Overexpression of the ATP binding cassette gene ABCA1 determines resistance to Curcumin in M14 melanoma cells - PubMed (original) (raw)

Overexpression of the ATP binding cassette gene ABCA1 determines resistance to Curcumin in M14 melanoma cells

Beatrice E Bachmeier et al. Mol Cancer. 2009.

Abstract

Background: Curcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma.

Results: We show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NFkappaB signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NFkappaB dependent manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NFkappaB. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression.

Conclusion: Resistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker.

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Figures

Figure 1

Figure 1

Melanoma Cells are resistant to Curcumin induced Apoptosis. Fluorescence micrographs of annexin V and propidium iodide stained human melanoma cells show that Curcumin induces only weakly apoptosis in M14 cells as evidenced by a very faint annexin V positive (green) staining. After 24 h of Curcumin treatment only very few M14 cells reach irreversible apoptosis and thereby a propidium iodide positive stage (red).

Figure 2

Figure 2

Curcumin does not down-regulate NFκB in Melanoma cells. a) Analysis of the binding of nuclear proteins from M14 melanoma cells to an oligo specific to the p65 binding site present in various promoters by EMSA revealed that Curcumin treatment did not alter p65 activity. There was no clear difference between the bands of treated (lane 2: 2 h; lane 3: 4 h; lane 4: 6 h) and untreated (lane 1) M14 cells using 10 μg of nuclear protein incubated with the 32P-labeled oligonucleotide. b) Immunofluorescent NFκB translocation assays using specific antibodies against the p65 unit show that NFκB is located both in the nucleus and the cytoplasm without any difference between Curcumin treated (lower panel) and untreated (upper panel) cells. (A, F: DAPI; B, G: p65; C, H: DAPI&p65). Experiments were performed in triplicates. Magnification ×200

Figure 3

Figure 3

a) Gene expression values of CXCL1 in normal skin (NS), normal human melanocytes (NHM), melanomas in situ (MIS), primary (PM) and metastatic (MM) melanomas were obtained from oligonucleotide microarray data. The expression values of single samples (symbols) and the mean expression level (lines) are reported. b) CXCL1 expression during progression from normal human melanocytes (1) to melanoma in situ (2), primary (3) and metastatic (4) melanoma. There is a significant trend to increased CXCL1 expression during melanoma progression. c) qRT-PCR analysis of M14 melanoma cells shows that treatment with Curcumin for several time periods (2, 4, 6, 15 and 24 h) did not effect CXCL1 expression. d) Well in line with RT-PCR results, CXCL1 expression visualized here by Western Blots of cell culture supernatants was not altered in M14 cells treated for several time intervals (6 h, 15 h, 24 h) with Curcumin (lanes indicated with "+") versus respective un-treated (lanes indicated with "-") cells. Equal amounts of total protein were applied per lane and experiments were performed in triplicate.

Figure 4

Figure 4

Expression of ABC genes in M14 cells as compared to the Curcumin sensitive cell line MDA-MB-231. Gene expression was analyzed using Affymetrix HU133plus2 arrays in M14 and MDA-MB-231 cells. The ratio of intensity levels of all probesets encoding ABC genes above the arbitrary threshold of 100 in M14 versus MDA-MB-231 cells were plotted on a log2 scale (A). All probesets encoding for ABC genes were used for a correlation plot (B).

Figure 5

Figure 5

Involvement of ABCA1 in Curcumin induced resistance. The effect of Curcumin on p65 (A), the apoptosis related factors bcl2 (B) and surviving (C) and cell death (increase of free nucleosomes, D) was analyzed in a series of double modulation experiments. Silencing of the multidrug resistance gene ABCA1 led to a reduction of p65 expression down to 50% of the original level already after 15 h (panel A, M14ABCA1si (triangle down)). The simultaneous treatment with 25 μM Curcumin further reduced p65 expression down to about 15% of the initial value after 24 h treatment (panel A, M14ABCA1si Cur (diamond)). Expression of the NFκB regulated anti-apoptotic factors bcl2 and survivin was reduced after ABCA1 silencing: bcl2 expression was reduced 10% in ABCA1-silenced cells 24 h after transfection with siRNA (panel B, M14ABCA1si (triangle down)). Treatment of M14 cells carrying the ABCA1 knock-downs with 25 μM Curcumin led to a significant reduction of bcl2 expression of about 35% with (panel B, M14ABCA1si Cur (diamond)). Hence Curcumin reduces bcl2 expression in M14 cells transfected with ABCA1 siRNAs by about 25%. Expression of survivin was diminished upon silencing of ABCA1 in M14 cells to about 50% of the basic value 24 h after transfection with siRNAs (panel C, M14ABCA1si (triangle down)). The addition of 25 μM Curcumin to ABCA1 silenced cells further reduced survivin expression significantly in M14 cells down to about 40% of initial expression levels (figure 5C, M14ABCA1si Cur (diamond)). Accordingly Curcumin treatment diminishes survivin expression in ABCA1 silenced M14 cells to 10%. Apoptosis as measured by means of increased numbers of free nucleosomes in cell lysates was induced in ABCA1 silenced M14 cells 24 h after transfection with siRNAs (panel D, M14ABCA1si (triangle down)). Curcumin treatment of the ABCA1 silenced M14 cells dramatically induced apoptosis already after 2 h with increasing effect up to 24 h (panel D, M14ABCA1si Cur (diamond)). Transfection of an unspecific nonsilencing siRNA control alone (M14nonsi) or in combination with 25 μM Curcumin (M14nonsi Cur) did neither alter the expression of p65, bcl2 and survivin nor induce apoptosis in M14 cells (panels A, B, C, D, M14nonsi (triangle up), M14nonsi Cur (square))

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