Radiosensitization of melanoma cells through combined inhibition of protein regulators of cell survival

Abstract

The incidence of melanoma continues to dramatically increase in most Western countries with predominantly Caucasian populations. However, only limited therapies for the metastatic stage of the disease are currently available. The main purpose of this study is to determine approaches that can substantially increase radiosensitivity of melanoma cells. The PI3K-AKT, NF-kappaB and COX-2 pathways, which are involved in the radioprotective response, are highly active in melanoma cells. Pharmacological suppression of COX-2 and PI3K-AKT, or RNAi-mediated knockdown of COX-2, substantially increased levels of G2/M arrest of the cell cycle and decreased clonogenic survival of gamma-irradiated melanomas, predominantly via a necrotic mechanism. On the other hand, resveratrol, a polyphenolic phytoalexin, selectively targets numerous cell signaling pathways, decreasing clonogenic survival primarily via an apoptotic mechanism. In melanoma cells, resveratrol inhibits STAT3 and NF-kappaB-dependent transcription, culminating in suppression of cFLIP and Bcl-xL expression, while activating the MAPK- and the ATM-Chk2-p53 pathways. Resveratrol also upregulates TRAIL promoter activity and induces TRAIL surface expression in some melanoma cell lines, resulting in a rapid development of apoptosis. Sequential treatment of melanoma cells, first with gamma-irradiation to upregulate TRAIL-R surface expression, and then with resveratrol to suppress antiapoptotic proteins cFLIP and Bcl-xL and induce TRAIL surface expression, had dramatic effects on upregulation of apoptosis in some melanoma lines, including SW1 and WM35. However, for melanoma lines exhibiting suppressed translocation of TRAIL to the cell surface, a necrotic mechanism of cell death was primarily involved in radiation response. Hence, surface expression of TRAIL induced by resveratrol appears to be a decisive event, one which determines an apoptotic versus a necrotic response of melanoma cells to sequential treatment.

Fig. 1

Radiation resistance of melanoma cell lines. (a) Cell cycle-apoptosis analysis of melanoma lines WM35 48 h after γ-irradiation using PI staining DNA and flow cytometry. Gamma-irradiation induces G2/M arrest of the cell cycle, while levels of apoptosis (Apop) are relatively low. (b, c) Clonogenic survival assay of human WM35, WM9, LU1205, HHMSX and mouse SW1 melanoma lines 12 days after γ-irradiation of 1.25–5 Gy. Error bars represent mean ± SD from four independent experiments. Results of a typical experiment are shown in (b). (d) Western blot analysis of phospho-AKT (Ser473), total AKT and COX-2 levels in melanoma cell lines

Fig. 2

Inhibition of COX-2 and PI3K-AKT activities further upregulates the G2/M arrest WM35 melanoma cells following γ-irradiation. (a) Western blot analysis of indicated proteins 6 h after irradiation. (b) NF-κB DNA-binding activity determined by EMSA 6 h after γ-irradiation of WM35 cells. (c) Effects of γ-radiation, COX-2 inhibitor NS398 (50 μM) and PI3K-AKT inhibitor LY294002 (50 μM) alone or in combination on the cell cycle of WM35 melanoma. Cells were stained with PI and analyzed by flow cytometry. Results of a typical experiment (one of three) are presented. (d) Effects of NS398 and LY294002 (50 μM) on γ-irradiation-induced death of WM35 cells. Apoptosis-necrosis analysis was performed by Annexin V-FITC + PI staining and the flow cytometry. Results of a typical experiment (one of four) are presented

Fig. 3

Inhibition of COX-2 and PI3K-AKT decreased melanoma cell survival following γ-irradiation. (a–c) Clonogenic survival assay of WM35, WM9 and LU1205 cells 12 days after γ-irradiation of 1.25–5 Gy alone or in combination with either NS398 (50 μM) or LY294002 (50 μM). Error bars represent mean ± SD from three independent experiments

