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Adenovirus-mediated siRNA targeting Bcl-xL inhibits proliferation, reduces invasion and enhances radiosensitivity of human colorectal cancer cells.

Yang J, Sun M, Zhang A, Lv C, De W, Wang Z - World J Surg Oncol (2011)

Bottom Line: Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells.Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells.Targeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China.

ABSTRACT

Introduction: Bcl-xL, an important member of anti-apoptotic Bcl-2 family, plays critical roles in tumor progression and development. Previously, we have reported that overexpression of Bcl-xL was correlated with prognosis of colorectal cancer (CRC) patients. The aim of this study was to investigate the association of Bcl-xL expression with invasion and radiosensitivity of human CRC cells.

Methods: RT-PCR and Western blot assays were performed to determine the expression of Bcl-xL mRNA and protein in CRC cells and normal human intestinal epithelial cell line. Then, adenovirus-mediated RNA interference technique was employed to inhibit the expression of Bcl-xL gene in CRC cells. The proliferation of CRC cells was analyzed by MTT and colony formation assay. The migration and invasion of CRC cells was determined by wound-healing and tranwell invasion assays. Additionally, the in vitro and in vivo radiosensitivity of CRC cells was determined by clonogenic cell survival assay and murine xnograft model, respectively.

Results: The levels of Bcl-xL mRNA and protein expression were significantly higher in human CRC cells than in normal human intestinal epithelial cell line. Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells. Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells. Ad/shBcl-xL could significantly suppress migration and invasion of CRC cells. Moreover, Ad/shBcl-xL could enhance in vitro and in vivo radiosensitivity of CRC cells by increasing caspase-dependent apoptosis.

Conclusions: Targeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.

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Effects of Ad/shBcl-xL on the in vitro sensitivity of LoVo cells to irradiation. A. Clonogenic survival after varying doses of irradiation exposure. The mock, Ad/shcontrol or Ad/shBcl-xL-infected LoVo cells were irradiated followed by a further incubation for 24 h at 37°C before trypsinization and plating for clonogenic survival. After 10-14 days incubation, colonies were stained, and the surviving fraction was determined. The log survival was formed to the number of cells plated, after correcting for plating efficiency. B. Flow cytometry analysis of apoptosis in mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL. C. Flow cytometry analysis of apoptosis in mock, Ad/shcontrol or Ad/shBcl-xL-infected LoVo combined with irradiation treatment (8.0 Gy). Error bars, the mean ± SD in three experiments (n = 3). *P < 0.05 vs mock.
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Figure 5: Effects of Ad/shBcl-xL on the in vitro sensitivity of LoVo cells to irradiation. A. Clonogenic survival after varying doses of irradiation exposure. The mock, Ad/shcontrol or Ad/shBcl-xL-infected LoVo cells were irradiated followed by a further incubation for 24 h at 37°C before trypsinization and plating for clonogenic survival. After 10-14 days incubation, colonies were stained, and the surviving fraction was determined. The log survival was formed to the number of cells plated, after correcting for plating efficiency. B. Flow cytometry analysis of apoptosis in mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL. C. Flow cytometry analysis of apoptosis in mock, Ad/shcontrol or Ad/shBcl-xL-infected LoVo combined with irradiation treatment (8.0 Gy). Error bars, the mean ± SD in three experiments (n = 3). *P < 0.05 vs mock.

Mentions: Previously, we have reported that the overexpression of Bcl-xL could affect the sensitivity of osteosarcoma cells to irradiation, but the association of Bcl-xL expression with the radiosensitivity of CRC cells is still unclear. To investigate the radiosensitizing effects of adenovirus-mediated siRNA targeting Bcl-xL on CRC cells, a colony-forming assay was performed. The surviving fraction of the cells infected with Ad/shBcl-xL at a MOI of 80 was significantly lower than that of mock LoVo or Ad/shcontrol-infected LoVo cells at doses of 4.0 and 10.0 Gy (Figure 5A). Then, flow cytometry was performed to analyze the changes of apoptosis in mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL combined with or without irradiation (8.0 Gy). As shown in Figure 5B, the apoptotic rate of Ad/shBcl-xL-infected LoVo cells was obviously increased by approximately 11.4% compared with mock LoVo cells (P < 0.05). The apoptotic rates of mock LoVo or Ad/shcontrol-infected LoVo cells treated with 8.0-Gy irradiation alone were approximately 12.6% and 14.2%, respectively. However, the apoptotic rate of Ad/shBcl-xL-infected LoVo cells increased to 23.6% (P < 0.05; Figure 5C). Thus, adenovirus-mediated siRNA targeting Bcl-xL could enhance the radiosensitivity of CRC cells by the increase of radiation-induced apoptosis.


