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Mitochondrially targeted vitamin E succinate efficiently kills breast tumour-initiating cells in a complex II-dependent manner.

Yan B, Stantic M, Zobalova R, Bezawork-Geleta A, Stapelberg M, Stursa J, Prokopova K, Dong L, Neuzil J - BMC Cancer (2015)

Bottom Line: This model was verified by stem cell marker expression, tumour initiation capacity and chemotherapeutic resistance.Mammospheres derived from NeuTL and MCF7 breast cancer cells were enriched in the level of stemness, and the sphere cells featured altered mitochondrial function.Sphere cultures were resistant to several established anti-cancer agents while they were susceptible to MitoVES.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Science, Griffith University, Southport, Qld, 4222, Australia. bing.yan@griffithuni.edu.au.

ABSTRACT

Background: Accumulating evidence suggests that breast cancer involves tumour-initiating cells (TICs), which play a role in initiation, metastasis, therapeutic resistance and relapse of the disease. Emerging drugs that target TICs are becoming a focus of contemporary research. Mitocans, a group of compounds that induce apoptosis of cancer cells by destabilising their mitochondria, are showing their potential in killing TICs. In this project, we investigated mitochondrially targeted vitamin E succinate (MitoVES), a recently developed mitocan, for its in vitro and in vivo efficacy against TICs.

Methods: The mammosphere model of breast TICs was established by culturing murine NeuTL and human MCF7 cells as spheres. This model was verified by stem cell marker expression, tumour initiation capacity and chemotherapeutic resistance. Cell susceptibility to MitoVES was assessed and the cell death pathway investigated. In vivo efficacy was studied by grafting NeuTL TICs to form syngeneic tumours.

Results: Mammospheres derived from NeuTL and MCF7 breast cancer cells were enriched in the level of stemness, and the sphere cells featured altered mitochondrial function. Sphere cultures were resistant to several established anti-cancer agents while they were susceptible to MitoVES. Killing of mammospheres was suppressed when the mitochondrial complex II, the molecular target of MitoVES, was knocked down. Importantly, MitoVES inhibited progression of syngeneic HER2(high) tumours derived from breast TICs by inducing apoptosis in tumour cells.

Conclusions: These results demonstrate that using mammospheres, a plausible model for studying TICs, drugs that target mitochondria efficiently kill breast tumour-initiating cells.

No MeSH data available.


Related in: MedlinePlus

Mitochondria play a role in high TIC killing activity of MitoVES. NeuTL adherent and sphere cells were exposed to 2 μM MitoVES for the times shown and ROS evaluated by flow cytometry using DCF (A) or MitoSOX (B), and expressed as relative mean fluorescence intensity (MFI). The histograms on the right are representative of individual readings. (C) Adherent and sphere NeuTL cells were assessed for routine respiration in the absence or presence of MitoVES at the concentrations shown (μM). (D) Adherent and sphere NeuTL cells were probed by WB for the levels of mitochondrial markers with actin as loading control. Adherent and sphere NeuTL (E) and MCF7 cells (F) were evaluated for ΔΨm,i using TMRM and flow cytometry. The histogram in panel E on the right is an example of a reading for NeuTL cells. (G) Adherent and sphere NeuTL cells were labelled with Hoechst to visualise nuclei and TMRM to document ΔΨm,i, and inspected by confocal microscopy. Sphere NeuTL (H) and MCF7 (I) cells were exposed to 2 μM MitoVES for 24 h in the absence or presence of 10 μM FCCP and apoptosis evaluated. The histogram in panel H on the right is an example of reading for NeuTL cells. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A-C, E and F indicates statistically significant differences for adherent and sphere cells with p < 0.05. The symbol ‘*’ in panels H and I indicates statistically significant differences in apoptosis induced in the presence and absence of FCCP with p < 0.05. Images in panels C and D are representative of three independent experiments
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Fig3: Mitochondria play a role in high TIC killing activity of MitoVES. NeuTL adherent and sphere cells were exposed to 2 μM MitoVES for the times shown and ROS evaluated by flow cytometry using DCF (A) or MitoSOX (B), and expressed as relative mean fluorescence intensity (MFI). The histograms on the right are representative of individual readings. (C) Adherent and sphere NeuTL cells were assessed for routine respiration in the absence or presence of MitoVES at the concentrations shown (μM). (D) Adherent and sphere NeuTL cells were probed by WB for the levels of mitochondrial markers with actin as loading control. Adherent and sphere NeuTL (E) and MCF7 cells (F) were evaluated for ΔΨm,i using TMRM and flow cytometry. The histogram in panel E on the right is an example of a reading for NeuTL cells. (G) Adherent and sphere NeuTL cells were labelled with Hoechst to visualise nuclei and TMRM to document ΔΨm,i, and inspected by confocal microscopy. Sphere NeuTL (H) and MCF7 (I) cells were exposed to 2 μM MitoVES for 24 h in the absence or presence of 10 μM FCCP and apoptosis evaluated. The histogram in panel H on the right is an example of reading for NeuTL cells. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A-C, E and F indicates statistically significant differences for adherent and sphere cells with p < 0.05. The symbol ‘*’ in panels H and I indicates statistically significant differences in apoptosis induced in the presence and absence of FCCP with p < 0.05. Images in panels C and D are representative of three independent experiments

