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

Abstract

Most metastatic melanoma patients fail to respond to available therapy, underscoring the need to develop more effective treatments. We screened 2,000 compounds from the Spectrum Library in human melanoma cell lines to identify compounds that enhanced the cytotoxic effect of temozolomide, a drug used to treat metastatic melanoma. Screening was done with the temozolomide-resistant melanoma cell line SK-MEL-19, and six compounds were identified that had little or no inherent cytotoxicity but significantly enhanced growth-inhibition by temozolomide. These compounds were tested in five additional melanoma cell lines. Cell proliferation and death assays were used to compare the efficacy of single agent temozolomide versus combination treatments. Effects of combination treatment on levels of DNA double-strand breaks, the DNA repair protein O 6 -methylguanine-DNA-methyltransferase, apoptosis[measured by cleaved caspase-3 and poly(ADP-ribose) polymerase], and cell cycle were examined. Pyrimethamine, an antiparasitic, sensitized melanoma cells to temozolomide. Temozolomide combined with Pyrimethamine synergistically inhibited cell proliferation in melanoma cells with combination index values of 0.7 or less. In addition, combination treatment induced cell cycle arrest and increased both DNA damage and apoptosis. The increase in cell death due to combination treatment was rescued by leucovorin. Other folate antagonists were also effective enhancers of temozolomide-induced cytotoxicity, and the effects of antifolates were also evident in gliomas. Our screening approach led to the identification of Pyrimethamine, an orally available drug that efficiently crosses the blood-brain barrier, as a potent enhancer of the efficacy of temozolomide as an antineoplastic agent via inhibition of folate metabolism. (Mol Cancer Res 2009;7(5):703-12)

New derivatives of the antimalarial drug Pyrimethamine in the control of melanoma tumor growth: an in vitro and in vivo study

“…Parallel analyses were carried out in cells treated with Pyr and the chemotherapeutic agent TMZ at a clinically relevant concentration (100 μmol/L) [30]. We tested Pyr-analogues using the same dosage of Pyr (8 μg/ml), which is known to induce cell death in melanoma cells [10, 11] at different time points, i.e. after 24, 48 and 72 h of treatment.…”

“…Previous studies have also demonstrated that Pyr is a potent pro-apoptotic inducer in cancer cells, e.g. in metastatic melanoma cells [10, 11]. It has been suggested that the mechanism underlying this activity involves both the activation of the caspase cascade (e.g.…”

siRNA Knockdown of Ribonucleotide Reductase Inhibits Melanoma Cell Line Proliferation Alone or Synergistically with Temozolomide

“…However, at more ''clinically relevant'' concentrations no appreciable effect on cell cycle, proliferation, or temozolomide-induced DNA damage has been observed following temozolomide treatment in human melanoma cell lines (Chen et al, 2009), which may not be surprising as this agent has a low response rate in the clinic (Rietschel et al, 2008). Several studies have suggested that methyl-guanine methyl transferase and RR may be involved in the temozolomide-resistance mechanism in cancer (Aghi et al, 2006;Augustine et al, 2009).…”

Celastrol Synergistically Enhances Temozolomide Cytotoxicity in Melanoma Cells

“…Previous work has described the activation of NF-κB by the mitogen-activated protein kinase (MAPK) pathway in human melanoma cells (10). To investigate whether alterations in the MAPK pathway are also involved in the mechanism of CEL/TMZ-induced cytotoxicity, we examined the effect of TMZ/CEL treatment on expression levels of key molecules in the MAPK pathway.…”

“…In an effort to reduce these costs, we have performed screens of chemical libraries consisting of marketed drugs and natural compounds with the goal of identifying agents with antitumor effects that can be expedited to clinical trials. We recently identified a group of antifolates that enhance the cytotoxicity of TMZ in melanoma cells (10). The use of antifolates is limited in some patients, however, due to intolerable side effects, thus there is a need to identify alternatives.…”