with the increased expression of apoptosis genes.…”