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Liposome encapsulated Disulfiram inhibits NFκB pathway and targets breast cancer stem cells in vitro and in vivo.

Liu P, Wang Z, Brown S, Kannappan V, Tawari PE, Jiang W, Irache JM, Tang JZ, Armesilla AL, Darling JL, Tang X, Wang W - Oncotarget (2014)

Bottom Line: This prompted us to develop a liposome-encapsulated DS (Lipo-DS) and examine its anticancer effect and mechanisms in vitro and in vivo.Mice tolerated the treatment very well and no significant in vivo nonspecific toxicity was observed.Further study may translate DS into cancer therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Research Institute in Healthcare Science, Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton, UK.

ABSTRACT
Breast cancer stem cells (BCSCs) are pan-resistant to different anticancer agents and responsible for cancer relapse. Disulfiram (DS), an antialcoholism drug, targets CSCs and reverses pan-chemoresistance. The anticancer application of DS is limited by its very short half-life in the bloodstream. This prompted us to develop a liposome-encapsulated DS (Lipo-DS) and examine its anticancer effect and mechanisms in vitro and in vivo. The relationship between hypoxia and CSCs was examined by in vitro comparison of BC cells cultured in spheroid and hypoxic conditions. To determine the importance of NFκB activation in bridging hypoxia and CSC-related pan-resistance, the CSC characters and drug sensitivity in BC cell lines were observed in NFκB p65 transfected cell lines. The effect of Lipo-DS on the NFκB pathway, CSCs and chemosensitivity was investigated in vitro and in vivo. The spheroid cultured BC cells manifested CSC characteristics and pan-resistance to anticancer drugs. This was related to the hypoxic condition in the spheres. Hypoxia induced activation of NFκB and chemoresistance. Transfection of BC cells with NFκB p65 also induced CSC characters and pan-resistance. Lipo-DS blocked NFκB activation and specifically targeted CSCs in vitro. Lipo-DS also targeted the CSC population in vivo and showed very strong anticancer efficacy. Mice tolerated the treatment very well and no significant in vivo nonspecific toxicity was observed. Hypoxia induced NFκB activation is responsible for stemness and chemoresistance in BCSCs. Lipo-DS targets NFκB pathway and CSCs. Further study may translate DS into cancer therapeutics.

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Lipo-DS/Cu induces ROS-MAPK and inhibits NFκB pathways and specifically targets BC cellsA. Top: Flow cytometric detection of Lipo-DS/Cu-induced ROS activity in BC cells. Dot-line: control, Solid-line: Lipo-DS/Cu. Bottom: Lipo-DS/Cu-induced ROS activity was reversed by NAC. +VE: positive control (Pyocyanin 200μM). B. The Lipo-DS/Cu-induced cytotoxicity was reversed by NAC. C. Lipo-DS/Cu specifically induced ROS activity in BC cell lines. Relative ROS activity = (Treated/untreated) × 100%. D. Lipo-DS/Cu demonstrated specific cytotoxicity in BC cell lines. Relative viability = (Treated/untreated) × 100%. E. Clonogenic assay shows that Lipo-DS/Cu abolished colony-forming ability in BC cell lines but no effect on normal cell lines. The cells were exposed to drugs for 1 hour and released in drug free medium for 72 hours (MTT) and 10 days (clonogenic assay). F. The effect of different treatments on JNK, p38 and ERK pathways. G. Lipo-DS/Cu blocked NFκB p65 nuclear translocation. H. Lipo-DS/Cu induced IκBα expression and inhibited phosphorylation of AKT and NFκB p65. For F – H, 6-day-cultured spheres were trypsinized and exposed to different treatments (Lipo-DS 1μM, Cu 10μM, Lipo-DS/Cu) for 4 hours and release in drug-free medium for 24 hours.
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Figure 4: Lipo-DS/Cu induces ROS-MAPK and inhibits NFκB pathways and specifically targets BC cellsA. Top: Flow cytometric detection of Lipo-DS/Cu-induced ROS activity in BC cells. Dot-line: control, Solid-line: Lipo-DS/Cu. Bottom: Lipo-DS/Cu-induced ROS activity was reversed by NAC. +VE: positive control (Pyocyanin 200μM). B. The Lipo-DS/Cu-induced cytotoxicity was reversed by NAC. C. Lipo-DS/Cu specifically induced ROS activity in BC cell lines. Relative ROS activity = (Treated/untreated) × 100%. D. Lipo-DS/Cu demonstrated specific cytotoxicity in BC cell lines. Relative viability = (Treated/untreated) × 100%. E. Clonogenic assay shows that Lipo-DS/Cu abolished colony-forming ability in BC cell lines but no effect on normal cell lines. The cells were exposed to drugs for 1 hour and released in drug free medium for 72 hours (MTT) and 10 days (clonogenic assay). F. The effect of different treatments on JNK, p38 and ERK pathways. G. Lipo-DS/Cu blocked NFκB p65 nuclear translocation. H. Lipo-DS/Cu induced IκBα expression and inhibited phosphorylation of AKT and NFκB p65. For F – H, 6-day-cultured spheres were trypsinized and exposed to different treatments (Lipo-DS 1μM, Cu 10μM, Lipo-DS/Cu) for 4 hours and release in drug-free medium for 24 hours.

