3-O-methylfunicone, a secondary metabolite produced by Penicillium pinophilum, induces growth arrest and apoptosis in HeLa cells

Abstract

3-O-Methylfunicone (OMF) is a secondary metabolite produced by the soil fungus Penicillium pinophilum which has cytostatic properties. The aim of this study was to investigate the mechanisms by which such properties are exerted, with special reference to any anti-proliferative and apoptotic potential, on HeLa cells. OMF treatment caused about 44% inhibition of cell growth after 24 h, and modifications in the tubulin fibre organization. In addition, a significant increase in p21 mRNA expression and a decrease in cyclin D1 and Cdk4 mRNA expression resulted at the same time. Apoptosis induction was demonstrated by the annexin V assay, cytofluorimetric analysis of the DNA content of the sub-G1 fraction and DNA laddering. Taken together, our data showed that the compound inhibits proliferation of HeLa cells by several mechanisms, such as disruption of tubulin fibres, cell cycle arrest and apoptosis induction. The capacity of the compound to affect the cell cycle and to modulate apoptosis is indicative of a potential for the development of a new agent for cancer chemotherapy.

Figure 1

Chemical structure of 3‐O‐methylfunicone.

Figure 2

Effect of OMF treatment on cell growth proliferation and on morphology of HeLa cells determined by trypan blue dye exclusion assay (a) and by phase contrast microscopy (b–f). (b) Untreated cells (72 h), (c) cells treated for 24 h with OMF, (d) cells treated for 48 h with OMF, (e) cells treated for 72 h with OMF, (f) cells treated for 72 h with absolute ethanol. Initial magnification 20 ×.

Figure 3

OMF treatment induces tubulin fibre modification. Tubulin fibres were visualized using FITC‐conjugated monoclonal antibody anti‐α tubulin. (a) Untreated cells (24 h), (b) cells treated for 24 h with OMF, (c) cells treated for 24 h with absolute ethanol. Initial magnification: 100 ×.

Figure 4

OMF treatment induces apoptosis. (a) DNA fragmentation induced in OMF‐treated cells after 48 and 72 h (lanes 3 and 4, respectively). Absolute ethanol does not induce DNA fragmentation (lane 5). Lane 1, untreated cells; lane 2, cells treated for 24 h. M, 100 bp ladder (Roche Diagnostics) used as MW marker. (b, c) Annexin V assay of OMF‐treated cells after 48 and 72 h, respectively. (d, e, f) DNA content of the sub‐G₁ fraction. HeLa cells, stained with propidium iodide, were analysed by FACS. When OMF‐treated cells were analysed after 48 h of treatment, over 30% of cells were observed in the sub‐G₁ phase (e). (f) FACS analysis at 72 h of treatment. Control cells (d). The results are representative of five different experiments.

Figure 5

RT‐PCR analysis using specific primers for p21 mRNA expression. Lane 1, untreated‐cell mRNA; lanes 2–6, mRNA from HeLa cells treated with OMF for 6, 17, 24, 48 and 72 h, respectively; lane 7, mRNA from HeLa cells treated with absolute ethanol for 72 h. Densitometric analysis of ethidium‐bromide‐stained agarose gel (NIH image V 1.6) was performed to quantify the amount of specific mRNAs present in the total RNA prepared from the different cell samples. The p21/GAPDH FI (fluorescence intensity) ratios were as follows: lanes 1, 2, 3, 4, 5, 6 and 7, 0.7 ± 0.12, 1.7 ± 0.25, 1.86 ± 0.32, 1.75 ± 0.36, 1.42 ± 0.26, 1.4 ± 0.37 and 0.53 ± 0.12, respectively. The values are the mean of at least five independent experiments ± SD (P < 0.05).

Figure 6

Induction of p21 protein after treatment with OMF. Lane 1, untreated‐cells; lanes 2–6, HeLa cells treated with OMF for 6, 17, 24, 48 and 72 h, respectively; lane 7, HeLa cells treated with absolute ethanol for 72 h. The values are the mean of at least five independent experiments ± SD (P < 0.05).

Figure 7

RT‐PCR analysis using specific primers for Cdk4 and cyclin D1 mRNA expression. (a, b) Lane 1, untreated‐cell mRNA; lane 2, mRNA from HeLa cells treated with OMF for 24 h; lane 3, mRNA from HeLa cells treated with absolute ethanol for 24 h. The Cdk4/GAPDH FI ratios were as follows: lanes 1, 2 and 3, 3.1 ± 0.54, 1.97 ± 0.35 and 3.2 ± 0.65, respectively. The cyclin D1/β‐actin FI ratios were as follows: lanes 1, 2 and 3, 3.74 ± 0.37, 1.96 ± 0.25 and 3.67 ± 0.41, respectively. The values are the mean of at least five independent experiments ± SD (P < 0.05).

Figure 8

Multiplex RT‐PCR analysis using specific primers for caspase‐1, c‐myc, p53, bcl2. Lane 1, untreated‐cell mRNA; lane 2, mRNA from HeLa cells treated with OMF for 72 h; lane 3, mRNA from HeLa cells treated with absolute ethanol for 72 h. The caspase‐1/GAPDH FI ratios were as follows: lanes 1, 2 and 3, 1.15 ± 0.07, 1.19 ± 0.02 and 1.2 ± 0.06, respectively. The c‐myc/GAPDH FI ratios were as follows: lanes 1, 2 and 3, 0.63 ± 0.09, 0.57 ± 0.05 and 0.61 ± 0.05, respectively. The bcl‐2/GAPDH FI ratios were as follows: lanes 1, 2 and 3, 0.85 ± 0.17, 0.24 ± 0.08 and 0.79 ± 0.11, respectively. The p53/GAPDH FI ratios were as follows: lanes 1, 2 and 3, 0.61 ± 0.13, 0.65 ± 0.11 and 0.57 ± 0.09, respectively. The values are the mean of at least five independent experiments ± SD (P < 0.05).

Figure 9

Western blot analysis relative to p53 analysis. Lane 1, untreated‐cells; lane 2, HeLa cells treated with OMF for 72 h; lane 3, HeLa cells treated with absolute ethanol for 72 h. The values are the mean of at least five independent experiments ± SD (P < 0.05).

Figure 10

Western blot analysis relative to Bcl‐2 analysis. Lane 1, untreated‐cells; lane 2, HeLa cells treated with OMF for 72 h; lane 3, HeLa cells treated with absolute ethanol for 72 h. The values are the mean of at least five independent experiments ± SD (P < 0.05).

Figure 11

RT‐PCR analysis using specific primers for Bax mRNA expression. Lane 1, untreated‐cell mRNA; lane 2, mRNA from HeLa cells treated with OMF for 72 h; lane 3, mRNA from HeLa cells treated with absolute ethanol for 72 h. The Bax/βactin FI ratios were as follows: lanes 1, 2 and 3, 0.61 ± 0.24, 1.2 ± 0.41 and 0.55 ± 0.19, respectively. The values are the mean of at least five independent experiments ± SD (P < 0.05).

Figure 12

RT‐PCR analysis using specific primers for hsp70B mRNA expression. Lane 1, untreated‐cell mRNA; lane 2 mRNA from Hela cells treated with OMF for 72 h; lane 3, mRNA from HeLa cells treated with absolute alcohol for 72 h. The hsp70B/β‐actin FI ratios were as follows: lanes 1, 2 and 3, 0.43 ± 0.09, 1.4 ± 0.23 and 0.58 ± 0.12, respectively. The values are the mean of at least five independent experiments ± SD (P < 0.05).