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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 induced apoptosis in Caco-2 cells. Caco-2 cells were transduced with Ad5/F35-scFv-irrelevant or Ad5/F35-scFv-M6-1B9. Transduced GFP positive cells were used for the apoptosis studies. (A) FACS analysis with PI staining for the detection of apoptotic cells. The histogram presents the percentage of apoptotic cells in sub-G1 peak, which was calculated as the percentage of all the transduced cells. (B) Detection of phosphatidylserine externalization by Annexin V assay. The dual parametric dot plot shows the viable cell population in the bottom left quadrant (Annexin V- PI-), the early apoptotic cells in the bottom right quadrant (Annexin V+ PI-), the late apoptotic cells in the top right quadrant (Annexin V+ PI+), and the necrotic cells in the top left quadrant (Annexin V- PI+). Identification of DNA fragmentation by TUNEL assay, using (C) CLSM and (D) FACS analysis. Representative fluorescent images of TUNEL showed DAPI (blue) stained nuclei, transduced cells (green), and TUNEL-positive cells (red). *p<0.05, as determined by one-way ANOVA.
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Figure 2: EMMPRIN down-regulation induced apoptosis in Caco-2 cells. Caco-2 cells were transduced with Ad5/F35-scFv-irrelevant or Ad5/F35-scFv-M6-1B9. Transduced GFP positive cells were used for the apoptosis studies. (A) FACS analysis with PI staining for the detection of apoptotic cells. The histogram presents the percentage of apoptotic cells in sub-G1 peak, which was calculated as the percentage of all the transduced cells. (B) Detection of phosphatidylserine externalization by Annexin V assay. The dual parametric dot plot shows the viable cell population in the bottom left quadrant (Annexin V- PI-), the early apoptotic cells in the bottom right quadrant (Annexin V+ PI-), the late apoptotic cells in the top right quadrant (Annexin V+ PI+), and the necrotic cells in the top left quadrant (Annexin V- PI+). Identification of DNA fragmentation by TUNEL assay, using (C) CLSM and (D) FACS analysis. Representative fluorescent images of TUNEL showed DAPI (blue) stained nuclei, transduced cells (green), and TUNEL-positive cells (red). *p<0.05, as determined by one-way ANOVA.

Mentions: To determine whether EMMPRIN down-regulation via scFv-M6-1B9 intrabody could influence apoptosis, apoptotic induction in Caco-2 cells transduced with Ad5/F35-scFv-irrelevant and Ad5/F35-scFv-M6-1B9 was analyzed using flow cytometry. Transduced GFP-positive cells were gated for further analysis of PI staining. Apoptotic cells with DNA content being lessthan the G1-phase cells, coupled with the reduced DNA content of the apoptotic nuclei, resulted in the hypodiploid DNA peak observed in the DNA fluorescence histogram. The percentage of apoptosis in the Caco-2 cells expressing scFv-M6-1B9 intrabody (43.6%) was observed to be significantly higher than the percentage in the scFv-irrelevant control (4.2%), as shown in Figure 2A.


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 induced apoptosis in Caco-2 cells. Caco-2 cells were transduced with Ad5/F35-scFv-irrelevant or Ad5/F35-scFv-M6-1B9. Transduced GFP positive cells were used for the apoptosis studies. (A) FACS analysis with PI staining for the detection of apoptotic cells. The histogram presents the percentage of apoptotic cells in sub-G1 peak, which was calculated as the percentage of all the transduced cells. (B) Detection of phosphatidylserine externalization by Annexin V assay. The dual parametric dot plot shows the viable cell population in the bottom left quadrant (Annexin V- PI-), the early apoptotic cells in the bottom right quadrant (Annexin V+ PI-), the late apoptotic cells in the top right quadrant (Annexin V+ PI+), and the necrotic cells in the top left quadrant (Annexin V- PI+). Identification of DNA fragmentation by TUNEL assay, using (C) CLSM and (D) FACS analysis. Representative fluorescent images of TUNEL showed DAPI (blue) stained nuclei, transduced cells (green), and TUNEL-positive cells (red). *p<0.05, as determined by one-way ANOVA.
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Related In: Results  -  Collection

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Figure 2: EMMPRIN down-regulation induced apoptosis in Caco-2 cells. Caco-2 cells were transduced with Ad5/F35-scFv-irrelevant or Ad5/F35-scFv-M6-1B9. Transduced GFP positive cells were used for the apoptosis studies. (A) FACS analysis with PI staining for the detection of apoptotic cells. The histogram presents the percentage of apoptotic cells in sub-G1 peak, which was calculated as the percentage of all the transduced cells. (B) Detection of phosphatidylserine externalization by Annexin V assay. The dual parametric dot plot shows the viable cell population in the bottom left quadrant (Annexin V- PI-), the early apoptotic cells in the bottom right quadrant (Annexin V+ PI-), the late apoptotic cells in the top right quadrant (Annexin V+ PI+), and the necrotic cells in the top left quadrant (Annexin V- PI+). Identification of DNA fragmentation by TUNEL assay, using (C) CLSM and (D) FACS analysis. Representative fluorescent images of TUNEL showed DAPI (blue) stained nuclei, transduced cells (green), and TUNEL-positive cells (red). *p<0.05, as determined by one-way ANOVA.
Mentions: To determine whether EMMPRIN down-regulation via scFv-M6-1B9 intrabody could influence apoptosis, apoptotic induction in Caco-2 cells transduced with Ad5/F35-scFv-irrelevant and Ad5/F35-scFv-M6-1B9 was analyzed using flow cytometry. Transduced GFP-positive cells were gated for further analysis of PI staining. Apoptotic cells with DNA content being lessthan the G1-phase cells, coupled with the reduced DNA content of the apoptotic nuclei, resulted in the hypodiploid DNA peak observed in the DNA fluorescence histogram. The percentage of apoptosis in the Caco-2 cells expressing scFv-M6-1B9 intrabody (43.6%) was observed to be significantly higher than the percentage in the scFv-irrelevant control (4.2%), as shown in Figure 2A.

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