Mentions: We previously reported that DS/Cu activates ROS-MAPK and inhibits NFκB pathways in attached cells [21]. In this study, we examined the effect of Lipo-DS/Cu on MSCs. Lipo-DS/Cu induced ROS activity in MSCs was reversed by NAC, a ROS inhibitor. NAC also reversed Lipo-DS/Cu induced cytotoxicity (Fig. 4A and B). In comparison with normal cell lines, Lipo-DS/Cu selectively induced higher ROS activity and cytotoxicity in cancer cells (Fig. 4C and 4D). After exposure to Lipo-DS/Cu for one hour, the clonogenicity in cancer cell lines was completely abolished but no significant effect was observed in Lipo-DS/Cu treated normal cells (MCF10A and HeCV)(Fig. 4E). After exposure to Lipo-DS/Cu for 4 hours, the major MAPK pathway elements, e.g. phosphorylated JNK, phosphorylated C-JUN and phosphorylated p38 but not ERK, were significantly induced (Fig. 4F). In contrast to the MAPK pathway, Lipo-DS/Cu inhibited IκBα degradation and blocked NFκB p65 nuclear translocation in both MCF7 and T47D CSCs. The phosphorylation of NFκB p65 and AKT in the MSCs was also inhibited by Lipo-DS/Cu (Fig. 4G and 4H).


Liposome encapsulated Disulfiram inhibits NFκB pathway and targets breast cancer stem cells in vitro and in vivo.

Liu P, Wang Z, Brown S, Kannappan V, Tawari PE, Jiang W, Irache JM, Tang JZ, Armesilla AL, Darling JL, Tang X, Wang W - Oncotarget (2014)

Lipo-DS/Cu induces ROS-MAPK and inhibits NFκB pathways and specifically targets BC cellsA. Top: Flow cytometric detection of Lipo-DS/Cu-induced ROS activity in BC cells. Dot-line: control, Solid-line: Lipo-DS/Cu. Bottom: Lipo-DS/Cu-induced ROS activity was reversed by NAC. +VE: positive control (Pyocyanin 200μM). B. The Lipo-DS/Cu-induced cytotoxicity was reversed by NAC. C. Lipo-DS/Cu specifically induced ROS activity in BC cell lines. Relative ROS activity = (Treated/untreated) × 100%. D. Lipo-DS/Cu demonstrated specific cytotoxicity in BC cell lines. Relative viability = (Treated/untreated) × 100%. E. Clonogenic assay shows that Lipo-DS/Cu abolished colony-forming ability in BC cell lines but no effect on normal cell lines. The cells were exposed to drugs for 1 hour and released in drug free medium for 72 hours (MTT) and 10 days (clonogenic assay). F. The effect of different treatments on JNK, p38 and ERK pathways. G. Lipo-DS/Cu blocked NFκB p65 nuclear translocation. H. Lipo-DS/Cu induced IκBα expression and inhibited phosphorylation of AKT and NFκB p65. For F – H, 6-day-cultured spheres were trypsinized and exposed to different treatments (Lipo-DS 1μM, Cu 10μM, Lipo-DS/Cu) for 4 hours and release in drug-free medium for 24 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202137&req=5

