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Aberrant Wnt/β-catenin signaling and elevated expression of stem cell proteins are associated with osteosarcoma side population cells of high tumorigenicity.

Yi XJ, Zhao YH, Qiao LX, Jin CL, Tian J, Li QS - Mol Med Rep (2015)

Bottom Line: The results of subsequent western blot and reverse transcription‑quantitative polymerase chain reaction analyses revealed that the protein levels of β‑catenin and cyclin D1 were markedly upregulated in the fluorescence‑activated cell sorted osteosarcoma SP cells.In addition, the elevated expression levels of stem cell proteins, including CD133, nestin Oct‑4, Sox‑2 and Nanog were significantly higher in the SP cells, which contributed to self‑renewal and enhanced the proliferation rate of the SP cells.Taken together, these data suggested that the identification of novel anticancer drugs, which suppress the Wnt/β‑catenin signaling and its downstream pathway may assist in eradicating osteosarcoma stem cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Traumatology, Linyi People's Hospital, Linyi, Shandong 276000, P.R. China.

ABSTRACT
According to the cancer stem cell theory, the presence of a small sub‑population of cancer cells, termed cancer stem cells (CSCs), have a significant implication on cancer treatment and are responsible for tumor recurrence. Previous studies have reported that alterations in the Wnt/β‑catenin signaling are crucial in the maintenance of CSCs. In the present study, the characteristic features and activation of Wnt/β‑catenin signaling in CSCs from osteosarcoma, an aggressive human bone tumor, were investigated. In total, ~2.1% of the cancer stem‑like side population (SP) cells were identified in the osteosarcoma samples. The results of subsequent western blot and reverse transcription‑quantitative polymerase chain reaction analyses revealed that the protein levels of β‑catenin and cyclin D1 were markedly upregulated in the fluorescence‑activated cell sorted osteosarcoma SP cells. In addition, the elevated expression levels of stem cell proteins, including CD133, nestin Oct‑4, Sox‑2 and Nanog were significantly higher in the SP cells, which contributed to self‑renewal and enhanced the proliferation rate of the SP cells. Furthermore, the SP cells were found to be highly invasive and able to form tumors in vivo. Taken together, these data suggested that the identification of novel anticancer drugs, which suppress the Wnt/β‑catenin signaling and its downstream pathway may assist in eradicating osteosarcoma stem cells.

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Clone formation efficiency of osteosarcoma SP cells. (A) Total number of sarcospheres generated by osteosarcoma SP cells was significantly higher than those generated by the non-SP cells. (B) Representative images of monoclonal spheres formed by osteosarcoma SP cells (magnification, ×100). The sarcospheres were generated rapidly on day 3 (D3) and the size of the spheres were increased on day 6 (D6). (C) SP cell invasiveness was measured using a Matrigel assay. SP and non-SP cells (4×105) were seeded and incubated for 72 h. The graph represents the number of cells, which invaded across the membrane. The data are expressed as the mean ± standard deviation. **P<0.01; *P<0.05 vs. non-SP cells. SP, side population.
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f4-mmr-12-04-5042: Clone formation efficiency of osteosarcoma SP cells. (A) Total number of sarcospheres generated by osteosarcoma SP cells was significantly higher than those generated by the non-SP cells. (B) Representative images of monoclonal spheres formed by osteosarcoma SP cells (magnification, ×100). The sarcospheres were generated rapidly on day 3 (D3) and the size of the spheres were increased on day 6 (D6). (C) SP cell invasiveness was measured using a Matrigel assay. SP and non-SP cells (4×105) were seeded and incubated for 72 h. The graph represents the number of cells, which invaded across the membrane. The data are expressed as the mean ± standard deviation. **P<0.01; *P<0.05 vs. non-SP cells. SP, side population.

Mentions: In order to compare the clone formation efficiency of the FACS-sorted SP and non-SP cells, sphere formation and invasion assays were performed. The total number of sarcospheres generated by the osteosarcoma SP cells was significantly higher, compared with the non-SP cells (Fig. 4A). Similarly, the sarcospheres generated by the SP cells were increased in size following continuous culture, and attained maximal size on day 6 (Fig. 4B). However, the non-SP cells did not attain a mature size. In addition, the in vitro Matrigel invasion assay demonstrated that FACS-sorted SP cells were significantly more invasive, compared with the non-SP cells (Fig. 4C). Taken together, the SP cells were capable of initiating tumor growth and causing tumor invasion.


