3-O-methylfunicone, a metabolite of Penicillium pinophilum, inhibits proliferation of human melanoma cells by causing G(2) + M arrest and inducing apoptosis

Abstract

Objectives: Melanoma cells take advantage of impaired ability to undergo programmed cell death in response to different external stimuli and chemotherapeutic drugs; this makes prevention of tumour progression very difficult. The aim of this study was to demonstrate whether 3-O-methylfunicone (OMF), a metabolite of Penicillium pinophilum, has the ability to arrest cell population growth and to induce apoptosis in A375P (parental) and A375M (metastasis derivatived) melanoma cell lines.

Materials and methods: Cell proliferation and apoptosis were analysed by flow cytometry, DNA fragmentation, caspase-3 and caspase-9 activation, and PARP-1 cleavage.

Results: We demonstrated that OMF affected cell proliferation in a time- and dose-dependent manner, reaching the best effect at concentration of 80 microg/ml for 24 h. Flow cytometry revealed that OMF caused significant G(2) phase arrest, which was associated with marked decrease in cyclin B1/p34(cdc2) complex and p21 induction. OMF also induced marked decrease of survivin expression. Reduced levels of apoptosis were evident after silencing p21 expression in both cell lines. Finally, the effect exercised by OMF on hTERT and TEP-1 gene expression confirmed the ability of this molecule to interfere with replicative ability of cells.

Conclusions: The results reported here seem to suggest that OMF as a promising molecule to include in strategies for treatment of melanoma.

Figure 1

Effect of 3‐O‐methylfunicone (OMF) treatment on cell proliferation (a and b, MTT assay) and on cell morphology of A375P (c–e) and A375M cells (f–h) defined by phase‐contrast microscopy. (a and b) A375P and A375M cells were treated with various concentrations of OMF for 12, 24, 48, and 72 h. Data are presented as means ± standard deviations of the results of five independent experiments. *Significantly different compared to control (P < 0.05). (c) untreated A375P cells (24 h); (d) 80 µg/ml OMF‐treated A375P cells for 24 h; (e) 100 µg/ml OMF‐treated A375P cells for 24 h; (f) untreated A375M cells (24 h); (g) 80 µg/ml OMF‐treated A375M cells for 24 h; and (h) 100 µg/ml OMF‐treated A375M cells for 24 h. Viability was determined by MTT assay (magnification ×20).

Figure 2

Effect of 3‐O‐methylfunicone (OMF) on DNA fragmentation. Lane 1, untreated A375P cells; lanes 2–5, A375P treated with 40, 60, 80, 100 µg/ml OMF for 24 h, respectively; lane 6, untreated A375M cells; lanes 7–10, A375M treated with 40, 60, 80, 100 µg/ml OMF for 24 h, respectively. M, 100‐bp ladder MW marker (Roche Diagnostics). Data shown are representative of five different experiments.

Figure 3

Effect of 3‐O‐methylfunicone (OMF) treatment on normal human epidermal melanocyte (NHEM) cell proliferation (MTT assay). NHEM cells were treated with various concentrations of OMF for 12, 24, 48, and 72 h. Data are presented as mean ± standard deviation of the results of five independent experiments.

Figure 4

RT‐PCR analysis using specific primers for cyclin B1 and p34^cdc2 mRNA expression (a and c) and Western blot analysis (b and d). (a and c) Top: Lane 1, untreated A375P cells mRNA; lane 2, mRNA from A375P treated with 80 µg/ml 3‐O‐methylfunicone (OMF) for 24 h; lane 3, untreated A375M cells mRNA; lane 4, mRNA from A375M treated with 80 µg/ml OMF for 24 h. M, 100‐bp ladder MW marker (Roche Diagnostics). Bottom: Quantitative measurements of the band intensities. (b and d) Lane 1, untreated A375P cells; lane 2, A375P treated with 80 µg/ml OMF for 24 h; lane 3, untreated A375M cells; lane 4, A375M treated with 80 µg/ml OMF for 24 h. Data shown are representative of five different experiments (± standard deviation). *Significantly different compared with control (P < 0.05).

