Ingenol-3-Angelate Suppresses Growth of Melanoma Cells and Skin Tumor Development by Downregulation of NF-κB-Cox2 Signaling

Abstract

BACKGROUND A recent focus in skin cancer prevention intervenes though modulating molecular links between inflammation and cell growth signaling, such as NF-κB. This study elucidates the effect of a non-tumor promoting phorbol ester, ingenol-3-angelate (I3A), on the growth of human melanoma cells and on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation and 7,12-Dimethylbenz(a)anthracene (DMBA)-induced skin carcinoma in mice. MATERIAL AND METHODS Cell viability was assessed by MTT assay, cell proliferation by clonogenic assay, apoptosis and cell cycle arrest was analyzed by flow cytometry, protein expression was studied by IHC and Western blotting, and gene expression by qPCR. RESULTS I3A suppressed the survival and proliferation of human melanoma cells with estimated IC50 values around 38 and 46 μM for A2058 and HT144 cell, respectively. I3A activated the protein levels of PKCδ and PKCε, which induced apoptosis by activating caspase-9 and caspace-3 followed by lowering of mitochondrial membrane potential and enhancing DNA fragmentation. I3A induced G1 phase cell cycle arrest as well as G2/M phase arrest in both cell lines. I3A inhibited the levels of NFκB p65 protein as well as phosphorylation of p65 and its nuclear translocation. I3A suppressed the gene expression of NF-κB, COX-2 and iNOS. I3A inhibited TPA-induced inflammation and epidermal hyperplasia in female ICR mice by downregulating NF-κB and iNOS. I3A suppressed the growth of skin tumor in DMBA-induced mice in dose-dependent manner. CONCLUSIONS The mechanism of I3A induces apoptosis in human melanoma cells and suppresses skin inflammation and carcinoma via downregulation of NF-κB-iNOS-COX-2 signaling.

Figure 1

Effect of I3A on cell survival and proliferation. (A) A2058 and HT144 cells were treated with I3A at indicated concentrations and cell viability as assayed by MTT. Cell viability is represented as percent of control. (B) A2058 and HT144 cells treated with or without I3A were visualized under a light microscope (40×). (C) A2058 and HT144 cells treated with or without I3A and clonogenic assay was performed. Percent colony formation is represented as compared to control. (D) Western blot analysis of PKC protein isoforms from A2058 and HT144 cells treated with or without I3A. Each quantitative experiment was performed in triplicate. I3A – ingenol-3-angelate; * P<0.05 vs. control.

Figure 2

I3A induces apoptotic cells death and cell cycle arrest. (A) Flow cytometry analysis of A2058 and HT144 cells treated with or without I3A after annexin V/PI-FITC staining. (B) ELISA microplate measurement of mitochondrial membrane potential using JC-10 staining of A2058 and HT144 cells treated with or without I3A. Data represent percent of control from experimental triplicate. (C) Agarose gel electrophoresis of genomic DNA isolated from A2058 and HT144 cells treated with or without I3A. Percent colony formation is represented as compared to control. (D) Western blot analysis of apoptosis-related proteins from A2058 and HT144 cells treated with or without I3A. (E) Flow cytometry analysis of cell cycle from A2058 and HT144 cells treated with or without I3A after PI staining. Each quantitative experiment was performed in triplicate. I3A – ingenol-3-angelate; * P<0.05 vs. control.

Figure 3

I3A induces intrinsic apoptosis pathway. (A, B) Western blot analysis of apoptosis related proteins from A2058 and HT144 cells treated with or without I3A. (C) MTT cell viability assay for A2058 and HT144 cells treated with or without I3A. Cell viability is represented as percent of control from experimental triplicates. I3A-1 – ingenol-3-angelate 1 μM; I3A-5 – ingenol-3-angelate 5 μM; z-VAD-fmk concentration 5 μM; * P<0.05 vs. control.

Figure 4

I3A downregulates NF-κB signaling. (A, B, D) Western blot analysis of proteins from A2058 and HT144 cells treated with or without I3A. (C) Quantitative real-time PCR analysis of NF-κB, iNOS, and Cox-2 genes from A2058 and HT144 cells treated with or without I3A. The data represented are fold changes (mean ±SD) of genes expression in I3A-treated samples vs. control (one-fold) from experimental triplicates. (E) MTT cell viability assay for A2058 and HT144 cells treated with or without I3A. Cell viability is represented as percent of control from experimental triplicates. I3A – ingenol-3-angelate; Cyt – cytosolic fraction; Nuc – nuclear fraction; prostratin concentration 100 nmol; * P<0.05 vs. control; ** P<0.05 vs. prostratin group.

Figure 5

I3A suppresses TPA-induce inflammation in mouse skin. (A) H&E staining of skin sections of mice (n=3) induced with TPA (10 nmol) and treated with I3A at indicated concentrations. Quantitative epidermal thickness measurement (in μM) from mouse skin. a, vehicle control group; b, TPA (10 nmol); c, TPA+I3A (25 nmol); d, TPA+I3A (50 nmol). (B) IHC analysis of PCNA, Cox-2 and iNOS from skin sections of mice (n=3) induced with TPA (10 nmol) and treated with I3A at indicated concentrations. The graph represents the quantitative expression of staining of proteins vs. control (100%). (C) Quantitative real-time PCR analysis of Cox-2, iNOS, NF-κB, and AP-1 genes from skin of mice (n=3) induced with TPA (10 nmol) and treated with I3A at indicated concentrations. The data represented are fold changes (mean ±SD) of genes expression in I3A-treated samples vs. control (one-fold). I3A – ingenol-3-angelate; VH – vehicle control; * P<0.05 vs. control; ** P<0.05 vs. TPA group.

Figure 6

I3A suppresses skim tumor development in DMBA-induced mice. The tumor count from mouse skin was performed as described in the Methodology section. The data represented are mean ±SD of tumor counts in mouse groups at 2 weeks (n=5). I3A – ingenol-3-angelate; DMBA concentration 100 μg/100 μL of acetone. * P<0.05 vs. control.

Figure 7

I3A downregulates NF-κB signaling in DMBA-induced mice. IHC analysis of PCNA (A), activated caspase-3 (B), and TUNEL staining (C) from skin sections of mice (n=3) induced with DMBA (100 μg) and treated with I3A at indicated concentrations. The graph represents the quantitative expression of staining of proteins vs. control (100%). (D) Quantitative real-time PCR analysis of caspase-3, PARP, NF-κB, and TNF-α genes from skin of mice (n=3) induced with DMBA (100 μg) and treated with I3A at indicated concentrations. The data represented is fold change (mean ±SD) of genes expression in I3A-treated samples vs. control (one-fold). I3A – ingenol-3-angelate; VH – vehicle control; * P<0.05 vs. control; ** P<0.05 vs. DMBA group.