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Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody.

Thammasit P, Sangboonruang S, Suwanpairoj S, Khamaikawin W, Intasai N, Kasinrerk W, Tayapiwatana C, Tragoolpua K - J Cancer (2015)

Bottom Line: The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression.In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3.In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level.

View Article: PubMed Central - PubMed

Affiliation: 1. Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.

ABSTRACT
Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer.

No MeSH data available.


Related in: MedlinePlus

Down-regulation of EMMPRIN cell surface expression via scFv-M6-1B9 intrabody and its accumulation inside Caco-2 cells. (A) Western blotting analysis of EMMPRIN protein expression and (B) densitometry analysis of EMMPRIN expression. The white, gray, and black bars represent the protein fractions from untransduced, Ad5/F35-scFv-irrelevant, and Ad5/F35-scFv-M6-1B9 transduced Caco-2 cells, respectively. Y-axis is the fold change of the EMMPRIN expression which was normalized with GAPDH. * p<0.05, as determined by one-way ANOVA.
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Figure 1: Down-regulation of EMMPRIN cell surface expression via scFv-M6-1B9 intrabody and its accumulation inside Caco-2 cells. (A) Western blotting analysis of EMMPRIN protein expression and (B) densitometry analysis of EMMPRIN expression. The white, gray, and black bars represent the protein fractions from untransduced, Ad5/F35-scFv-irrelevant, and Ad5/F35-scFv-M6-1B9 transduced Caco-2 cells, respectively. Y-axis is the fold change of the EMMPRIN expression which was normalized with GAPDH. * p<0.05, as determined by one-way ANOVA.

Mentions: Our previous study revealed for the first time that scFv-M6-1B9 intrabody has the ability to down-regulate EMMPRIN cell surface expression on colorectal cancer cell, Caco-2, via flow cytometry 31. To confirm the reduction of the EMMPRIN cell surface expression due to its accumulation inside the cells, immunoblotting was performed. The reduction of EMMPRIN cell surface expression had been demonstrated in the membrane fraction protein of the Caco-2 cells expressing scFv-M6-1B9 intrabody by 50%, compared to untransduced and irrelevant control. Interestingly, increasing accumulation of EMMPRIN was found in the cytoplasmic fraction protein of the Caco-2 cells expressing scFv-M6-1B9 intrabody, as shown in Figure 1. This indicates that the scFv-M6-1B9 intrabody dramatically reduced EMMPRIN cell surface expression and that these molecules accumulated inside the Caco-2 cells.


Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody.

Thammasit P, Sangboonruang S, Suwanpairoj S, Khamaikawin W, Intasai N, Kasinrerk W, Tayapiwatana C, Tragoolpua K - J Cancer (2015)

Down-regulation of EMMPRIN cell surface expression via scFv-M6-1B9 intrabody and its accumulation inside Caco-2 cells. (A) Western blotting analysis of EMMPRIN protein expression and (B) densitometry analysis of EMMPRIN expression. The white, gray, and black bars represent the protein fractions from untransduced, Ad5/F35-scFv-irrelevant, and Ad5/F35-scFv-M6-1B9 transduced Caco-2 cells, respectively. Y-axis is the fold change of the EMMPRIN expression which was normalized with GAPDH. * p<0.05, as determined by one-way ANOVA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4317764&req=5

Figure 1: Down-regulation of EMMPRIN cell surface expression via scFv-M6-1B9 intrabody and its accumulation inside Caco-2 cells. (A) Western blotting analysis of EMMPRIN protein expression and (B) densitometry analysis of EMMPRIN expression. The white, gray, and black bars represent the protein fractions from untransduced, Ad5/F35-scFv-irrelevant, and Ad5/F35-scFv-M6-1B9 transduced Caco-2 cells, respectively. Y-axis is the fold change of the EMMPRIN expression which was normalized with GAPDH. * p<0.05, as determined by one-way ANOVA.
Mentions: Our previous study revealed for the first time that scFv-M6-1B9 intrabody has the ability to down-regulate EMMPRIN cell surface expression on colorectal cancer cell, Caco-2, via flow cytometry 31. To confirm the reduction of the EMMPRIN cell surface expression due to its accumulation inside the cells, immunoblotting was performed. The reduction of EMMPRIN cell surface expression had been demonstrated in the membrane fraction protein of the Caco-2 cells expressing scFv-M6-1B9 intrabody by 50%, compared to untransduced and irrelevant control. Interestingly, increasing accumulation of EMMPRIN was found in the cytoplasmic fraction protein of the Caco-2 cells expressing scFv-M6-1B9 intrabody, as shown in Figure 1. This indicates that the scFv-M6-1B9 intrabody dramatically reduced EMMPRIN cell surface expression and that these molecules accumulated inside the Caco-2 cells.

Bottom Line: The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression.In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3.In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level.

View Article: PubMed Central - PubMed

Affiliation: 1. Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.

ABSTRACT
Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer.

No MeSH data available.


Related in: MedlinePlus