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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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Hypoxia induces expression of stem cell markers in BC cell linesA. The morphology of spheres cultured in serum-free (MSC) and serum-containing (SUS) medium (40× magnification). B. Flow cytometric analysis of ALDH activity and expression of CD24, CD44, Oct4, Sox2 and Nanog proteins in monolayer- and suspension-cultured cells. In comparison with the attached cells, all of these markers are expressed significantly higher in MSC and SUS (p<0.01). C. Specificity of ALDEFUOR in detection of ALDH activity in BC cell lines. DEAB: specific inhibitor of ALDH. D. Flow cytometric detection of hypoxic cells stained with Hypoxyprobe. Significantly higher population of hypoxic cells was detected in MSC and SUS cells (p<0.01). E. Confocal microscopy images of the hypoxic cells detected by Hypoxyprobe in serum-free (MSC) and serum-containing (SUS) medium cultured BC cells (×400 magnification). F and G. Hypoxyprobe stained hypoxic population in hypoxia-cultured BC cells was detected by flow cytometry (F) and immunocytochemistry (G) respectively. H. Flow cytometric comparison of ALDH activity and expression levels of CD24, CD44, Oct4, Sox2 and Nanog proteins in normoxia- and hypoxia-cultured cells. In comparison with the normoxic cells, stem cell markers were detected in significantly higher population of the hypoxic cells (p<0.01). ATT: monolayer culture; MSC: serum-free stem cell culture; SUS: serum-containing medium culture; Norm: normoxia; Hypo: hypoxia. The numbers in the frame represent Mean (SD) from three experiments.
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Figure 1: Hypoxia induces expression of stem cell markers in BC cell linesA. The morphology of spheres cultured in serum-free (MSC) and serum-containing (SUS) medium (40× magnification). B. Flow cytometric analysis of ALDH activity and expression of CD24, CD44, Oct4, Sox2 and Nanog proteins in monolayer- and suspension-cultured cells. In comparison with the attached cells, all of these markers are expressed significantly higher in MSC and SUS (p<0.01). C. Specificity of ALDEFUOR in detection of ALDH activity in BC cell lines. DEAB: specific inhibitor of ALDH. D. Flow cytometric detection of hypoxic cells stained with Hypoxyprobe. Significantly higher population of hypoxic cells was detected in MSC and SUS cells (p<0.01). E. Confocal microscopy images of the hypoxic cells detected by Hypoxyprobe in serum-free (MSC) and serum-containing (SUS) medium cultured BC cells (×400 magnification). F and G. Hypoxyprobe stained hypoxic population in hypoxia-cultured BC cells was detected by flow cytometry (F) and immunocytochemistry (G) respectively. H. Flow cytometric comparison of ALDH activity and expression levels of CD24, CD44, Oct4, Sox2 and Nanog proteins in normoxia- and hypoxia-cultured cells. In comparison with the normoxic cells, stem cell markers were detected in significantly higher population of the hypoxic cells (p<0.01). ATT: monolayer culture; MSC: serum-free stem cell culture; SUS: serum-containing medium culture; Norm: normoxia; Hypo: hypoxia. The numbers in the frame represent Mean (SD) from three experiments.

Mentions: In this study, we examined if the traditional stem cell culture system is essential for maintaining the stemness in vitro. Two breast cancer cell lines were cultured in both classical serum-free spheroid stem cell culture system and serum-rich (10%) medium in parallel. After 7 days culture, BC cells formed typical mammospheres in both conditions. The serum-free medium cultured cells formed numerous relatively smaller spheres which loosely aggregated together. In contrast, the cells cultured in the serum-rich medium formed markedly larger and tighter spheres (Fig. 1A). Furthermore, we compared the expression of stem cell markers and CSC-related embryonic proteins in these cells. Both MSCs and SUS cells have significantly higher proportion of cells expressing stem cell markers (ALDH+ and CD24low/CD44high) and CSC-related embryonic proteins (Sox2, Nanog and Oct4). In comparison with the MSCs, most of these proteins are expressed at higher levels in the SUS cells (Fig. 1B). CSCs commonly possess de novo resistance to a wide range of anticancer drugs [1]. Furthermore we examined the chemosensitivity in these cells. Table 1 shows that resistance of BC cells to three first line anti-BC drugs was induced in both culture systems. These results suggest that the stemness and chemosensitivity in BC cells were not governed by the components in the culture medium. It has been reported that the hypoxic condition in the stem cell niche is essential for maintaining the stemness and chemoresistance [6]. We hypothesized that the hypoxic condition in the mammospheres may play the role in maintenance of stemness and chemoresistance. Fig. 1D and 1E demonstrate that in comparison with the adherent cells, high population of hypoxic cells were detected in both MSC and SUS cells by HypoxyProbe. Furthermore we cultured both cell lines in hypoxic condition (1% O2) for 5 days to determine the relationship between hypoxia and MSC characteristics. Fig. 1F to 1H show that the hypoxia-cultured monolayer cells express MSC markers and embryonic proteins. Similar to the MSC and SUS cells, the cells cultured in hypoxic condition are significantly resistant to chemotherapeutic agents (Table 1). All of these data indicate that hypoxia may play a key role in determination of stemness and chemosentivity in BC cells.


