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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

EMMPRIN down-regulation by scFv-M6-1B9 intrabody affecting decline of CEA level. Caco-2 cells transduced for 7 days and 10 days were collected as the supernatant, extracted cells for total RNA, and reverse transcribed into cDNA. (A) CEA secretions in the Caco-2 cell supernatant. The amount of CEA released into the medium was determined using an automated analyzer. (B) The expression of CEA mRNA was analyzed using real-time RT-PCR. GAPDH mRNA was used as the internal control. The normalized mRNA expression was analyzed using the comparative CT method. The white and black bars represent day 7 and day 10 of incubation, respectively. The data are presented as mean±s.e.m. *p<0.05, as determined by one-way ANOVA.
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Figure 4: EMMPRIN down-regulation by scFv-M6-1B9 intrabody affecting decline of CEA level. Caco-2 cells transduced for 7 days and 10 days were collected as the supernatant, extracted cells for total RNA, and reverse transcribed into cDNA. (A) CEA secretions in the Caco-2 cell supernatant. The amount of CEA released into the medium was determined using an automated analyzer. (B) The expression of CEA mRNA was analyzed using real-time RT-PCR. GAPDH mRNA was used as the internal control. The normalized mRNA expression was analyzed using the comparative CT method. The white and black bars represent day 7 and day 10 of incubation, respectively. The data are presented as mean±s.e.m. *p<0.05, as determined by one-way ANOVA.

Mentions: The objective was to examine the effect of EMMPRIN down-regulation by scFv-M6-1B9 intrabody on CEA release from Caco-2 cells. CEA secretion was examined by measuring the CEA concentration in the Caco-2 cell culture supernatant. Noteworthily, more than 50% inhibition of the CEA release was produced in the Caco-2 cells expressing scFv-M6-1B9 intrabody after 10 days of incubation, as demonstrated in Figure 4A. Contrary to this observation, the untransduced and irrelevant control cells both showed high CEA levels in the supernatant.


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)

EMMPRIN down-regulation by scFv-M6-1B9 intrabody affecting decline of CEA level. Caco-2 cells transduced for 7 days and 10 days were collected as the supernatant, extracted cells for total RNA, and reverse transcribed into cDNA. (A) CEA secretions in the Caco-2 cell supernatant. The amount of CEA released into the medium was determined using an automated analyzer. (B) The expression of CEA mRNA was analyzed using real-time RT-PCR. GAPDH mRNA was used as the internal control. The normalized mRNA expression was analyzed using the comparative CT method. The white and black bars represent day 7 and day 10 of incubation, respectively. The data are presented as mean±s.e.m. *p<0.05, as determined by one-way ANOVA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4317764&req=5

Figure 4: EMMPRIN down-regulation by scFv-M6-1B9 intrabody affecting decline of CEA level. Caco-2 cells transduced for 7 days and 10 days were collected as the supernatant, extracted cells for total RNA, and reverse transcribed into cDNA. (A) CEA secretions in the Caco-2 cell supernatant. The amount of CEA released into the medium was determined using an automated analyzer. (B) The expression of CEA mRNA was analyzed using real-time RT-PCR. GAPDH mRNA was used as the internal control. The normalized mRNA expression was analyzed using the comparative CT method. The white and black bars represent day 7 and day 10 of incubation, respectively. The data are presented as mean±s.e.m. *p<0.05, as determined by one-way ANOVA.
Mentions: The objective was to examine the effect of EMMPRIN down-regulation by scFv-M6-1B9 intrabody on CEA release from Caco-2 cells. CEA secretion was examined by measuring the CEA concentration in the Caco-2 cell culture supernatant. Noteworthily, more than 50% inhibition of the CEA release was produced in the Caco-2 cells expressing scFv-M6-1B9 intrabody after 10 days of incubation, as demonstrated in Figure 4A. Contrary to this observation, the untransduced and irrelevant control cells both showed high CEA levels in the supernatant.

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