Molecular changes induced by the curcumin analogue D6 in human melanoma cells

Abstract

Background: In a previous report, we described the in vitro and in vivo antiproliferative and proapoptotic activity of a hydroxylated biphenyl (D6), a structural analogue of curcumin, on malignant melanoma and neuroblastoma tumours. In this paper, we investigated the molecular changes induced by such a compound, underlying cell growth arrest and apoptosis in melanoma cells.

Results: To shed light on the mechanisms of action of D6, we firstly demonstrated its quick cellular uptake and subsequent block of cell cycle in G2/M phase transition. A gene expression profile analysis of D6-treated melanoma cells and fibroblasts was then carried out on high density microarrays, to assess gene expression changes induced by this compound. The expression profile study evidenced both an induction of stress response pathways and a modulation of cell growth regulation mechanisms. In particular, our data suggest that the antiproliferative and proapoptotic activities of D6 in melanoma could be partially driven by up-regulation of the p53 signalling pathways as well as by down-regulation of the PI3K/Akt and NF-kB pathways. Modulation of gene expression due to D6 treatment was verified by western blot analysis for single proteins of interest, confirming the results from the gene expression profile analysis.

Conclusions: Our findings contribute to the understanding of the mechanisms of action of D6, through a comprehensive description of the molecular changes induced by this compound at the gene expression level, in agreement with the previously reported anti-tumour effects on melanoma cells.

Figure 1

D6 cellular uptake. A: molecular structure of the curcumin-related biphenyl compound, the αβ-unsaturated ketone D6 (3E,3'E)-4,4'-(5,5',6,6'-tetramethoxy-[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one); B: intracellular D6 mean concentrations following incubation of LB24 cells with 10 μM D6 for the indicated times. Mean values were calculated by the LC/MS method.

Figure 2

Cell cycle progression analysis. Cytofluorimetric analysis results from LB24 cells, untreated or treated with D6 for 24 hours. A: the table summarizes the percentage mean values ± SD of each cell cycle phase cell populations, obtained in three independent experiments. T-test was performed independently for each phase considering untreated (0 μM) versus treated (5 and 10 μM D6 respectively) cells. * P< 0.05 , ** P< 0.001; B: DNA content frequency histograms representing cells from untreated (0 μM) or 5 and 10 μM D6 treated LB24 cells. The histograms are referred to one, representative of three independent experiments.

Figure 3

Melanoma significant pathways. Diagram of most representative pathways modulated by D6 treatment in melanoma cells. X axes: representative molecular pathways. Pathways are numbered according to Table 3; Y axes: percentage of D6 modulated genes for each pathway. Numbers on top of the columns represent the total number of genes involved in each pathway.

Figure 4

Western blot analysis. Validation by western blotting of selected gene products expression. LB24 melanoma cells were grown to semi-confluence, treated (+) or not (−) for 24 hours with 10 μM D6, then harvested and lysed as described in Methods. Cell lysates were analysed by western blotting for the expression levels of selected proteins modulated by D6 treatment: (A) p53 targets: p21, GADD45A and Noxa; (B) cell cycle regulators: cyclin B, cdc25, cyclin F; (C) growth signals stimulating proteins: PI3K, p-Akt, c-kit; (D) proapoptotic proteins: DDIT3, Bcl10. Protein levels were quantified by Image J Software and normalized over (ratio) the GAPDH protein levels used as internal control. Data are reported as percentage of protein expression in D6 treated cells compared to untreated ones (100%).

Figure 5

Summary. The diagram summarizes the major gene expression changes induced by D6 in melanoma cells, outlining their possible role in determining cell fate. Up-regulated proteins are represented in red gradations, while down-regulated ones are in green. Arrow-head lines indicate molecular activation; blunt-head lines indicate inhibition.