A method for regulating cancer stem cell growth by inhibiting phosphorylation of 120th threonine residue of tspyl5 protein, a composition containing the peptide sequence functioning to inhibit the phosphorylation and a use thereof

a technology of tspyl5 and phosphorylation, which is applied in the direction of peptide/protein ingredients, peptide sources, drug compositions, etc., can solve the problems of poor cancer prognosis, increased p53 degradation, and still technical limit in developing an inhibitor of radiation-sensitive or radiation-resistant cancer stem cells, so as to inhibit growth and metastasis

Active Publication Date: 2018-11-08
KOREA ATOMIC ENERGY RES INST
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  • Abstract
  • Description
  • Claims
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Benefits of technology

[0022]The present invention relates to a peptide inhibiting the phosphorylation of threonine, the 120th residue of TSPYL5(testis-specific Y-like protein 5). The said peptide was confirmed to inhibit the growth and metastasis of lung cancer cells and the sphere formation as well, so that the peptide composed of the nucleotide sequence represented by SEQ. ID. NO: 43 or NO: 44 that can inhibit the phosphorylation of threonine, the 120th residue of TSPYL5, can be effectively used as an inhibitor of cancer cell growth, metastasis, or cancer stem cell growth.

Problems solved by technology

It has been found that TSPYL5 acts as an inhibitor of USP7, and thereby increases p53 degradation, which results in the poor cancer prognosis(M. T. Epping, et al., Nat.
However, there is a still technical limit in developing an inhibitor of radiation-sensitive or radiation-resistant cancer stem cells.

Method used

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  • A method for regulating cancer stem cell growth by inhibiting phosphorylation of 120th threonine residue of tspyl5 protein, a composition containing the peptide sequence functioning to inhibit the phosphorylation and a use thereof
  • A method for regulating cancer stem cell growth by inhibiting phosphorylation of 120th threonine residue of tspyl5 protein, a composition containing the peptide sequence functioning to inhibit the phosphorylation and a use thereof
  • A method for regulating cancer stem cell growth by inhibiting phosphorylation of 120th threonine residue of tspyl5 protein, a composition containing the peptide sequence functioning to inhibit the phosphorylation and a use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Over-Expressing Cells

[0079] Construction of Vectors Over-Expressing TSPYL5, PTEN, and AKT1 Genes

[0080]The cells over-expressing TSPYL5, PTEN, and AKT1 genes were constructed as follows. First, the TSPYL5, PTEN, and AKT1 genes were obtained by reverse transcription from the mRNA originated from the lung cancer cell line A549 or H460.

[0081]Particularly, 1 ml of trisol was added to the lung cancer cell line A549 or H460, which was well mixed for 5 minutes. 200 μl of chloroform reagent was added thereto, which was well mixed again for 5 minutes. The mixture was centrifuged at 4° C. for 10 minutes. 200 μl of the supernatant was transferred into a new tube. 500 μl of isopropanol was added thereto, followed by reaction for 10 minutes at room temperature. Centrifugation was performed again at 4° C. to eliminate the supernatant. The precipitate was washed with DEPC solution containing 75% ethanol, followed by centrifugation again to eliminate the supernatant. The precipitate ...

example 2

Inhibition of the Expressions of TSPYL5, AKT, and PTEN

[0090]The following experiment using siRNA was performed to inhibit the intracellular expressions of TSPYL5, AKT, and PTEN.

[0091]Particularly, the expressions of TSPYL5 and AKT were inhibited in A549 cells, and the expression of PTEN was inhibited in H460 cells. 2×105 cells were transfected with 100 nM of each gene specific siRNA and Scrambled Stealth™ RNA molecule(negative control: siControl) in penicillin-streptomycin solution free medium using Lipofectamine RNAi MAX(Invitrogen) according to the manufacturer's protocol. After 4˜6 hour reaction, the medium was replaced with the medium supplemented with 100 units / mt of penicillin-streptomycin, followed by culture for 72 hours.

