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Pronounced cancer resistance in a subterranean rodent, the blind mole-rat, Spalax: in vivo and in vitro evidence.

Manov I, Hirsh M, Iancu TC, Malik A, Sotnichenko N, Band M, Avivi A, Shams I - BMC Biol. (2013)

Bottom Line: This was accompanied by decreased cancer cell viability, reduced colony formation in soft agar, disturbed cell cycle progression, chromatin condensation and mitochondrial fragmentation.Spalax fibroblast conditioned media had no effect on proliferation of noncancerous cells.Obviously, along with adaptation to hypoxia, Spalax has evolved efficient anti-cancer mechanisms yet to be elucidated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Evolution, University of Haifa, Haifa 31095, Israel.

ABSTRACT

Background: Subterranean blind mole rats (Spalax) are hypoxia tolerant (down to 3% O2), long lived (>20 years) rodents showing no clear signs of aging or aging related disorders. In 50 years of Spalax research, spontaneous tumors have never been recorded among thousands of individuals. Here we addressed the questions of (1) whether Spalax is resistant to chemically-induced tumorigenesis, and (2) whether normal fibroblasts isolated from Spalax possess tumor-suppressive activity.

Results: Treating animals with 3-Methylcholantrene (3MCA) and 7,12-Dimethylbenz(a) anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA), two potent carcinogens, confirmed Spalax high resistance to chemically induced cancers. While all mice and rats developed the expected tumors following treatment with both carcinogens, among Spalax no tumors were observed after DMBA/TPA treatment, while 3MCA induced benign fibroblastic proliferation in 2 Spalax individuals out of12, and only a single animal from the advanced age group developed malignancy 18 months post-treatment. The remaining animals are still healthy 30 months post-treatment. In vitro experiments showed an extraordinary ability of normal Spalax cultured fibroblasts to restrict malignant behavior in a broad spectrum of human-derived and in newly isolated Spalax 3MCA-induced cancer cell lines. Growth of cancer cells was inhibited by either direct interaction with Spalax fibroblasts or with soluble factors released into culture media and soft agar. This was accompanied by decreased cancer cell viability, reduced colony formation in soft agar, disturbed cell cycle progression, chromatin condensation and mitochondrial fragmentation. Cells from another cancer resistant subterranean mammal, the naked mole rat, were also tested for direct effect on cancer cells and, similar to Spalax, demonstrated anti-cancer activity. No effect on cancer cells was observed using fibroblasts from mouse, rat or Acomys. Spalax fibroblast conditioned media had no effect on proliferation of noncancerous cells.

Conclusions: This report provides pioneering evidence that Spalax is not only resistant to spontaneous cancer but also to experimentally induced cancer, and shows the unique ability of Spalax normal fibroblasts to inhibit growth and kill cancer cells, but not normal cells, either through direct fibroblast-cancer cell interaction or via soluble factors. Obviously, along with adaptation to hypoxia, Spalax has evolved efficient anti-cancer mechanisms yet to be elucidated. Exploring the molecular mechanisms allowing Spalax to survive in extreme environments and to escape cancer as well as to kill homologous and heterologous cancer cells may hold the key for understanding the molecular nature of host resistance to cancer and identify new anti-cancer strategies for treating humans.

