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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

Breast TICs are resistant to chemotherapeutic drugs but sensitive to MitoVES. Adherent and sphere NeuTL (A) and MCF7 cells (B) were exposed to different concentrations of the agents for 24 h and viability assessed by the MTT assay. (C) NeuTL adherent and sphere cells were exposed to 50 μM α-TOS or 2 μM MitoVES for 24 h and inspected by light microscopy. Adherent or sphere NeuTL (D) or MCF7 cells (E) were exposed to α-TOS (50 μM), MitoVES (2 μM) or parthenolide (PTL; 10 μM) for 12 h and apoptosis evaluated using the annexin V/PI method. (F) Adherent NeuTL cells were exposed to 2 μM MitoVES for 24 h and evaluated for cell cycle distribution. (G) NeuTL sphere and adherent cells were exposed to 5 μM MitoVES for 12 h and full length and cleaved PARP, caspase-9 (C9), caspase-3 (C3) and caspase-8 assessed using WB with actin as loading control. The level of full length and cleaved proteins was evaluated by densitometry and related to actin. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A, B, D-F indicates statistically significant differences for adherent and sphere cells with p < 0.05. The symbol ‘*’ in panel G indicates statistically significant differences in the expression of the full length and cleaved protein with p < 0.05. Images in panel C are representative of three independent experiments
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Fig2: Breast TICs are resistant to chemotherapeutic drugs but sensitive to MitoVES. Adherent and sphere NeuTL (A) and MCF7 cells (B) were exposed to different concentrations of the agents for 24 h and viability assessed by the MTT assay. (C) NeuTL adherent and sphere cells were exposed to 50 μM α-TOS or 2 μM MitoVES for 24 h and inspected by light microscopy. Adherent or sphere NeuTL (D) or MCF7 cells (E) were exposed to α-TOS (50 μM), MitoVES (2 μM) or parthenolide (PTL; 10 μM) for 12 h and apoptosis evaluated using the annexin V/PI method. (F) Adherent NeuTL cells were exposed to 2 μM MitoVES for 24 h and evaluated for cell cycle distribution. (G) NeuTL sphere and adherent cells were exposed to 5 μM MitoVES for 12 h and full length and cleaved PARP, caspase-9 (C9), caspase-3 (C3) and caspase-8 assessed using WB with actin as loading control. The level of full length and cleaved proteins was evaluated by densitometry and related to actin. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A, B, D-F indicates statistically significant differences for adherent and sphere cells with p < 0.05. The symbol ‘*’ in panel G indicates statistically significant differences in the expression of the full length and cleaved protein with p < 0.05. Images in panel C are representative of three independent experiments

Mentions: Figure 2 A documents that NeuTL spheres are more resistant to doxorubicin and paclitaxel compared to their adherent counterparts, consistent with their TIC nature. α-TOS killed adherent and sphere NeuTL and MCF7 cells with similar efficacy, while MitoVES was more efficient in killing sphere cells (Fig. 2 A, B). The IC50 values were higher for killing sphere cells by doxorubicin and paclitaxel, while they were significantly lower for MitoVES (Table 1). As the MTT assay used for cell viability partially relies on the oxidative capacity of mitochondria, the above results of α-TOS and MitoVES may be affected to some extent. Therefore further cell death assessment was carried on by flow cytometry using PI and Annexin IV staining. We can see that MitoVES also induced more cell death by apoptosis in sphere vs. adherent cells, while α-TOS was inefficient (Fig. 2 C-E). At 2 μM, MitoVES was more efficient in inducing apoptosis in MCF7 sphere cells that 10 μM parthenolide. While MitoVES at 2 μM was not very efficient in causing apoptosis in adherent NeuTL cells, it arrested their cell cycle (Fig. 2 F). The apoptotic nature of cell death induced in sphere cells by MitoVES is documented in Fig. 2 G. Apart from apoptotic proteins activated by MitoVES treatment, there were also certain amounts of cleaved Caspase-8 and cleaved Caspase-9 documented in the control group, which may be due to a small population of cells undergoing apoptosis among the whole cell culture.Fig. 2


