Nutjeera Intasai

Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells.

BackgroundExpression of intracellular antibodies (intrabodies) has become a broadly applicable technology for generation of phenotypic knockouts in vivo. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer.ResultsThe recombinant antibody fragments, Fab and scFv, of the murine monoclonal antibody (clone M6-1B9) reacted specifically to CD147 by indirect enzyme-linked immunosorbent assays (ELISA) using a recombinant CD147-BCCP as a target. This indicated that the Fab- and scFv-M6-1B9 displaying on phage surfaces were correctly folded and functionally active. We subsequently constructed a CD147-specific scFv, scFv-M6-1B9-intrabody, in 293A cells. The expression of CD147 on 293A cell surface was monitored at 36 h after transduction by flow cytometry and demonstrated remarkable reduction. Colocalization of scFv-M6-1B9 intrabody with CD147 in the ER network was depicted using a 3D deconvolution microscopy system.ConclusionThe results suggest that our approach can generate antibody fragments suitable for decreasing the expression of CD147 on 293A cells. This study represents a step toward understanding the role of the cell surface protein, CD147.

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

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.

EMMPRIN reduction via scFv-M6-1B9 intrabody affects α3β1-integrin and MCT1 functions and results in suppression of progressive phenotype in the colorectal cancer cell line Caco-2

Extracellular matrix metalloproteinase inducer (EMMPRIN) exhibits overexpression in various cancers and promotes cancer progression and metastasis via the interaction with its associated molecules. The scFv-M6-1B9 intrabody has a potential ability to reduce EMMPRIN cell surface expression. However, the subsequent effect of scFv-M6-1B9 intrabody-mediated EMMPRIN abatement on its related molecules, α3β1-integrin, MCT1, MMP-2 and MMP-9, is undefined. Our results demonstrated that the scFv-M6-1B9 intrabody efficiently decreased α3β1-integrin cell surface expression levels. In addition, intracellular accumulation of MCT1 and lactate were increased. These results lead to suppression of features characteristic for tumor progression, including cell migration, proliferation and invasion, in a colorectal cancer cell line (Caco-2) although there was no difference in MMP expression. Thus, EMMPRIN represents an attractive target molecule for the disruption of cancer proliferation and metastasis. An scFv-M6-1B9 intrabody-based approach could be relevant for cancer gene therapy.

Downregulation of Extracellular Matrix Metalloproteinase Inducer by scFv-M6-1B9 Intrabody Suppresses Cervical Cancer Invasion Through Inhibition of Urokinase-Type Plasminogen Activator

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.

Recombinant Multivalent EMMPRIN Extracellular Domain Induces U937 Human Leukemia Cell Apoptosis by Downregulation of Monocarboxylate Transporter 1 and Activation of Procaspase-9

This study was carried out to understand the effect of the recombinant multivalent extracellular matrix metalloproteinase inducer (EMMPRIN) extracellular domain, designated as rmEMMPRINex, on the apoptotic cell death of human leukemia U937 cells. Expression of monocarboxylate transporter 1 (MCT1) and caspase-9 in U937 treated with rmEMMPRINex was investigated in this study. Levels of membrane MCT1 and intracellular procaspase-9 were decreased in rmEMMPRINex-treated cells in comparison to controls. However, the expression of activated caspase-9 was undetectable. rmEMMPRINex also induced DNA fragmentation and apoptosis in U937 cells. Taken together, we concluded that interaction of rmEMMPRINex with U937 cells leads to inhibition of MCT1 membrane expression, intracellular activation of procaspase-9, followed by DNA fragmentation and apoptosis. This may contribute to the conceptual development of novel cancer drugs in the future.

Clusterin as a Blood Biomarker for Diagnosis of Mild Cognitive Impairment and Alzheimer's Disease

With increasing global life expectancy, Alzheimers disease will become an increasingly prevalent health problem. The development of biomarkers that predict risk for both Alzheimers disease and mild cognitive impairment will be useful for early diagnosis of dementia. To date, no surrogate blood biomarker exists to classify between Alzheimers disease/mild cognitive impairment and normal controls or Alzheimers disease and mild cognitive impairment/normal control as a diagnostic parameter. In this study, we analyzed serum levels of amyloid-β 40 (Aβ 40 ), amyloid-β 42 (Aβ 42 ), clusterin (CLU) and p97 using ELISA kits from 157 subjects with normal cognition, mild cognitive impairment and Alzheimers disease. We found a significant increase in serum levels of Aβ 42 (P<0.05) and serum clusterin (P<0.001) between normal and Alzheimers disease subjects and between normal and mild cognitively impaired subjects. In contrast, serum Aβ 40 and p97 levels did not differ significantly between all groups. We also used receiver operating characteristic curves to determine the cut-off point of Aβ 42 and clusterin to differentiate either cognitively normal from cognitively impaired subjects (both Alzheimers disease and mild cognitive impairment) or cognitively normal and mild cognitively impaired subjects from those with Alz- heimers disease. Only clusterin with 84% sensitivity, 75% specificity and good accuracy of diagnosis showed promise for diagnosing patients with cognitive impairment (Alzheimers disease and mild cognitive impairment).

Abstract 5433: Binding of multivalent CD147 extracellular domain on U937 cells induces apoptosis via activation of caspase-9 and downregulation of MCT-1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA CD147 is highly expressed in activated lymphocytes and cancer cells. CD147 plays many important roles in cellular functions. We have previously reported that binding of multivalent CD147 extracellular domain displayed on phage particles (multivalent CD147Ex phages) induced apoptosis in a monocytic cell line, U937, by triggering caspase-3 activation. Activation of caspase-8, caspase-9 and MCT-1 was investigated in this study. Levels of intracellular procaspase-9 and membrane MCT-1 were noticeably decreased in multivalent CD147Ex phage-induced U937 cells in comparison to controls. However, activation of caspase-8 was not obvious. Multivalent CD147Ex phages also induced DNA fragmentation and growth arrest of U937 cells. Taken together with previous studies, we hypothesize that the CD147 extracellular domain on phage particles may bind to its ligand(s) on the cell surface of U937 leading to inhibition of MCT-1 cell surface expression, intracellular activation of caspase-9, followed by caspase-3 activation, DNA fragmentation and growth arrest. We identified a mechanism of CD147 in triggering its ligand(s) on U937 cells leading to U937 apoptotic cell death. This may contribute to the conceptual development of novel cancer drugs based on CD147 polymers. Citation Format: Nutjeera Intasai, Supansa Pata, Khajornsak Tragoolpua, Chatchai Tayapiwatana. Binding of multivalent CD147 extracellular domain on U937 cells induces apoptosis via activation of caspase-9 and downregulation of MCT-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5433. doi:10.1158/1538-7445.AM2014-5433