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Effects of conditioned media (CM) on viability of cancerous and non-cancerous cells. (A) Hep3B cells were seeded in a 96-well plate at a density of 5 × 103 and 1 × 103 cells/well in RPMI-DMEM/F12 medium conditioned by Spalax or mouse skin newborn fibroblasts (SpNbF and MNbF, respectively). Hep3B cells were incubated for four days; viability was estimated by PrestoBlue® Reagent. (B,C) Hep3B cells (1 × 104 cell/well) were cultured in six-well plates under conditioned medium of Spalax adult skin fibroblasts (B) or grown in medium generated by Hep3B cells (C). After nine days, the cells’ survival rates were assessed by a Countess® cell counter (Life Technologies); red: dead cells, blue: viable cells. (D) Hep3B and HepG2 cells were incubated under Spalax CM for four days, followed by changing the media either to fresh media or new Spalax CM. After two days, viability was estimated by PrestoBlue® Reagent. (E)Spalax fibrosarcoma cells (SpFS2240) were incubated for three or seven days in full medium or under CM of Spalax adult skin normal fibroblasts (SpAdF CM), Hep3B (Hep3B CM), Spalax fibrosarcoma (SpFS2240 CM). Cell viability was evaluated by using PrestoBlue® reagent. Results are presented as percentage of control (SpFS2240 CM); mean ± S.D. (F) Effects of CM generated by Spalax or mouse normal fibroblasts (SpNbF CM and MNbF CM, respectively) on the growth of non-cancerous cells. The viability was estimated after four days by PrestoBlue® reagent; mean ± S.D. (G) Heat treatment of conditioned media. Seven-day CM, generated by Spalax or rat fibroblasts, was heat-treated at 56°C for 10 minutes and 30 minutes prior to addition to Hep3B cancer cells (2,000 cell/well) in 96-well plates. Cells were incubated for seven days followed by PrestoBlue® test. All results were obtained from three independent experiments performed in three to six technical repeats.
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Figure 7: Effects of conditioned media (CM) on viability of cancerous and non-cancerous cells. (A) Hep3B cells were seeded in a 96-well plate at a density of 5 × 103 and 1 × 103 cells/well in RPMI-DMEM/F12 medium conditioned by Spalax or mouse skin newborn fibroblasts (SpNbF and MNbF, respectively). Hep3B cells were incubated for four days; viability was estimated by PrestoBlue® Reagent. (B,C) Hep3B cells (1 × 104 cell/well) were cultured in six-well plates under conditioned medium of Spalax adult skin fibroblasts (B) or grown in medium generated by Hep3B cells (C). After nine days, the cells’ survival rates were assessed by a Countess® cell counter (Life Technologies); red: dead cells, blue: viable cells. (D) Hep3B and HepG2 cells were incubated under Spalax CM for four days, followed by changing the media either to fresh media or new Spalax CM. After two days, viability was estimated by PrestoBlue® Reagent. (E)Spalax fibrosarcoma cells (SpFS2240) were incubated for three or seven days in full medium or under CM of Spalax adult skin normal fibroblasts (SpAdF CM), Hep3B (Hep3B CM), Spalax fibrosarcoma (SpFS2240 CM). Cell viability was evaluated by using PrestoBlue® reagent. Results are presented as percentage of control (SpFS2240 CM); mean ± S.D. (F) Effects of CM generated by Spalax or mouse normal fibroblasts (SpNbF CM and MNbF CM, respectively) on the growth of non-cancerous cells. The viability was estimated after four days by PrestoBlue® reagent; mean ± S.D. (G) Heat treatment of conditioned media. Seven-day CM, generated by Spalax or rat fibroblasts, was heat-treated at 56°C for 10 minutes and 30 minutes prior to addition to Hep3B cancer cells (2,000 cell/well) in 96-well plates. Cells were incubated for seven days followed by PrestoBlue® test. All results were obtained from three independent experiments performed in three to six technical repeats.

Mentions: To determine whether the anti-cancer activity of Spalax fibroblasts was mediated by fibroblast-secreted soluble factors, conditioned media (CM) obtained from Spalax, mouse and rat monolayers were tested. Cancer cells of different origins were incubated under CM of normal fibroblasts, which had never been exposed to cancer cells or other stimuli. Effects of CM generated by cancer cells were also tested (Figure 7). As demonstrated in Figure 7A, exposure of Hep3B cells to CM from cultured newborn Spalax fibroblasts decreased cancer cell viability as measured by mitochondrial respiratory function. Exposure to mouse CM hardly had an effect on cancer cell viability. Similarly, nine-day exposure of Hep3B cells to CM generated by adult (>5.5 years old) Spalax fibroblasts obviously reduced cancer cell viability as was determined by a trypan blue extrusion assay (Figure 7B,C): cancer cells exposed to Spalax fibroblast-conditioned CM reached 49% death, whereas unexposed cells remained completely adherent and viable (Figure 7C).


