Novel use of ubiquitin c-terminal hydrolase-l1

A technology of UCH-L1 and its use, which is applied in the field of compositions for diagnosing cancer metastasis, and can solve problems such as poor understanding of specific characteristics

Inactive Publication Date: 2008-12-03
EWHA UNIV IND COLLABORATION FOUND +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, little is known about the substrate specificity of the UCH-L1 enzyme and the specificity characteristics of the isoforms of the DUB family (Christopher N. et al., "substrate specificity of deubiquitinating enzymes; Ubiquitin C-terminal hydrolase," Biochemistry, 37 , 3358-3368, 1998)

Method used

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  • Novel use of ubiquitin c-terminal hydrolase-l1
  • Novel use of ubiquitin c-terminal hydrolase-l1
  • Novel use of ubiquitin c-terminal hydrolase-l1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0103] Embodiment 1 utilizes UCH-L1 to diagnose cancer transfer, suppress cancer transfer and screen the inhibitor of cancer transfer

[0104] I-1. Screening of proteins associated with cancer metastasis to identify target molecules

[0105] In order to identify proteins associated with cancer metastasis, the inventors of the present application analyzed the H157 (squamous cell carcinoma) with invasive ability, the H358 (bronchoalveolar carcinoma) cancer cell line without invasive ability, and the normal lung cell line WI38 ( Differentially expressed proteins in intracellular or extracellular fractions of VA-13 subclone 2RA (fibroblasts).

[0106] H157, H358 and WI38 cells were maintained in RPMI-1640 supplemented with 10% (v / v) fetal bovine serum (FBS), 100 units / mL penicillin, 100 μg / mL streptomycin, 3.75 μg / mL sodium bicarbonate and 10 mM HEPES (pH 7.3, containing 2mM L-glutamine).

[0107] I-1-1) Using Matrigel TM Coated transwell for invasion detection

[0108...

Embodiment II

[0156] B16F10 mouse melanoma stable cells expressing UCH-L1-specific RNAi in Example II decrease in cell migration

[0157] Through the metastasis experiments using animal models, the inventors of the present application tried to clarify whether UCH-L1 is a key target molecule related to cancer metastasis. Normal B16F10 mouse melanoma cells are highly invasive, and their first target organ during metastasis is the lung. Using a lentivirus-based system, the inventors of the present application generated B16F10 stable cells in which UCH-L1 was knocked out.

[0158] After the B16F10 stable cells were prepared, the B16F10 stable cells were injected intravenously into the tail vein to check whether the lung metastases were reduced.

[0159] Figure 11 is a schematic diagram showing a metastasis experiment using an animal model.

[0160] II-1. Preparation of B16F10 mouse melanoma stable cells expressing UCH-L1-specific RNAi

[0161] The expression level of UCH-L1 in the B16F...

Embodiment III

[0174] Embodiment III utilizes UCH-L1 to screen the inhibitor of tumor metastasis

[0175] To screen for chemical agents that inhibit the expression or hydrolase activity of UCH-L1 (inhibitors for cancer metastasis), High Performance Screening (HTS) can be used ( FIGS. 18 and 19 ).

[0176] As shown in Figure 18, ubiquitin-AMC (Ub-AMC, a C-terminal 7-amino-4-methylcoumarin derivative of ubiquitin) can be used as an artificial fluorescent substrate for UCH-L1. The "hit ratio" of UCH-L1 inhibitors can be detected by monitoring the ratio of UCH-L1 released from Ub-AMC to free AMC (relative fluorescence intensity).

[0177] In detail, when the mixture of UCH-L1 (5 ~ 10nM) and Ub-AMC (0 ~ 1000nM) was mixed with different candidate chemical preparations (different concentrations) as inhibitors in UCH assay buffer (50mM Tris-HCl [pH 7.6] , 0.5MmEDTA, 0.1mg / mL albumin, with or without 5mM dithiothreitol), the relative fluorescence intensity can be monitored by HTS to verify the hydro...

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Abstract

The present invention relates to a composition for diagnosing cancer metastasis comprising Ubiquitin C-terminal hydrolase-L1 (UCH-L1), a use of UCH-L1 for diagnosing cancer metastasis and a method for diagnosing cancer metastasis using the same; a composition for suppressing cancer metastasis comprising an inhibitor of UCH-L1, a use of an inhibitor of UCH-L1 for suppressing cancer metastasis and a method of suppressing cancer metastasis using the same; a composition for screening a cancer metastasis inhibitor comprising UCH-L1 protein, a use of UCH-L1 protein for screening a cancer metastasis inhibitor and a method of screening cancer metastasis inhibitor using the same; a composition for screening a cancer metastasis inhibitor comprising UCH-L1 gene, a use of UCH-L1 gene for screening a cancer metastasis inhibitor, a method of screening cancer metastasis inhibitor using the same. UCH-L1 is a key molecule to modulate cell migration including the cancer invasion according to their expression level. Thus, the monoclonal and polyclonal antibodies and the substrate of UCH-L1 can be used for diagnosis of cancer metastasis. Also, the cancer metastasis can be suppressed by inhibiting of the expression of UCH-L1 or activity of the enzyme, thus we can screen and develop an inhibitor of UCH-L1 and use it as an adjuvant drug for anticancer therapy.

Description

technical field [0001] The present invention relates to a composition for diagnosing cancer metastasis comprising ubiquitin C-terminal hydrolase L1 (UCH-L1), the use of UCH-L1 for diagnosing cancer metastasis and a method for diagnosing cancer metastasis using UCH-L1; comprising UCH-L1 Composition of L1 inhibitor for inhibiting cancer metastasis, use of UCH-L1 inhibitor for inhibiting cancer metastasis and method for inhibiting cancer metastasis by using UCH-L1 inhibitor; UCH-L1 protein-containing composition for screening cancer metastasis inhibition Composition of agent, UCH-L1 protein is used for the purpose of screening cancer metastasis inhibitor and the method for utilizing UCH-L1 protein to screen cancer metastasis inhibitor; Composition for screening cancer metastasis inhibitor comprising UCH-L1 gene, UCH -A use of L1 gene for screening cancer metastasis inhibitors and a method for screening cancer metastasis inhibitors using UCH-L1 gene. Background technique [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00A61K31/70
CPCC07K14/4703C12N2310/14C12N15/1137C12N2310/53C12Q1/6886C12Q2600/136A61K31/70A61P35/04G01N33/574G01N33/53
Inventor 李公珠金贤晶赵政宇孟哲永南俞琼林相美金荣美
Owner EWHA UNIV IND COLLABORATION FOUND
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