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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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Phenotypic characterization of FACS-sorted osteosarcoma SP cells. (A) In vitro cell proliferation assay. The cell proliferation rates of the SP cells were significantly higher than those of the non-SP cells. The x-axis represents days (D) 1–7, the y-axis indicates the corresponding OD value at 450 nm. (B) Comparison of cell survival rates of the SP cells and non-SP cells following treatment with etoposide, gemcitabine, 5-FU, cisplatin, paclitaxel and oxaliplatin. The SP cells exhibited increased resistance and increased survival rates (>80%), compared with the non-SP cells (<35%). (C) Immunocytochemistry analysis of the sorted SP cells (magnification, x100). SP cells exhibited enhanced expression of ABCG2 (stained red), compared with the non-SP cells. Cells were counterstained with hematoxylin. The data are expressed as the mean ± standard deviation. *P<0.05; **P<0.01 vs. non-SP cells. SP, side population; 5-FU, 5-fluorouracil; OD, optical density.
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f2-mmr-12-04-5042: Phenotypic characterization of FACS-sorted osteosarcoma SP cells. (A) In vitro cell proliferation assay. The cell proliferation rates of the SP cells were significantly higher than those of the non-SP cells. The x-axis represents days (D) 1–7, the y-axis indicates the corresponding OD value at 450 nm. (B) Comparison of cell survival rates of the SP cells and non-SP cells following treatment with etoposide, gemcitabine, 5-FU, cisplatin, paclitaxel and oxaliplatin. The SP cells exhibited increased resistance and increased survival rates (>80%), compared with the non-SP cells (<35%). (C) Immunocytochemistry analysis of the sorted SP cells (magnification, x100). SP cells exhibited enhanced expression of ABCG2 (stained red), compared with the non-SP cells. Cells were counterstained with hematoxylin. The data are expressed as the mean ± standard deviation. *P<0.05; **P<0.01 vs. non-SP cells. SP, side population; 5-FU, 5-fluorouracil; OD, optical density.

Mentions: In order to characterize the FACS-sorted SP cells, the SP and non-SP cells were subsequently subjected to in vitro cell proliferation and multi-drug resistance assays. Notably, the osteosarcoma SP cells (Fig. 2A) underwent rapid cell proliferation, beginning on the third day (D3), and became more confluent on the eight day (data not shown). However, the growth rates of the non-SP cells were significantly lower, compared with the SP cells (Fig. 2A). Similarly, the SP cells were resistant to uptake drugs, including etoposide, gemcitabine, 5-flurouracil (5-FU), cisplatin, paclitaxel and oxaliplatin. Upon treatment with these drugs, the survival rate of the SP cells was significantly higher, compared with the non-SP cells (Fig. 2B). The increased drug resistance of the SP cells was most likely due to overexpression of ABCG2 in the SP cells. As shown in the Fig. 2C, the SP cells were more positive to ABCG2 than the non-SP cells. Therefore, these findings demonstrated that the osteosarcoma SP cells underwent marked proliferation and exhibited enhanced survival rates following chemotherapy.


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)

Phenotypic characterization of FACS-sorted osteosarcoma SP cells. (A) In vitro cell proliferation assay. The cell proliferation rates of the SP cells were significantly higher than those of the non-SP cells. The x-axis represents days (D) 1–7, the y-axis indicates the corresponding OD value at 450 nm. (B) Comparison of cell survival rates of the SP cells and non-SP cells following treatment with etoposide, gemcitabine, 5-FU, cisplatin, paclitaxel and oxaliplatin. The SP cells exhibited increased resistance and increased survival rates (>80%), compared with the non-SP cells (<35%). (C) Immunocytochemistry analysis of the sorted SP cells (magnification, x100). SP cells exhibited enhanced expression of ABCG2 (stained red), compared with the non-SP cells. Cells were counterstained with hematoxylin. The data are expressed as the mean ± standard deviation. *P<0.05; **P<0.01 vs. non-SP cells. SP, side population; 5-FU, 5-fluorouracil; OD, optical density.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-mmr-12-04-5042: Phenotypic characterization of FACS-sorted osteosarcoma SP cells. (A) In vitro cell proliferation assay. The cell proliferation rates of the SP cells were significantly higher than those of the non-SP cells. The x-axis represents days (D) 1–7, the y-axis indicates the corresponding OD value at 450 nm. (B) Comparison of cell survival rates of the SP cells and non-SP cells following treatment with etoposide, gemcitabine, 5-FU, cisplatin, paclitaxel and oxaliplatin. The SP cells exhibited increased resistance and increased survival rates (>80%), compared with the non-SP cells (<35%). (C) Immunocytochemistry analysis of the sorted SP cells (magnification, x100). SP cells exhibited enhanced expression of ABCG2 (stained red), compared with the non-SP cells. Cells were counterstained with hematoxylin. The data are expressed as the mean ± standard deviation. *P<0.05; **P<0.01 vs. non-SP cells. SP, side population; 5-FU, 5-fluorouracil; OD, optical density.
Mentions: In order to characterize the FACS-sorted SP cells, the SP and non-SP cells were subsequently subjected to in vitro cell proliferation and multi-drug resistance assays. Notably, the osteosarcoma SP cells (Fig. 2A) underwent rapid cell proliferation, beginning on the third day (D3), and became more confluent on the eight day (data not shown). However, the growth rates of the non-SP cells were significantly lower, compared with the SP cells (Fig. 2A). Similarly, the SP cells were resistant to uptake drugs, including etoposide, gemcitabine, 5-flurouracil (5-FU), cisplatin, paclitaxel and oxaliplatin. Upon treatment with these drugs, the survival rate of the SP cells was significantly higher, compared with the non-SP cells (Fig. 2B). The increased drug resistance of the SP cells was most likely due to overexpression of ABCG2 in the SP cells. As shown in the Fig. 2C, the SP cells were more positive to ABCG2 than the non-SP cells. Therefore, these findings demonstrated that the osteosarcoma SP cells underwent marked proliferation and exhibited enhanced survival rates following chemotherapy.

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