Subject: Molecular Biology studies of the Hypoxia- and Cancer- Tolerance of the Subterranean Mole Rat, Spalax
The mole rat, Spalax, lives permanently in sealed underground tunnels, and evolved a unique adaptive complex for living underground, one of which is to cope with hypoxia and hypercapnia endowing it cancer-resistance and anti-cancer activity. Our main goal is to unravel the molecular mechanisms that confer the Spalax with the ability to tolerate sharp and abrupt fluctuations in oxygen in its habitat and their connections to its anti-cancer capabilities
We are working with the hypoxia-tolerant (6% O2 measured in the field and down to 3% O2 in laboratory experiments), long-lived (>20 years) subterranean blind mole-rat, Spalax, which shows an outstanding cancer-resistance and anti-cancer capabilities. Observations of thousands of individuals at our Institute have never noticed a spontaneous malignant tumor. Furthermore, among a growing long list of hypoxia-controlled genes that show difference in structure and expression between Spalax and rat, and under different hypoxic-stress and normoxic conditions, we have found in Spalax different structure and function of major genes related to cancer (VEGF, p53, heparanase , genes of antioxidant defense and DNA-repair genes). Genome-wide sequencing and assessment of Spalax tanscriptome assembly and expression data has revealed enrichment of genes that overlap cancer resistance ,apoptosis, angiogenesis, and hypoxia-tolerance and elicits much wider and stronger expression in Spalax than in rat .
Moreover, Spalax has shown extremely high cancer-resistance to chemical carcinogens that induced cancer in 100% of mice and rats. Most intriguing, fibroblast cells only from Spalax, but not from other species, inhibit growth and kill cancer cells, but not normal cells, from various tissues and species, most importantly a wide range of human cancer cells. This is exhibited in both co-culture system or by exposure to factors secreted into conditioned media harvested from Spalax fibroblasts. Decreased cancer cell viability and proliferation, reduced colony formation in soft agar, disturbed cell cycle progression, chromatin condensation, nuclei deformation and mitochondrial fragmentation were reproducibly observed .
Our present objectives is to identify and isolate the substances secreted by Spalax cells, resolve with which components they interact that are active only on cancer cells, in order to unravel the biological mechanisms and pathways that evolved in Spalax cell machinery and ultimately lead to the death of cancer-cells. The study may attest to be a breakthrough in the conservative paradigm of cancer research, completely dependent on laboratory, inbred rodents. Achieving this goal will open new horizons in cancer studies, and may prove that the long lived, hypoxia- and cancer-tolerant Spalax is a significant biological resource to biomedical research as an organism for treatment and prevention of cancer in humans.
Effects of conditioned media generated by Spalax fibroblasts on viability of cancerous and non-cancerous cells.
Hep3B cells (1 × 104 cell/well) were cultured in 6-well plates under conditioned medium of Spalax adult skin fibroblasts (B) or grown in medium generated by Hep3B cells (C). After 9-days, cells were collected, and their survival rates were assessed by trypan blue extrusion assay and analyzed by using a Countess® automatic cell counter (Life Technologies); red: dead cells, blue: viable cells.
Spalax fibroblast-conditioned medium compromises cell cycle, causes nuclear and mitochondrial fragmentation in Hep3B cells.
Hep3B cells were grown on cover slips under medium conditioned by Spalax or rat fibroblasts for 7 days. Representative phase-contrast images demonstrating morphological changes (×200) are depicted. Cells were harvested and stained with PI, and cell cycles were analyzed by flow cytometry. Representative flow cytometry histograms of three independent experiments performed in duplicate are presented. Hep3B cells were stained with MitoTracker®Red, fixed with formaldehyde and counterstained with DAPI. Representative fluorescence microscopy images demonstrating nuclear and mitochondrial changes are present. White arrows point fragmented nuclei; empty arrows point chromatin condensation. Scale bars represent 10 μm.
Effect of Spalax, rat and mouse fibroblasts on Spalax-derived fibrosarcoma cells colony formation.
(A) Cancer cells were grown in soft agar on top of monolayers of mouse, rat, and Spalax fibroblasts. After 3 weeks colonies were counted. At least 10 fields were recorded for each observation. Two representative images demonstrating effects of different fibroblasts on colony-formation are shown (×40). (B) Colony numbers and cumulative total colony area (μm2) from 5 fields were calculated to demonstrate the effects of the fibroblasts monolayer on the cancer cell colony formation and growth.