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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 the expression of apoptosis or metastasis-related proteins in LoVo cells. A. Western blot detection of active caspase-9 (p47 and p37), caspase-3 (p32 and p17), PARP (p116 and p85) and uPA protein expression. B. Analysis of activity of uPA in mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL. The cells in 24-well plates (5.0 × 105/well) were cultured, starved in serum-free medium overnightand. Measurements were made in three separate experiments, and data are shown as mean ± standard deviation. *P < 0.05 vs mock. OD: optical density.
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Figure 7: Effects of Ad/shBcl-xL on the expression of apoptosis or metastasis-related proteins in LoVo cells. A. Western blot detection of active caspase-9 (p47 and p37), caspase-3 (p32 and p17), PARP (p116 and p85) and uPA protein expression. B. Analysis of activity of uPA in mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL. The cells in 24-well plates (5.0 × 105/well) were cultured, starved in serum-free medium overnightand. Measurements were made in three separate experiments, and data are shown as mean ± standard deviation. *P < 0.05 vs mock. OD: optical density.

Mentions: The Bcl-2 family members are important regulators of the mitochondrial pathway of apoptosis. Then, we analyzed the effect of siRNA-mediated Bcl-xL inhibition on the expression of caspase-9, caspase-3 and PARP protein. Western blot assay showed that Ad/shBcl-xL could significantly induce activation of caspase-9, caspase-3 and PARP (Figure 7A). Additionally, we showed that Ad/shBcl-xL could significantly inhibit the expression of uPA protein compared with control cells (Figure 7A). Then, we analyzed the changes of uPA activity. As shown in Figure 7B, compared with mock LoVo cells, the activity of uPA in Ad/Bcl-xL-infected LoVo cells was significantly reduced by approximately 54.6% (Figure 7B). Therefore, the changes of those proteins might be involved in Bcl-xL-induced malignant phenotypes 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 the expression of apoptosis or metastasis-related proteins in LoVo cells. A. Western blot detection of active caspase-9 (p47 and p37), caspase-3 (p32 and p17), PARP (p116 and p85) and uPA protein expression. B. Analysis of activity of uPA in mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL. The cells in 24-well plates (5.0 × 105/well) were cultured, starved in serum-free medium overnightand. Measurements were made in three separate experiments, and data are shown as mean ± standard deviation. *P < 0.05 vs mock. OD: optical density.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Effects of Ad/shBcl-xL on the expression of apoptosis or metastasis-related proteins in LoVo cells. A. Western blot detection of active caspase-9 (p47 and p37), caspase-3 (p32 and p17), PARP (p116 and p85) and uPA protein expression. B. Analysis of activity of uPA in mock LoVo or LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL. The cells in 24-well plates (5.0 × 105/well) were cultured, starved in serum-free medium overnightand. Measurements were made in three separate experiments, and data are shown as mean ± standard deviation. *P < 0.05 vs mock. OD: optical density.
Mentions: The Bcl-2 family members are important regulators of the mitochondrial pathway of apoptosis. Then, we analyzed the effect of siRNA-mediated Bcl-xL inhibition on the expression of caspase-9, caspase-3 and PARP protein. Western blot assay showed that Ad/shBcl-xL could significantly induce activation of caspase-9, caspase-3 and PARP (Figure 7A). Additionally, we showed that Ad/shBcl-xL could significantly inhibit the expression of uPA protein compared with control cells (Figure 7A). Then, we analyzed the changes of uPA activity. As shown in Figure 7B, compared with mock LoVo cells, the activity of uPA in Ad/Bcl-xL-infected LoVo cells was significantly reduced by approximately 54.6% (Figure 7B). Therefore, the changes of those proteins might be involved in Bcl-xL-induced malignant phenotypes 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