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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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Related in: MedlinePlus

Dot-plot analysis of FACS for sorting of osteosarcoma SP cells. (A) Cells were stained with Hoechst 33342 dye and SP cells of 2.1% were identified and outlined (gated population). (B) Percentage of SP cells was significantly reduced to 0.3% in the presence of verapamil. (C) Graphical representation of the SP cells with and without verapamil treatment. This quantitative graph was constructed with data from the dot blot analysis using FACS. SP, side population; FACS, fluorescence-activated cell sorting.
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f1-mmr-12-04-5042: Dot-plot analysis of FACS for sorting of osteosarcoma SP cells. (A) Cells were stained with Hoechst 33342 dye and SP cells of 2.1% were identified and outlined (gated population). (B) Percentage of SP cells was significantly reduced to 0.3% in the presence of verapamil. (C) Graphical representation of the SP cells with and without verapamil treatment. This quantitative graph was constructed with data from the dot blot analysis using FACS. SP, side population; FACS, fluorescence-activated cell sorting.

Mentions: The present study investigated the presence of cancer stem-like SP cells in osteosarcoma samples. Using FACS, a small population of SP cells of ~2.1% were found to exclude the Hoechst 33342 dye (Fig. 1A; gated region). The action of pumping out Hoechst 33342 dye is actively performed by over-expression of the ABC transporter protein, ABCG2, which was confirmed by treatment of cells with verapamil. Upon verapamil treatment, the population of SP cells were significantly reduced to 0.7% (Fig. 1B; gated region). These data indicated that the SP cells were resistant to drug uptake due to the overexpression of ABC transporter proteins (Fig. 1C).


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)

Dot-plot analysis of FACS for sorting of osteosarcoma SP cells. (A) Cells were stained with Hoechst 33342 dye and SP cells of 2.1% were identified and outlined (gated population). (B) Percentage of SP cells was significantly reduced to 0.3% in the presence of verapamil. (C) Graphical representation of the SP cells with and without verapamil treatment. This quantitative graph was constructed with data from the dot blot analysis using FACS. SP, side population; FACS, fluorescence-activated cell sorting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-mmr-12-04-5042: Dot-plot analysis of FACS for sorting of osteosarcoma SP cells. (A) Cells were stained with Hoechst 33342 dye and SP cells of 2.1% were identified and outlined (gated population). (B) Percentage of SP cells was significantly reduced to 0.3% in the presence of verapamil. (C) Graphical representation of the SP cells with and without verapamil treatment. This quantitative graph was constructed with data from the dot blot analysis using FACS. SP, side population; FACS, fluorescence-activated cell sorting.
Mentions: The present study investigated the presence of cancer stem-like SP cells in osteosarcoma samples. Using FACS, a small population of SP cells of ~2.1% were found to exclude the Hoechst 33342 dye (Fig. 1A; gated region). The action of pumping out Hoechst 33342 dye is actively performed by over-expression of the ABC transporter protein, ABCG2, which was confirmed by treatment of cells with verapamil. Upon verapamil treatment, the population of SP cells were significantly reduced to 0.7% (Fig. 1B; gated region). These data indicated that the SP cells were resistant to drug uptake due to the overexpression of ABC transporter proteins (Fig. 1C).

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