TABLE 3SEQ.PrimerSizeSequence(5′-3′)ID. NOsiTSPYL525 merAAAGGUAGAACUGCA17InvitrogenAGGGAUUGGGCCCAAUCCCUUGCAG18UUCUACCUUUsiPTEN21 merGAUAUCAAGAGGAUG19BioneerGAUU(dTdT)AAUCCAUCCUCUUGA20UAUC(dTdT)siAKT21 merGACUGACACCAGGUA21BioneerUUUU(dTdT)AAAUACCUGGUGUCA22GUC...

experimental example 1

Regulation Effect of PTEN on TSPYL5 Expression

[0093] Changes in the Expressions of ALDH and CD44 According to the Over-Expression or Inhibition of PTEN

[0094]The expressions of the cancer stem cell markers ALDH1 and CD44 were measured by flow cytometry after ALDEFLUOR staining and CD44 antibody staining to investigate the changes of cancer stem cell characteristics according to the over-expression or inhibition of PTEN.

[0095]Particularly, A549 cells(VC), PTEN over-expressing A549 cells(PTEN(+)), H460 cells(si-CTL), and PTEN suppressed H460 cells(si-PTEN) were added with 0.5 ml of ALDEFLUOR assay buffer, resulting in 1×106cell / ml of mixture. 5 μl of the lysed cells and the activated ALDEFLUOR substrate was loaded respectively in two empty tubes. 500 μl of the mixture was loaded in the control tube. 5 μl of DEAB(diethylaminobenzaldehyde), the ALDH1 activity inhibitor, was added only to the control. Then, reaction was induced at 37° C. for 30 minutes. Centrifugation was performed to eli...

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Abstract

The present invention relates to a peptide suppressing the phosphorylation of threonine(T120), the 120th residue of TSPYL5 (testis-specific Y-like protein 5), which is specifically as follows. The present inventors constructed T120D, the mutant of the 120th residue threonine(T120) of TSPYL5, and T120A-TSPYL5 gene and then transfected cells with them in order to investigate the effect of phosphorylation on T120 residue. As a result, wild-type TSPYL5 and T120D moved into nucleus and stayed there. But in the case of T120A-TSPYL5, TSPYL5 did not move into nucleus and instead it was expressed only in cytoplasm. The protein could not bind to AKT, either. Instead, ubiquitination of TSPYL5 was increased but SUMOylation was inhibited. Also, the expressions of ALDH1-A1, -A3, CD44 gene and protein were reduced, and thereby the growth and metastasis of lung cancer cells were suppressed and sphere formation was reduced. Based on the observation above, the inventors constructed the peptide composed of the amino acid sequences represented by SEQ. ID. NO: 43 or NO: 44 that could inhibit phosphorylation of the 120th residue threonine of TSPYL5. The said peptide can be effectively used as a composition for the inhibition of cancer cell growth, metastasis, and cancer stem cell growth.

Description

TECHNICAL FIELD[0001]The present invention relates to a peptide inhibiting phosphorylation of the 120th residue threonine of TSPYL5(testis-specific protein, Y-encoded-like 5) and a composition comprising the same for the inhibition of cancer cell growth, metastasis, or cancer stem cell growth.BACKGROUND ART[0002]Cancer stem cells are a self-renewal small cell group displaying pluripotent potency enabling the differentiation into various tissues and cells like general stem cells. So, with a small number of these cells, a tumor can be induced in an experimental animal model. Cancer stem cells are also highly resistant against chemotherapy and radiotherapy (B. M. Boman, M. S. Wicha, Cancer stem cells: A step toward the cure, J. Clin. Oncol.(2008) 26:2795-2799).[0003]Cancer stem cells were first reported in acute myeloid leukemia, and later were found in general solid cancers including breast cancer, leading to the identification of solid cancer stem cells(D. Bonnet, J. E. Dick, Human a...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/02C07K14/47C07K7/08A61P35/04
CPCA61K38/02A61P35/04C07K7/08C07K14/4702A61K38/00A61K38/10C12N5/0695
Inventor KIM, IN GYUKIM, SEO YEONLEE, JEI HACHOI, SOO IMKIM, MIN SIKKIM, JUNG YULSHIN, BYUNGCHULJUNG, UHEE
Owner KOREA ATOMIC ENERGY RES INST
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