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Compositions and methods for inhibiting tumor growth and metastasis

a technology of plasminogen and cell surface receptor, which is applied in the field of modulating the activity of p11 protein, can solve the problem of rate-limiting binding of plasminogen to its cell surface receptor, and achieve the effect of reducing the production of a61

Inactive Publication Date: 2007-10-04
WAISMAN DAVID M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] According to the present invention, it was discovered that an annexin II heterotetramer or its subunits stimulates the conversion of plasminogen to A61 in vitro. It was also discovered that an annexin II heterotetramer or its subunits (p36, p11) possesses an intrinsic plasmin reductase activity, and that the cysteinyl residues of both subunits of the ann...

Problems solved by technology

Binding of plasminogen to its cell surface receptors is thought to be rate-limiting for efficient activation of plasminogen by uPA (Stephens et al., 1989; Namiranian et al., 1995, Biochem J 309:977-982).

Method used

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  • Compositions and methods for inhibiting tumor growth and metastasis
  • Compositions and methods for inhibiting tumor growth and metastasis
  • Compositions and methods for inhibiting tumor growth and metastasis

Examples

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example 1

Stimulation of A61 Production

[0091] A61 is an internal fragment of plasminogen that encompasses the sequence Lys78-Lys468 (SEQ ID NO:1). The release of A61 from plasmin is facilitated by the reduction of the Cys462-Cys541 disulfide bond of plasmin. Therefore, the release of A61 generates a free sulfhydryl residue at Cys462. Since plasminogen and plasmin contain only disulfides, A61 can be discriminated from these proteins on the basis of its reactivity with free sulfhydryl-reactive reagents such as 3-(N-maleimidylpropionyl)biocytin (MPB). The reaction of free-sulfhydryl-containing proteins with MPB results in the biotinylation of the protein which allows easy detection with streptavadin-HRP.

[0092] As shown in FIG. 1A (lane 2), the incubation of u-PA with plasminogen resulted in the generation of plasmin. As expected, the plasmin generated by this reaction did not contain a free cysteinyl residue and therefore did not react with MPB (FIG. 1B, lane 2). However, the addition of AIIt ...

example 2

Stimulation of A61 Production by Other Disulfide Reductases

[0100] Protein disulfide isomerase, thioredoxin and phosphoglycerate kinase are three protein disulfide reductases that are secreted by cultured cells (Lay et al., 2000, Nature 408:869-873; Rubartelli et al., 1992, J Biol Chem 267:24161-24164; Terada et al., 1995, J Biol Chem 270:20410-20416; Soderberg et al., 2000, Cancer Res 60:2281-2289). The three reductases have been shown to act as plasmin reductases (Stathakis et al., 1997, J Biol Chem 272:20641-20645; Lay et al., 2000). As shown in FIG. 6, under the assay conditions AIIt was a more potent plasmin reductase than the other reductases in vitro.

[0101] Thioredoxin and protein disulfide isomerase share a common sequence, Trp-Cys-Gly-Pro-Cys-Lys (SEQ ID NO:4), which participates in the cleavage, formation and reshuffling of disulfide bonds. This sequence is not present in phosphoglycerate kinase or AIIt, suggesting that these reductases have distinct catalytic mechanisms....

example 3

Down-Regulation of AIIt Blocks A61 Generation

[0102] HT1080 fibrosarcoma cells were stably transfected (transduced) with a pLin retroviral vector encoding a p11 gene in the sense (pLin-p11S) or antisense (pLin-p11AS) orientation, or an empty pLin vector (pLin-V). The pLin-p11AS transduced cells showed a decrease in both p11 and p36 subunits on the cell surface whereas pLin-p11S transduced cells showed an increase in both p11 and p36 subunits (FIG. 7A). As shown in FIG. 7B, incubation of the pLin-p11S transduced cells with plasminogen resulted in enhanced A61 formation compared to the pLin-V control cells. In contrast, the pLin-p11AS transduced cells failed to produce A61. Additionally, HeLa cells transfected with a p11 antisense expressing vector also failed to convert plasminogen to A61.

[0103] The data in FIG. 7B establishes a role for AIIt in A61 formation in HT 1080 fibrosarcoma cells. However, it is unclear if plasmin could be directly reduced or if plasmin autoproteolysis prec...

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Abstract

Disclosed are compositions and methods useful in the reduction of p11 protein activity in cancer cells. P11 protein is demonstrated to affect plasmin production and activity, MMP activity, plasminogen activation, antiangiogenic plasmin fragment production, cell invasion, tumor development and metastasis. Compositions that modulate levels of p11 either up or down are demonstrated to be effective in reducing tumor development. Also disclosed are cancer treatment methods that employ compositions that modulate p11 activity and clonal cell lines and assays useful for the identification of compositions that modulate p11 activity.

Description

PARENT CASE TEXT [0001] This application is a divisional of U.S. patent application Ser. No. 10 / 735,577, filed on Dec. 12, 2003, which claims benefit of priority to U.S. Provisional Patent Application 60 / 433,140, filed Dec. 13, 2002, and is a continuation-in-part of U.S. patent application Ser. No. 10 / 304,287, filed on Nov. 26, 2002, which claims priority to U.S. Provisional Patent Application 60 / 333,866, filed Nov. 28, 2001, which is now abandoned.GOVERNMENT SUPPORT [0002] This work was supported in part by a grant from the National Institutes of Health (CA78639). The United States Government has certain rights in this invention.SEQUENCE LISTING [0003] A paper copy of the sequence listing and a computer readable form of the same sequence listing are appended below and herein incorporated by reference. The information recorded in computer readable form is identical to the written sequence listing, according to 37 C.F.R. 1.821 (f). BACKGROUND OF THE INVENTION [0004] 1. Field of the I...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N9/64C12N15/113
CPCC12N15/1137C12N15/1138C12N2310/53C12N2310/111C12N2310/14C12N2310/11A61P35/00
Inventor WAISMAN, DAVID M.
Owner WAISMAN DAVID M
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