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Recombinant disintegrin (r-Cam-dis) from Crotalus adamanteus inhibits adhesion of human pancreatic cancer cell lines to laminin-1 and vitronectin.

Suntravat M, Barret HS, Jurica CA, Lucena SE, Perez JC, Sánchez EE - J Venom Res (2015)

Bottom Line: The r-Cam-dis showed potent binding to αvβ3 integrin, but was moderate to weak with αvβ5, αvβ6, α2β1, and α6β1.Interestingly, the inhibition of r-Cam-dis on pancreatic cancer cell lines adhesion to laminin-1 was more effective than that to vitronectin.Based on our binding results to integrin receptors and previous adhesion studies using function-blocking monoclonal antibodies, it is suggested that r-Cam-dis could be inhibiting adhesion of pancreatic cancer cell lines through integrins α2β1, α6β1, αvβ5, and αvβ6.

View Article: PubMed Central - PubMed

Affiliation: National Natural Toxins Research Center, Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA.

ABSTRACT
Pancreatic cancer is a malignant cancer common worldwide having poor prognosis, even when diagnosed at its early stage. Cell adhesion plays a critical role in cancer invasion and metastasis. Integrins are major mediators of cell adhesion and play an important role in invasion and metastatic growth of human pancreatic cancer cells. Snake disintegrins are the most potent ligands of several integrins and have potential therapeutic applications for cancers. We have previously cloned and expressed a new recombinant RGD-disintegrin from Crotalus adamanteus (r-Cam-dis). This recently published r-Cam-dis has an extra nine amino acids derived from the vector (SPGARGSEF) at the N-terminus end and has strong anti-platelet activity. However, this r-Cam-dis contains the contamination of the cleavage of the N-terminal end of the pET-43.1a cloning vector. In this study, we have cloned r-Cam-dis in a different cloning vector (pGEX-4T-1) showing five different amino acids (GSPEF) at the N-terminal part. This new r-Cam-dis was expressed and tested for inhibition of platelet aggregation, specific binding activity with seven different integrins, and inhibition of adhesion of three different pancreatic cancer cell lines on laminin-1 and vitronectin. The r-Cam-dis showed potent binding to αvβ3 integrin, but was moderate to weak with αvβ5, αvβ6, α2β1, and α6β1. Interestingly, the inhibition of r-Cam-dis on pancreatic cancer cell lines adhesion to laminin-1 was more effective than that to vitronectin. Based on our binding results to integrin receptors and previous adhesion studies using function-blocking monoclonal antibodies, it is suggested that r-Cam-dis could be inhibiting adhesion of pancreatic cancer cell lines through integrins α2β1, α6β1, αvβ5, and αvβ6.

No MeSH data available.


Related in: MedlinePlus

Interaction of immobilized r-Cam-dis with integrins A) αvβ3, B) αvβ5, C) αvβ6, D) α2β1, E) α6β1, F) α3β1, and G) α5β1. Integrin binding was measured by indirect ELISA assay as described in Materials and Methods. Absorbance at 450 nm of the individual well was measured to determine the binding activity. The error bars represent the standard deviation from two independent experiments with n=2.
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Figure 3: Interaction of immobilized r-Cam-dis with integrins A) αvβ3, B) αvβ5, C) αvβ6, D) α2β1, E) α6β1, F) α3β1, and G) α5β1. Integrin binding was measured by indirect ELISA assay as described in Materials and Methods. Absorbance at 450 nm of the individual well was measured to determine the binding activity. The error bars represent the standard deviation from two independent experiments with n=2.

Mentions: To confirm that r-Cam-dis is capable of direct integrin binding, we employed indirect ELISA assay. As shown in Figure 3, r-Cam-dis was able to bind to integrins αvβ3, αvβ5, αvβ6, α2β1, and α6β1 (Figure 3A-E) but not to α3β1 and α5β1 (Figure 3F and 3G). The binding activity was most potent in the presence of integrin αvβ3. Echistatin, a well-known RGD-disintegrin that bind preferentially to the integrin αvβ3, showed binding specificity to only integrins αvβ3 and αvβ5 and was considerably less effective when compared with r-Cam-dis.