Pronounced cancer resistance in a subterranean rodent, the blind mole-rat, Spalax: in vivo and in vitro evidence.

Manov I, Hirsh M, Iancu TC, Malik A, Sotnichenko N, Band M, Avivi A, Shams I - BMC Biol. (2013)

Effects of conditioned media (CM) on viability of cancerous and non-cancerous cells. (A) Hep3B cells were seeded in a 96-well plate at a density of 5 × 103 and 1 × 103 cells/well in RPMI-DMEM/F12 medium conditioned by Spalax or mouse skin newborn fibroblasts (SpNbF and MNbF, respectively). Hep3B cells were incubated for four days; viability was estimated by PrestoBlue® Reagent. (B,C) Hep3B cells (1 × 104 cell/well) were cultured in six-well plates under conditioned medium of Spalax adult skin fibroblasts (B) or grown in medium generated by Hep3B cells (C). After nine days, the cells’ survival rates were assessed by a Countess® cell counter (Life Technologies); red: dead cells, blue: viable cells. (D) Hep3B and HepG2 cells were incubated under Spalax CM for four days, followed by changing the media either to fresh media or new Spalax CM. After two days, viability was estimated by PrestoBlue® Reagent. (E)Spalax fibrosarcoma cells (SpFS2240) were incubated for three or seven days in full medium or under CM of Spalax adult skin normal fibroblasts (SpAdF CM), Hep3B (Hep3B CM), Spalax fibrosarcoma (SpFS2240 CM). Cell viability was evaluated by using PrestoBlue® reagent. Results are presented as percentage of control (SpFS2240 CM); mean ± S.D. (F) Effects of CM generated by Spalax or mouse normal fibroblasts (SpNbF CM and MNbF CM, respectively) on the growth of non-cancerous cells. The viability was estimated after four days by PrestoBlue® reagent; mean ± S.D. (G) Heat treatment of conditioned media. Seven-day CM, generated by Spalax or rat fibroblasts, was heat-treated at 56°C for 10 minutes and 30 minutes prior to addition to Hep3B cancer cells (2,000 cell/well) in 96-well plates. Cells were incubated for seven days followed by PrestoBlue® test. All results were obtained from three independent experiments performed in three to six technical repeats.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 7: Effects of conditioned media (CM) on viability of cancerous and non-cancerous cells. (A) Hep3B cells were seeded in a 96-well plate at a density of 5 × 103 and 1 × 103 cells/well in RPMI-DMEM/F12 medium conditioned by Spalax or mouse skin newborn fibroblasts (SpNbF and MNbF, respectively). Hep3B cells were incubated for four days; viability was estimated by PrestoBlue® Reagent. (B,C) Hep3B cells (1 × 104 cell/well) were cultured in six-well plates under conditioned medium of Spalax adult skin fibroblasts (B) or grown in medium generated by Hep3B cells (C). After nine days, the cells’ survival rates were assessed by a Countess® cell counter (Life Technologies); red: dead cells, blue: viable cells. (D) Hep3B and HepG2 cells were incubated under Spalax CM for four days, followed by changing the media either to fresh media or new Spalax CM. After two days, viability was estimated by PrestoBlue® Reagent. (E)Spalax fibrosarcoma cells (SpFS2240) were incubated for three or seven days in full medium or under CM of Spalax adult skin normal fibroblasts (SpAdF CM), Hep3B (Hep3B CM), Spalax fibrosarcoma (SpFS2240 CM). Cell viability was evaluated by using PrestoBlue® reagent. Results are presented as percentage of control (SpFS2240 CM); mean ± S.D. (F) Effects of CM generated by Spalax or mouse normal fibroblasts (SpNbF CM and MNbF CM, respectively) on the growth of non-cancerous cells. The viability was estimated after four days by PrestoBlue® reagent; mean ± S.D. (G) Heat treatment of conditioned media. Seven-day CM, generated by Spalax or rat fibroblasts, was heat-treated at 56°C for 10 minutes and 30 minutes prior to addition to Hep3B cancer cells (2,000 cell/well) in 96-well plates. Cells were incubated for seven days followed by PrestoBlue® test. All results were obtained from three independent experiments performed in three to six technical repeats.
Mentions: To determine whether the anti-cancer activity of Spalax fibroblasts was mediated by fibroblast-secreted soluble factors, conditioned media (CM) obtained from Spalax, mouse and rat monolayers were tested. Cancer cells of different origins were incubated under CM of normal fibroblasts, which had never been exposed to cancer cells or other stimuli. Effects of CM generated by cancer cells were also tested (Figure 7). As demonstrated in Figure 7A, exposure of Hep3B cells to CM from cultured newborn Spalax fibroblasts decreased cancer cell viability as measured by mitochondrial respiratory function. Exposure to mouse CM hardly had an effect on cancer cell viability. Similarly, nine-day exposure of Hep3B cells to CM generated by adult (>5.5 years old) Spalax fibroblasts obviously reduced cancer cell viability as was determined by a trypan blue extrusion assay (Figure 7B,C): cancer cells exposed to Spalax fibroblast-conditioned CM reached 49% death, whereas unexposed cells remained completely adherent and viable (Figure 7C).

