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Production of Monoclonal Antibodies in Plants for Cancer Immunotherapy
Moussavou, Ghislain,Ko, Kisung,Lee, Jeong-Hwan,Choo, Young-Kug Hindawi Publishing Corporation 2015 BioMed research international Vol.2015 No.-
<P>Plants are considered as an alternative platform for recombinant monoclonal antibody (mAb) production due to the improvement and diversification of transgenic techniques. The diversity of plant species offers a multitude of possibilities for the valorization of genetic resources. Moreover, plants can be propagated indefinitely, providing cheap biomass production on a large scale in controlled conditions. Thus, recent studies have shown the successful development of plant systems for the production of mAbs for cancer immunotherapy. However, their several limitations have to be resolved for efficient antibody production in plants.</P>
Anti-cancer Effects of Multiple Monoclonal Antibodies (mAbPC×B) using Solanaceae
Ghislain Moussavou,Sung-Youn Heo,Malg-Um Lim,Mi-Ran Hwang,Kyung-A Hwang,Kisung Ko,Young-Kug Choo 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1
The human colorectal carcinoma-associated GA733 antigen epithelial cell adhesion molecule (EpCAM) was initially described as a cell surface protein selectively expressed in some myeloid cancers. The glycoprotein was originally defined by anti-GA733, anti-CO17-1A, and anti-EpCAM mAbs, which bind to different epitopes on this antigen. We demonstrated that treatment with plant-derived multiple antibodies (mAbPC×B; anti-colorectal cancer mAb, mAbp CO17-1A and anti-breast cancer mAb, BR 55 mAb) with RAW264.7 cells significantly inhibited cell growth in SW 620 and MCF 7 cells. Expression of p53 and p21 increased, whereas the expression of G1 phase-related proteins, cyclin D1, CDK4, cyclin E and CDK, decreased. In addition, plant-derived multiple antibodies with RAW264.7 cells treatment decreased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-a, caspase-8, caspase-9, caspase and caspase-6 increased. We observed that plant-derived multiple antibodies significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. These results suggest that plant-derived multiple monoclonal antibodies could have an anti-cancer effect for colorectal and breast cancer. Further clinical investigation should be conducted on plant-derived multiple monoclonal antibodies to determine its possible chemopreventive and/or therapeutic efficacy for the treatment of human colon and breast cancer.
( Ghislain Moussavou ),( Dong Hoon Kwak ),( Malg Um Lim ),( Ji Su Kim ),( Sun Uk Kim ),( Kyu Tae Chang ),( Young Kug Choo ) 생화학분자생물학회(구 한국생화학분자생물학회) 2013 BMB Reports Vol.46 No.11
Gangliosides are complex glycosphingolipids that are the major component of cytoplasmic cell membranes, and play a role in the control of biological processes. Human mesenchymal stem cells (hMSCs) have received considerable attention as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. In this study, we focus on various functional roles of gangliosides in the differentiation of hMSCs into osteoblasts or neuronal cells. A relationship between gangliosides and epidermal growth factor receptor (EGFR) activation during osteoblastic differentiation of hMSCs was observed, and the gangliosides may play a major role in the regulation of the differentiation. The roles of gangliosides in osteoblast differentiation are dependent on the origin of hMSCs. The reduction of ganglioside biosynthesis inhibited the neuronal differentiation of hMSCs during an early stage of the differentiation process, and the ganglioside expression can be used as a marker for the identification of neuronal differentiation from hMSCs. [BMB Reports 2013; 46(11): 527-532]
Ghislain Moussavou,이정환,LuQIAO,노유훈,신용규,이태진,이승호,고기성 한국곤충학회 2018 Entomological Research Vol.48 No.1
The baculovirus expression system has been considered as a highly efficient tool for the production of recombinant biopharmaceutical proteins. The recombinant antigenic glycoprotein GA733 is a cell surface protein that is strongly expressed in human colorectal cancer. Efficient virus titration should be established to achieve optimal multiplicity of infection (MOI) conditions, which are in turn essential for strong expression of the recombinant GA733 fused to the human immunoglobulin IgG Fc fragment (GA733‐Fc) in the baculovirus‐insect system. In the present study, the Sf9 cell line was transfected with plasmid DNA containing the GA733‐Fc expression cassette under the control of the baculovirus polyhedron promoter. MOI values (0.05, 0.1, 0.5, 1, and 3) were calculated based on both microscope observations and results of titration assay and then used to determine the optimum recombinant expression and harvested sample [cell culture media (CM) or cell lysate (CL)]. The pFastBac dual vector carrying the GA733‐Fc gene was constructed to express GA733‐Fc and used to generate recombinant baculoviruses. Western blotting results showed that recombinant protein expression was dependent on the MOI. In addition, CM and CL showed significant differences in protein synthesis and protein secretion capacities. Our findings suggested that our proposed titration method can be used for reliable calculation of MOI values, which significantly influence recombinant GA733‐Fc protein expression in the baculovirus‐insect cell system.
