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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

Effects of r-Cam-dis on adhesion of BxPC-3, Panc-1, and AsPC-1 pancreatic cancer cell lines on vitronectin and laminin-1. A) BxPC-3, B) Panc-1, and C) AsPC-1 were seeded in 96-well plates, which were pre-coated with vitronectin or laminin-1 in the absence (PBS added), or presence of various concentrations of r-Cam-dis. Cell adhesion was measured by MTT technique and the results were expresses as percent of inhibition. The results are expressed as mean±SD (n=3). An asterisk (*) indicates the significant difference between the inhibition of vitronectin adhesion and laminin-1 adhesion in each cell type by r-Cam-dis at P<0.05.
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Figure 4: Effects of r-Cam-dis on adhesion of BxPC-3, Panc-1, and AsPC-1 pancreatic cancer cell lines on vitronectin and laminin-1. A) BxPC-3, B) Panc-1, and C) AsPC-1 were seeded in 96-well plates, which were pre-coated with vitronectin or laminin-1 in the absence (PBS added), or presence of various concentrations of r-Cam-dis. Cell adhesion was measured by MTT technique and the results were expresses as percent of inhibition. The results are expressed as mean±SD (n=3). An asterisk (*) indicates the significant difference between the inhibition of vitronectin adhesion and laminin-1 adhesion in each cell type by r-Cam-dis at P<0.05.

Mentions: The r-Cam-dis inhibited BxPC-3 adhesion to vitronectin and laminin-1, in a concentration-dependent manner with IC50 values of 10.73μM and 6.85μM, respectively (Figure 4A). Adhesion to vitronectin in Panc-1 was dose-dependently inhibited up to 28.9±5.2% (Figure 4B). The inhibition of Panc-1 binding to laminin-1 was not investigated because untreated Panc-1 control cells did not adhere to laminin-1. The r-Cam-dis also inhibited AsPC-1 adhesion to vitronectin and laminin-1 by 19.3±1% and 45±1%, respectively (Figure 4C).


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)

Effects of r-Cam-dis on adhesion of BxPC-3, Panc-1, and AsPC-1 pancreatic cancer cell lines on vitronectin and laminin-1. A) BxPC-3, B) Panc-1, and C) AsPC-1 were seeded in 96-well plates, which were pre-coated with vitronectin or laminin-1 in the absence (PBS added), or presence of various concentrations of r-Cam-dis. Cell adhesion was measured by MTT technique and the results were expresses as percent of inhibition. The results are expressed as mean±SD (n=3). An asterisk (*) indicates the significant difference between the inhibition of vitronectin adhesion and laminin-1 adhesion in each cell type by r-Cam-dis at P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effects of r-Cam-dis on adhesion of BxPC-3, Panc-1, and AsPC-1 pancreatic cancer cell lines on vitronectin and laminin-1. A) BxPC-3, B) Panc-1, and C) AsPC-1 were seeded in 96-well plates, which were pre-coated with vitronectin or laminin-1 in the absence (PBS added), or presence of various concentrations of r-Cam-dis. Cell adhesion was measured by MTT technique and the results were expresses as percent of inhibition. The results are expressed as mean±SD (n=3). An asterisk (*) indicates the significant difference between the inhibition of vitronectin adhesion and laminin-1 adhesion in each cell type by r-Cam-dis at P<0.05.
Mentions: The r-Cam-dis inhibited BxPC-3 adhesion to vitronectin and laminin-1, in a concentration-dependent manner with IC50 values of 10.73μM and 6.85μM, respectively (Figure 4A). Adhesion to vitronectin in Panc-1 was dose-dependently inhibited up to 28.9±5.2% (Figure 4B). The inhibition of Panc-1 binding to laminin-1 was not investigated because untreated Panc-1 control cells did not adhere to laminin-1. The r-Cam-dis also inhibited AsPC-1 adhesion to vitronectin and laminin-1 by 19.3±1% and 45±1%, respectively (Figure 4C).

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