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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

MitoVES affects mitochondrial complexes. (A) NeuTL spheres were treated with 2 μM MitoVE for and 4 h, before they were harvested, permeablised with saponin and evaluated for respiration at the presence of substrates specific for CI and CII using the protocal indicated in more detail in Materials and Methods. The abbreviations in the top left line graph are: L, leak; CI, complex I; CII, complex II; ETS, electron transfer system (uncoupled resiraiton); CII’, uncoupled respiration via CII; ROX, residual respiration; PMG, pyruvate, malate and glutamate; cyt c, cytochrome c; succ, succinate; F, FCCP; rot, rotenone; ama, antimycin A. (B) The respiration via CI and CII, and the uncoupled respiration via CI (CI’) and CII (CII’) as derived from results shown in panel A is documented in control cells and cells exposed to 10 μM MitoVES for 2 and 4 h. (C) The mitochondrial fraction, prepared from control NeuTL cells or cells exposed to 10 μM MitoVES for 2 and 4 h, was lysed in the presence of digitonin and subjected to native blue gel electrophoresis as detailed in Materials and Methods. Specific subunits of individual complexes were detected using the antibodies as shown. HSP60 was used as a loading control. The symbol ‘*’ in panels indicates statistically significant differences (p < 0.05) for the respiration after cells were exposed to MitoVES
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Fig4: MitoVES affects mitochondrial complexes. (A) NeuTL spheres were treated with 2 μM MitoVE for and 4 h, before they were harvested, permeablised with saponin and evaluated for respiration at the presence of substrates specific for CI and CII using the protocal indicated in more detail in Materials and Methods. The abbreviations in the top left line graph are: L, leak; CI, complex I; CII, complex II; ETS, electron transfer system (uncoupled resiraiton); CII’, uncoupled respiration via CII; ROX, residual respiration; PMG, pyruvate, malate and glutamate; cyt c, cytochrome c; succ, succinate; F, FCCP; rot, rotenone; ama, antimycin A. (B) The respiration via CI and CII, and the uncoupled respiration via CI (CI’) and CII (CII’) as derived from results shown in panel A is documented in control cells and cells exposed to 10 μM MitoVES for 2 and 4 h. (C) The mitochondrial fraction, prepared from control NeuTL cells or cells exposed to 10 μM MitoVES for 2 and 4 h, was lysed in the presence of digitonin and subjected to native blue gel electrophoresis as detailed in Materials and Methods. Specific subunits of individual complexes were detected using the antibodies as shown. HSP60 was used as a loading control. The symbol ‘*’ in panels indicates statistically significant differences (p < 0.05) for the respiration after cells were exposed to MitoVES

Mentions: We tested the contribution of CI and CII to respiration of breast cancer cells and whether this is affected by MitoVES. As shown in Fig. 4 A & B, oxygen consumption was inhibited more at the level of CII, the target of the agent. This was observed for both coupled and uncoupled state of respiration. Native blue gel electrophoresis using a mild detergent followed by WB was employed to assess the change of mitochondrial respiratory complexes and supercomplexes upon MitoVES treatment. Some decrease in the level of supercomplexes in cells treated with MitoVES was observed after 2 and 4 h of exposure to the drug (Fig. 4 C).Fig. 4


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)

MitoVES affects mitochondrial complexes. (A) NeuTL spheres were treated with 2 μM MitoVE for and 4 h, before they were harvested, permeablised with saponin and evaluated for respiration at the presence of substrates specific for CI and CII using the protocal indicated in more detail in Materials and Methods. The abbreviations in the top left line graph are: L, leak; CI, complex I; CII, complex II; ETS, electron transfer system (uncoupled resiraiton); CII’, uncoupled respiration via CII; ROX, residual respiration; PMG, pyruvate, malate and glutamate; cyt c, cytochrome c; succ, succinate; F, FCCP; rot, rotenone; ama, antimycin A. (B) The respiration via CI and CII, and the uncoupled respiration via CI (CI’) and CII (CII’) as derived from results shown in panel A is documented in control cells and cells exposed to 10 μM MitoVES for 2 and 4 h. (C) The mitochondrial fraction, prepared from control NeuTL cells or cells exposed to 10 μM MitoVES for 2 and 4 h, was lysed in the presence of digitonin and subjected to native blue gel electrophoresis as detailed in Materials and Methods. Specific subunits of individual complexes were detected using the antibodies as shown. HSP60 was used as a loading control. The symbol ‘*’ in panels indicates statistically significant differences (p < 0.05) for the respiration after cells were exposed to MitoVES
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494715&req=5

Fig4: MitoVES affects mitochondrial complexes. (A) NeuTL spheres were treated with 2 μM MitoVE for and 4 h, before they were harvested, permeablised with saponin and evaluated for respiration at the presence of substrates specific for CI and CII using the protocal indicated in more detail in Materials and Methods. The abbreviations in the top left line graph are: L, leak; CI, complex I; CII, complex II; ETS, electron transfer system (uncoupled resiraiton); CII’, uncoupled respiration via CII; ROX, residual respiration; PMG, pyruvate, malate and glutamate; cyt c, cytochrome c; succ, succinate; F, FCCP; rot, rotenone; ama, antimycin A. (B) The respiration via CI and CII, and the uncoupled respiration via CI (CI’) and CII (CII’) as derived from results shown in panel A is documented in control cells and cells exposed to 10 μM MitoVES for 2 and 4 h. (C) The mitochondrial fraction, prepared from control NeuTL cells or cells exposed to 10 μM MitoVES for 2 and 4 h, was lysed in the presence of digitonin and subjected to native blue gel electrophoresis as detailed in Materials and Methods. Specific subunits of individual complexes were detected using the antibodies as shown. HSP60 was used as a loading control. The symbol ‘*’ in panels indicates statistically significant differences (p < 0.05) for the respiration after cells were exposed to MitoVES
Mentions: We tested the contribution of CI and CII to respiration of breast cancer cells and whether this is affected by MitoVES. As shown in Fig. 4 A & B, oxygen consumption was inhibited more at the level of CII, the target of the agent. This was observed for both coupled and uncoupled state of respiration. Native blue gel electrophoresis using a mild detergent followed by WB was employed to assess the change of mitochondrial respiratory complexes and supercomplexes upon MitoVES treatment. Some decrease in the level of supercomplexes in cells treated with MitoVES was observed after 2 and 4 h of exposure to the drug (Fig. 4 C).Fig. 4

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