Fig. 4

RNAi-mediated knockdown of COX-2 expression substantially increased levels of the G2/M arrested metastatic melanoma cells and decreased cancer cell survival. (a) Suppression COX-2 expression levels by specific RNAi affect p53 (P-Ser20) basal levels in LU1205 melanoma cells. (b) Effects of COX-2 knockdown on the cell cycle in LU1205 and WM9 melanoma cells. PI staining and FACS analysis were used. (c) Effects of γ-irradiation on surface TRAIL-R2/DR5 levels in the control and COX-2 knockdown LU1205 cells. Immunostaining with anti-DR5-PE and FACS analysis were used. (d) Clonogenic survival assay of the control and COX-2 knockdown LU1205 and WM9 cells after γ-irradiation. Error bars represent mean ± SD from three independent experiments

Fig. 5

Combined treatment of melanoma lines with γ-irradiation and TRAIL. (a) Effect of γ-irradiation on TRAIL-R2/DR5 surface expression. Immunostaining with anti-DR5-PE and FACS analysis were used. (b). Upregulation of TRAIL-induced apoptosis in WM35 melanoma cells after sequential treatment with γ-irradiation (5 Gy) and 16 h after irradiation with recombinant TRAIL (50 ng/ml) for an additional 24 h. Cell cycle-apoptosis analysis was performed using PI staining DNA and flow cytometry. (c) Clonogenic survival assay of WM35, WM9 and LU1205 cells 12 days after treatment with recombinant TRAIL (50 ng/ml) alone or in combination with γ-irradiation of 1.25–5 Gy. Error bars represent mean ± SD from three independent experiments

Fig. 6

Effects of resveratrol (RV) on cellular proteins controlling cell survival and apoptosis in SW1 melanoma cells. (a) Effects of RV on NF-κB-, AP-1, STAT- and p53-dependent luciferase reporter activities, TRAIL and FLIP promoter activities. (b) Effects of RV on TRAIL surface expression in SW1 melanoma cells. Immunostaining with anti-TRAIL-PE mAb and FACS analysis were used. (c) Effects of RV (50 μM) on basal nuclear NF-κB and NF-Y activities in SW1 cells determined by EMSA 6 h after treatment. Positions of DNA-binding complexes are indicated. Free labeled probes are not shown. Western blot analysis was performed for detection of total and phosphoprotein levels of STAT3, FOXO-3A, ERK1/2, JNK1/2, cJun, p38MAPK, ATF2, TRAIL, TRAIL-R, cFLIP, phospho-p53 (Ser20), total p53 and p21-WAF 6 h after treatment with RV. Western blot analysis of Bcl-xL, XIAP and β-actin levels 16 h after treatment with RV. (d) Levels of apoptosis induced by RV (50 μM) in melanoma lines 16 h after treatment. (e) Effects of anti-TRAIL (5 μg/ml) and anti-TNF (5 μg/ml) inhibitory mAbs on RV-induced apoptosis in SW1 cells. PI staining DNA and flow cytometry analysis were used for cell cycle-apoptosis analysis. (f) Clonogenic survival assay for combined treatment of SW1 cells by RV (0–100 μM) and Bay 11-7082 (5 μM). Error bars represent mean ± SD from three independent experiments

Fig. 7

Synergistic effects of RV and γ-irradiation on regulation of cell death in SW1 mouse and WM35 human melanomas. (a) Effects of γ-irradiation (5 Gy), RV (50 μM) and combined treatment on the cell cycle and apoptosis in SW1 cells. Cells were stained by PI 40 h after treatment. RSV was added 16 h after irradiation for an additional 24 h. Apoptosis (Apop) levels were determined as the percentage of cells with hypodiploid content of DNA in the pre-G0/G1 region using flow cytometry; % cells at the distinct phases of the cell cycle is indicated. Results of typical experiments (one of three) are presented. (b) Effects of γ-irradiation on TRAIL and TRAIL-R surface expression in SW1 cells. (c) Clonogenic survival assay of SW1 cells 12 days after indicated treatments: RV (25–100 μM) and γ-irradiation (2.5 Gy and 5 Gy) were used. Error bars represent mean ± SD from three independent experiments. (d, e) Clonogenic survival assay of WM35 and LU1205 cells 12 days after indicated treatments. Error bars represent mean ± SD from three independent experiments