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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

Schematic diagram of possible apoptotic mechanism underlying EMMPRIN down-regulation via scFv-M6-1B9 intrabody. (A) EMMPRIN facilitates proper expression of MCT1 at the cell surface. This association plays an important functional role in lactate transport. In cancer cells, an excess of lactic acid is produced by aerobic glycolysis, which must be rapidly exported across the plasma membrane to avoid a drop in pHi57, 65. (B) The scFv-M6-1B9 intrabody can specifically trap the EMMPRIN molecule within the ER, based on the ER retention signal. Suppression of EMMPRIN-MCT1 complex by the down-regulation of EMMPRIN, which resulted in the inhibition of the lactate transporter. (C) Blocking of lactate transport leads to the accumulation of intracellular lactate inside the cell 31. The acidic pHi can be stimulated by different pathways, which are as follows: (i) by altering the mitochondrial membrane potential (Δψm) directly or by increasing the permeability of the outer mitochondrial membrane by promoting the binding of Bcl-2 62; (ii) by activating nuclear localized endonucleases, a key enzyme that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals 63; and (iii) by activating caspase-3, a pro-apoptotic cysteine protease involved in the activation cascade of caspases responsible for apoptosis execution 64.
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Figure 5: Schematic diagram of possible apoptotic mechanism underlying EMMPRIN down-regulation via scFv-M6-1B9 intrabody. (A) EMMPRIN facilitates proper expression of MCT1 at the cell surface. This association plays an important functional role in lactate transport. In cancer cells, an excess of lactic acid is produced by aerobic glycolysis, which must be rapidly exported across the plasma membrane to avoid a drop in pHi57, 65. (B) The scFv-M6-1B9 intrabody can specifically trap the EMMPRIN molecule within the ER, based on the ER retention signal. Suppression of EMMPRIN-MCT1 complex by the down-regulation of EMMPRIN, which resulted in the inhibition of the lactate transporter. (C) Blocking of lactate transport leads to the accumulation of intracellular lactate inside the cell 31. The acidic pHi can be stimulated by different pathways, which are as follows: (i) by altering the mitochondrial membrane potential (Δψm) directly or by increasing the permeability of the outer mitochondrial membrane by promoting the binding of Bcl-2 62; (ii) by activating nuclear localized endonucleases, a key enzyme that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals 63; and (iii) by activating caspase-3, a pro-apoptotic cysteine protease involved in the activation cascade of caspases responsible for apoptosis execution 64.

Mentions: It has been well established that EMMPRIN acts as a chaperone for monocarboxylate transporter 1 (MCT1) which is an accompaniment during the maturation in the ER and the translocation to the plasma membrane 55. These interactions play an important role in lactate transport responsible for cellular pH regulation in relation to proliferation, transformation, and apoptosis 56-59. The elevation of lactate and the further decrease in the intracellular pH (pHi) are caused by the changes in the H+/lactate co-transporters which possess the ability to protect the cells from apoptosis by maintaining pHi 60. It is convincingly evident that acidic pHi triggers the apoptosis hallmarks such as nuclear condensation, cytoplasmic vacuolization, and endonucleosome DNA degradation 61. Our current study demonstrates that EMMPRIN and MCT1 translocation were defective in the Caco-2 cells expressing the scFv-M6-1B9 intrabody. These cells effectively restrain the clearance of intracellular lactate and, ultimately, acidosis facilitates the occurrence of apoptosis. The possible mechanism of the acidic pHi potentially playing important roles in activating apoptosis is shown in three pathways: (i) by shifting the mitochondrial membrane potential (Δψm) directly or promoting the binding of Bcl-2 to the Bcl-2 19 kDa interacting protein (BNIP3), which leads to increased mitochondrial membrane permeability 62; (ii) by activating nuclear localized endonucleases involved in DNA fragmentation 63; and (iii) by activating caspase-3, a pro-apoptotic protease that, in turn, inactivates proteins vital for normal cellular function 64. Thus, the down-regulation of EMMPRIN by scFv-M6-1B9 intrabody induces apoptosis cell death in the colorectal cancer cell, Caco-2, and this is clearly associated with an elevated intracellular acidosis and eventually the consequence of the activation of apoptosis signaling molecules, as presented in Figure 5.


