KR20160047735A - Composition for the cancer stem cells growth and Anti-Cancer metastasis containing TSPYL5 expression or activation inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising as an active ingredient an expression or activity inhibitor of TSPYL5 (testis-specific protein, Y-encoded-like 5) , The expression level of TSPYL5 (testis-specific protein, Y-encoded-like 5), and the cancer stem cell markers ALDH1 and CD44 were markedly increased and the expression or activity of TSPYL5 was inhibited by the expression of the cancer stem cell markers ALDH1 and CD44 TSPYL5 expression or activity inhibitor has been shown to inhibit the growth of cancer stem cells and cancer in a variety of tumors by confirming that the EMT function, which is an important pathway of cancer stem cell growth and metastasis, It can be usefully used as an effective ingredient of a composition for inhibiting metastasis.

Description

[0001] The present invention relates to a composition for inhibiting cancer stem cell growth and cancer metastasis containing TSPYL5 expression or activity inhibitor as an active ingredient,

The present invention relates to a composition for inhibiting cancer stem cell growth and cancer metastasis comprising an expression or activity inhibitor of TSPYL5 (testis-specific protein, Y-encoded-like 5) as an active ingredient and a composition for inhibiting tumor growth and cancer metastasis by using the TSPYL5 and cancer stem cell marker ALDH And a screening method for candidate substances for inhibiting metastasis.

In recent years, the number of patients with cancer has increased, and the number of treatments through chemotherapy and radiation has also increased. About 30 to 50% of patients receive radiation therapy during the cancer treatment period. Radiation therapy, which is used to reduce tumor size through preoperative radiotherapy and to remove residual malignant cells after surgery, has been used to treat low-dose radiation with a relatively low dose of radiation in order to reduce adverse effects on high- Radiation therapy is used, and its effectiveness differs through the nature of the cancer, its combination with patients and anticancer agents. In general, radiotherapy is performed on uterine, lung, parenchymal, brain, breast, colorectal, laryngeal, and head and neck cancer. Or recurrence of cancer and worsening, despite the success of initial treatment.

Lung cancer is the leading cause of cancer mortality and has the lowest rate of cure. It has been reported that the probability of survival for 5 years is less than 15% even if the initial treatment response is good (Jemal A, Siegel R, Cancer statistics. CA. Cancer . J. Clin . 56 (2006) 106-130). In the present invention, lung cancer refers to cancer originating from lung cancer cells, and lung cancer is classified into small cell lung cancer and non-small cell lung cancer. Of these, small-cell lung cancer accounts for about 15% of lung cancer, and it develops in the bronchial and bronchial regions and has a faster progression than non-small cell lung cancer. Non-small cell lung cancer is divided into three types: adenocarcinoma, squamous cell carcinoma (SCC), and large cell carcinoma. Non-small cell lung cancer accounts for approximately 40% It is produced in peripheral bronchi. Squamous cell carcinomas are present in about 25% of non-small cell lung cancers and are generally thought to originate from the central bronchus, and hyaline cell carcinomas may be derived from neuroendocrine cells and may be mixed with other non-small cell lung cancers. Small cell or non-small cell lung cancer As such with many differences in genetic, histological feature, immuno phenotype and clinical treatment surface (Travis WD Pathology of lung cancer Clin Chest Med 32 (2011) 669692;..... Spira A, Ettinger DS, Multidisciplinary management of lung cancer, N Engl J. Med ., 350 (2004) 379-392).