Mentions: MitoVES was more efficient in ROS generation in sphere than adherent cells, in particular when assessed with DCF (Fig. 3 A, B). MitoVES also more efficiently suppressed respiration in sphere compared to adherent cells (Fig. 3 C). That the two types of cells do not differ in mitochondrial mass was confirmed by WB (Fig. 3 D). Both NeuTL and MCF7 spheres showed considerably higher ΔΨm,i potential than their adherent conterparts (Fig. 3 E-G). Important role of ΔΨm,i in apoptosis induction by MitoVES follows from an experiment, in which the mitochondrial uncoupler FCCP inhibited MitoVES-induced killing in NeuTL and MCF7 spheres (Fig. 3 H, I). The higher ΔΨm,i in sphere cells may enrich more MitoVES into their mitochondrial, which contribute to the high susceptibility of spheres upon MitoVES treatment in comparison with their adherent counterparts. Moreover, it is also found that NeuTL sphere cells have higher expression of mitochondrial complexes (unpublished data), some of which function as the molecular targets of MitoVES.Fig. 3


Mitochondrially targeted vitamin E succinate efficiently kills breast tumour-initiating cells in a complex II-dependent manner.

Yan B, Stantic M, Zobalova R, Bezawork-Geleta A, Stapelberg M, Stursa J, Prokopova K, Dong L, Neuzil J - BMC Cancer (2015)