Recombinant disintegrin (r-Cam-dis) from Crotalus adamanteus inhibits adhesion of human pancreatic cancer cell lines to laminin-1 and vitronectin.

Suntravat M, Barret HS, Jurica CA, Lucena SE, Perez JC, Sánchez EE - J Venom Res (2015)

Interaction of immobilized r-Cam-dis with integrins A) αvβ3, B) αvβ5, C) αvβ6, D) α2β1, E) α6β1, F) α3β1, and G) α5β1. Integrin binding was measured by indirect ELISA assay as described in Materials and Methods. Absorbance at 450 nm of the individual well was measured to determine the binding activity. The error bars represent the standard deviation from two independent experiments with n=2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440708&req=5

Figure 3: Interaction of immobilized r-Cam-dis with integrins A) αvβ3, B) αvβ5, C) αvβ6, D) α2β1, E) α6β1, F) α3β1, and G) α5β1. Integrin binding was measured by indirect ELISA assay as described in Materials and Methods. Absorbance at 450 nm of the individual well was measured to determine the binding activity. The error bars represent the standard deviation from two independent experiments with n=2.
Mentions: To confirm that r-Cam-dis is capable of direct integrin binding, we employed indirect ELISA assay. As shown in Figure 3, r-Cam-dis was able to bind to integrins αvβ3, αvβ5, αvβ6, α2β1, and α6β1 (Figure 3A-E) but not to α3β1 and α5β1 (Figure 3F and 3G). The binding activity was most potent in the presence of integrin αvβ3. Echistatin, a well-known RGD-disintegrin that bind preferentially to the integrin αvβ3, showed binding specificity to only integrins αvβ3 and αvβ5 and was considerably less effective when compared with r-Cam-dis.

Bottom Line: The r-Cam-dis showed potent binding to αvβ3 integrin, but was moderate to weak with αvβ5, αvβ6, α2β1, and α6β1.Interestingly, the inhibition of r-Cam-dis on pancreatic cancer cell lines adhesion to laminin-1 was more effective than that to vitronectin.Based on our binding results to integrin receptors and previous adhesion studies using function-blocking monoclonal antibodies, it is suggested that r-Cam-dis could be inhibiting adhesion of pancreatic cancer cell lines through integrins α2β1, α6β1, αvβ5, and αvβ6.

View Article: PubMed Central - PubMed

Affiliation: National Natural Toxins Research Center, Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA.

ABSTRACT
Pancreatic cancer is a malignant cancer common worldwide having poor prognosis, even when diagnosed at its early stage. Cell adhesion plays a critical role in cancer invasion and metastasis. Integrins are major mediators of cell adhesion and play an important role in invasion and metastatic growth of human pancreatic cancer cells. Snake disintegrins are the most potent ligands of several integrins and have potential therapeutic applications for cancers. We have previously cloned and expressed a new recombinant RGD-disintegrin from Crotalus adamanteus (r-Cam-dis). This recently published r-Cam-dis has an extra nine amino acids derived from the vector (SPGARGSEF) at the N-terminus end and has strong anti-platelet activity. However, this r-Cam-dis contains the contamination of the cleavage of the N-terminal end of the pET-43.1a cloning vector. In this study, we have cloned r-Cam-dis in a different cloning vector (pGEX-4T-1) showing five different amino acids (GSPEF) at the N-terminal part. This new r-Cam-dis was expressed and tested for inhibition of platelet aggregation, specific binding activity with seven different integrins, and inhibition of adhesion of three different pancreatic cancer cell lines on laminin-1 and vitronectin. The r-Cam-dis showed potent binding to αvβ3 integrin, but was moderate to weak with αvβ5, αvβ6, α2β1, and α6β1. Interestingly, the inhibition of r-Cam-dis on pancreatic cancer cell lines adhesion to laminin-1 was more effective than that to vitronectin. Based on our binding results to integrin receptors and previous adhesion studies using function-blocking monoclonal antibodies, it is suggested that r-Cam-dis could be inhibiting adhesion of pancreatic cancer cell lines through integrins α2β1, α6β1, αvβ5, and αvβ6.

No MeSH data available.


Related in: MedlinePlus