Composition and Method for Treatment of Tumors

a tumor and tumor technology, applied in the field of tumor tumors, can solve the problems of uncontrolled tissue growth, use of members of the tnf superfamily, and metabolic failure of the cell's mitochondria,

Inactive Publication Date: 2013-03-07
SANOFI SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is related to a method for testing whether a compound can stop a protein called TNF-suppressing activity. The method involves growing cells in the presence or absence of TNF superfamily members, and then testing the viability of these cells with or without the compound. The results can show whether the compound is a TNF inhibitor or not.

Problems solved by technology

If the apoptotic process of a cell is disturbed, the cell may continue to divide, thus leading to uncontrolled tissue growth.
The pores formed by Bax (Bcl-2-associated X protein) and Bak (Bcl-2-antagonist / killer 1) cause the loss of mitochondrial membrane polarization and release of cytochrome c, resulting in the metabolic failure of the cell's mitochondria.
Treatment using members of the TNF superfamily has, however, some limitations in that various tumor cells are resistant to apoptosis induced by members of the TNF superfamily, including TRAIL.

Method used

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  • Composition and Method for Treatment of Tumors
  • Composition and Method for Treatment of Tumors
  • Composition and Method for Treatment of Tumors

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0089]This example describes the discovery that Core 1 is involved in resistance to TRAIL-mediated apoptosis.

[0090]A functional genetic screen of a mouse E14 cDNA library g HCT116 colon carcinoma line, a cell line known to be sensitive to TRAIL-induced apoptosis, was conducted [FIG. 1A]. The screen identified UQCRC1, which encodes Core 1, as being involved in resistance to TRAIL-mediated apoptosis.

[0091]HCT116 cells (American Type Culture Collection, Manassas, Va.) and 293 EBNA packaging cell line (Invitrogen) were grown separately in Dulbecco's modified Eagle's Medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco) at 37° C. and 5% CO2.

[0092]A mouse embryonic day-14 retroviral cDNA library (gift of George Daley, MIT, Cambridge, Mass.) was cloned into pEYK retroviral vector and transfected into the above 293 EBNA packaging cell line using Lipofectamine (Invitrogen). Forty-eight to 72 hours after the transfection, the supernatant was c...

example 2

[0094]To further characterize the ability of Core 1 to protect against TRAIL-induced apoptosis, the pEYK vector containing the partial cDNA of UQCRC1 isolated from the screen was used to directly infect HCT116 cells. Cells expressing dominant-negative FADD (dnFADD), which inhibits the signal from the TRAIL receptors, was used as a control. Cells were treated for 48 hours with 0, 10 or 50 ng / mL of TRAIL and assayed for Annexin V, an early marker of apoptosis [FIG. 2A]. Expression of dnFADD inhibited TRAIL-induced apoptosis, with 78% of cells Annexin V negative or non-apoptotic at 50 ng / ml TRAIL. In cells expressing Green Fluorescent Protein (GFP), only 2% were Annexin V negative at 50 ng / ml, compared to 14.5% in Core 1 expressing cells, a 7 fold increase in protection from TRAIL induced apoptosis. This suggests that expression of the partial Core 1 protein provides some protection, but does not completely inhibit TRAIL induced apoptosis.

example 3

[0095]As Core 1 is a subunit of the mitochondrial respiratory Complex III, work was conducted to determine whether expression of Core 1 protects against mitochondrial depolarization, a critical step in mitochondrial mediated apoptosis. Changes in mitochondrial membrane potential during TRAIL treatment were studied using the JC-1 Assay Kit (Molecular Probes). JC-1 (5, 5′,6,6′,-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine) is a cationic dye which has a diffuse green fluorescence in the cytoplasm. The accumulation of JC-1 in the mitochondria, which depends on intact, polarized mitochondria, results in the formation of red fluorescent aggregates. The ratio of green to red fluorescence was measured for the cells described in Example 2 above by Fluorescence-activated Cell Sorter (FACS) analysis. FACS is a method to sort cells based on specific cellular surface markers (e.g., red or green fluorescence). The above ratio is plotted as a percentage of polarized mitochondria.

[009...

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Abstract

The present invention relates to a composition which is useful in the treatment of a tumor, a method for making such a composition, and a method for using such a composition. The invention relates also to a method for assaying for inhibitors of the activity of Core 1 protein and / or other proteins of the respiratory complex III of mitochondria.

Description

FIELD OF THE INVENTION[0001]A tumor is an abnormal growth of tissue which may be malignant or benign. A benign tumor grows only locally whereas a malignant tumor spreads to other tissues.[0002]Cancer is a genetic disease. The ability to resist apoptosis is a hallmark of cancer cells. Many genetic lesions are known to confer resistance to programmed cell death (apoptosis) in cancer cells. During apoptosis, enzymes called caspases carry out the process of breaking down the cell. Initiator caspases cleave inactive pro-forms of effector caspases, thus activating them. The activated effector caspases then proceed to cleave proteins within the cell. In normal tissue, the rate of apoptosis balances the rate of cellular growth in the tissue, thus regulating the growth of the tissue. If the apoptotic process of a cell is disturbed, the cell may continue to divide, thus leading to uncontrolled tissue growth.[0003]Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) is a promising t...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/18C12Q1/68C12N15/113
CPCA61K9/0019A61K31/7105A61K48/00C12N9/0055C12Y110/02002C12N15/1137C12N2310/14C12N2799/027C12N15/1136A61P35/00A61K39/395
Inventor AUGUST, PAULHUANG, WAAN JENGNATESAN, SRIDARANLIEBERMAN, SOYAN
Owner SANOFI SA
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