Recently, the claim that cancer stem cell is the main cause of cancer recurrence after radiation therapy is attracting attention. Cancer cells can be self-renewed constantly and can produce tumors in experimental animal models with a small number of cell populations, such as stem cells, which have various potential capacity (pulprint potency) (BM Boman, MS Wicha, Cancer stem cells: a step toward the cure, J. Clin . Oncol . 26 (2008) 2795-2799). The presence of cancer stem cells was first demonstrated in acute myelogenous leukemia (leukemia), and in the case of general solid tumors including breast cancer, the existence of stem cells was confirmed in solid tumor types by demonstrating cancer stem cells (D. Bonnet, JE Dick, ... leukemia is organized as hierachy that originates from a primitive hematopoietic cell Nat Med 3 (1997) 730- 737;.. M. Al-Hajj, MF Clarke, Self-renewal and tumor stem cells Oncogene 23 (2003) 7274- 7284). In particular, in the brain tumor, the transmembrane protein CD133 (prominin-1 or AC133) was used as a marker to recognize and isolate cancer stem cells. Thus, when 100 transgenic CD133 + cells were transplanted into NOD mice, new tumors were formed (SK Singh, C. Hawkins, ID Clarke, et al., Identification of human brain tumor initiating cells, Nature 432 (2004) 401). Another penetrating protein, CD44 (hyaluronate receptor or P-glycoprotein 1), has also been used as a marker for cancer stem cells in solid tumors. For example, in breast cancer, cells isolated by the combination of CD44 and other markers induced xenograft tumor growth (M. Al-Hajj, MS Wicha, A. Bentino-Hernandez et al, prospective identification of tumorigenic breast cancer cells. Proc . Natl. Acad. Sci. USA. 100 (2003) 3983-3988). Along with these membrane-penetrating proteins CD133 and CD44, a new promising cancer stem cell marker is aldehyde dehydrogenase 1 (ALDH1).

ALDH1 is a detoxifying enzyme that oxidizes intracellular aldehydes and is highly resistant to alkylating agents or oxidative stress (M. Magni, S. Shammah, R. Schiro. Et al., Induction . of cyclophosphamide-resistance by aldehyde- dehydrogenase gene transfer Blood 87 (1996) 1097-1103; NA Sophos, V. Vasiliou, Aldehyde dehydrogenase gene superfamily:.... the 2002 update, Chem Biol Interact 143-144 (2003) 5 -22). In addition, ALDH converts retinol (Vitamine A) to retinoic acid, the most active form of retinoids important for the treatment and prevention of cancer, affecting cell growth and proliferation. ALDH is generally known to have 19 genes and is classified into ALDH 1, II, and III classes. Of these, only ALDH1 has retinal dehydrogenase activity that converts retinal to retinoic acid, and ALDH1A1 and retinaldehyde dehydrogenase 3 (ALDH1A3) have the most efficient activity (A. Sima, M . Parisotto, S. Mader, PV Bhat , kinetic. characterization of recombinant mouse retinal dehydrogenase type 3 and 4 for retinal substrates. Biochim. Biophys. Acta 1790 (2009) 1660-1664). Therefore, the activity of ALDH1 is important for the isolation of subpopulations of cancer stem cells in various cancer cell lines including lung cancer cells (J. Feng, Q. Qiu, K. Abha et al, Aldehyde dehydrogenase 1 is a tumor stem cell -associated marker in lung cancer, Mol. Cancer. Res. 7 (2009) 330-338). Therefore, in order to avoid the recurrence and metastasis of cancer cells and completely eliminate the cancer, development of an anticancer drug which removes the cancer stem cells by targeting the cancer stem cells having the characteristics of the stem cells, need.

The TSPYL5 gene is located in the chromosome 8q22, one of the testis-specific protein, Y-encoded-like (TSPY-L) family. It is one of the 70 genes that are predominantly expressed in breast cancer and are expected to play an important role in the carcinogenesis process of breast cancer (van't Veer, LJ et al. Nature . 415: 530-536, 2002). In addition, it has been reported to be one of 10 possible genes as a classification gene to distinguish between head and neck squamous cell carcinoma and lung squamous cell carcinoma (Kim, TY et al., Clin . Cancer Res. 13 (10): 2905-2915, 2007) . However, the cell physiological function of the TSPYL5 gene is still unknown.

