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Disulfiram modulated ROS-MAPK and NFκB pathways and targeted breast cancer cells with cancer stem cell-like properties.

Yip NC, Fombon IS, Liu P, Brown S, Kannappan V, Armesilla AL, Xu B, Cassidy J, Darling JL, Wang W - Br. J. Cancer (2011)

Bottom Line: The effect of DS on BC cell lines and BCSCs was determined by MTT, western blot, CSCs culture and CSCs marker analysis.Disulfiram/copper significantly enhanced (3.7-15.5-fold) cytotoxicity of paclitaxel (PAC).Combination index isobologram analysis demonstrated a synergistic effect between DS/Cu and PAC.

View Article: PubMed Central - PubMed

Affiliation: Research Institute in Healthcare Science, School of Applied Sciences, University of Wolverhampton, Wolverhampton WV1 1LY, UK.

ABSTRACT

Background: Previous studies indicate that disulfiram (DS), an anti-alcoholism drug, is cytotoxic to cancer cell lines and reverses anticancer drug resistance. Cancer stem cells (CSCs) are the major cause of chemoresistance leading to the failure of cancer chemotherapy. This study intended to examine the effect of DS on breast cancer stem cells (BCSCs).

Methods: The effect of DS on BC cell lines and BCSCs was determined by MTT, western blot, CSCs culture and CSCs marker analysis.

Results: Disulfiram was highly toxic to BC cell lines in vitro in a copper (Cu)-dependent manner. In Cu-containing medium (1 μM), the IC(50) concentrations of DS in BC cell lines were 200-500 nM. Disulfiram/copper significantly enhanced (3.7-15.5-fold) cytotoxicity of paclitaxel (PAC). Combination index isobologram analysis demonstrated a synergistic effect between DS/Cu and PAC. The increased Bax and Bcl2 protein expression ratio indicated that intrinsic apoptotic pathway may be involved in DS/Cu-induced apoptosis. Clonogenic assay showed DS/Cu-inhibited clonogenicity of BC cells. Mammosphere formation and the ALDH1(+VE) and CD24(Low)/CD44(High) CSCs population in mammospheres were significantly inhibited by exposure to DS/Cu for 24 h. Disulfiram/copper induced reactive oxygen species (ROS) generation and activated its downstream apoptosis-related cJun N-terminal kinase and p38 MAPK pathways. Meanwhile, the constitutive NFκB activity in BC cell lines was inhibited by DS/Cu.

Conclusion: Disulfiram/copper inhibited BCSCs and enhanced cytotoxicity of PAC in BC cell lines. This may be caused by simultaneous induction of ROS and inhibition of NFκB.

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The effect of DS/Cu on the clonogenicity and CSCs in BC cell lines. (A) Clonogenic assay. The cells exposed to Cu1 μ, PAC40 n, DS (250 n for MDA-MB-231 and T47D, 200 n for MCF7), DS/Cu or PAC/DS/Cu for 24 h were cultured in drug-free medium in six-well plates at a cell density of 2.5 × 103 per well for 7–10 days. The colonies were stained with crystal violet, counted and photographed as described in Materials and Methods. (B) DS/Cu and PAC/DS/Cu inhibited mammosphere formation. The BC cells were treated with PAC40 n, DS250 n, Cu1 μ, DS/Cu or PAC/DS/Cu for 48 h and then sub-cultured in drug-free SCM in ultra-low attachment six-well plates (5000 cells per well) for 7 days and photographed at × 40 magnification. (C) DS/Cu inhibited ALDH expression in mammospheres. The ALDH+VE population was flow cytometrically determined in mammospheres exposed to drugs (Cu1 μ, DS1 μ or DS1 μ/Cu1 μ) for 16 h. (D) DS/Cu abolished CD24Low/CD44High population in mammospheres. The expression of CD24 and CD44 was examined after 16 h exposure to Cu1 μ, DS1 μ, PAC100 n or DS/Cu/PAC. The inserted numbers in the frame represent percentage of ALDH+VE or CD24Low/CD44High cells (mean±s.d. from three experiments, **P<0.01).
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fig4: The effect of DS/Cu on the clonogenicity and CSCs in BC cell lines. (A) Clonogenic assay. The cells exposed to Cu1 μ, PAC40 n, DS (250 n for MDA-MB-231 and T47D, 200 n for MCF7), DS/Cu or PAC/DS/Cu for 24 h were cultured in drug-free medium in six-well plates at a cell density of 2.5 × 103 per well for 7–10 days. The colonies were stained with crystal violet, counted and photographed as described in Materials and Methods. (B) DS/Cu and PAC/DS/Cu inhibited mammosphere formation. The BC cells were treated with PAC40 n, DS250 n, Cu1 μ, DS/Cu or PAC/DS/Cu for 48 h and then sub-cultured in drug-free SCM in ultra-low attachment six-well plates (5000 cells per well) for 7 days and photographed at × 40 magnification. (C) DS/Cu inhibited ALDH expression in mammospheres. The ALDH+VE population was flow cytometrically determined in mammospheres exposed to drugs (Cu1 μ, DS1 μ or DS1 μ/Cu1 μ) for 16 h. (D) DS/Cu abolished CD24Low/CD44High population in mammospheres. The expression of CD24 and CD44 was examined after 16 h exposure to Cu1 μ, DS1 μ, PAC100 n or DS/Cu/PAC. The inserted numbers in the frame represent percentage of ALDH+VE or CD24Low/CD44High cells (mean±s.d. from three experiments, **P<0.01).

