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Growth suppression of colorectal cancer by plant-derived multiple mAb CO17-1A × BR55 via inhibition of ERK1/2 phosphorylation.

Kwak DH, Moussavou G, Lee JH, Heo SY, Ko K, Hwang KA, Jekal SJ, Choo YK - Int J Mol Sci (2014)

Bottom Line: The mAb(P) 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.In vivo, the mAb(P) CO17-1A × BR55 significantly and efficiently inhibited the growth of colon tumors compared to another mAbs.Therefore, this study results suggest that multiple mAb(P) CO17-1A × BR55 has a significant effect on apoptosis-mediated anticancer by suppression of ERK1/2 phosphorylation in colon cancer compared to another mAbs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Glycoscience, Wonkwang University, Iksan, Jeonbuk 570-749, Korea. velvety7@nate.com.

ABSTRACT
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(P)) CO17-1A and mAb(P) CO17-1A × BR55) with RAW264.7 cells significantly inhibited the cell growth in SW620 cancer cells. In particular, multi mAb(P) 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(P) CO17-1A × BR55-treated. The mAb(P) 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. In vivo, the mAb(P) 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(P) CO17-1A × BR55. In addition, the mAb(P) 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(P) 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(P) CO17-1A × BR55 to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

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The inhibitory effect of multiple mAbP CO17-1A × BR55 on cells proliferation by apoptosis in SW620 cells. (A) SW620 cells were seeded at 3 × 104 cells/well in 96-well plates and treated with mAbP CO17-1A or anti-epithelial cellular adhesion molecule (EpCAM) or multiple mAbP CO17-1A × BR55, and RAW264.7 cells for 24 h. Cell proliferation was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; (B) Up-regulating expression of and Bax, and down-regulating expression of BCl-2. Expression of apoptosis-related proteins including cleaved caspase-3, BCl-2-associated X protein (Bax) and B-Cell lymphoma-2 (BCl-2) in SW620 cells. The expression of cleaved caspase-3, Bax, BCl-2 and β-actin proteins was measured by western blot analysis using specific antibodies; (C) Apoptotic cell death was determined by 4',6-diamidino-2-phenylindole (DAPI) staining and dUTP nick end labeling (TUNEL) assay. The TUNEL-positive (green) cells are an apoptotic cells (200× magnification). Apoptotic cells (DAPI-stained TUNEL-positive cells) were estimated by direct counting of fragmented nuclei after DAPI and TUNEL staining. Each band is representative of three independent experiments. Values represent mean (SD) of three experiments, each performed in triplicate. *** p < 0.001, ** p < 0.01, * p < 0.05 indicates a statistically significant difference from the control group.
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ijms-15-21105-f002: The inhibitory effect of multiple mAbP CO17-1A × BR55 on cells proliferation by apoptosis in SW620 cells. (A) SW620 cells were seeded at 3 × 104 cells/well in 96-well plates and treated with mAbP CO17-1A or anti-epithelial cellular adhesion molecule (EpCAM) or multiple mAbP CO17-1A × BR55, and RAW264.7 cells for 24 h. Cell proliferation was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; (B) Up-regulating expression of and Bax, and down-regulating expression of BCl-2. Expression of apoptosis-related proteins including cleaved caspase-3, BCl-2-associated X protein (Bax) and B-Cell lymphoma-2 (BCl-2) in SW620 cells. The expression of cleaved caspase-3, Bax, BCl-2 and β-actin proteins was measured by western blot analysis using specific antibodies; (C) Apoptotic cell death was determined by 4',6-diamidino-2-phenylindole (DAPI) staining and dUTP nick end labeling (TUNEL) assay. The TUNEL-positive (green) cells are an apoptotic cells (200× magnification). Apoptotic cells (DAPI-stained TUNEL-positive cells) were estimated by direct counting of fragmented nuclei after DAPI and TUNEL staining. Each band is representative of three independent experiments. Values represent mean (SD) of three experiments, each performed in triplicate. *** p < 0.001, ** p < 0.01, * p < 0.05 indicates a statistically significant difference from the control group.

Mentions: Anticancer effects of mAb appear via binding to receptors of immune cells, which causes cancer cells death by antibody-dependent cell-mediated cytotoxicity (ADCC). To determine whether the immunoreaction of mAbs (anti-EpCAM mAb, mAbP CO17-1A, mAbP BR55 and multiple mAbP CO17-1A × BR55) with RAW264.7 cells is inhibited to SW620 cancer cell growth, the inhibitory effect of mAbs (anti-EpCAM mAb, mAbP CO17-1A, mAbP BR55 and multiple mAbP CO17-1A × BR55) on SW620 cancer cell growth was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell growth inhibition by immunoreaction of mAbs (anti-EpCAM mAb, mAbP CO17-1A, mAbP BR55 and multiple mAbP CO17-1A × BR55) with RAW264.7 cells was also confirmed by trypan blue dye exclusion. SW620 cells viability was significantly decreased after treatments with anti-EpCAM mAb, mAbP CO17-1A, and multiple mAbP CO17-1A × BR55 compared to the untreated control. Moreover, treatment of SW620 cells with multiple mAbP CO17-1A × BR55 (40 μM) and RAW264.7 cells resulted in significantly suppressed cell growth (Figure 2A). However, treatment with either mAbP BR55 alone did not markedly inhibit growth of SW620 cancer cells (Figure 2A).