Currently, chemotherapy and radiation therapy are mainly used for chemotherapy. In particular, except for surgery, chemotherapy and radiotherapy use various preparations and radiation, but their application is limited to the extent that the human body can afford. The limited application of these chemotherapy and radiation therapies has the disadvantage of limiting the range of application and limiting efficacy in clinical trials no matter how good the efficacy is in animal tests. This is due to adverse effects caused by the appearance of cancer cells with anticancer drug resistance or cytotoxicity of each therapy. For example, cisplatin is one of the most effective anti-cancer agents among the over 30 types of cancer drugs currently being used in clinical practice. It is one of the most useful drugs for cancer such as testicular cancer, ovarian cancer, lung cancer, head and neck cancer, bladder cancer, gastric cancer, (Teni Boulikas, Oncology < RTI ID = 0.0 > Reports , 10: 1663-1682, 2003). However, recently, many cancers resistant to cisplatin have been reported, and cancer cells having such resistance are currently being challenged, and there is a risk of recurrence after cancer treatment, so that a combination therapy of cisplatin with other chemicals or cisplatin and intracellular expressed protein (Tito Fojo, Oncogene , 22: 7512-7523, 2003). In the present study, Radiation therapy is an anti-cancer therapy that treats various cancers occurring in the human body by irradiating an appropriate amount of radiation to the affected part. Such a radiation therapy lowers the hematopoietic function of the human body and suppresses the immune system, There is a problem of lowering. Furthermore, since cancer cells acquire resistance through repetitive irradiation, these radiation therapies also have limitations in chemotherapy.

As a result of the efforts to solve the above problems, the present inventors have found that A549 cells, which are non-small cell carcinoma cells, are irradiated with dividing radiation, and the amount of TSPYL5 and ALDH1 and CD44 are significantly increased and expression of TSPYL5 Confirming that activation inhibition significantly reduces EMT function, which is a crucial pathway for cancer stem cell growth and metastasis in various cell lines, confirming that the TSPYL5 expression or activity inhibitor can be effectively used as an active ingredient of a composition for inhibiting cancer metastasis Thus completing the present invention.

It is an object of the present invention to provide a pharmaceutical composition for preventing and inhibiting cancer metastasis comprising as an active ingredient an expression or activity inhibitor of TSPYL5 (testis-specific protein, Y-encoded-like 5).

In order to accomplish the object of the present invention, the present invention provides a pharmaceutical composition for preventing and inhibiting cancer metastasis comprising as an active ingredient an expression or activity inhibitor of TSPYL5 (testis-specific protein, Y-encoded-like 5).

In addition, the present invention provides a kit for inhibiting cancer stem cell growth comprising TSPYL5 expression or activity inhibitor.

The present invention also provides a method for inhibiting cancer stem cell growth comprising treating cancer cells with the expression or activity inhibitor of TSPYL5.

In addition,

1) preparing a TSPYL5 expressing cell line;

2) treating the cell line of step 1) with the test substance;

3) measuring the expression or activity of ALDH in the cell line; And

4) screening a candidate substance for cancer metastasis inhibitor, which comprises selecting a test substance whose expression or activity level of ALDH is lower than that of a control group not treated with the test substance.

In addition,

1) preparing a TSPYL5 expressing cell line;

2) treating the cell line of step 1) with the test substance;

3) measuring the expression or activity of ALDH in the cell line; And

4) selecting a test substance whose expression or activity level of ALDH is lower than that of a control group in which the test substance is not treated, in a screening method for a cancer stem cell growth inhibitor candidate substance.

The present invention is based on the finding that TSPYL5 (testis-specific protein, Y-encoded-like 5), and the expression levels of ALDH1 and CD44, which are cancer stem cell markers, are significantly increased by TSPYL5 Expression or activity inhibition significantly inhibited the expression of ALDH1 and CD44, which are cancer stem cell markers, and thereby significantly inhibited the EMT function, which is an important pathway for cancer stem cell growth and metastasis. By confirming that the growth of malignant cells is inhibited, The TSPYL5 expression or activity inhibitor may be usefully used as an active ingredient of a composition for inhibiting cancer stem cell growth and cancer metastasis in various tumors.