Figure 5

RT‐PCR analysis using specific primers for p21, p27 and p53 mRNA expression (a–c) and Western blot analysis (d). (a, c) Top: Lane 1, untreated A375P cells mRNA; lane 2, mRNA from A375P treated with 80 µg/ml 3‐O‐methylfunicone (OMF) for 24 h; lane 3, mRNA from p53‐siRNA A375P; lane 4, mRNA from p53‐siRNA A375P treated with 80 µg/ml OMF for 24; lane 5, untreated A375M cells mRNA; lane 6, mRNA from A375M treated with 80 µg/ml OMF for 24 h; lane 7, mRNA from p53‐siRNA A375M; lane 8, mRNA from p53‐siRNA A375M treated with 80 µg/ml OMF for 24 h M, 100‐bp ladder MW marker (Roche Diagnostics). (b) Top: Lane 1, untreated A375P cells mRNA; lane 2, mRNA from A375P treated with 80 µg/ml OMF for 24 h; lane 3, untreated A375M cells mRNA; lane 4, mRNA from A375M treated with 80 µg/ml OMF for 24 h. M, 100‐bp ladder MW marker (Roche Diagnostics). Bottom: Quantitative measurements of band intensities. (d) Lane 1, untreated A375P cells; lane 2, A375P treated with 80 µg/ml OMF for 24 h; lane 3, untreated A375M cells; lane 4, A375M treated with 80 µg/ml OMF for 24 h. Data shown are representative of five different experiments (± standard deviation). *Significantly different compared with control (P < 0.05).

Figure 6

RT‐PCR analysis using specific primers for survivin mRNA expression. Top: Lane 1, untreated A375P cells mRNA; lane 2, mRNA from A375P treated with 80 µg/ml 3‐O‐methylfunicone (OMF) for 24 h; lane 3, untreated A375M cells mRNA; lane 4, mRNA from A375M treated with 80 µg/ml OMF for 24 h. M, 100‐bp ladder MW marker (Roche Diagnostics). Bottom: Quantitative measurements of band intensities. Data shown are representative of five different experiments (± standard deviation). *Significantly different compared with control (P < 0.05).

Figure 7

Effect of p21 silencing on cells treated with 3‐O‐methylfunicone (OMF). (a–c) RT‐PCR analysis using specific primers for p21, cyclin B1 and p34 mRNA expression. Top: Lane 1, untreated A375P cells mRNA; lane 2, mRNA from p21‐siRNA A375P; lane 3, mRNA from A375P treated with 80 µg/ml OMF for 24 h; lane 4, mRNA from p21‐siRNA A375P treated with 80 µg/ml OMF for 24 h; lane 5, untreated A375M cells mRNA; lane 2, mRNA from p21‐siRNA A375M; lane 3, mRNA from A375M treated with 80 µg/ml OMF for 24 h; lane 4, mRNA from p21‐siRNA A375M treated with 80 µg/ml OMF for 24 h; M, 100‐bp ladder MW marker (Roche Diagnostics). Bottom: Quantitative measurements of band intensities. Data shown are representative of five different experiments (± standard deviation). *Significantly different compared with control (P < 0.05).

Figure 8

Effect of 3‐O‐methylfunicone (OMF) on PARP‐1 cleavage in A375P and A375M cells. Lane 1, untreated A375P cells; lane 2, A375P treated with 80 µg/ml OMF for 24 h; lane 3, untreated A375M cells; lane 4, A375M treated with 80 µg/ml OMF for 24 h. Data shown are representative of five different experiments (± standard deviation). *Significantly different compared with control (P < 0.05).

Figure 9

Multiplex RT‐PCR analysis using specific primers for caspase mRNA expression (a) and Western blot analysis (b). Lane 1, untreated A375P cell mRNA; lane 2, mRNA from A375P treated with 80 µg/ml OMF for 24 h; lane 3, untreated A375P cell mRNA; lane 4, mRNA from A375M treated with 80 µg/ml OMF for 24 h; C+, positive control; M, 100‐bp ladder MW marker (Roche Diagnostics). (d,e) Quantitative measurements of the band intensities relative to caspase‐8 (c), caspase‐3 (d) and caspase‐9 (e) RT‐PCR. Data shown are representative of five different experiments (± standard deviation). *Significantly different compared with control (P < 0.05).

Figure 10

RT‐PCR analysis using specific primers for h‐TERT and TEP‐1 mRNA expression. (a,b) Top: Lane 1, untreated A375P cells mRNA; lane 2, mRNA from A375P treated with 80 µg/ml OMF for 24 h; lane 3, untreated A375M cells mRNA; lane 4, mRNA from A375M treated with 80 µg/ml OMF for 24 h. M, 100‐bp ladder MW marker (Roche Diagnostics). Bottom: Quantitative measurements of band intensities. Data shown are representative of five different experiments (± standard deviation). *Significantly different compared with control (P < 0.05).

Figure 11

3‐O‐methylfunicone (OMF) affects cell replicative ability. Day 0: OMF‐treated cells for 24 h and replated in OMF‐free medium. Data shown are representative of five different experiments (± standard deviation). *Significantly different compared with control (P < 0.05).