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)

Hypoxia induces expression of stem cell markers in BC cell linesA. The morphology of spheres cultured in serum-free (MSC) and serum-containing (SUS) medium (40× magnification). B. Flow cytometric analysis of ALDH activity and expression of CD24, CD44, Oct4, Sox2 and Nanog proteins in monolayer- and suspension-cultured cells. In comparison with the attached cells, all of these markers are expressed significantly higher in MSC and SUS (p<0.01). C. Specificity of ALDEFUOR in detection of ALDH activity in BC cell lines. DEAB: specific inhibitor of ALDH. D. Flow cytometric detection of hypoxic cells stained with Hypoxyprobe. Significantly higher population of hypoxic cells was detected in MSC and SUS cells (p<0.01). E. Confocal microscopy images of the hypoxic cells detected by Hypoxyprobe in serum-free (MSC) and serum-containing (SUS) medium cultured BC cells (×400 magnification). F and G. Hypoxyprobe stained hypoxic population in hypoxia-cultured BC cells was detected by flow cytometry (F) and immunocytochemistry (G) respectively. H. Flow cytometric comparison of ALDH activity and expression levels of CD24, CD44, Oct4, Sox2 and Nanog proteins in normoxia- and hypoxia-cultured cells. In comparison with the normoxic cells, stem cell markers were detected in significantly higher population of the hypoxic cells (p<0.01). ATT: monolayer culture; MSC: serum-free stem cell culture; SUS: serum-containing medium culture; Norm: normoxia; Hypo: hypoxia. The numbers in the frame represent Mean (SD) from three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Hypoxia induces expression of stem cell markers in BC cell linesA. The morphology of spheres cultured in serum-free (MSC) and serum-containing (SUS) medium (40× magnification). B. Flow cytometric analysis of ALDH activity and expression of CD24, CD44, Oct4, Sox2 and Nanog proteins in monolayer- and suspension-cultured cells. In comparison with the attached cells, all of these markers are expressed significantly higher in MSC and SUS (p<0.01). C. Specificity of ALDEFUOR in detection of ALDH activity in BC cell lines. DEAB: specific inhibitor of ALDH. D. Flow cytometric detection of hypoxic cells stained with Hypoxyprobe. Significantly higher population of hypoxic cells was detected in MSC and SUS cells (p<0.01). E. Confocal microscopy images of the hypoxic cells detected by Hypoxyprobe in serum-free (MSC) and serum-containing (SUS) medium cultured BC cells (×400 magnification). F and G. Hypoxyprobe stained hypoxic population in hypoxia-cultured BC cells was detected by flow cytometry (F) and immunocytochemistry (G) respectively. H. Flow cytometric comparison of ALDH activity and expression levels of CD24, CD44, Oct4, Sox2 and Nanog proteins in normoxia- and hypoxia-cultured cells. In comparison with the normoxic cells, stem cell markers were detected in significantly higher population of the hypoxic cells (p<0.01). ATT: monolayer culture; MSC: serum-free stem cell culture; SUS: serum-containing medium culture; Norm: normoxia; Hypo: hypoxia. The numbers in the frame represent Mean (SD) from three experiments.
Mentions: In this study, we examined if the traditional stem cell culture system is essential for maintaining the stemness in vitro. Two breast cancer cell lines were cultured in both classical serum-free spheroid stem cell culture system and serum-rich (10%) medium in parallel. After 7 days culture, BC cells formed typical mammospheres in both conditions. The serum-free medium cultured cells formed numerous relatively smaller spheres which loosely aggregated together. In contrast, the cells cultured in the serum-rich medium formed markedly larger and tighter spheres (Fig. 1A). Furthermore, we compared the expression of stem cell markers and CSC-related embryonic proteins in these cells. Both MSCs and SUS cells have significantly higher proportion of cells expressing stem cell markers (ALDH+ and CD24low/CD44high) and CSC-related embryonic proteins (Sox2, Nanog and Oct4). In comparison with the MSCs, most of these proteins are expressed at higher levels in the SUS cells (Fig. 1B). CSCs commonly possess de novo resistance to a wide range of anticancer drugs [1]. Furthermore we examined the chemosensitivity in these cells. Table 1 shows that resistance of BC cells to three first line anti-BC drugs was induced in both culture systems. These results suggest that the stemness and chemosensitivity in BC cells were not governed by the components in the culture medium. It has been reported that the hypoxic condition in the stem cell niche is essential for maintaining the stemness and chemoresistance [6]. We hypothesized that the hypoxic condition in the mammospheres may play the role in maintenance of stemness and chemoresistance. Fig. 1D and 1E demonstrate that in comparison with the adherent cells, high population of hypoxic cells were detected in both MSC and SUS cells by HypoxyProbe. Furthermore we cultured both cell lines in hypoxic condition (1% O2) for 5 days to determine the relationship between hypoxia and MSC characteristics. Fig. 1F to 1H show that the hypoxia-cultured monolayer cells express MSC markers and embryonic proteins. Similar to the MSC and SUS cells, the cells cultured in hypoxic condition are significantly resistant to chemotherapeutic agents (Table 1). All of these data indicate that hypoxia may play a key role in determination of stemness and chemosentivity in BC cells.

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