Kwak, Dong Hoon,Moussavou, Ghislain,Lee, Ju Hyoung,Heo, Sung Youn,Ko, Kisung,Hwang, Kyung-A,Jekal, Seung-Joo,Choo, Young-Kug MDPI 2014 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.15 No.11
<P>We have generated the transgenic Tabaco plants expressing multiple monoclonal antibody (mAb) CO7-1A × BR55 by cross-pollinating with mAb CO17-1A and mAb BR55. We have demonstrated the anti-cancer effect of plant-derived multiple mAb CO17-1A × BR55. We find that co-treatment of colorectal mAbs (anti-epithelial cellular adhesion molecule (EpCAM), plant-derived monoclonal antibody (mAb<SUP>P</SUP>) CO17-1A and mAb<SUP>P</SUP> CO17-1A × BR55) with RAW264.7 cells significantly inhibited the cell growth in SW620 cancer cells. In particular, multi mAb<SUP>P</SUP> CO17-1A × BR55 significantly and efficiently suppressed the growth of SW620 cancer cells compared to another mAbs. Apoptotic death-positive cells were significantly increased in the mAb<SUP>P</SUP> CO17-1A × BR55-treated. The mAb<SUP>P</SUP> CO17-1A × BR55 treatment significantly decreased the expression of B-Cell lymphoma-2 (BCl-2), but the expression of Bcl-2-associated X protein (Bax), and cleaved caspase-3 were markedly increased. <I>In vivo</I>, the mAb<SUP>P</SUP> CO17-1A × BR55 significantly and efficiently inhibited the growth of colon tumors compared to another mAbs. The apoptotic cell death and inhibition of pro-apoptotic proteins expression were highest by treatment with mAb<SUP>P</SUP> CO17-1A × BR55. In addition, the mAb<SUP>P</SUP> CO17-1A × BR55 significantly inhibited the extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in cancer cells and tumors. Therefore, this study results suggest that multiple mAb<SUP>P</SUP> CO17-1A × BR55 has a significant effect on apoptosis-mediated anticancer by suppression of ERK1/2 phosphorylation in colon cancer compared to another mAbs. In light of these results, further clinical investigation should be conducted on mAb<SUP>P</SUP> CO17-1A × BR55 to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.</P>
Effect of gangliosides on LPS stimulation and nitric oxide release in porcine kidney cell line PK15
이주택,고기성,임맑음,Ghislain Moussavou,김지수,장규태,추영국 한국통합생물학회 2013 Animal cells and systems Vol.17 No.5
Gangliosides, which are glycosphingolipids containing sialic acid, play important regulatory roles in cell proliferation and adhesion, survival and immunosuppressive activity. In this study, we investigated whether gangliosides can affect cell viability in the porcine kidney (PK) cell line, PK15, when stimulated with lipopolysaccharide (LPS). As the amount of LPS that PK15 cells were treated with was increased, the cell proliferation decreased, whereas nitric oxide (NO) production increased. High-performance thin-layer chromatography (HPTLC) and immunofluorescence analyses showed that GM3 and GM2 ganglioside expression significantly decreased in LPS-stimulated PK15 compared to unstimulated PK15. UDP-glucose ceramide glucosyltransferase (Ugcg), which catalyzes the initial step in the glycosphingolipid biosynthesis pathway, was knocked-down in PK15 by using short hairpin RNA (shRNA). Western blot and HPTLC analyses showed that the Ugcg protein expression decreased and the ganglioside expression decreased in the Ugcg-knockdown (UKD) PK15. There was a greater decrease in cell proliferation in LPS-stimulated UKD PK15 cells than in LPS-stimulated PK15 cells without the UKD. However, the increase in NO release was greater in LPS-stimulated UKD PK15 cells than in LPS-stimulated PK15 cells without the UKD. These findings suggest that gangliosides may interact with components of the inflammatory response pathway and, thus, are relevant for the design of future therapeutic strategies intended to prolong xenotransplantation.