Mentions: Clonogenic assays (Franken et al, 2006) were performed to examine the ability of DS/Cu to induce ‘reproductive death' in BC cells. After 16 h exposure to PAC (40 n: 4–18-fold higher than IC50 concentration), DS (200–250 n: sub-IC50 concentration)/Cu1 μ or PAC and DS/Cu in combination, the treated cells were collected and cultured in drug-free medium for 7–14 days. The colony number was reduced by exposure to PAC, DS or Cu alone. The colony number in PAC-, DS- and Cu-treated groups was decreased, which was caused by slow growth of the surviving cells leading to the cell number in some colonies not reaching the counting threshold (50 cells). In contrast, the clonogenicity of BC cell lines was significantly inhibited by DS/Cu and totally eradicated by exposure to PAC plus DS/Cu (Figure 4A).


Disulfiram modulated ROS-MAPK and NFκB pathways and targeted breast cancer cells with cancer stem cell-like properties.

Yip NC, Fombon IS, Liu P, Brown S, Kannappan V, Armesilla AL, Xu B, Cassidy J, Darling JL, Wang W - Br. J. Cancer (2011)

The effect of DS/Cu on the clonogenicity and CSCs in BC cell lines. (A) Clonogenic assay. The cells exposed to Cu1 μ, PAC40 n, DS (250 n for MDA-MB-231 and T47D, 200 n for MCF7), DS/Cu or PAC/DS/Cu for 24 h were cultured in drug-free medium in six-well plates at a cell density of 2.5 × 103 per well for 7–10 days. The colonies were stained with crystal violet, counted and photographed as described in Materials and Methods. (B) DS/Cu and PAC/DS/Cu inhibited mammosphere formation. The BC cells were treated with PAC40 n, DS250 n, Cu1 μ, DS/Cu or PAC/DS/Cu for 48 h and then sub-cultured in drug-free SCM in ultra-low attachment six-well plates (5000 cells per well) for 7 days and photographed at × 40 magnification. (C) DS/Cu inhibited ALDH expression in mammospheres. The ALDH+VE population was flow cytometrically determined in mammospheres exposed to drugs (Cu1 μ, DS1 μ or DS1 μ/Cu1 μ) for 16 h. (D) DS/Cu abolished CD24Low/CD44High population in mammospheres. The expression of CD24 and CD44 was examined after 16 h exposure to Cu1 μ, DS1 μ, PAC100 n or DS/Cu/PAC. The inserted numbers in the frame represent percentage of ALDH+VE or CD24Low/CD44High cells (mean±s.d. from three experiments, **P<0.01).
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fig4: The effect of DS/Cu on the clonogenicity and CSCs in BC cell lines. (A) Clonogenic assay. The cells exposed to Cu1 μ, PAC40 n, DS (250 n for MDA-MB-231 and T47D, 200 n for MCF7), DS/Cu or PAC/DS/Cu for 24 h were cultured in drug-free medium in six-well plates at a cell density of 2.5 × 103 per well for 7–10 days. The colonies were stained with crystal violet, counted and photographed as described in Materials and Methods. (B) DS/Cu and PAC/DS/Cu inhibited mammosphere formation. The BC cells were treated with PAC40 n, DS250 n, Cu1 μ, DS/Cu or PAC/DS/Cu for 48 h and then sub-cultured in drug-free SCM in ultra-low attachment six-well plates (5000 cells per well) for 7 days and photographed at × 40 magnification. (C) DS/Cu inhibited ALDH expression in mammospheres. The ALDH+VE population was flow cytometrically determined in mammospheres exposed to drugs (Cu1 μ, DS1 μ or DS1 μ/Cu1 μ) for 16 h. (D) DS/Cu abolished CD24Low/CD44High population in mammospheres. The expression of CD24 and CD44 was examined after 16 h exposure to Cu1 μ, DS1 μ, PAC100 n or DS/Cu/PAC. The inserted numbers in the frame represent percentage of ALDH+VE or CD24Low/CD44High cells (mean±s.d. from three experiments, **P<0.01).