Brief Description of the Drawings Figure 1 shows the results of irradiating lung cancer cell lines with split radiation:
(A) shows the expression of TSPYL5 and the expression of ALDH1A1, ALDH1A3, CD44, Sox2, Oct4, PTEN, Jagged1 and β-catenin in lung cancer cell lines by western blotting B) shows the result of immunofluorescence analysis of TSPYL5 and ALDH1A1 expression by irradiating the lung cancer cell line with split radiation (2 Gy x 3).
Figure 2 shows the EMT correlations of lung cancer cell lines by split radiation:
(A) shows the results of migration and mobility invasion by irradiating the lung cancer cell lines with split radiation, (B) irradiates the lung cancer cell line with dividing radiation to detect EMT markers N-cadherin, E-cadherin and Expression of Snail1 was confirmed by Western blotting.
FIG. 3 (A) shows separation of ALDH1 active and ALDH1 inactive cells by AACSF using a FACS machine after Aldefluor staining on A549 cells, (B) ALDH1 (+, high) activity indicating the activity of lung cancer stem cells (C) shows the results of colony formation assay of cells with ALDH1 (-, low) inactive cells and ALDH1A1 (+, high) activated cells and ALDH1 (ALDH1A3, CD44 and CD133) in the ALDH1 (+, high) and ALDH1 (-, low) inactive cells. And the results are shown.
FIG. 4 (A) shows the result of PCR and Western blotting to examine the correlation between the overexpression and inhibition of the TSPYL5 gene and ALDH1 by the overexpression and inhibition of the TSPYL5 gene in the lung cancer cell line, and (B) (C) shows the change of ALDH1 expression during TSPYL5 overexpression and inhibition using FACS after Aldeflour staining, (D) shows the change of TSPYL5 overexpression Western blot and colony formation analysis through inhibition of TSPYL5 in ALDH1 (+, high) activated cells.
FIG. 5 shows the results of confirming the ability of the TSPYL5 gene to inhibit and overexpress cell metastasis in a lung cancer cell line:
(A) and (C) show the expression of the EMT markers N-cadherin, E-cadherin, Twist, Vimentin and Snail1 by inhibition and overexpression of the TSPYL5 gene in lung cancer cell lines, (D) shows the inhibition and overexpression of the TSPYL5 gene in lung cancer cell lines to confirm infiltration and mobility.
FIG. 6 is a graph showing the results of inhibition and overexpression of TSPYL5 gene in lung cancer cell line.
(A) shows the results of sphere formation and Western blotting of A549 cells continuously suppressing TSPYL5 through A549 cells and shRNA-TSPYL5, (B) shows the sphere formation of H460 cells overexpressing H460 cells and TSPYL5 The result is confirmed by Western blot experiment.
FIG. 7 shows the results of confirming the expression of TSPYL5 protein and cancer stem cell markers after inhibiting or overexpressing the ALDH1 gene to confirm the relationship between ALDH1 and TSPYL5, the lung cancer stem cell markers:
(A) shows the results of western blot analysis of the expression of CD44, CD133, Sox2, Oct3 / 4, Nanog and β-catenin and TSPYL5, which are cancer stem cell markers ALDH1A1 and ALDH1A3, (C) overexpresses the human stem cell markers ALDH1A1 and ALDH1A3 and inhibits the expression of CD44, CD133, Sox2, Oct3 / 4, Nanog, and β-catenin and TSPYL5 expression were confirmed by western blotting. (D) shows the results of confirming cell growth through analysis of colony formation after overexpression of ALDH1A1 and ALDH1A3.
8 is a graph showing the results of confirming the ability of the ALDH1 gene to inhibit and overexpress cell metastasis in a lung cancer cell line:
(A) and (C) show the expression of the EMT markers N-cadherin, E-cadherin, Twist, Vimentin and Snail1 by inhibition and overexpression of the ALDH1 gene in lung cancer cell lines, (D) shows the inhibition and overexpression of the ALDH1 gene in lung cancer cell lines to confirm infiltration and mobility.
FIG. 9 shows the sphere formation experiments of the cancer stem cell growth during TSPYL5 inhibition in the lung cancer cell line Calu3 cell, hepatocellular cell HepG2 cell and pancreatic cancer cell line Panc1 cell with a large number of TSPYL5 genes, and Western blot analysis of the cancer stem cell markers ALDH1A1 and ALDH1A3 Fig.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing and inhibiting cancer metastasis comprising, as an active ingredient, an expression or activity inhibitor of TSPYL5 (testis-specific protein, Y-encoded-like 5).

The TSPYL5 preferably has the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

The TSPYL5 expression inhibitor is preferably an antisense nucleotide complementary to the mRNA of the TSPYL5 gene, a small interfering RNA or shRNA (short haripin RNA), and the activity inhibitor of TSPYL5 is complementary to the TSPYL5 protein Binding compound, peptide, peptide mimetics, and an antibody.

