Editor's Highlight: PPARβ/δ and PPARγ Inhibit Melanoma Tumorigenicity by Modulating Inflammation and Apoptosis

Abstract

Skin tumorigenesis results from DNA damage, increased inflammation, and evasion of apoptosis. The peroxisome proliferator-activated receptors (PPARs) can modulate these mechanisms in non-melanoma skin cancer. However, limited data exists regarding the role of PPARs in melanoma. This study examined the effect of proliferator-activated receptor-β/δ (PPARβ/δ) and PPARγ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the UACC903 human melanoma cell line. Stable overexpression of either PPARβ/δ or PPARγ enhanced ligand-induced expression of a PPARβ/δ/PPARγ target gene in UACC903 cell lines as compared with controls. The induction of target gene expression by ligand activation of PPARγ was not altered by overexpression of PPARβ/δ, or vice versa. Stable overexpression of either PPARβ/δ or PPARγ reduced the percentage of cells in the G1 and S phase of the cell cycle, and increased the percentage of cells in the G2/M phase of the cell cycle in UACC903 cell lines as compared with controls. Ligand activation of PPARβ/δ did not further alter the distribution of cells within each phase of the cell cycle. By contrast, ligand activation of PPARγ enhanced these changes in stable UACC903 cells overexpressing PPARγ compared with controls. Stable overexpression of either PPARβ/δ or PPARγ and/or ligand activation of either PPARβ/δ or PPARγ inhibited cell proliferation, and anchorage-dependent clonogenicity of UACC903 cell lines as compared with controls. Further, overexpression of either PPARβ/δ or PPARγ and/or ligand activation of either PPARβ/δ or PPARγ inhibited ectopic xenograft tumorigenicity derived from UACC903 melanoma cells as compared with controls, and this was likely due in part to induction of apoptosis. Results from these studies demonstrate the antitumorigenic effects of both PPARβ/δ and PPARγ and suggest that targeting these receptors may be useful for primary or secondary melanoma chemoprevention.

Keywords: (PPARβ/δ); (PPARγ); cancer; cell proliferation; melanoma; peroxisome proliferator-activated receptor-β/δ; peroxisome proliferator-activated receptor-γ; xenografts.

Figure 1

Characterization of a human malignant melanoma cell line (UACC903) overexpressing PPARβ/δ or PPARγ. A, Representative photographs of control UACC903 cells, UACC903-Migr1 control cells (Migr1), UACC903-hPPARβ/δ cells (hPPARβ/δ), and UACC903-hPPARγ cells (hPPARγ) examined by fluorescent microscopy (upper panels) or light microscopy (lower panels). qPCR analysis for mRNA expression of PPARβ/δ (B) and PPARγ (C) in the UACC903 cell lines, normalized to the GAPDH mRNA. D, Western blot analysis of PPARβ/δ and PPARγ in the UACC903 cell lines, normalized to ACTIN expression. + indicates positive control: cell lysate from COS-1 cells transfected with a hPPARβ/δ or a hPPARγ expression vector. qPCR analysis of ANGPTL4 (E,G) and PDPK1 (F,H) mRNA expression in response to 0.0 – 10.0 µM of the PPARβ/δ ligand GW0742 (E,F) for 8 h or the PPARγ ligand rosiglitazone (G,H) for 24 h, normalized to the GAPDH mRNA. Data represents triplicate independent sample means ± SEM. Values with different letters are significantly different (p ≤ .05) using ANOVA with Bonferroni’s multiple comparison. The color image is available in the online version of the article.

Figure 2

Effect of ligand activation and/or overexpression of PPARβ/δ or PPARγ on cell cycle progression. Cell cycle progression was examined in control UACC903, UACC903-Migr1 cells (Migr1), UACC903-hPPARβ/δ cells (hPPARβ/δ), and UACC903-hPPARγ cells (hPPARγ) by flow cytometry. A, Effect of PPARβ/δ or PPARγ overexpression on cell cycle progression. B, Effect of PPARβ/δ ligand activation on cell cycle progression. Cells were treated for 24 h with 0.0 – 10.0 µM GW0742. C, Effect of PPARγ ligand activation on cell cycle progression. Cells were treated for 24 h with 0.0–10.0 µM rosiglitazone. Data represents triplicate independent sample means ± SEM. *Significantly higher compared with UACC903 control (p ≤ .05) by ANOVA with Bonferroni’s multiple comparison. #Significantly lower compared with UACC903 control (p ≤ .05) by ANOVA with Bonferroni’s multiple comparison.