Mentions: Clonogenic assays (Franken et al, 2006) were performed to examine the ability of DS/Cu to induce ‘reproductive death' in BC cells. After 16 h exposure to PAC (40 n: 4–18-fold higher than IC50 concentration), DS (200–250 n: sub-IC50 concentration)/Cu1 μ or PAC and DS/Cu in combination, the treated cells were collected and cultured in drug-free medium for 7–14 days. The colony number was reduced by exposure to PAC, DS or Cu alone. The colony number in PAC-, DS- and Cu-treated groups was decreased, which was caused by slow growth of the surviving cells leading to the cell number in some colonies not reaching the counting threshold (50 cells). In contrast, the clonogenicity of BC cell lines was significantly inhibited by DS/Cu and totally eradicated by exposure to PAC plus DS/Cu (Figure 4A).

Bottom Line: The effect of DS on BC cell lines and BCSCs was determined by MTT, western blot, CSCs culture and CSCs marker analysis.Disulfiram/copper significantly enhanced (3.7-15.5-fold) cytotoxicity of paclitaxel (PAC).Combination index isobologram analysis demonstrated a synergistic effect between DS/Cu and PAC.

View Article: PubMed Central - PubMed

Affiliation: Research Institute in Healthcare Science, School of Applied Sciences, University of Wolverhampton, Wolverhampton WV1 1LY, UK.

ABSTRACT

Background: Previous studies indicate that disulfiram (DS), an anti-alcoholism drug, is cytotoxic to cancer cell lines and reverses anticancer drug resistance. Cancer stem cells (CSCs) are the major cause of chemoresistance leading to the failure of cancer chemotherapy. This study intended to examine the effect of DS on breast cancer stem cells (BCSCs).

Methods: The effect of DS on BC cell lines and BCSCs was determined by MTT, western blot, CSCs culture and CSCs marker analysis.

Results: Disulfiram was highly toxic to BC cell lines in vitro in a copper (Cu)-dependent manner. In Cu-containing medium (1 μM), the IC(50) concentrations of DS in BC cell lines were 200-500 nM. Disulfiram/copper significantly enhanced (3.7-15.5-fold) cytotoxicity of paclitaxel (PAC). Combination index isobologram analysis demonstrated a synergistic effect between DS/Cu and PAC. The increased Bax and Bcl2 protein expression ratio indicated that intrinsic apoptotic pathway may be involved in DS/Cu-induced apoptosis. Clonogenic assay showed DS/Cu-inhibited clonogenicity of BC cells. Mammosphere formation and the ALDH1(+VE) and CD24(Low)/CD44(High) CSCs population in mammospheres were significantly inhibited by exposure to DS/Cu for 24 h. Disulfiram/copper induced reactive oxygen species (ROS) generation and activated its downstream apoptosis-related cJun N-terminal kinase and p38 MAPK pathways. Meanwhile, the constitutive NFκB activity in BC cell lines was inhibited by DS/Cu.

Conclusion: Disulfiram/copper inhibited BCSCs and enhanced cytotoxicity of PAC in BC cell lines. This may be caused by simultaneous induction of ROS and inhibition of NFκB.

Show MeSH
Related in: MedlinePlus