The siRNA is composed of a sense sequence of 15 to 30 mers selected in the nucleotide sequence of the mRNA of the gene encoding the human TSPYL5 protein (SEQ ID NO: 2) and an antisense sequence complementarily binding to the sense sequence At this time, the sense sequence is preferably composed of 25 bases, and most preferably has the nucleotide sequence of SEQ ID NO: 17 or 18, although it is not particularly limited thereto.

The shRNA refers to double-stranded RNA of a hairpin structure having a loop region of 2 to 10 bases, and bases of such a loop region can be those known in the art (Proc. Natl. Acad. Sci. USA Nature Biotechnology 20: 505-508, 2002; Nature Biotechnology 20: 500-505, 2002; Nat Cell Biol. 5: 489-490, 2003; Proc. Natl. Acad. Sci USA 99 (9): 6047-6052, 2002). The double stranded portion of the shRNA can be constructed similarly to the siRNA.

The antisense nucleotide binds (hybridizes) to a complementary base sequence of DNA, immature-mRNA or mature mRNA, as defined in the Watson-click base pair, to interfere with the flow of genetic information as a protein in DNA. The nature of antisense nucleotides that are specific for the target sequence makes them exceptionally multifunctional. Because antisense nucleotides are long chains of monomeric units they can be readily synthesized against the target RNA sequence. Many recent studies have demonstrated the utility of antisense nucleotides as biochemical means for studying target proteins (Rothenberg et al., J. Natl . Cancer Inst . , ≪ / RTI > 81: 1539-1544, 1999). The use of antisense nucleotides can be considered as a novel type of inhibitor since there has been much progress in the field of oligonucleotide chemistry and in the field of nucleotide synthesis showing improved cell adsorption, target binding affinity and nuclease resistance.

The peptide mimetics inhibit the activity of the TSPYL5 protein as peptides or non-peptides that inhibit the binding domain of the TSPYL5 protein. Major residues of non-hydrolyzable peptide analogs include the beta-turn dipeptide core (Nagai et al. Tetrahedron Lett., 26: 647, 1985), keto-methylene pseudopeptides (Ewenson et al. J Med Chem 29: 295, 1986; and Ewenson et al. In Peptides: Proceedings of the 9th American Peptide Symposium, Pierce Chemical Co. Rockland, IL, 1985), Huffman et al in Peptides (Chemistry and Biology, GR Marshall ed ESCOM Publisher: Leiden, Netherlands, 1988), β-aminoalcohols (Gordon et al., Biochem Biophys Res Commun 126: 419 1985) and a substituted gamma-lactam ring (Chemie and Biology, GR Marshell ed., ESCOM Publisher: Leiden, Netherlands, 1988).

The TSPYL5 expression or activity inhibitor preferably inhibits the growth and metastasis of cancer stem cells but is not limited thereto.

Wherein the cancer is selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, It is preferably any one selected from the group consisting of cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor. , Uterine cancer, colon cancer, renal cancer, liver cancer, lung cancer, ovarian cancer, stomach cancer and pancreatic cancer, but is not limited thereto.

In a specific example of the present invention, the present inventors confirmed the increase in expression of TSPYL5 by dividing radiation, and it was confirmed that the expression level of TSPYL5 protein and cancer stem cell marker and related protein was increased during irradiation, Gy) irradiation, the TSPYL5 protein and the cancer stem cell marker and the related protein were overexpressed in the 3 times split irradiation (refer to FIG. 1).

In addition, the infiltration and mobility of cancer cells due to the split radiation were examined. As a result, the infiltration and mobility of A549 cells irradiated with the split radiation were further increased, and the epithelial cell marker The expression level of E-cadherin decreased and the expression level of N-cadherin and Snail, which are mesenchymal cell markers, increased (see FIG. 2).

In addition, ALDH1-activated cells and ALDH1-inactivated cells isolated from A549 cells were found to be well grown, but inactive cells did not grow. In ALDH1-activated cells, ALDH1A1, ALDH1A3, CD44 and CD133, and TSPYL5, and conversely PTEN was found to be more abundant in the ALDH1 inactive cells than in the active cells (see FIG. 3).