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)

Schematic diagram of possible apoptotic mechanism underlying EMMPRIN down-regulation via scFv-M6-1B9 intrabody. (A) EMMPRIN facilitates proper expression of MCT1 at the cell surface. This association plays an important functional role in lactate transport. In cancer cells, an excess of lactic acid is produced by aerobic glycolysis, which must be rapidly exported across the plasma membrane to avoid a drop in pHi57, 65. (B) The scFv-M6-1B9 intrabody can specifically trap the EMMPRIN molecule within the ER, based on the ER retention signal. Suppression of EMMPRIN-MCT1 complex by the down-regulation of EMMPRIN, which resulted in the inhibition of the lactate transporter. (C) Blocking of lactate transport leads to the accumulation of intracellular lactate inside the cell 31. The acidic pHi can be stimulated by different pathways, which are as follows: (i) by altering the mitochondrial membrane potential (Δψm) directly or by increasing the permeability of the outer mitochondrial membrane by promoting the binding of Bcl-2 62; (ii) by activating nuclear localized endonucleases, a key enzyme that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals 63; and (iii) by activating caspase-3, a pro-apoptotic cysteine protease involved in the activation cascade of caspases responsible for apoptosis execution 64.
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Related In: Results  -  Collection

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Figure 5: Schematic diagram of possible apoptotic mechanism underlying EMMPRIN down-regulation via scFv-M6-1B9 intrabody. (A) EMMPRIN facilitates proper expression of MCT1 at the cell surface. This association plays an important functional role in lactate transport. In cancer cells, an excess of lactic acid is produced by aerobic glycolysis, which must be rapidly exported across the plasma membrane to avoid a drop in pHi57, 65. (B) The scFv-M6-1B9 intrabody can specifically trap the EMMPRIN molecule within the ER, based on the ER retention signal. Suppression of EMMPRIN-MCT1 complex by the down-regulation of EMMPRIN, which resulted in the inhibition of the lactate transporter. (C) Blocking of lactate transport leads to the accumulation of intracellular lactate inside the cell 31. The acidic pHi can be stimulated by different pathways, which are as follows: (i) by altering the mitochondrial membrane potential (Δψm) directly or by increasing the permeability of the outer mitochondrial membrane by promoting the binding of Bcl-2 62; (ii) by activating nuclear localized endonucleases, a key enzyme that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals 63; and (iii) by activating caspase-3, a pro-apoptotic cysteine protease involved in the activation cascade of caspases responsible for apoptosis execution 64.
Mentions: It has been well established that EMMPRIN acts as a chaperone for monocarboxylate transporter 1 (MCT1) which is an accompaniment during the maturation in the ER and the translocation to the plasma membrane 55. These interactions play an important role in lactate transport responsible for cellular pH regulation in relation to proliferation, transformation, and apoptosis 56-59. The elevation of lactate and the further decrease in the intracellular pH (pHi) are caused by the changes in the H+/lactate co-transporters which possess the ability to protect the cells from apoptosis by maintaining pHi 60. It is convincingly evident that acidic pHi triggers the apoptosis hallmarks such as nuclear condensation, cytoplasmic vacuolization, and endonucleosome DNA degradation 61. Our current study demonstrates that EMMPRIN and MCT1 translocation were defective in the Caco-2 cells expressing the scFv-M6-1B9 intrabody. These cells effectively restrain the clearance of intracellular lactate and, ultimately, acidosis facilitates the occurrence of apoptosis. The possible mechanism of the acidic pHi potentially playing important roles in activating apoptosis is shown in three pathways: (i) by shifting the mitochondrial membrane potential (Δψm) directly or promoting the binding of Bcl-2 to the Bcl-2 19 kDa interacting protein (BNIP3), which leads to increased mitochondrial membrane permeability 62; (ii) by activating nuclear localized endonucleases involved in DNA fragmentation 63; and (iii) by activating caspase-3, a pro-apoptotic protease that, in turn, inactivates proteins vital for normal cellular function 64. Thus, the down-regulation of EMMPRIN by scFv-M6-1B9 intrabody induces apoptosis cell death in the colorectal cancer cell, Caco-2, and this is clearly associated with an elevated intracellular acidosis and eventually the consequence of the activation of apoptosis signaling molecules, as presented in Figure 5.

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