Aberrant Wnt/β-catenin signaling and elevated expression of stem cell proteins are associated with osteosarcoma side population cells of high tumorigenicity.

Yi XJ, Zhao YH, Qiao LX, Jin CL, Tian J, Li QS - Mol Med Rep (2015)

Clone formation efficiency of osteosarcoma SP cells. (A) Total number of sarcospheres generated by osteosarcoma SP cells was significantly higher than those generated by the non-SP cells. (B) Representative images of monoclonal spheres formed by osteosarcoma SP cells (magnification, ×100). The sarcospheres were generated rapidly on day 3 (D3) and the size of the spheres were increased on day 6 (D6). (C) SP cell invasiveness was measured using a Matrigel assay. SP and non-SP cells (4×105) were seeded and incubated for 72 h. The graph represents the number of cells, which invaded across the membrane. The data are expressed as the mean ± standard deviation. **P<0.01; *P<0.05 vs. non-SP cells. SP, side population.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581745&req=5

f4-mmr-12-04-5042: Clone formation efficiency of osteosarcoma SP cells. (A) Total number of sarcospheres generated by osteosarcoma SP cells was significantly higher than those generated by the non-SP cells. (B) Representative images of monoclonal spheres formed by osteosarcoma SP cells (magnification, ×100). The sarcospheres were generated rapidly on day 3 (D3) and the size of the spheres were increased on day 6 (D6). (C) SP cell invasiveness was measured using a Matrigel assay. SP and non-SP cells (4×105) were seeded and incubated for 72 h. The graph represents the number of cells, which invaded across the membrane. The data are expressed as the mean ± standard deviation. **P<0.01; *P<0.05 vs. non-SP cells. SP, side population.
Mentions: In order to compare the clone formation efficiency of the FACS-sorted SP and non-SP cells, sphere formation and invasion assays were performed. The total number of sarcospheres generated by the osteosarcoma SP cells was significantly higher, compared with the non-SP cells (Fig. 4A). Similarly, the sarcospheres generated by the SP cells were increased in size following continuous culture, and attained maximal size on day 6 (Fig. 4B). However, the non-SP cells did not attain a mature size. In addition, the in vitro Matrigel invasion assay demonstrated that FACS-sorted SP cells were significantly more invasive, compared with the non-SP cells (Fig. 4C). Taken together, the SP cells were capable of initiating tumor growth and causing tumor invasion.

Bottom Line: The results of subsequent western blot and reverse transcription‑quantitative polymerase chain reaction analyses revealed that the protein levels of β‑catenin and cyclin D1 were markedly upregulated in the fluorescence‑activated cell sorted osteosarcoma SP cells.In addition, the elevated expression levels of stem cell proteins, including CD133, nestin Oct‑4, Sox‑2 and Nanog were significantly higher in the SP cells, which contributed to self‑renewal and enhanced the proliferation rate of the SP cells.Taken together, these data suggested that the identification of novel anticancer drugs, which suppress the Wnt/β‑catenin signaling and its downstream pathway may assist in eradicating osteosarcoma stem cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Traumatology, Linyi People's Hospital, Linyi, Shandong 276000, P.R. China.

ABSTRACT
According to the cancer stem cell theory, the presence of a small sub‑population of cancer cells, termed cancer stem cells (CSCs), have a significant implication on cancer treatment and are responsible for tumor recurrence. Previous studies have reported that alterations in the Wnt/β‑catenin signaling are crucial in the maintenance of CSCs. In the present study, the characteristic features and activation of Wnt/β‑catenin signaling in CSCs from osteosarcoma, an aggressive human bone tumor, were investigated. In total, ~2.1% of the cancer stem‑like side population (SP) cells were identified in the osteosarcoma samples. The results of subsequent western blot and reverse transcription‑quantitative polymerase chain reaction analyses revealed that the protein levels of β‑catenin and cyclin D1 were markedly upregulated in the fluorescence‑activated cell sorted osteosarcoma SP cells. In addition, the elevated expression levels of stem cell proteins, including CD133, nestin Oct‑4, Sox‑2 and Nanog were significantly higher in the SP cells, which contributed to self‑renewal and enhanced the proliferation rate of the SP cells. Furthermore, the SP cells were found to be highly invasive and able to form tumors in vivo. Taken together, these data suggested that the identification of novel anticancer drugs, which suppress the Wnt/β‑catenin signaling and its downstream pathway may assist in eradicating osteosarcoma stem cells.

Show MeSH
Related in: MedlinePlus