In addition, when TSPYL5 was overexpressed, the expression of ALDH1, which is a cancer stem cell marker, was further increased and more cell proliferation was confirmed. As a result, when TSPYL5 expression was suppressed, a cancer stem cell marker (TSPYL5) and ALDH gene and protein were suppressed in the ALDH-activated cells and the cancer cell growth was also inhibited (see FIG. 4). The expression of TSPYL5 by the infiltration / As a result of confirming the transfer ability through the mobility analysis, it was confirmed that the inhibition of TSPYL5 expression inhibited the metastasis and increased the transduction capacity through overexpression of TSPYL5 (see FIG. 5).

In addition, when the expression of TSPYL5 was inhibited, the size of sphere, which is a main characteristic of malignant transformation, was relatively small, and cell growth was inhibited. In the case of overexpression of TSPYL5 expression, sphere The size was increased and the cell growth was increased (see FIG. 6).

When ALDH1 expression was inhibited, the expression level of TSPYL5 was not changed, and cell growth was significantly reduced. When ALDH1 was overexpressed, expression of TSPYL5 (Fig. 7). When the expression of ALDH1 was inhibited, TSPYL5 was found to be upregulated by invasion / mobility analysis The expression of N-cadherin, Vimentin, Twist, Snail1 and Slug, which are the protein markers of mesenchymal cells, is decreased and the expression of E-cadherin, a marker of epithelial cells, is increased. The expression of N-cadherin, Vimentin, Twist, Snail1, and Slug, which are protein markers of mesenchymal cells, as an EMT marker, was increased by ALDH1 overexpression Exerts, marker expression of E-cadherin epithelial cells was confirmed that the reduced (see Fig. 8).

In addition, TSPYL5 expression was suppressed in Calu3 cells (adenocarcinoma), hepatocarcinoma HepG2 cells and pancreatic cancer Panc1 cells, in which TSPYL5 gene is expressed more than non-small cell lung cancer cell, A549 cell, and the main characteristic of malignant transformation The size of sphere, which is the main characteristic of malignant transformation, was relatively small when the expression of TSPYL5 was suppressed in the lung cancer cell Calu3 cell, hepatocellular HepG2 cell and pancreatic cancer cell line Panc1 cell similarly to A549 cell It was confirmed that the growth of cancer stem cells was inhibited by the decrease of the expression amount of ALDH1, which is a major cancer stem cell marker (see FIG. 9).

Therefore, in the present invention, when A549 cells, which are non-small cell carcinoma cells, were irradiated with the split radiation, the expression level of TSPYL5 and the cancer stem cell markers ALDH1 and CD44 were markedly increased, and the expression or activity inhibition of TSPYL5 was inhibited by the cancer stem cell markers ALDH1 TSPYL5 expression or activity inhibitor was shown to inhibit the growth of malignant cells in various types of cancer by confirming that EMT function, which is an important pathway of cancer stem cell growth and metastasis, is suppressed by suppressing the expression level of CD44, It can be used as a pharmaceutical composition for suppressing cancer metastasis.

The composition of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The composition of the present invention can be administered orally or parenterally, and it is preferable to select an external or intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection, But is not limited thereto.

The composition of the present invention can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to conventional methods have. Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate (calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at a dose of 0.0001 to 1 g / kg per day, preferably 0.001 to 200 mg / kg per day, but is not limited thereto. The above administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

In addition, the present invention provides a kit for inhibiting cancer stem cell growth comprising TSPYL5 expression or activity inhibitor.

The TSPYL5 preferably has the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

Wherein the cancer is selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, It is preferably any one selected from the group consisting of cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor. , Uterine cancer, colon cancer, renal cancer, liver cancer, lung cancer, ovarian cancer, stomach cancer and pancreatic cancer, but is not limited thereto.

The kit may further include a washing solution or an eluting solution capable of removing the substrate to be color-developed with the enzyme and the unbound protein and retaining only the combined marker. The samples used for the analysis include biological samples capable of identifying disease-specific polypeptides that can be distinguished from normal conditions such as serum, urine, and saliva. Preferably from a biological fluid sample, for example blood, serum, plasma, more preferably serum. The sample may be prepared to increase the detection sensitivity of the marker. For example, a serum sample obtained from a patient may be analyzed by anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, sequential extraction, or gel electrophoresis. However, the present invention is not limited thereto.

