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pp-GalNAc-T13 induces high metastatic potential of murine Lewis lung cancer by generating trimeric Tn antigen

Matsumoto, Y;Zhang, Q;Akita, K;Nakada, H;Hamamura, K;Tokuda, N;Tsuchida, A;Matsubara, T;Hori, T;Okajima, T;Furukawa, K;Urano, T;Furukawa, K;
Product: Cholera Toxin B Subunit Biotin Conjugate

In order to analyze the mechanisms for cancer metastasis, high metastatic sublines (H7-A, H7-Lu, H7-O, C4-sc, and C4-ly) were obtained by repeated injection of mouse Lewis lung cancer sublines H7 and C4 into C57BL/6 mice. These sublines exhibited increased proliferation and invasion activity in vitro. Ganglioside profiles exhibited lower expression of GM1 in high metastatic sublines than the parent lines. Then, we established GM1-Si-1 and GM1-Si-2 by stable silencing of GM1 synthase in H7 cells. These GM1-knockdown clones exhibited increased proliferation and invasion. Then, we explored genes that markedly altered in the expression levels by DNA microarray in the combination of C4 vs. C4-ly or H7 vs. H7 (GM1-Si). Consequently, pp-GalNAc-T13 gene was identified as up-regulated genes in the high metastatic sublines. Stable transfection of pp-GalNAc-T13 cDNA into C4 (T13-TF) resulted in increased invasion and motility. Then, immunoblotting and flow cytometry using various antibodies and lectins were performed. Only anti-trimeric Tn antibody (mAb MLS128), showed increased expression levels of trimeric Tn antigen in T13-TF clones. Moreover, immunoprecipitation/immunoblotting was performed by mAb MLS128, leading to the identification of an 80 kDa band carrying trimeric Tn antigen, i.e. Syndecan-1. Stable silencing of endogenous pp-GalNAc-T13 in C4-sc (T13-KD) revealed that primary tumors generated by subcutaneous injection of T13-KD clones showed lower coalescence to fascia and peritoneum, and significantly reduced lung metastasis than control clones. These data suggested that high expression of pp-GalNAc-T13 gene generated trimeric Tn antigen on Syndecan-1, leading to the enhanced metastasis.

PubMed ID: 22306014
251325132017-06-052017-06-0511:03:0311:03:032018-03-302018-03-3011:42:4011:42:40Matsumoto, Y;Zhang, Q;Akita, K;Nakada, H;Hamamura, K;Tokuda, N;Tsuchida, A;Matsubara, T;Hori, T;Okajima, T;Furukawa, K;Urano, T;Furukawa, K;Matsumoto, Y;Zhang, Q;Akita, K;Nakada, H;Hamamura, K;Tokuda, N;Tsuchida, A;Matsubara, T;Hori, T;Okajima, T;Furukawa, K;Urano, T;Furukawa, K;20122012pp-GalNAc-T13 induces high metastatic potential of murine Lewis lung cancer by generating trimeric Tn antigenpp-GalNAc-T13 induces high metastatic potential of murine Lewis lung cancer by generating trimeric Tn antigenBiochemical And Biophysical Research CommunicationsBiochemical And Biophysical Research Communications7-137-13419419112230601422306014

In order to analyze the mechanisms for cancer metastasis, high metastatic sublines (H7-A, H7-Lu, H7-O, C4-sc, and C4-ly) were obtained by repeated injection of mouse Lewis lung cancer sublines H7 and C4 into C57BL/6 mice. These sublines exhibited increased proliferation and invasion activity in vitro. Ganglioside profiles exhibited lower expression of GM1 in high metastatic sublines than the parent lines. Then, we established GM1-Si-1 and GM1-Si-2 by stable silencing of GM1 synthase in H7 cells. These GM1-knockdown clones exhibited increased proliferation and invasion. Then, we explored genes that markedly altered in the expression levels by DNA microarray in the combination of C4 vs. C4-ly or H7 vs. H7 (GM1-Si). Consequently, pp-GalNAc-T13 gene was identified as up-regulated genes in the high metastatic sublines. Stable transfection of pp-GalNAc-T13 cDNA into C4 (T13-TF) resulted in increased invasion and motility. Then, immunoblotting and flow cytometry using various antibodies and lectins were performed. Only anti-trimeric Tn antibody (mAb MLS128), showed increased expression levels of trimeric Tn antigen in T13-TF clones. Moreover, immunoprecipitation/immunoblotting was performed by mAb MLS128, leading to the identification of an 80 kDa band carrying trimeric Tn antigen, i.e. Syndecan-1. Stable silencing of endogenous pp-GalNAc-T13 in C4-sc (T13-KD) revealed that primary tumors generated by subcutaneous injection of T13-KD clones showed lower coalescence to fascia and peritoneum, and significantly reduced lung metastasis than control clones. These data suggested that high expression of pp-GalNAc-T13 gene generated trimeric Tn antigen on Syndecan-1, leading to the enhanced metastasis.

