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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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Disulfiram was cytotoxic to BC cells in a copper-dependent manner and synergistically enhanced cytotoxicity of PAC in BC cell lines. (A) MTT cytotoxicity assay. The BC cells were exposed to different treatments for 72 h. (B) The morphology ( × 100 magnification) of BC cell lines after 72 h drug exposure (DS: 1 μ of DS in serum-free medium, Cu: CuCl2 1 μ, DS/Cu: DS 1 μ + Cu 1 μ). (C) The DNA contents of BC cells after 72 h drug exposure (DS: 1 μ of DS in serum-free medium, Cu: CuCl2 1 μ, DS/Cu: DS 1 μ+Cu 1 μ). The DNA contents in the treated cells (10 000 events) were determined. The sub-G1 population represents the apoptotic cells (**P<0.01, n=3). The cleavage of PARP protein (D) and the expression levels of Bcl2 and Bax (E) after 72 h drug exposure were determined by western blot. Tub: α-tubulin used as a loading control. (F) MTT analysis of the combined effect of PAC and DS/Cu1 μ. PAC:DS/Cu1 μ=1:62.5. (G) The PAC-induced apoptosis was enhanced by DS/Cu. The DNA contents in the cell lines treated for 72 h with PAC (1 n) or PAC plus DS/Cu (DS: MCF7, 100 n; MDA-MB-231 and T47D, 150 n; Cu: CuCl2 1 μ) were determined by flow cytometry. (**P<0.01, n=3).
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fig1: Disulfiram was cytotoxic to BC cells in a copper-dependent manner and synergistically enhanced cytotoxicity of PAC in BC cell lines. (A) MTT cytotoxicity assay. The BC cells were exposed to different treatments for 72 h. (B) The morphology ( × 100 magnification) of BC cell lines after 72 h drug exposure (DS: 1 μ of DS in serum-free medium, Cu: CuCl2 1 μ, DS/Cu: DS 1 μ + Cu 1 μ). (C) The DNA contents of BC cells after 72 h drug exposure (DS: 1 μ of DS in serum-free medium, Cu: CuCl2 1 μ, DS/Cu: DS 1 μ+Cu 1 μ). The DNA contents in the treated cells (10 000 events) were determined. The sub-G1 population represents the apoptotic cells (**P<0.01, n=3). The cleavage of PARP protein (D) and the expression levels of Bcl2 and Bax (E) after 72 h drug exposure were determined by western blot. Tub: α-tubulin used as a loading control. (F) MTT analysis of the combined effect of PAC and DS/Cu1 μ. PAC:DS/Cu1 μ=1:62.5. (G) The PAC-induced apoptosis was enhanced by DS/Cu. The DNA contents in the cell lines treated for 72 h with PAC (1 n) or PAC plus DS/Cu (DS: MCF7, 100 n; MDA-MB-231 and T47D, 150 n; Cu: CuCl2 1 μ) were determined by flow cytometry. (**P<0.01, n=3).

Mentions: In CuCl2 (1 μ)-supplemented medium, DS was highly cytotoxic to BC cell lines (IC50_72h: 110–476 n; Figure 1A; Table 1). Disulfiram was also toxic to cancer cell lines in the complete medium without CuCl2 supplement with higher IC50s (456–1100 n; Figure 1A; Table 1). A biphasic effect was observed in two out of three BC cell lines. The cancer cells appeared to be protected at higher concentrations of DS. Disulfiram alone in serum-free medium (to rule out the influence of trace amount of Cu contained in FCS) or Cu alone was not toxic to BC cell lines even at a very high concentration (20 μ). The drug-induced morphological changes are shown in Figure 1B. The flow cytometric DNA content analysis demonstrated significant increase of apoptosis (sub-G1 population) in 72 h DS/Cu treated, but not other groups (Figure 1C). The cleaved PARP protein, an indicator of caspase activation, was detected in DS/Cu-treated cells (Figure 1D). Disulfiram/copper significantly inhibited Bcl2 and induced Bax expression; therefore, the Bax/Bcl2 ratio was increased in DS/Cu-treated cells (Figure 1E).