In the present invention, when TSPYL5 is overexpressed in the lung cancer cell line, expression of the cancer stem cell marker, ALDH1, is increased. On the contrary, when the expression of TSPYL5 is inhibited by siRNA, expression of the cancer stem cell marker, ALDH1 is decreased It is possible to use various kinds of cancer stem cell growth inhibition kits by selecting test substances whose expression or activity level of ALDH1 is decreased according to the expression or inhibition of TSPYL5 expression.

The present invention also provides a method for inhibiting cancer stem cell growth comprising treating cancer cells with the expression or activity inhibitor of TSPYL5.

The TSPYL5 preferably has the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

Wherein the cancer is selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, It is preferably any one selected from the group consisting of cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor. , Uterine cancer, colon cancer, renal cancer, liver cancer, lung cancer, ovarian cancer, stomach cancer and pancreatic cancer, but is not limited thereto.

In the present invention, when TSPYL5 is overexpressed in the lung cancer cell line, expression of the cancer stem cell marker, ALDH1, is increased. On the contrary, when the expression of TSPYL5 is inhibited by siRNA, expression of the cancer stem cell marker, ALDH1 is decreased It is possible to use various methods for inhibiting the growth of cancer stem cells by selecting a test substance whose expression or activity level of ALDH1 is decreased according to the expression or inhibition of TSPYL5 expression.

In addition,

1) preparing a TSPYL5 expressing cell line;

2) treating the cell line of step 1) with the test substance;

3) measuring the expression or activity of ALDH (aldehyde dehydrogenase) of the cell line; And

4) screening a candidate substance for cancer metastasis inhibitor, which comprises selecting a test substance whose expression or activity level of ALDH is lower than that of a control group not treated with the test substance.

In addition,

1) preparing a TSPYL5 expressing cell line;

2) treating the cell line of step 1) with the test substance;

3) measuring the expression or activity of ALDH in the cell line; And

4) selecting a test substance whose expression or activity level of ALDH is lower than that of a control group in which the test substance is not treated, in a screening method for a cancer stem cell growth inhibitor candidate substance.

In the above method, the TSPYL5 expressing cell line of step 1) is preferably prepared by irradiating a cancer cell line with radiation.

It is preferable to irradiate the above-mentioned radiation with a single irradiation of 2 Gy or 4 Gy, or 2 to 5 divided radiation of 2 Gy, and more preferably, three divided irradiation of 2 Gy.

The TSPYL5 preferably has the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

Wherein the cancer is selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, It is preferably any one selected from the group consisting of cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor. , Uterine cancer, colon cancer, renal cancer, liver cancer, lung cancer, ovarian cancer, stomach cancer and pancreatic cancer, but is not limited thereto.

In the present invention, when TSPYL5 is overexpressed in the lung cancer cell line, expression of the cancer stem cell marker, ALDH1, is increased. On the contrary, when the expression of TSPYL5 is inhibited by siRNA, expression of the cancer stem cell marker, ALDH1 is decreased As a result, various kinds of tumor metastasis inhibitor and cancer stem cell growth inhibitor candidates can be screened by selecting test substances whose expression or activity level of ALDH1 is decreased according to expression or inhibition of TSPYL5 expression.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Radiation-induced lung cancer cell lines TSPYL5  Check gene expression level

<1-1> Non-small cell Lung cancer cell line  By culture and split radiation TSPYL5  Check the expression level

To confirm the increased expression of TSPYL5 by dividing radiation, gamma ray was irradiated to the lung cancer cell line using the gamma ray irradiation facility (source: ⁶⁰ C ₀ ) of the Korea Atomic Energy Research Institute and Western blotting experiment was performed.