In order to analyze the mechanisms for cancer metastasis, high metastatic sublines (H7-A, H7-Lu, H7-O, C4-sc, and C4-ly) were obtained by repeated injection of mouse Lewis lung cancer sublines H7 and C4 into C57BL/6 mice. These sublines exhibited increased proliferation and invasion activity in vitro. Ganglioside profiles exhibited lower expression of GM1 in high metastatic sublines than the parent lines. Then, we established GM1-Si-1 and GM1-Si-2 by stable silencing of GM1 synthase in H7 cells. These GM1-knockdown clones exhibited increased proliferation and invasion. Then, we explored genes that markedly altered in the expression levels by DNA microarray in the combination of C4 vs. C4-ly or H7 vs. H7 (GM1-Si). Consequently, pp-GalNAc-T13 gene was identified as up-regulated genes in the high metastatic sublines. Stable transfection of pp-GalNAc-T13 cDNA into C4 (T13-TF) resulted in increased invasion and motility. Then, immunoblotting and flow cytometry using various antibodies and lectins were performed. Only anti-trimeric Tn antibody (mAb MLS128), showed increased expression levels of trimeric Tn antigen in T13-TF clones. Moreover, immunoprecipitation/immunoblotting was performed by mAb MLS128, leading to the identification of an 80 kDa band carrying trimeric Tn antigen, i.e. Syndecan-1. Stable silencing of endogenous pp-GalNAc-T13 in C4-sc (T13-KD) revealed that primary tumors generated by subcutaneous injection of T13-KD clones showed lower coalescence to fascia and peritoneum, and significantly reduced lung metastasis than control clones. These data suggested that high expression of pp-GalNAc-T13 gene generated trimeric Tn antigen on Syndecan-1, leading to the enhanced metastasis.

2.2812.281

... Cholera toxin B (CTB) subunit-biotin conjugates was from List Biological Laboratories, Inc. (Campbell, CA). ...

... Cholera toxin B (CTB) subunit-biotin conjugates was from List Biological Laboratories, Inc. (Campbell, CA). ...

http://www.sciencedirect.com/science/article/pii/S0006291X12001337http://www.sciencedirect.com/science/article/pii/S0006291X120013372012-03-022012-03-0210.1016/j.bbrc.2012.01.08610.1016/j.bbrc.2012.01.086Cholera Toxin B Subunit Biotin ConjugateCholera Toxin B Subunit Biotin Conjugatekoichi@med.nagoya-u.ac.jpkoichi@med.nagoya-u.ac.jpActivity;Analyze;Anti;Antibodies;Antibody;Antigen;Biological;Biotin;Cancer;Cholera;Control;CTB;DNA;Expression;Flow Cytometry;Ganglioside;Gene;GM1;Identification;Immunoprecipitation;In vitro;Injection;kDa;List;List Biological;Lung;Microarray;Mouse;Murine;Proliferation;Subunit;Toxin;Toxin B;Transduction;Biochemical And Biophysical Research CommunicationsActivity;Analyze;Anti;Antibodies;Antibody;Antigen;Biological;Biotin;Cancer;Cholera;Control;CTB;DNA;Expression;Flow Cytometry;Ganglioside;Gene;GM1;Identification;Immunoprecipitation;In vitro;Injection;kDa;List;List Biological;Lung;Microarray;Mouse;Murine;Proliferation;Subunit;Toxin;Toxin B;Transduction;Biochemical And Biophysical Research Communications112112pp-galnac-t13-induces-high-metastatic-potentialpp-galnac-t13-induces-high-metastatic-potential