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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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NFκB is responsible for maintaining CSC characters in suspension- and hypoxia-cultured cellsA. High levels of NFκB p65 and HIF2α protein were detected in the nuclear protein extracted from suspension- and hypoxia-cultured cells. Nuc: nucleolin used as a loading standard. B. Phosphorylated AKT, NFκB p65_Ser276 and degradation of IκBα were detected in suspension- and hypoxia-cultured cells by western blot. α-Tub: α-tubulin used as a loading control. C. High NFκB DNA binding activity was detected by EMSA. Mut and WT: mutant and wild type probe competition. D and E. High NFκB p65 protein (D) and transcriptional activity (E) were detected in p65 transfected clones (C1, C3, P1 and C2) by western blot and luciferase reporter gene assay respectively. Mock: empty vector transfected cells. F. High ALDH+ and CD24low/CD44high population was detected in NFκB p65 transfected clones.
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Figure 2: NFκB is responsible for maintaining CSC characters in suspension- and hypoxia-cultured cellsA. High levels of NFκB p65 and HIF2α protein were detected in the nuclear protein extracted from suspension- and hypoxia-cultured cells. Nuc: nucleolin used as a loading standard. B. Phosphorylated AKT, NFκB p65_Ser276 and degradation of IκBα were detected in suspension- and hypoxia-cultured cells by western blot. α-Tub: α-tubulin used as a loading control. C. High NFκB DNA binding activity was detected by EMSA. Mut and WT: mutant and wild type probe competition. D and E. High NFκB p65 protein (D) and transcriptional activity (E) were detected in p65 transfected clones (C1, C3, P1 and C2) by western blot and luciferase reporter gene assay respectively. Mock: empty vector transfected cells. F. High ALDH+ and CD24low/CD44high population was detected in NFκB p65 transfected clones.

Mentions: High HIF2α nuclear protein was detected in the cells cultured in hypoxia, MSC and SUS conditions. IκBα degradation and NFκB p65 nuclear translocation were detected (Fig. 2A and 2B). NFκB p65 and AKT Phosphorylation and increased NFκB DNA binding activity were also detected in these cells (Fig. 2B and 2C). These results indicate that NFκB may be the pivotal factor in hypoxia-induced CSC characteristics. To examine the importance of NFκB in maintenance of stemness and chemosensitivity, both MCF7 and T47D cell lines were transfected with NFκB p65 subunit. High p65 protein levels and transcriptional activity of NFκB were detected in the transformed clones (Fig. 2D and 2E). Fig. 2F shows that the transformed clones possess significantly higher (p<0.01) population of CSCs (ALDH+, CD24low/CD44high). In comparison with the mock-transfected cells, the p65 transfected cell lines are highly resistant to three first line anti-BC drugs [doxorubicin (Dox), PTX and dFdC] (Table 1). These data suggest that NFκB plays a key role in hypoxia-induced CSCs and chemoresistance.


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)

NFκB is responsible for maintaining CSC characters in suspension- and hypoxia-cultured cellsA. High levels of NFκB p65 and HIF2α protein were detected in the nuclear protein extracted from suspension- and hypoxia-cultured cells. Nuc: nucleolin used as a loading standard. B. Phosphorylated AKT, NFκB p65_Ser276 and degradation of IκBα were detected in suspension- and hypoxia-cultured cells by western blot. α-Tub: α-tubulin used as a loading control. C. High NFκB DNA binding activity was detected by EMSA. Mut and WT: mutant and wild type probe competition. D and E. High NFκB p65 protein (D) and transcriptional activity (E) were detected in p65 transfected clones (C1, C3, P1 and C2) by western blot and luciferase reporter gene assay respectively. Mock: empty vector transfected cells. F. High ALDH+ and CD24low/CD44high population was detected in NFκB p65 transfected clones.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: NFκB is responsible for maintaining CSC characters in suspension- and hypoxia-cultured cellsA. High levels of NFκB p65 and HIF2α protein were detected in the nuclear protein extracted from suspension- and hypoxia-cultured cells. Nuc: nucleolin used as a loading standard. B. Phosphorylated AKT, NFκB p65_Ser276 and degradation of IκBα were detected in suspension- and hypoxia-cultured cells by western blot. α-Tub: α-tubulin used as a loading control. C. High NFκB DNA binding activity was detected by EMSA. Mut and WT: mutant and wild type probe competition. D and E. High NFκB p65 protein (D) and transcriptional activity (E) were detected in p65 transfected clones (C1, C3, P1 and C2) by western blot and luciferase reporter gene assay respectively. Mock: empty vector transfected cells. F. High ALDH+ and CD24low/CD44high population was detected in NFκB p65 transfected clones.
Mentions: High HIF2α nuclear protein was detected in the cells cultured in hypoxia, MSC and SUS conditions. IκBα degradation and NFκB p65 nuclear translocation were detected (Fig. 2A and 2B). NFκB p65 and AKT Phosphorylation and increased NFκB DNA binding activity were also detected in these cells (Fig. 2B and 2C). These results indicate that NFκB may be the pivotal factor in hypoxia-induced CSC characteristics. To examine the importance of NFκB in maintenance of stemness and chemosensitivity, both MCF7 and T47D cell lines were transfected with NFκB p65 subunit. High p65 protein levels and transcriptional activity of NFκB were detected in the transformed clones (Fig. 2D and 2E). Fig. 2F shows that the transformed clones possess significantly higher (p<0.01) population of CSCs (ALDH+, CD24low/CD44high). In comparison with the mock-transfected cells, the p65 transfected cell lines are highly resistant to three first line anti-BC drugs [doxorubicin (Dox), PTX and dFdC] (Table 1). These data suggest that NFκB plays a key role in hypoxia-induced CSCs and chemoresistance.

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