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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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Related in: MedlinePlus

Effects of Ad/shBcl-xL on migration and invasion of LoVo cells. A. Scratch wound-healing assay. The cells were infected with mock, Ad/shcontrol or Ad/shBcl-xL as indicated. 72 h after wounding, cells with extended membrane protrusion moved into the wounded areas. Mock: no adenovirus. B. Transwell invasion assay. The invasive LoVo cells were stained and counted under microscope and quantitative results for the transmembrane ability of each group of cells (n = 3). *P < 0.05 vs mock.
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Figure 4: Effects of Ad/shBcl-xL on migration and invasion of LoVo cells. A. Scratch wound-healing assay. The cells were infected with mock, Ad/shcontrol or Ad/shBcl-xL as indicated. 72 h after wounding, cells with extended membrane protrusion moved into the wounded areas. Mock: no adenovirus. B. Transwell invasion assay. The invasive LoVo cells were stained and counted under microscope and quantitative results for the transmembrane ability of each group of cells (n = 3). *P < 0.05 vs mock.

Mentions: Prior researches show that Bcl-xL is an anti-apoptotic protein of Bcl-2 family involved in the regulation and promotion of tumor cell survival, but whether Bcl-xL affects the process of tumor invasion and metastasis is still unclear. To investigate the effect of Bcl-xL inhibition on migration and invasion of CRC cells, we performed the scratch-wound and matrigel transwell assays. Scratch-wound assay on confluent monolayers was used as a way of determining cell migration. The mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL were reseeded in the six-well culture wells with the same cell number for the wound healing assay. At 48 h after wounding, the healing ability of Ad/shBcl-xL-infected LoVo cells significantly lagged behind the mock LoVo or Ad/shcontrol-infected LoVo cells (Figure 4A). Matrigel transwell assay was done to analyze the changes of in vitro invasion capacity of LoVo cells (Figure 4B). Ad/shBcl-xL-infected LoVo cells showed a significantly decreased invasion capacity compared with mock LoVo cells or Ad/shcontrol-infected LoVo cells. A representative experiment demonstrates a marked reduction in the migration of LoVo cells after Ad/shBcl-xL infection compared with mock or Ad/shcontrol infection (P < 0.05). The results were quantified from three independent experiments confirming significant decrease in the number of Bcl-xL-ablated LoVo migrating through collagen. These data indicated that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit in vitro migration and invasion of CRC cells.


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 migration and invasion of LoVo cells. A. Scratch wound-healing assay. The cells were infected with mock, Ad/shcontrol or Ad/shBcl-xL as indicated. 72 h after wounding, cells with extended membrane protrusion moved into the wounded areas. Mock: no adenovirus. B. Transwell invasion assay. The invasive LoVo cells were stained and counted under microscope and quantitative results for the transmembrane ability of each group of cells (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 4: Effects of Ad/shBcl-xL on migration and invasion of LoVo cells. A. Scratch wound-healing assay. The cells were infected with mock, Ad/shcontrol or Ad/shBcl-xL as indicated. 72 h after wounding, cells with extended membrane protrusion moved into the wounded areas. Mock: no adenovirus. B. Transwell invasion assay. The invasive LoVo cells were stained and counted under microscope and quantitative results for the transmembrane ability of each group of cells (n = 3). *P < 0.05 vs mock.
Mentions: Prior researches show that Bcl-xL is an anti-apoptotic protein of Bcl-2 family involved in the regulation and promotion of tumor cell survival, but whether Bcl-xL affects the process of tumor invasion and metastasis is still unclear. To investigate the effect of Bcl-xL inhibition on migration and invasion of CRC cells, we performed the scratch-wound and matrigel transwell assays. Scratch-wound assay on confluent monolayers was used as a way of determining cell migration. The mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL were reseeded in the six-well culture wells with the same cell number for the wound healing assay. At 48 h after wounding, the healing ability of Ad/shBcl-xL-infected LoVo cells significantly lagged behind the mock LoVo or Ad/shcontrol-infected LoVo cells (Figure 4A). Matrigel transwell assay was done to analyze the changes of in vitro invasion capacity of LoVo cells (Figure 4B). Ad/shBcl-xL-infected LoVo cells showed a significantly decreased invasion capacity compared with mock LoVo cells or Ad/shcontrol-infected LoVo cells. A representative experiment demonstrates a marked reduction in the migration of LoVo cells after Ad/shBcl-xL infection compared with mock or Ad/shcontrol infection (P < 0.05). The results were quantified from three independent experiments confirming significant decrease in the number of Bcl-xL-ablated LoVo migrating through collagen. These data indicated that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit in vitro migration and invasion of CRC cells.

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