Growth suppression of colorectal cancer by plant-derived multiple mAb CO17-1A × BR55 via inhibition of ERK1/2 phosphorylation.

Kwak DH, Moussavou G, Lee JH, Heo SY, Ko K, Hwang KA, Jekal SJ, Choo YK - Int J Mol Sci (2014)

The inhibitory effect of multiple mAbP CO17-1A × BR55 on cells proliferation by apoptosis in SW620 cells. (A) SW620 cells were seeded at 3 × 104 cells/well in 96-well plates and treated with mAbP CO17-1A or anti-epithelial cellular adhesion molecule (EpCAM) or multiple mAbP CO17-1A × BR55, and RAW264.7 cells for 24 h. Cell proliferation was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; (B) Up-regulating expression of and Bax, and down-regulating expression of BCl-2. Expression of apoptosis-related proteins including cleaved caspase-3, BCl-2-associated X protein (Bax) and B-Cell lymphoma-2 (BCl-2) in SW620 cells. The expression of cleaved caspase-3, Bax, BCl-2 and β-actin proteins was measured by western blot analysis using specific antibodies; (C) Apoptotic cell death was determined by 4',6-diamidino-2-phenylindole (DAPI) staining and dUTP nick end labeling (TUNEL) assay. The TUNEL-positive (green) cells are an apoptotic cells (200× magnification). Apoptotic cells (DAPI-stained TUNEL-positive cells) were estimated by direct counting of fragmented nuclei after DAPI and TUNEL staining. Each band is representative of three independent experiments. Values represent mean (SD) of three experiments, each performed in triplicate. *** p < 0.001, ** p < 0.01, * p < 0.05 indicates a statistically significant difference from the control group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4264215&req=5

ijms-15-21105-f002: The inhibitory effect of multiple mAbP CO17-1A × BR55 on cells proliferation by apoptosis in SW620 cells. (A) SW620 cells were seeded at 3 × 104 cells/well in 96-well plates and treated with mAbP CO17-1A or anti-epithelial cellular adhesion molecule (EpCAM) or multiple mAbP CO17-1A × BR55, and RAW264.7 cells for 24 h. Cell proliferation was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; (B) Up-regulating expression of and Bax, and down-regulating expression of BCl-2. Expression of apoptosis-related proteins including cleaved caspase-3, BCl-2-associated X protein (Bax) and B-Cell lymphoma-2 (BCl-2) in SW620 cells. The expression of cleaved caspase-3, Bax, BCl-2 and β-actin proteins was measured by western blot analysis using specific antibodies; (C) Apoptotic cell death was determined by 4',6-diamidino-2-phenylindole (DAPI) staining and dUTP nick end labeling (TUNEL) assay. The TUNEL-positive (green) cells are an apoptotic cells (200× magnification). Apoptotic cells (DAPI-stained TUNEL-positive cells) were estimated by direct counting of fragmented nuclei after DAPI and TUNEL staining. Each band is representative of three independent experiments. Values represent mean (SD) of three experiments, each performed in triplicate. *** p < 0.001, ** p < 0.01, * p < 0.05 indicates a statistically significant difference from the control group.
Mentions: Anticancer effects of mAb appear via binding to receptors of immune cells, which causes cancer cells death by antibody-dependent cell-mediated cytotoxicity (ADCC). To determine whether the immunoreaction of mAbs (anti-EpCAM mAb, mAbP CO17-1A, mAbP BR55 and multiple mAbP CO17-1A × BR55) with RAW264.7 cells is inhibited to SW620 cancer cell growth, the inhibitory effect of mAbs (anti-EpCAM mAb, mAbP CO17-1A, mAbP BR55 and multiple mAbP CO17-1A × BR55) on SW620 cancer cell growth was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell growth inhibition by immunoreaction of mAbs (anti-EpCAM mAb, mAbP CO17-1A, mAbP BR55 and multiple mAbP CO17-1A × BR55) with RAW264.7 cells was also confirmed by trypan blue dye exclusion. SW620 cells viability was significantly decreased after treatments with anti-EpCAM mAb, mAbP CO17-1A, and multiple mAbP CO17-1A × BR55 compared to the untreated control. Moreover, treatment of SW620 cells with multiple mAbP CO17-1A × BR55 (40 μM) and RAW264.7 cells resulted in significantly suppressed cell growth (Figure 2A). However, treatment with either mAbP BR55 alone did not markedly inhibit growth of SW620 cancer cells (Figure 2A).

Bottom Line: The mAb(P) 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.In vivo, the mAb(P) CO17-1A × BR55 significantly and efficiently inhibited the growth of colon tumors compared to another mAbs.Therefore, this study results suggest that multiple mAb(P) CO17-1A × BR55 has a significant effect on apoptosis-mediated anticancer by suppression of ERK1/2 phosphorylation in colon cancer compared to another mAbs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Glycoscience, Wonkwang University, Iksan, Jeonbuk 570-749, Korea. velvety7@nate.com.

ABSTRACT
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(P)) CO17-1A and mAb(P) CO17-1A × BR55) with RAW264.7 cells significantly inhibited the cell growth in SW620 cancer cells. In particular, multi mAb(P) 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(P) CO17-1A × BR55-treated. The mAb(P) 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. In vivo, the mAb(P) 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(P) CO17-1A × BR55. In addition, the mAb(P) 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(P) 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(P) CO17-1A × BR55 to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

Show MeSH
Related in: MedlinePlus