Bottom Line: This was accompanied by decreased cancer cell viability, reduced colony formation in soft agar, disturbed cell cycle progression, chromatin condensation and mitochondrial fragmentation.Spalax fibroblast conditioned media had no effect on proliferation of noncancerous cells.Obviously, along with adaptation to hypoxia, Spalax has evolved efficient anti-cancer mechanisms yet to be elucidated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Evolution, University of Haifa, Haifa 31095, Israel.

ABSTRACT

Background: Subterranean blind mole rats (Spalax) are hypoxia tolerant (down to 3% O2), long lived (>20 years) rodents showing no clear signs of aging or aging related disorders. In 50 years of Spalax research, spontaneous tumors have never been recorded among thousands of individuals. Here we addressed the questions of (1) whether Spalax is resistant to chemically-induced tumorigenesis, and (2) whether normal fibroblasts isolated from Spalax possess tumor-suppressive activity.

Results: Treating animals with 3-Methylcholantrene (3MCA) and 7,12-Dimethylbenz(a) anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA), two potent carcinogens, confirmed Spalax high resistance to chemically induced cancers. While all mice and rats developed the expected tumors following treatment with both carcinogens, among Spalax no tumors were observed after DMBA/TPA treatment, while 3MCA induced benign fibroblastic proliferation in 2 Spalax individuals out of12, and only a single animal from the advanced age group developed malignancy 18 months post-treatment. The remaining animals are still healthy 30 months post-treatment. In vitro experiments showed an extraordinary ability of normal Spalax cultured fibroblasts to restrict malignant behavior in a broad spectrum of human-derived and in newly isolated Spalax 3MCA-induced cancer cell lines. Growth of cancer cells was inhibited by either direct interaction with Spalax fibroblasts or with soluble factors released into culture media and soft agar. This was accompanied by decreased cancer cell viability, reduced colony formation in soft agar, disturbed cell cycle progression, chromatin condensation and mitochondrial fragmentation. Cells from another cancer resistant subterranean mammal, the naked mole rat, were also tested for direct effect on cancer cells and, similar to Spalax, demonstrated anti-cancer activity. No effect on cancer cells was observed using fibroblasts from mouse, rat or Acomys. Spalax fibroblast conditioned media had no effect on proliferation of noncancerous cells.

Conclusions: This report provides pioneering evidence that Spalax is not only resistant to spontaneous cancer but also to experimentally induced cancer, and shows the unique ability of Spalax normal fibroblasts to inhibit growth and kill cancer cells, but not normal cells, either through direct fibroblast-cancer cell interaction or via soluble factors. Obviously, along with adaptation to hypoxia, Spalax has evolved efficient anti-cancer mechanisms yet to be elucidated. Exploring the molecular mechanisms allowing Spalax to survive in extreme environments and to escape cancer as well as to kill homologous and heterologous cancer cells may hold the key for understanding the molecular nature of host resistance to cancer and identify new anti-cancer strategies for treating humans.

Show MeSH
Related in: MedlinePlus