Methotrexate inhibits the viability of human melanoma cell lines and enhances Fas/Fas-ligand expression, apoptosis and response to interferon-alpha: rationale for its use in combination therapy

Abstract

Melanoma, a highly aggressive form of cancer, is notoriously resistant to available therapies. Methotrexate (MTX), an antifolate, competitively inhibits DNA synthesis and is effective for several types of cancer. In cutaneous T-cell lymphoma (CTCL), MTX increases Fas death receptor by decreasing Fas promoter methylation by blocking the synthesis of SAM, the principal methyl donor for DNMTs, resulting in enhanced Fas-mediated apoptosis. The objective of this study was to explore the effects of MTX in human melanoma. MTX variably inhibited the survival of melanoma cells and induced apoptosis as evident by annexin V positivity and senescence associated β-galactosidase activity induction. Furthermore, MTX caused increased transcript and protein levels of extrinsic apoptotic pathway factors Fas and Fas-ligand, albeit at different levels in different cell lines. Our pyrosequencing studies showed that this increased expression of Fas was associated with Fas promoter demethylation. Overall, the ability of MTX to up-regulate Fas/FasL and enhance melanoma apoptosis through extrinsic as well as intrinsic pathways might make it a useful component of novel combination therapies designed to affect multiple melanoma targets simultaneously. In support of this concept, combination therapy with MTX and interferon-alpha (IFNα) induced significantly greater apoptosis in the aggressive A375 cell line than either agent alone.

Keywords: Apoptosis; DNA methylation; Fas/FasL; Melanoma; Methotrexate.

Fig. 1

MTX reduces viability and induces apoptosis and senescence in melanoma cell lines. Panels A and B show the effect on growth and viability of MTX at 1 μM dose for 72 h. This was assessed using the luminescence based Cyto-Tox-Glo assay system. The Y-axes show relative luminescence units (RLU) of live and dead cells and the asterisk (*) represents statistical significance (p < 0.05) when compared with no treatment (NT) vehicle controls. Data represent mean ± standard deviation of three experiments with similar results. Panel C shows the morphological changes in controls versus 5 μM MTX treatment. Cells were photographed using Nikon Eclipse Ti microscope and a high performance CCD camera at 20× magnification. Panel D shows that at 48 h post-treatment, MTX induces apoptosis in a dose dependent manner as assessed by annexin V-FITC/PI staining. Data represent total annexin V-positive cells (mean ± standard deviation of three experiments with similar results), with statistical significance (p < 0.05) when compared with NT (*), 1 μM MTX (!) and 5 μM MTX (^). Panel E shows that MTX induced senescence in melanoma lines at 48 h post-treatments as assessed by β-galactosidase staining (blue) and counter stained with nuclear fast red at 40× using Evos (AMG) microscope.

Fig. 2

MTX upregulates Fas death receptor in melanoma cell lines. Panel A shows that MTX treatment upregulates Fas in melanoma cell lines as demonstrated by overlaid flow cytometry histograms. Filled grey curve is isotype control staining; dotted line is DMSO treated control; and heavy black line is MTX treated cells. Panel B shows the histograms of the mean fluorescence intensity (MFI) of Fas on Y-axis (as in Panel A). Data represent the mean ± standard deviation of three experiments with similar results when compared with vehicle treated control (*p < 0.05).

Fig. 3

MTX upregulates FasL in melanoma cell lines. Panel A shows upregulation of FasL in melanoma cell with MTX treatment by overlaid flow cytometry histograms as in Fig. 2A. Panel B shows the histogram of the MFI of FasL as in Fig. 2B. Data represented as mean ± standard deviation of three experiments with similar results compared with vehicle treated control (*p < 0.05).

Fig. 4

MTX upregulates Fas/FasL transcripts in melanoma cell lines. Histogram of Q-RT/PCR data shows upregulation of Fas and FasL transcript among all the melanoma cell lines studied. Data represent mean ± standard deviation of three experiments with similar results relative to the baseline transcript of vehicle treated controls that were set as 1(*p < 0.05).

Fig. 5

MTX induces apoptosis through both the extrinsic and intrinsic pathways in melanoma cell lines. Immunoblots show that both cleaved caspase-8 and -9 are induced by MTX treatment. GAPDH is the loading control.

Fig. 6

MTX demethylates the FAS promoter in melanoma cell lines. Line graph shows pyrographs of Fas promoter CpG sites in melanoma cell lines before and after MTX treatment. Y axis represents% methylation and X axis represents selected CpG sites (11–18).

Fig. 7

MTX/IFNα2b combination treatment enhances apoptosis of A375 melanoma cells significantly more than either agent alone. Methotrexate functions as a demethylating agent and together with interferon-alfa2b, induces superior apoptotic response in A375 melanoma cells. Histogram shows% apoptotic cells (total annexin V-positive) on the Y axis. Overlaid flow cytometry plots show the distribution of annexin V positivity. Filled grey filled histogram represents NT control; dashed line is MTX treated cells; dotted line is IFN treated cells; and heavy black line is MTX + IFN treated cells. Data represent mean ± standard deviation of three experiments with similar results (*p < 0.05) when compared with NT (*), 5 μM MTX (^) and 1000 U/ml IFNα2b (!).