Adenovirus-mediated siRNA targeting Bcl-xL inhibits proliferation, reduces invasion and enhances radiosensitivity of human colorectal cancer cells.

Yang J, Sun M, Zhang A, Lv C, De W, Wang Z - World J Surg Oncol (2011)

Effects of Ad/shBcl-xL on the in vitro sensitivity of LoVo cells to irradiation. A. Clonogenic survival after varying doses of irradiation exposure. The mock, Ad/shcontrol or Ad/shBcl-xL-infected LoVo cells were irradiated followed by a further incubation for 24 h at 37°C before trypsinization and plating for clonogenic survival. After 10-14 days incubation, colonies were stained, and the surviving fraction was determined. The log survival was formed to the number of cells plated, after correcting for plating efficiency. B. Flow cytometry analysis of apoptosis in mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL. C. Flow cytometry analysis of apoptosis in mock, Ad/shcontrol or Ad/shBcl-xL-infected LoVo combined with irradiation treatment (8.0 Gy). Error bars, the mean ± SD in three experiments (n = 3). *P < 0.05 vs mock.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198948&req=5

Figure 5: Effects of Ad/shBcl-xL on the in vitro sensitivity of LoVo cells to irradiation. A. Clonogenic survival after varying doses of irradiation exposure. The mock, Ad/shcontrol or Ad/shBcl-xL-infected LoVo cells were irradiated followed by a further incubation for 24 h at 37°C before trypsinization and plating for clonogenic survival. After 10-14 days incubation, colonies were stained, and the surviving fraction was determined. The log survival was formed to the number of cells plated, after correcting for plating efficiency. B. Flow cytometry analysis of apoptosis in mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL. C. Flow cytometry analysis of apoptosis in mock, Ad/shcontrol or Ad/shBcl-xL-infected LoVo combined with irradiation treatment (8.0 Gy). Error bars, the mean ± SD in three experiments (n = 3). *P < 0.05 vs mock.
Mentions: Previously, we have reported that the overexpression of Bcl-xL could affect the sensitivity of osteosarcoma cells to irradiation, but the association of Bcl-xL expression with the radiosensitivity of CRC cells is still unclear. To investigate the radiosensitizing effects of adenovirus-mediated siRNA targeting Bcl-xL on CRC cells, a colony-forming assay was performed. The surviving fraction of the cells infected with Ad/shBcl-xL at a MOI of 80 was significantly lower than that of mock LoVo or Ad/shcontrol-infected LoVo cells at doses of 4.0 and 10.0 Gy (Figure 5A). Then, flow cytometry was performed to analyze the changes of apoptosis in mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL combined with or without irradiation (8.0 Gy). As shown in Figure 5B, the apoptotic rate of Ad/shBcl-xL-infected LoVo cells was obviously increased by approximately 11.4% compared with mock LoVo cells (P < 0.05). The apoptotic rates of mock LoVo or Ad/shcontrol-infected LoVo cells treated with 8.0-Gy irradiation alone were approximately 12.6% and 14.2%, respectively. However, the apoptotic rate of Ad/shBcl-xL-infected LoVo cells increased to 23.6% (P < 0.05; Figure 5C). Thus, adenovirus-mediated siRNA targeting Bcl-xL could enhance the radiosensitivity of CRC cells by the increase of radiation-induced apoptosis.

Bottom Line: Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells.Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells.Targeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China.

ABSTRACT

Introduction: Bcl-xL, an important member of anti-apoptotic Bcl-2 family, plays critical roles in tumor progression and development. Previously, we have reported that overexpression of Bcl-xL was correlated with prognosis of colorectal cancer (CRC) patients. The aim of this study was to investigate the association of Bcl-xL expression with invasion and radiosensitivity of human CRC cells.

Methods: RT-PCR and Western blot assays were performed to determine the expression of Bcl-xL mRNA and protein in CRC cells and normal human intestinal epithelial cell line. Then, adenovirus-mediated RNA interference technique was employed to inhibit the expression of Bcl-xL gene in CRC cells. The proliferation of CRC cells was analyzed by MTT and colony formation assay. The migration and invasion of CRC cells was determined by wound-healing and tranwell invasion assays. Additionally, the in vitro and in vivo radiosensitivity of CRC cells was determined by clonogenic cell survival assay and murine xnograft model, respectively.

Results: The levels of Bcl-xL mRNA and protein expression were significantly higher in human CRC cells than in normal human intestinal epithelial cell line. Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells. Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells. Ad/shBcl-xL could significantly suppress migration and invasion of CRC cells. Moreover, Ad/shBcl-xL could enhance in vitro and in vivo radiosensitivity of CRC cells by increasing caspase-dependent apoptosis.

Conclusions: Targeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.

Show MeSH
Related in: MedlinePlus