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)

Disulfiram was cytotoxic to BC cells in a copper-dependent manner and synergistically enhanced cytotoxicity of PAC in BC cell lines. (A) MTT cytotoxicity assay. The BC cells were exposed to different treatments for 72 h. (B) The morphology ( × 100 magnification) of BC cell lines after 72 h drug exposure (DS: 1 μ of DS in serum-free medium, Cu: CuCl2 1 μ, DS/Cu: DS 1 μ + Cu 1 μ). (C) The DNA contents of BC cells after 72 h drug exposure (DS: 1 μ of DS in serum-free medium, Cu: CuCl2 1 μ, DS/Cu: DS 1 μ+Cu 1 μ). The DNA contents in the treated cells (10 000 events) were determined. The sub-G1 population represents the apoptotic cells (**P<0.01, n=3). The cleavage of PARP protein (D) and the expression levels of Bcl2 and Bax (E) after 72 h drug exposure were determined by western blot. Tub: α-tubulin used as a loading control. (F) MTT analysis of the combined effect of PAC and DS/Cu1 μ. PAC:DS/Cu1 μ=1:62.5. (G) The PAC-induced apoptosis was enhanced by DS/Cu. The DNA contents in the cell lines treated for 72 h with PAC (1 n) or PAC plus DS/Cu (DS: MCF7, 100 n; MDA-MB-231 and T47D, 150 n; Cu: CuCl2 1 μ) were determined by flow cytometry. (**P<0.01, n=3).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101904&req=5

fig1: Disulfiram was cytotoxic to BC cells in a copper-dependent manner and synergistically enhanced cytotoxicity of PAC in BC cell lines. (A) MTT cytotoxicity assay. The BC cells were exposed to different treatments for 72 h. (B) The morphology ( × 100 magnification) of BC cell lines after 72 h drug exposure (DS: 1 μ of DS in serum-free medium, Cu: CuCl2 1 μ, DS/Cu: DS 1 μ + Cu 1 μ). (C) The DNA contents of BC cells after 72 h drug exposure (DS: 1 μ of DS in serum-free medium, Cu: CuCl2 1 μ, DS/Cu: DS 1 μ+Cu 1 μ). The DNA contents in the treated cells (10 000 events) were determined. The sub-G1 population represents the apoptotic cells (**P<0.01, n=3). The cleavage of PARP protein (D) and the expression levels of Bcl2 and Bax (E) after 72 h drug exposure were determined by western blot. Tub: α-tubulin used as a loading control. (F) MTT analysis of the combined effect of PAC and DS/Cu1 μ. PAC:DS/Cu1 μ=1:62.5. (G) The PAC-induced apoptosis was enhanced by DS/Cu. The DNA contents in the cell lines treated for 72 h with PAC (1 n) or PAC plus DS/Cu (DS: MCF7, 100 n; MDA-MB-231 and T47D, 150 n; Cu: CuCl2 1 μ) were determined by flow cytometry. (**P<0.01, n=3).
Mentions: In CuCl2 (1 μ)-supplemented medium, DS was highly cytotoxic to BC cell lines (IC50_72h: 110–476 n; Figure 1A; Table 1). Disulfiram was also toxic to cancer cell lines in the complete medium without CuCl2 supplement with higher IC50s (456–1100 n; Figure 1A; Table 1). A biphasic effect was observed in two out of three BC cell lines. The cancer cells appeared to be protected at higher concentrations of DS. Disulfiram alone in serum-free medium (to rule out the influence of trace amount of Cu contained in FCS) or Cu alone was not toxic to BC cell lines even at a very high concentration (20 μ). The drug-induced morphological changes are shown in Figure 1B. The flow cytometric DNA content analysis demonstrated significant increase of apoptosis (sub-G1 population) in 72 h DS/Cu treated, but not other groups (Figure 1C). The cleaved PARP protein, an indicator of caspase activation, was detected in DS/Cu-treated cells (Figure 1D). Disulfiram/copper significantly inhibited Bcl2 and induced Bax expression; therefore, the Bax/Bcl2 ratio was increased in DS/Cu-treated cells (Figure 1E).

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