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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 MAPK and NFκB pathways. The overnight cultured BC cells were exposed to PAC1 μ, DS1 μ/Cu1 μ or PAC1 μ/DS1 μ/Cu1 μ for indicated time lengths. The expression levels and phosphorylation status of proteins in JNK (A) and p38 (B) pathways were detected by western blot. (C) The activation of JNK and p38 pathways was reversed by NAC. The phosphorylation of cJun and p38 in BC cell lines was determined by western blot after exposure to DS/Cu or DS/Cu plus NAC (10 m) for 24 h. (D) NFκB DNA-binding activity was analysed by electrophoretic mobility-shift assay assay. Nu: western blot of nucleolin was used as a protein loading control. The BC cell lines were treated with PAC1 μ, DS1 μ/Cu1 μ or PAC/DS/Cu for 24 h. (E) NFκB transcriptional activity examined by luciferase reporter gene assay after exposure to PAC1 μ, DS1 μ/Cu1 μ or PAC/DS/Cu for 24 h.
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fig3: The effect of DS/Cu on MAPK and NFκB pathways. The overnight cultured BC cells were exposed to PAC1 μ, DS1 μ/Cu1 μ or PAC1 μ/DS1 μ/Cu1 μ for indicated time lengths. The expression levels and phosphorylation status of proteins in JNK (A) and p38 (B) pathways were detected by western blot. (C) The activation of JNK and p38 pathways was reversed by NAC. The phosphorylation of cJun and p38 in BC cell lines was determined by western blot after exposure to DS/Cu or DS/Cu plus NAC (10 m) for 24 h. (D) NFκB DNA-binding activity was analysed by electrophoretic mobility-shift assay assay. Nu: western blot of nucleolin was used as a protein loading control. The BC cell lines were treated with PAC1 μ, DS1 μ/Cu1 μ or PAC/DS/Cu for 24 h. (E) NFκB transcriptional activity examined by luciferase reporter gene assay after exposure to PAC1 μ, DS1 μ/Cu1 μ or PAC/DS/Cu for 24 h.

Mentions: Figure 3A shows the effect of PAC, DS/Cu and PAC/DS/Cu on the activation of the JNK pathway. Total JNK protein expression was not affected by the above treatments. However, the expression of phosphorylated JNK, cJun and total cJun was persistently (up to 24 h) induced by DS/Cu and PAC/DS/Cu. In contrast, the expression of these proteins was not or only very mildly up-regulated by PAC. High levels of phosphorylated p38 were also detected up to 24 h following DS/Cu and PAC/DS/Cu exposure (Figure 3B). To determine the causal relationship between ROS and JNK, p38 pathways, BC cell lines were exposed to DS/Cu for 24 h with or without addition of NAC. N-acetyl-cysteine significantly inhibited or totally blocked DS/Cu-induced cJun and p38 phosphorylation (Figure 3C). cJun N-terminal kinase and p38 are the major pathways responsible for ROS-induced apoptosis (McCubrey et al, 2006). Singly blocking JNK or p38 also reversed the DS/Cu-induced cytotoxicity, but at a significantly lower levels than NAC-induced ROS blocking (P<0.01; Figure 2B).


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 MAPK and NFκB pathways. The overnight cultured BC cells were exposed to PAC1 μ, DS1 μ/Cu1 μ or PAC1 μ/DS1 μ/Cu1 μ for indicated time lengths. The expression levels and phosphorylation status of proteins in JNK (A) and p38 (B) pathways were detected by western blot. (C) The activation of JNK and p38 pathways was reversed by NAC. The phosphorylation of cJun and p38 in BC cell lines was determined by western blot after exposure to DS/Cu or DS/Cu plus NAC (10 m) for 24 h. (D) NFκB DNA-binding activity was analysed by electrophoretic mobility-shift assay assay. Nu: western blot of nucleolin was used as a protein loading control. The BC cell lines were treated with PAC1 μ, DS1 μ/Cu1 μ or PAC/DS/Cu for 24 h. (E) NFκB transcriptional activity examined by luciferase reporter gene assay after exposure to PAC1 μ, DS1 μ/Cu1 μ or PAC/DS/Cu for 24 h.
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getmorefigures.php?uid=PMC3101904&req=5

fig3: The effect of DS/Cu on MAPK and NFκB pathways. The overnight cultured BC cells were exposed to PAC1 μ, DS1 μ/Cu1 μ or PAC1 μ/DS1 μ/Cu1 μ for indicated time lengths. The expression levels and phosphorylation status of proteins in JNK (A) and p38 (B) pathways were detected by western blot. (C) The activation of JNK and p38 pathways was reversed by NAC. The phosphorylation of cJun and p38 in BC cell lines was determined by western blot after exposure to DS/Cu or DS/Cu plus NAC (10 m) for 24 h. (D) NFκB DNA-binding activity was analysed by electrophoretic mobility-shift assay assay. Nu: western blot of nucleolin was used as a protein loading control. The BC cell lines were treated with PAC1 μ, DS1 μ/Cu1 μ or PAC/DS/Cu for 24 h. (E) NFκB transcriptional activity examined by luciferase reporter gene assay after exposure to PAC1 μ, DS1 μ/Cu1 μ or PAC/DS/Cu for 24 h.
Mentions: Figure 3A shows the effect of PAC, DS/Cu and PAC/DS/Cu on the activation of the JNK pathway. Total JNK protein expression was not affected by the above treatments. However, the expression of phosphorylated JNK, cJun and total cJun was persistently (up to 24 h) induced by DS/Cu and PAC/DS/Cu. In contrast, the expression of these proteins was not or only very mildly up-regulated by PAC. High levels of phosphorylated p38 were also detected up to 24 h following DS/Cu and PAC/DS/Cu exposure (Figure 3B). To determine the causal relationship between ROS and JNK, p38 pathways, BC cell lines were exposed to DS/Cu for 24 h with or without addition of NAC. N-acetyl-cysteine significantly inhibited or totally blocked DS/Cu-induced cJun and p38 phosphorylation (Figure 3C). cJun N-terminal kinase and p38 are the major pathways responsible for ROS-induced apoptosis (McCubrey et al, 2006). Singly blocking JNK or p38 also reversed the DS/Cu-induced cytotoxicity, but at a significantly lower levels than NAC-induced ROS blocking (P<0.01; Figure 2B).

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