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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 Spalax, mouse and rat conditioned media on morphology and cell cycle progression in HepG2 cells. HepG2 cells were incubated under conditioned media for eight days; thereafter, cell morphology was documented using phase contrast microscopy, harvested, stained with PI and analyzed by flow cytometry. Representative images (×200) and flow cytometry histograms are presented: (A) control media; (B) rat CM; (C) mouse CM; (D) Spalax CM; (E) BrdU incorporation assay: HepG2 were grown in 96-well plates (2000 cells/well) for four and seven days under Spalax-generated CM. BrdU Cell Proliferation ELISA (Exalpha) was used. Time-dependent decrease in cell proliferation under Spalax-generated CM is depicted. CM, Conditioned media; PI, Propidium iodide.
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Figure 9: Effects of Spalax, mouse and rat conditioned media on morphology and cell cycle progression in HepG2 cells. HepG2 cells were incubated under conditioned media for eight days; thereafter, cell morphology was documented using phase contrast microscopy, harvested, stained with PI and analyzed by flow cytometry. Representative images (×200) and flow cytometry histograms are presented: (A) control media; (B) rat CM; (C) mouse CM; (D) Spalax CM; (E) BrdU incorporation assay: HepG2 were grown in 96-well plates (2000 cells/well) for four and seven days under Spalax-generated CM. BrdU Cell Proliferation ELISA (Exalpha) was used. Time-dependent decrease in cell proliferation under Spalax-generated CM is depicted. CM, Conditioned media; PI, Propidium iodide.

Mentions: To investigate the mechanisms by which Spalax fibroblasts induce cancer cell death, we examined nuclear and mitochondrial shape dynamics, as well as cell cycle distributions in Hep3B and HepG2 cells. No changes in the morphology of cells, nuclei and mitochondria as well as in cell cycle distribution were found when Hep3B cells were incubated with rat CM (Figure 8, middle row) compared to Hep3B grown with their own medium (Figure 8, upper row; control). In contrast, following exposure to Spalax CM, Hep3B cells undergo phenotypic changes observed under phase contrast microscopy: cellular shrinkage, irregularities in the plasma membrane and blebs formation (Figure 8, lower row, phase-contrast). Cell cycle analysis revealed a noticeable accumulation of dead cells in sub-G1 (36.7% versus 16.4% in control), a reduction in the number of cells in G0/G1 (28.9% versus 49.6% in control), and a modest arrest of proliferation in G2/M (21.7% versus 17.1% in control) (Figure 8, lower row, cell cycle). Nuclear staining with DAPI of Hep3B cells that were grown with Spalax CM for eight days, revealed heterogeneous chromatin appearance within irregularly shaped nuclei, and in many cells extensive chromatin condensation and nuclear fragmentation were conspicuous (Figure 8, lower row, DAPI staining). On the other hand, homogeneous patterns with regular-shaped nuclei were mainly represented in the cells incubated with rat CM as well as in the control cells (Figure 8, upper and middle row, DAPI staining). To examine whether Spalax fibroblast CM could induce mitochondrial dynamic changes in cancer cells, Hep3B cells were stained with MitoTracker-Red® probe after eight days of incubation. Compared with control and rat CM, the mitochondrial network of cells after eight-day growth with Spalax CM demonstrated the presence of damaged fragmented mitochondria (Figure 8, lower row, MitoTracker® + DAPI). Similar to Hep3B cells, HepG2 cells under Spalax CM also showed morphological changes and accumulation of cells in sub-G0/G1 whereas mouse and rat CM did not affect cellular morphology and cell cycle distribution (Figure 9). BrdU incorporation into DNA, a marker for cell proliferation, confirmed a time-dependent anti proliferative effect of Spalax CM on HepG2 cancer cells (Figure 9E).


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 Spalax, mouse and rat conditioned media on morphology and cell cycle progression in HepG2 cells. HepG2 cells were incubated under conditioned media for eight days; thereafter, cell morphology was documented using phase contrast microscopy, harvested, stained with PI and analyzed by flow cytometry. Representative images (×200) and flow cytometry histograms are presented: (A) control media; (B) rat CM; (C) mouse CM; (D) Spalax CM; (E) BrdU incorporation assay: HepG2 were grown in 96-well plates (2000 cells/well) for four and seven days under Spalax-generated CM. BrdU Cell Proliferation ELISA (Exalpha) was used. Time-dependent decrease in cell proliferation under Spalax-generated CM is depicted. CM, Conditioned media; PI, Propidium iodide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750378&req=5

Figure 9: Effects of Spalax, mouse and rat conditioned media on morphology and cell cycle progression in HepG2 cells. HepG2 cells were incubated under conditioned media for eight days; thereafter, cell morphology was documented using phase contrast microscopy, harvested, stained with PI and analyzed by flow cytometry. Representative images (×200) and flow cytometry histograms are presented: (A) control media; (B) rat CM; (C) mouse CM; (D) Spalax CM; (E) BrdU incorporation assay: HepG2 were grown in 96-well plates (2000 cells/well) for four and seven days under Spalax-generated CM. BrdU Cell Proliferation ELISA (Exalpha) was used. Time-dependent decrease in cell proliferation under Spalax-generated CM is depicted. CM, Conditioned media; PI, Propidium iodide.
Mentions: To investigate the mechanisms by which Spalax fibroblasts induce cancer cell death, we examined nuclear and mitochondrial shape dynamics, as well as cell cycle distributions in Hep3B and HepG2 cells. No changes in the morphology of cells, nuclei and mitochondria as well as in cell cycle distribution were found when Hep3B cells were incubated with rat CM (Figure 8, middle row) compared to Hep3B grown with their own medium (Figure 8, upper row; control). In contrast, following exposure to Spalax CM, Hep3B cells undergo phenotypic changes observed under phase contrast microscopy: cellular shrinkage, irregularities in the plasma membrane and blebs formation (Figure 8, lower row, phase-contrast). Cell cycle analysis revealed a noticeable accumulation of dead cells in sub-G1 (36.7% versus 16.4% in control), a reduction in the number of cells in G0/G1 (28.9% versus 49.6% in control), and a modest arrest of proliferation in G2/M (21.7% versus 17.1% in control) (Figure 8, lower row, cell cycle). Nuclear staining with DAPI of Hep3B cells that were grown with Spalax CM for eight days, revealed heterogeneous chromatin appearance within irregularly shaped nuclei, and in many cells extensive chromatin condensation and nuclear fragmentation were conspicuous (Figure 8, lower row, DAPI staining). On the other hand, homogeneous patterns with regular-shaped nuclei were mainly represented in the cells incubated with rat CM as well as in the control cells (Figure 8, upper and middle row, DAPI staining). To examine whether Spalax fibroblast CM could induce mitochondrial dynamic changes in cancer cells, Hep3B cells were stained with MitoTracker-Red® probe after eight days of incubation. Compared with control and rat CM, the mitochondrial network of cells after eight-day growth with Spalax CM demonstrated the presence of damaged fragmented mitochondria (Figure 8, lower row, MitoTracker® + DAPI). Similar to Hep3B cells, HepG2 cells under Spalax CM also showed morphological changes and accumulation of cells in sub-G0/G1 whereas mouse and rat CM did not affect cellular morphology and cell cycle distribution (Figure 9). BrdU incorporation into DNA, a marker for cell proliferation, confirmed a time-dependent anti proliferative effect of Spalax CM on HepG2 cancer cells (Figure 9E).

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