Ju-Taek Lee,Jae-Sung Ryu,Ghislain Moussavou,Malg-Um Lim,Hyun-Ki Min,Yoon-Ju Na,Su-Bin Lee,In-Sun Kim,Ju-Hyoung Lee,Ji-Su Kim,Sun-Uk Kim,Kyu-Tae Chang,Young-Kug Choo 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
Glycosphingolipids including gangliosides play important regulatory roles in cell prolifera-tion and differentiation. UDP-glucose:ceramide glucosyltransferase(Ugcg) catalyze the initial step in glycosphingolipids biosynthesis pathway. In this study, Ugcg expression was reduced to approximately 80% by short hairpin RNAs(shRNAs) to evaluate the roles of glycosphingolipids in proliferation and gangliosdies differentiation of PK15. HPTLC/immunofluorescence analyses of shRNA transfected PK15 revealed that treatment with Ugcg-shRNA decreased expression of major gangliosides, GM1 and GD1b. Furthermore, MTT and western blot/immunofluorescence analyses demonstreated that inhibition of the Ugcg expression in PK15 resulted in decrease of cell proliferation. In addition to we showed that inflammatory respon-ses in PK15 and gangliosides knock-down PK15 cells stimulated with Escherichia coli lipopolysaccharide(LPS). The gangliosides GM3, GM2, GD3, GD1a and GD1b were detected in normal PK15 cell. However almost gangliosides undetected in gangliosides knock-down PK15 cell. In addition we found that the expression levels of TNF-α, indu-cible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and we measured the production of NO. The expression of inflammatory proteins, TNF- α, iNOS and COX-2 the elevated concentration in gangliosides knock-down PK15 cell. These results suggest that gangliosides interact with components of the proinflammatory response pathway and might, therefore, be relevant for designing future therapeutic strategies intended to prolong xenograft survival.
Expression of gangliosides during differentiation to endothelial cells of mouse embryonic stem cells
Malg-Um Lim,Jae-Sung Ryu,Ghislain Moussavou,Ju-Taek Lee,Hyun-Ki Mun,Yoon-Ju Na,Su-Bin Lee,In-Sun Kim,Ju-Hyoung Lee,Ji-Su Kim,Sun-Uk Kim,Kyu-Tae Chang,Young-Kug Choo 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
Malg-Um Lim,Jae-Sung Ryu,Ghislain Moussavou,Ju-Taek Lee,Hyun-Ki Min,Yoon-Ju Na,Chi-Hyun Ahn,In-Sun Kim,Ju-Hyoung Lee,Ji-Su Kim,Sun-Uk Kim,Kyu-Tae Chang,Kisung Ko,Deog-Bon Koo,Young-Kug Choo 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1
Gangliosides, play important regulatory roles in cell adhesion, survival and immunosuppressive activity. In this study, we investigated whether gangliosides can affect to immune responses in lipopolysaccharide (LPS)-stimulated porcine kidney cell line, PK15. Cell proliferation decreased, whefreas nitro oxide (NO) increased in 5 ㎍/ml of LPS-stimulated PK15. In high-performance thin layer chromatography (HPTLC) and immunofluorsence analysis showed that gangliosides GM3, GM2, GD3, GD1a and GD1b were detected, however, expression of GM3 and GM2 decreased in LPS-stimulated PK15. Furthermore, we knock- downed UDP-glucose:ceramide glucosyltransferase (Ugcg) by short hairpin (sh) RNA, which catalyzes the initial step in glycosphingolipids biosynthesis pathway. Western blot and HPTLC analyse showed that Ugcg protein and gangliosides expression decreased in Ugcg shRNA infected PK15. When we stimulated Ugcg gene knock-downed PK15 with LPS (5 ㎍/ml), cell proliferation was decreased, however, NO release increased compared to un-infected PK15. Moreover, we investigated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α), and these protein expression levels increased in LPS-stimulated Ugcg gene knock-downed PK15. These results suggest that gangliosides, specifically GM3 and GM2, may play a role in the interaction with components of the inflammatory response, and to be relevant for designing future therapeutic strategies intended to prolong xenotransplantation.