Figure 4: Lipo-DS/Cu induces ROS-MAPK and inhibits NFκB pathways and specifically targets BC cellsA. Top: Flow cytometric detection of Lipo-DS/Cu-induced ROS activity in BC cells. Dot-line: control, Solid-line: Lipo-DS/Cu. Bottom: Lipo-DS/Cu-induced ROS activity was reversed by NAC. +VE: positive control (Pyocyanin 200μM). B. The Lipo-DS/Cu-induced cytotoxicity was reversed by NAC. C. Lipo-DS/Cu specifically induced ROS activity in BC cell lines. Relative ROS activity = (Treated/untreated) × 100%. D. Lipo-DS/Cu demonstrated specific cytotoxicity in BC cell lines. Relative viability = (Treated/untreated) × 100%. E. Clonogenic assay shows that Lipo-DS/Cu abolished colony-forming ability in BC cell lines but no effect on normal cell lines. The cells were exposed to drugs for 1 hour and released in drug free medium for 72 hours (MTT) and 10 days (clonogenic assay). F. The effect of different treatments on JNK, p38 and ERK pathways. G. Lipo-DS/Cu blocked NFκB p65 nuclear translocation. H. Lipo-DS/Cu induced IκBα expression and inhibited phosphorylation of AKT and NFκB p65. For F – H, 6-day-cultured spheres were trypsinized and exposed to different treatments (Lipo-DS 1μM, Cu 10μM, Lipo-DS/Cu) for 4 hours and release in drug-free medium for 24 hours.
Mentions: We previously reported that DS/Cu activates ROS-MAPK and inhibits NFκB pathways in attached cells [21]. In this study, we examined the effect of Lipo-DS/Cu on MSCs. Lipo-DS/Cu induced ROS activity in MSCs was reversed by NAC, a ROS inhibitor. NAC also reversed Lipo-DS/Cu induced cytotoxicity (Fig. 4A and B). In comparison with normal cell lines, Lipo-DS/Cu selectively induced higher ROS activity and cytotoxicity in cancer cells (Fig. 4C and 4D). After exposure to Lipo-DS/Cu for one hour, the clonogenicity in cancer cell lines was completely abolished but no significant effect was observed in Lipo-DS/Cu treated normal cells (MCF10A and HeCV)(Fig. 4E). After exposure to Lipo-DS/Cu for 4 hours, the major MAPK pathway elements, e.g. phosphorylated JNK, phosphorylated C-JUN and phosphorylated p38 but not ERK, were significantly induced (Fig. 4F). In contrast to the MAPK pathway, Lipo-DS/Cu inhibited IκBα degradation and blocked NFκB p65 nuclear translocation in both MCF7 and T47D CSCs. The phosphorylation of NFκB p65 and AKT in the MSCs was also inhibited by Lipo-DS/Cu (Fig. 4G and 4H).

Bottom Line: This prompted us to develop a liposome-encapsulated DS (Lipo-DS) and examine its anticancer effect and mechanisms in vitro and in vivo.Mice tolerated the treatment very well and no significant in vivo nonspecific toxicity was observed.Further study may translate DS into cancer therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Research Institute in Healthcare Science, Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton, UK.

ABSTRACT
Breast cancer stem cells (BCSCs) are pan-resistant to different anticancer agents and responsible for cancer relapse. Disulfiram (DS), an antialcoholism drug, targets CSCs and reverses pan-chemoresistance. The anticancer application of DS is limited by its very short half-life in the bloodstream. This prompted us to develop a liposome-encapsulated DS (Lipo-DS) and examine its anticancer effect and mechanisms in vitro and in vivo. The relationship between hypoxia and CSCs was examined by in vitro comparison of BC cells cultured in spheroid and hypoxic conditions. To determine the importance of NFκB activation in bridging hypoxia and CSC-related pan-resistance, the CSC characters and drug sensitivity in BC cell lines were observed in NFκB p65 transfected cell lines. The effect of Lipo-DS on the NFκB pathway, CSCs and chemosensitivity was investigated in vitro and in vivo. The spheroid cultured BC cells manifested CSC characteristics and pan-resistance to anticancer drugs. This was related to the hypoxic condition in the spheres. Hypoxia induced activation of NFκB and chemoresistance. Transfection of BC cells with NFκB p65 also induced CSC characters and pan-resistance. Lipo-DS blocked NFκB activation and specifically targeted CSCs in vitro. Lipo-DS also targeted the CSC population in vivo and showed very strong anticancer efficacy. Mice tolerated the treatment very well and no significant in vivo nonspecific toxicity was observed. Hypoxia induced NFκB activation is responsible for stemness and chemoresistance in BCSCs. Lipo-DS targets NFκB pathway and CSCs. Further study may translate DS into cancer therapeutics.

Show MeSH
Related in: MedlinePlus