Expression of PAFR as part of a prosurvival response to chemotherapy: a novel target for combination therapy in melanoma

Abstract

Melanoma cells express the platelet-activating factor receptor (PAFR) and, thus, respond to PAF, a bioactive lipid produced by both tumour cells and those in the tumour microenvironment such as macrophages. Here, we show that treatment of a human melanoma SKmel37 cell line with cisplatin led to increased expression of PAFR and its accumulation. In the presence of exogenous PAF, melanoma cells were significantly more resistant to cisplatin-induced cell death. Inhibition of PAFR-dependent signalling pathways by a PAFR antagonist (WEB2086) showed chemosensitisation of melanoma cells in vitro. Nude mice were inoculated with SKmel37 cells and treated with cisplatin and WEB2086. Animals treated with both agents showed significantly decreased tumour growth compared to the control group and groups treated with only one agent. PAFR accumulation and signalling are part of a prosurvival program of melanoma cells, therefore constituting a promising target for combination therapy for melanomas.

Figure 1

Cisplatin-treated SKmel37 cells accumulate PAFR. (a) qPCR analysis of PAFR expression in SKmel37 cells exposed to cisplatin for 6 and 24 hours, relatively to expression levels of GAPDH. PAFR expression was significantly increased in cells treated with 5 mM cisplatin after 6 hours, but not in any other condition, as compared to the control (CTL, no treatment; **P < 0.01). (b) Western blot analysis of in vitro cisplatin-treated SKmel37 cells using anti-PAFR and anti-β-actin antibodies. At 6 h after treatment, cells exposed to cisplatin did not reveal significant increase in PAFR protein expression, while at 24 h, cells treated with increasing concentrations of cisplatin presented an up to 2.6-fold increase in PAFR levels.

Figure 2

PAF protects SKmel 37 cells from cisplatin-induced cell death in vitro. (a) Cell death was measured as the percentage of hypodiploid cells (in total cell population) at each condition. (b) Cell cycle analysis of the live cell population in each condition. CIS: cisplatin; WEB: WEB2086.

Figure 3

Combination therapy using cisplatin and the antagonist of PAFR is more efficient than either therapy alone (a) Average tumour volume in each experimental group (±SEM) measured daily. (b) Percentage of complete tumour regression in each experimental condition.