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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 in vivo sensitivity of LoVo cells to irradiation. A. Protein samples extracted from tumors at day 7 or 42 were determined using Western blot analysis for Bcl-xL expression levels. GAPDH was used as a loading control. B. Immunohistochemistry was performed to detect the expression of Bcl-xL protein in tissues of LoVo tumors treated with PBS, Ad/shcontrol or Ad/shBcl-xL at day 7 or 42. C. Proliferation of tumors in the mice injected with LoVo cells treated with PBS, Ad/shcontrol or Ad/shBcl-xL. D. Dectection of average tumor size at day 42 after the inoculation of LoVo cells treated with PBS, Ad/shcontrol or Ad/shBcl-xL. All experiments were performed in triplicate (n = 3). *P < 0.05 vs mock.
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Figure 6: Effects of Ad/shBcl-xL on the in vivo sensitivity of LoVo cells to irradiation. A. Protein samples extracted from tumors at day 7 or 42 were determined using Western blot analysis for Bcl-xL expression levels. GAPDH was used as a loading control. B. Immunohistochemistry was performed to detect the expression of Bcl-xL protein in tissues of LoVo tumors treated with PBS, Ad/shcontrol or Ad/shBcl-xL at day 7 or 42. C. Proliferation of tumors in the mice injected with LoVo cells treated with PBS, Ad/shcontrol or Ad/shBcl-xL. D. Dectection of average tumor size at day 42 after the inoculation of LoVo cells treated with PBS, Ad/shcontrol or Ad/shBcl-xL. All experiments were performed in triplicate (n = 3). *P < 0.05 vs mock.

Mentions: Firstly, Western blot assay was performed to detect the expression of Bcl-xL protein in LoVo xenografts on day 7 and 42 post injection of adenovirus. As shown in Figure 6A, the expression of Bcl-xL protein in the turmors in Ad/shBcl-xL-treated group was significantly downregulated compared with PBS or Ad/shcontrol-treated group. Immunohistochemistry assay was performed to analyze the expression of Bcl-xL protein in tumor tissues from LoVo xenografts. As shown in Figure 6B, the tumors in Ad/shBcl-xL-treated group showed significant decreases in the cytoplasmic immunostaining of Bcl-xL protein compared with PBS or Ad/shcontrol-treated group. Next, we attempted to investigate the effect of siRNA-mediated knockdown of Bcl-xL on the in vivo radiosensitivity of CRC cells. Nude mice with established tumors xenografts were treated with PBS or adenovirus (Ad/shBcl-xL or Ad/shcontrol), followed by 4.0Gy-local radiotherapy. A representative growth curve of LoVo xenografted tumors after various treatment was shown in Figure 6C. Ad/shBcl-xL plus irradiation led to a significant suppression of tumor growth compared with Ad/shcontrol plus irradiation or Ad/shBcl-xL alone (P < 0.05). On day 42, the tumor-inhibition rates of Ad/shBcl-xL group, Ad/shcontrol plus irradiation group and Ad/shBcl-xL plus irradiation group were 20.2, 35.1 and 56.8%, respectively (P < 0.05; Figure 6D). These experimental data showed that adenovirus-mediated siRNA targeting Bcl-xL could increase the in vivo radiosensitivity of CRC cells, which showed that combined Bcl-xL downregulation with radiotherapy could lead to a stronger anti-tumor effect for human CRC.


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 vivo sensitivity of LoVo cells to irradiation. A. Protein samples extracted from tumors at day 7 or 42 were determined using Western blot analysis for Bcl-xL expression levels. GAPDH was used as a loading control. B. Immunohistochemistry was performed to detect the expression of Bcl-xL protein in tissues of LoVo tumors treated with PBS, Ad/shcontrol or Ad/shBcl-xL at day 7 or 42. C. Proliferation of tumors in the mice injected with LoVo cells treated with PBS, Ad/shcontrol or Ad/shBcl-xL. D. Dectection of average tumor size at day 42 after the inoculation of LoVo cells treated with PBS, Ad/shcontrol or Ad/shBcl-xL. All experiments were performed in triplicate (n = 3). *P < 0.05 vs mock.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 6: Effects of Ad/shBcl-xL on the in vivo sensitivity of LoVo cells to irradiation. A. Protein samples extracted from tumors at day 7 or 42 were determined using Western blot analysis for Bcl-xL expression levels. GAPDH was used as a loading control. B. Immunohistochemistry was performed to detect the expression of Bcl-xL protein in tissues of LoVo tumors treated with PBS, Ad/shcontrol or Ad/shBcl-xL at day 7 or 42. C. Proliferation of tumors in the mice injected with LoVo cells treated with PBS, Ad/shcontrol or Ad/shBcl-xL. D. Dectection of average tumor size at day 42 after the inoculation of LoVo cells treated with PBS, Ad/shcontrol or Ad/shBcl-xL. All experiments were performed in triplicate (n = 3). *P < 0.05 vs mock.
Mentions: Firstly, Western blot assay was performed to detect the expression of Bcl-xL protein in LoVo xenografts on day 7 and 42 post injection of adenovirus. As shown in Figure 6A, the expression of Bcl-xL protein in the turmors in Ad/shBcl-xL-treated group was significantly downregulated compared with PBS or Ad/shcontrol-treated group. Immunohistochemistry assay was performed to analyze the expression of Bcl-xL protein in tumor tissues from LoVo xenografts. As shown in Figure 6B, the tumors in Ad/shBcl-xL-treated group showed significant decreases in the cytoplasmic immunostaining of Bcl-xL protein compared with PBS or Ad/shcontrol-treated group. Next, we attempted to investigate the effect of siRNA-mediated knockdown of Bcl-xL on the in vivo radiosensitivity of CRC cells. Nude mice with established tumors xenografts were treated with PBS or adenovirus (Ad/shBcl-xL or Ad/shcontrol), followed by 4.0Gy-local radiotherapy. A representative growth curve of LoVo xenografted tumors after various treatment was shown in Figure 6C. Ad/shBcl-xL plus irradiation led to a significant suppression of tumor growth compared with Ad/shcontrol plus irradiation or Ad/shBcl-xL alone (P < 0.05). On day 42, the tumor-inhibition rates of Ad/shBcl-xL group, Ad/shcontrol plus irradiation group and Ad/shBcl-xL plus irradiation group were 20.2, 35.1 and 56.8%, respectively (P < 0.05; Figure 6D). These experimental data showed that adenovirus-mediated siRNA targeting Bcl-xL could increase the in vivo radiosensitivity of CRC cells, which showed that combined Bcl-xL downregulation with radiotherapy could lead to a stronger anti-tumor effect for human CRC.

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