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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

Apoptosis induced by MitoVES is dependent on complex II. (A) Adherent and sphere MCF7 cells were transfected with non-silencing (NS) and SDHC shRNA and assessed for the level of SDHA and SDHC by qPCR and WB with actin as loading control. The graphs on the right show the level of the SDHA and SDHC proteins in the sub-lines related to actin. (B) Parental and SDHClow MCF7 cells were grown in serum-containing and ‘sphere’ medium and inspected by light microscopy. Parental and SDHClow MCF7 sphere cells were evaluated for SDH and SQR activities (C) and for the level of stemness genes related to their level in MCF7 adherent cells set as 1 (D). MCF7 sphere cells were exposed to MitoVES homologues at 5 μM for 1 h and assessed for ROS using MitoSOX (E) and for 12 h and assessed for apoptosis (F). Parental, NS and SDHClow MCF7 sphere cells, as shown, were exposed to MitoVES for 24 h (viability) or 12 h (apoptosis) and evaluated for viability using the MTT assay (G) and apoptosis by the annexin V/PI method (H). (I) Adherent and sphere MCF7 cells were evaluated for apoptosis after 24 h exposure to 5 μM MitoVES in the absence or presence of 10 μM TTFA. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A-C indicates statistically significant differences for parental and SDHClow MCF7 cells, in panel D for adherent and sphere cells, in panels E and F for control and treated cells, in panels G and H for parental and SDHClow MCF7 cells, and in panel I for cells treated in the absence and presence of TTFA, with p < 0.05. Images in panel B are representative of three independent experiments
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Fig6: Apoptosis induced by MitoVES is dependent on complex II. (A) Adherent and sphere MCF7 cells were transfected with non-silencing (NS) and SDHC shRNA and assessed for the level of SDHA and SDHC by qPCR and WB with actin as loading control. The graphs on the right show the level of the SDHA and SDHC proteins in the sub-lines related to actin. (B) Parental and SDHClow MCF7 cells were grown in serum-containing and ‘sphere’ medium and inspected by light microscopy. Parental and SDHClow MCF7 sphere cells were evaluated for SDH and SQR activities (C) and for the level of stemness genes related to their level in MCF7 adherent cells set as 1 (D). MCF7 sphere cells were exposed to MitoVES homologues at 5 μM for 1 h and assessed for ROS using MitoSOX (E) and for 12 h and assessed for apoptosis (F). Parental, NS and SDHClow MCF7 sphere cells, as shown, were exposed to MitoVES for 24 h (viability) or 12 h (apoptosis) and evaluated for viability using the MTT assay (G) and apoptosis by the annexin V/PI method (H). (I) Adherent and sphere MCF7 cells were evaluated for apoptosis after 24 h exposure to 5 μM MitoVES in the absence or presence of 10 μM TTFA. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A-C indicates statistically significant differences for parental and SDHClow MCF7 cells, in panel D for adherent and sphere cells, in panels E and F for control and treated cells, in panels G and H for parental and SDHClow MCF7 cells, and in panel I for cells treated in the absence and presence of TTFA, with p < 0.05. Images in panel B are representative of three independent experiments

Mentions: Whether MitoVES induces apoptosis in breast TICs via CII has not been tested. We therefore knocked down the SDHC subunit of CII in MCF7 cells and found that SDHClow MCF7 cells form spheres with low level of SDHC, while SDHA is unaffected (Fig. 6 B). SDH activity of CII, residing in SDHA, was only marginally affected, while SQR activity of CII that requires intact SDHC was suppressed (Fig. 6C). SDHClow MCF7 spheres feature high level of stemness, as documented by several TIC markers (Fig. 6D). Treatment of MCF7 sphere cells with MitoVES homologues differing in the length of the aliphatic chain linking the tocopheryl succinyl group TPP+ group revealed that the short-chain homologues are inefficient in ROS generation and apoptosis induction (Fig. 6 E, F), pointing to CII as a target. SDHClow MCF7 spheres showed higher viability in the presence of MitoVES than MCF7 spheres (Fig. 6G) with the IC50 value ~4-fold higher (Table 1). SDHClow spheres were also more resistant to MitoVES-induced apoptosis than their parental counterparts (Fig. 6H). Finally, thenoyltrifluoroacetate (TTFA), an agent binding to CII’s UbQ site, prevented apoptosis induced by MitoVES.Fig. 6


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)