Figure 3

Effect of ligand activation and/or overexpression of PPARβ/δ or PPARγ on cell proliferation. The growth of UACC903, UACC903-Migr1 vector control cells (Migr1), UACC903-hPPARβ/δ cells (hPPARβ/δ), or UACC903-hPPARγ cells (hPPARγ) was examined over a 72-h period by Coulter Counting as described in “Materials and Methods” section. Cells were treated with indicated concentration of ligand, GW0742 (A,C,E,G) or rosiglitazone (B,D,F,H), at Time 0. Data represent triplicate independent sample means ± SEM. *Significantly different value (p ≤ .05) from cell line-specific vehicle control at the particular time point, as determined by ANOVA with Bonferroni’s multiple comparison. (I) Calculated doubling time from the 24- to 72-h growth period of the vehicle control in each cell line, as described in the “Materials and Methods” section. Values with different letters are significantly different (p ≤ 05) using ANOVA with Bonferroni’s multiple comparison.

Figure 4

Effect of ligand activation and/or overexpression of PPARβ/δ or PPARγ on anchorage-dependent clonogenicity. Anchorage-dependent clonogenicity was examined in control UACC903, UACC903-Migr1 vector control cells (Migr1), UACC903-hPPARβ/δ cells (hPPARβ/δ), or UACC903-hPPARγ cells (hPPARγ). Effect of GW0742 (A) or rosiglitazone (B) on clonal expansion. Cells were plated and treated as described in “Materials and Methods” section, and the dishes were left undisturbed for 14 days. The plating efficiency for each cell line is presented. Values with different letters are significantly different (p ≤ .05) using ANOVA with Bonferroni’s multiple comparison. Surviving fraction quantification from administration of GW0742 (C) or rosiglitazone (D). *Significantly different compared with cell line-specific vehicle control (p ≤ .05) by ANOVA with Bonferroni’s multiple comparison.

Figure 5

Ligand activation and/or overexpression of PPARβ/δ suppresses ectopic xenografts derived from UACC903 melanoma cancer cell lines. A, Representative photomicrographs of xenografts derived from UACC903-Migr1 (Migr1) or UACC903-hPPARβ/δ (hPPARβ/δ) cells. B, Average tumor volumes over time. C, Relative expression of the PPARβ/δ target gene ADRP in tumors. D, Average tumor area in paraffin-embedded tumor sections. E, Representative photomicrographs of H&E-stained xenografts. Tumor cells frequently exhibited mitotic figures (white arrows) and rosette structure (black arrowheads). Magnification = 12.5 × (left) and 400 × (right). F, Average tumor weight at the end of the study. G, Apoptosis in xenograft tumors was determined by TUNEL assay. Left panel, apoptotic tumor cells are indicated by white arrowheads. Magnification = 1000×. Bar = 10 μm. Right panel, quantification of apoptotic index in tumor sections. Control = (Con); GW0742-treated (2.5 mg/kg/d) = (GW). Values represent the mean ± SEM. Values with different superscript letters are significantly different at p ≤ .05. *Significantly different than control, p ≤ .05. The color image is available in the online version of the article.

Figure 6

Ligand activation and/or overexpression of PPARγ suppresses ectopic xenografts derived from UACC903 melanoma cancer cell lines. A, Representative photomicrographs of xenografts derived from UACC903-Migr1 (Migr1) or UACC903-hPPARγ (hPPARγ) cells. B, Average tumor volumes over time. C, Relative expression of the PPARβ/δ target gene ADRP in tumors. D, Average tumor area in paraffin-embedded tumor sections. E, Representative photomicrographs of H&E-stained xenografts. Tumor cells frequently exhibited mitotic figures (white arrows) and rosette structure (black arrowheads). Magnification = 12.5 × (left) and 400 × (right). F, Average tumor weight at the end of the study. G, Apoptosis in xenograft tumors was determined by TUNEL assay. Left panel, apoptotic tumor cells were indicated by white arrowheads. Magnification = 1000×. Bar = 10 μm. Right panel, quantification of apoptotic index in tumor sections. Control = (Con); Rosiglitazone-treated (10 mg/kg/d) = (Rosi). Values represent the mean ± SEM. Values with different superscript letters are significantly different at p ≤ .05. *Significantly different than control, p ≤ .05. The color image is available in the online version of the article.