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)

Breast TICs are resistant to chemotherapeutic drugs but sensitive to MitoVES. Adherent and sphere NeuTL (A) and MCF7 cells (B) were exposed to different concentrations of the agents for 24 h and viability assessed by the MTT assay. (C) NeuTL adherent and sphere cells were exposed to 50 μM α-TOS or 2 μM MitoVES for 24 h and inspected by light microscopy. Adherent or sphere NeuTL (D) or MCF7 cells (E) were exposed to α-TOS (50 μM), MitoVES (2 μM) or parthenolide (PTL; 10 μM) for 12 h and apoptosis evaluated using the annexin V/PI method. (F) Adherent NeuTL cells were exposed to 2 μM MitoVES for 24 h and evaluated for cell cycle distribution. (G) NeuTL sphere and adherent cells were exposed to 5 μM MitoVES for 12 h and full length and cleaved PARP, caspase-9 (C9), caspase-3 (C3) and caspase-8 assessed using WB with actin as loading control. The level of full length and cleaved proteins was evaluated by densitometry and related to actin. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A, B, D-F indicates statistically significant differences for adherent and sphere cells with p < 0.05. The symbol ‘*’ in panel G indicates statistically significant differences in the expression of the full length and cleaved protein with p < 0.05. Images in panel C are representative of three independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Breast TICs are resistant to chemotherapeutic drugs but sensitive to MitoVES. Adherent and sphere NeuTL (A) and MCF7 cells (B) were exposed to different concentrations of the agents for 24 h and viability assessed by the MTT assay. (C) NeuTL adherent and sphere cells were exposed to 50 μM α-TOS or 2 μM MitoVES for 24 h and inspected by light microscopy. Adherent or sphere NeuTL (D) or MCF7 cells (E) were exposed to α-TOS (50 μM), MitoVES (2 μM) or parthenolide (PTL; 10 μM) for 12 h and apoptosis evaluated using the annexin V/PI method. (F) Adherent NeuTL cells were exposed to 2 μM MitoVES for 24 h and evaluated for cell cycle distribution. (G) NeuTL sphere and adherent cells were exposed to 5 μM MitoVES for 12 h and full length and cleaved PARP, caspase-9 (C9), caspase-3 (C3) and caspase-8 assessed using WB with actin as loading control. The level of full length and cleaved proteins was evaluated by densitometry and related to actin. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A, B, D-F indicates statistically significant differences for adherent and sphere cells with p < 0.05. The symbol ‘*’ in panel G indicates statistically significant differences in the expression of the full length and cleaved protein with p < 0.05. Images in panel C are representative of three independent experiments
Mentions: Figure 2 A documents that NeuTL spheres are more resistant to doxorubicin and paclitaxel compared to their adherent counterparts, consistent with their TIC nature. α-TOS killed adherent and sphere NeuTL and MCF7 cells with similar efficacy, while MitoVES was more efficient in killing sphere cells (Fig. 2 A, B). The IC50 values were higher for killing sphere cells by doxorubicin and paclitaxel, while they were significantly lower for MitoVES (Table 1). As the MTT assay used for cell viability partially relies on the oxidative capacity of mitochondria, the above results of α-TOS and MitoVES may be affected to some extent. Therefore further cell death assessment was carried on by flow cytometry using PI and Annexin IV staining. We can see that MitoVES also induced more cell death by apoptosis in sphere vs. adherent cells, while α-TOS was inefficient (Fig. 2 C-E). At 2 μM, MitoVES was more efficient in inducing apoptosis in MCF7 sphere cells that 10 μM parthenolide. While MitoVES at 2 μM was not very efficient in causing apoptosis in adherent NeuTL cells, it arrested their cell cycle (Fig. 2 F). The apoptotic nature of cell death induced in sphere cells by MitoVES is documented in Fig. 2 G. Apart from apoptotic proteins activated by MitoVES treatment, there were also certain amounts of cleaved Caspase-8 and cleaved Caspase-9 documented in the control group, which may be due to a small population of cells undergoing apoptosis among the whole cell culture.Fig. 2

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