Mitochondria play a role in high TIC killing activity of MitoVES. NeuTL adherent and sphere cells were exposed to 2 μM MitoVES for the times shown and ROS evaluated by flow cytometry using DCF (A) or MitoSOX (B), and expressed as relative mean fluorescence intensity (MFI). The histograms on the right are representative of individual readings. (C) Adherent and sphere NeuTL cells were assessed for routine respiration in the absence or presence of MitoVES at the concentrations shown (μM). (D) Adherent and sphere NeuTL cells were probed by WB for the levels of mitochondrial markers with actin as loading control. Adherent and sphere NeuTL (E) and MCF7 cells (F) were evaluated for ΔΨm,i using TMRM and flow cytometry. The histogram in panel E on the right is an example of a reading for NeuTL cells. (G) Adherent and sphere NeuTL cells were labelled with Hoechst to visualise nuclei and TMRM to document ΔΨm,i, and inspected by confocal microscopy. Sphere NeuTL (H) and MCF7 (I) cells were exposed to 2 μM MitoVES for 24 h in the absence or presence of 10 μM FCCP and apoptosis evaluated. The histogram in panel H on the right is an example of reading for NeuTL cells. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A-C, E and F indicates statistically significant differences for adherent and sphere cells with p < 0.05. The symbol ‘*’ in panels H and I indicates statistically significant differences in apoptosis induced in the presence and absence of FCCP with p < 0.05. Images in panels C and D are representative of three independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig3: Mitochondria play a role in high TIC killing activity of MitoVES. NeuTL adherent and sphere cells were exposed to 2 μM MitoVES for the times shown and ROS evaluated by flow cytometry using DCF (A) or MitoSOX (B), and expressed as relative mean fluorescence intensity (MFI). The histograms on the right are representative of individual readings. (C) Adherent and sphere NeuTL cells were assessed for routine respiration in the absence or presence of MitoVES at the concentrations shown (μM). (D) Adherent and sphere NeuTL cells were probed by WB for the levels of mitochondrial markers with actin as loading control. Adherent and sphere NeuTL (E) and MCF7 cells (F) were evaluated for ΔΨm,i using TMRM and flow cytometry. The histogram in panel E on the right is an example of a reading for NeuTL cells. (G) Adherent and sphere NeuTL cells were labelled with Hoechst to visualise nuclei and TMRM to document ΔΨm,i, and inspected by confocal microscopy. Sphere NeuTL (H) and MCF7 (I) cells were exposed to 2 μM MitoVES for 24 h in the absence or presence of 10 μM FCCP and apoptosis evaluated. The histogram in panel H on the right is an example of reading for NeuTL cells. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A-C, E and F indicates statistically significant differences for adherent and sphere cells with p < 0.05. The symbol ‘*’ in panels H and I indicates statistically significant differences in apoptosis induced in the presence and absence of FCCP with p < 0.05. Images in panels C and D are representative of three independent experiments
Mentions: MitoVES was more efficient in ROS generation in sphere than adherent cells, in particular when assessed with DCF (Fig. 3 A, B). MitoVES also more efficiently suppressed respiration in sphere compared to adherent cells (Fig. 3 C). That the two types of cells do not differ in mitochondrial mass was confirmed by WB (Fig. 3 D). Both NeuTL and MCF7 spheres showed considerably higher ΔΨm,i potential than their adherent conterparts (Fig. 3 E-G). Important role of ΔΨm,i in apoptosis induction by MitoVES follows from an experiment, in which the mitochondrial uncoupler FCCP inhibited MitoVES-induced killing in NeuTL and MCF7 spheres (Fig. 3 H, I). The higher ΔΨm,i in sphere cells may enrich more MitoVES into their mitochondrial, which contribute to the high susceptibility of spheres upon MitoVES treatment in comparison with their adherent counterparts. Moreover, it is also found that NeuTL sphere cells have higher expression of mitochondrial complexes (unpublished data), some of which function as the molecular targets of MitoVES.Fig. 3

Bottom Line: This model was verified by stem cell marker expression, tumour initiation capacity and chemotherapeutic resistance.Mammospheres derived from NeuTL and MCF7 breast cancer cells were enriched in the level of stemness, and the sphere cells featured altered mitochondrial function.Sphere cultures were resistant to several established anti-cancer agents while they were susceptible to MitoVES.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Science, Griffith University, Southport, Qld, 4222, Australia. bing.yan@griffithuni.edu.au.

ABSTRACT

Background: Accumulating evidence suggests that breast cancer involves tumour-initiating cells (TICs), which play a role in initiation, metastasis, therapeutic resistance and relapse of the disease. Emerging drugs that target TICs are becoming a focus of contemporary research. Mitocans, a group of compounds that induce apoptosis of cancer cells by destabilising their mitochondria, are showing their potential in killing TICs. In this project, we investigated mitochondrially targeted vitamin E succinate (MitoVES), a recently developed mitocan, for its in vitro and in vivo efficacy against TICs.

Methods: The mammosphere model of breast TICs was established by culturing murine NeuTL and human MCF7 cells as spheres. This model was verified by stem cell marker expression, tumour initiation capacity and chemotherapeutic resistance. Cell susceptibility to MitoVES was assessed and the cell death pathway investigated. In vivo efficacy was studied by grafting NeuTL TICs to form syngeneic tumours.

Results: Mammospheres derived from NeuTL and MCF7 breast cancer cells were enriched in the level of stemness, and the sphere cells featured altered mitochondrial function. Sphere cultures were resistant to several established anti-cancer agents while they were susceptible to MitoVES. Killing of mammospheres was suppressed when the mitochondrial complex II, the molecular target of MitoVES, was knocked down. Importantly, MitoVES inhibited progression of syngeneic HER2(high) tumours derived from breast TICs by inducing apoptosis in tumour cells.

Conclusions: These results demonstrate that using mammospheres, a plausible model for studying TICs, drugs that target mitochondria efficiently kill breast tumour-initiating cells.

No MeSH data available.


Related in: MedlinePlus