Apoptosis induced by MitoVES is dependent on complex II. (A) Adherent and sphere MCF7 cells were transfected with non-silencing (NS) and SDHC shRNA and assessed for the level of SDHA and SDHC by qPCR and WB with actin as loading control. The graphs on the right show the level of the SDHA and SDHC proteins in the sub-lines related to actin. (B) Parental and SDHClow MCF7 cells were grown in serum-containing and ‘sphere’ medium and inspected by light microscopy. Parental and SDHClow MCF7 sphere cells were evaluated for SDH and SQR activities (C) and for the level of stemness genes related to their level in MCF7 adherent cells set as 1 (D). MCF7 sphere cells were exposed to MitoVES homologues at 5 μM for 1 h and assessed for ROS using MitoSOX (E) and for 12 h and assessed for apoptosis (F). Parental, NS and SDHClow MCF7 sphere cells, as shown, were exposed to MitoVES for 24 h (viability) or 12 h (apoptosis) and evaluated for viability using the MTT assay (G) and apoptosis by the annexin V/PI method (H). (I) Adherent and sphere MCF7 cells were evaluated for apoptosis after 24 h exposure to 5 μM MitoVES in the absence or presence of 10 μM TTFA. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A-C indicates statistically significant differences for parental and SDHClow MCF7 cells, in panel D for adherent and sphere cells, in panels E and F for control and treated cells, in panels G and H for parental and SDHClow MCF7 cells, and in panel I for cells treated in the absence and presence of TTFA, with p < 0.05. Images in panel B are representative of three independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494715&req=5

Fig6: Apoptosis induced by MitoVES is dependent on complex II. (A) Adherent and sphere MCF7 cells were transfected with non-silencing (NS) and SDHC shRNA and assessed for the level of SDHA and SDHC by qPCR and WB with actin as loading control. The graphs on the right show the level of the SDHA and SDHC proteins in the sub-lines related to actin. (B) Parental and SDHClow MCF7 cells were grown in serum-containing and ‘sphere’ medium and inspected by light microscopy. Parental and SDHClow MCF7 sphere cells were evaluated for SDH and SQR activities (C) and for the level of stemness genes related to their level in MCF7 adherent cells set as 1 (D). MCF7 sphere cells were exposed to MitoVES homologues at 5 μM for 1 h and assessed for ROS using MitoSOX (E) and for 12 h and assessed for apoptosis (F). Parental, NS and SDHClow MCF7 sphere cells, as shown, were exposed to MitoVES for 24 h (viability) or 12 h (apoptosis) and evaluated for viability using the MTT assay (G) and apoptosis by the annexin V/PI method (H). (I) Adherent and sphere MCF7 cells were evaluated for apoptosis after 24 h exposure to 5 μM MitoVES in the absence or presence of 10 μM TTFA. Data are mean values ± S.D. (n = 3). The symbol ‘*’ in panels A-C indicates statistically significant differences for parental and SDHClow MCF7 cells, in panel D for adherent and sphere cells, in panels E and F for control and treated cells, in panels G and H for parental and SDHClow MCF7 cells, and in panel I for cells treated in the absence and presence of TTFA, with p < 0.05. Images in panel B are representative of three independent experiments
Mentions: Whether MitoVES induces apoptosis in breast TICs via CII has not been tested. We therefore knocked down the SDHC subunit of CII in MCF7 cells and found that SDHClow MCF7 cells form spheres with low level of SDHC, while SDHA is unaffected (Fig. 6 B). SDH activity of CII, residing in SDHA, was only marginally affected, while SQR activity of CII that requires intact SDHC was suppressed (Fig. 6C). SDHClow MCF7 spheres feature high level of stemness, as documented by several TIC markers (Fig. 6D). Treatment of MCF7 sphere cells with MitoVES homologues differing in the length of the aliphatic chain linking the tocopheryl succinyl group TPP+ group revealed that the short-chain homologues are inefficient in ROS generation and apoptosis induction (Fig. 6 E, F), pointing to CII as a target. SDHClow MCF7 spheres showed higher viability in the presence of MitoVES than MCF7 spheres (Fig. 6G) with the IC50 value ~4-fold higher (Table 1). SDHClow spheres were also more resistant to MitoVES-induced apoptosis than their parental counterparts (Fig. 6H). Finally, thenoyltrifluoroacetate (TTFA), an agent binding to CII’s UbQ site, prevented apoptosis induced by MitoVES.Fig. 6

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