The cells were treated with 10% fetal bovine serum (Hyclone) and 100 units / ml penicillin-streptomycin solution (Hyclone) in RPMI-1640 medium (Hyclone) Mixed and cultured. The cells were incubated at 37 ° C in a 5% CO ₂ , And the cells were cultured in 75-well flasks at 1 × 10 ⁶ cells / well. Two days after irradiation, 2 Gy of radiation was irradiated three times, and 2 Gy and 4 Gy were irradiated to A549 cells prepared as a comparative group on the last 3 irradiation days. The irradiated cells were incubated at 37 ° C in a 5% CO ₂ Lt; / RTI &gt; for 7 days. Each of the cells was collected, and 50 μl of protein lysis solution was added thereto, followed by reaction at 4 ° C for 30 minutes. Then, the pellet and supernatant were separated by centrifugation at 13000 rpm at 4 ° C. The above supernatant was loaded onto SDS-gel in 40 ug of each protein using a protein quantitation kit (sigma). Then, the proteins loaded on the SDS gel were transferred to a nitrocellulose membrane and reacted with BSA buffer for 30 minutes at room temperature to prevent other antibodies from binding. The primary antibodies ALDH1A1, ALDH1A3 (Abcam), TSPYL5, PTEN and β-catenin Rabbit, Anti-Mouse (Jurkat), Jagged1 (santa cruz), Oct4 (millipore), CD44, Sox2 and β-actin signaling) was reacted in PBS buffer diluted 1: 10000 for 1 hour. The nitrocellulose membrane was then washed 5 times with PBS and then sensitized to a film with a detection solution.

As a result, as shown in Fig. 1A, it was confirmed that the expression level of TSPYL5 protein and cancer stem cell marker and related protein was increased by irradiation. TSPYL5 protein was overexpressed at 3 Gy irradiation at 2 Gy than single radiation (2, 4 Gy) irradiation, and both the cancer stem cell marker and related proteins were overexpressed in the split radiation (FIG. 1A).

<1-2> Intracellular staining ( Intracellular staining ) Was used after the split radiation TSPYL5  Expression and localization

In order to confirm the increased expression of TSPYL5 by dividing radiation, gamma ray was irradiated to the lung cancer cell line using the gamma ray irradiation facility (source: ⁶⁰ C ₀ ) of the Korea Atomic Energy Research Institute and immunofluorescence was used for intracellular staining Respectively.

Lung cancer cell line A549 and A549 cells irradiated with split radiation were cultured in 1 × 10 ⁵ cells in a cover glass dish and cultured for 24 hours. Cells were fixed with 4% paraformaldehyde solution for 20 minutes After washing the coverslips with PBS three times, they were reacted with 0.3-0.5% Triton X-100 solution for 5 minutes and washed three times with PBS. After incubation with 1% BSA for 20 min, the cells were incubated for 2 hours in a 1: 100 dilution of primary antibody TSPYL5 (santa cruz) and ALDH1A1 (cell signaling) The cells were incubated with 1: 1000 dilution buffer for 1 hour, washed three times with PBS, and stained with DAPI solution for 5 min. The increase in TSPYL5 expression was observed by electron microscopy. I could confirm.

As a result, as shown in Fig. 1B, TSPYL5 and ALHD1A1 expression levels were increased in A549 cells irradiated with split radiation, and TSPYL5 was generally present in the cytoplasm, but the expression level in the nucleus was increased by dividing irradiation (Fig. 1B ).

< Example  2> Invasion / Mobility Assay ( Migration / Invasion assay Analysis of cancer cell metastasis ability by split radiation irradiation

Matrigel kit and Western blot experiments were conducted to confirm the infiltration and mobility of cancer cells due to the split irradiation.

Specifically, 0.8 μm pore size Transwell (Falcon, USA) was used to measure the metastatic ability of cancer cells due to split radiation. In the migration assay, A549 cells and A549 cells were irradiated with 5 × 10 ⁴ cells in 100 μl of serum-free RPMI-1640 medium, and then transferred to the upper chamber of the Transwell. The lower chamber contained 7% 500 [mu] l of RPMI1640 medium supplemented with FBS was added to bind the two chambers. After incubation for about 40 hours at 37 ° C in a 5% CO ₂ incubator, the membrane of the Upper chamber is wiped with cotton swabs and then stained with a crystal violet solution. Respectively.

Invasion assay was performed by coating Matrigel (20 ug / well; BD GBiosciences) 100 ul into the Transwell upper chamber, and then performing the same method as the infiltration assay. Cells stained with crystal violet solution were eluted with 500 μl of 10% acetic acid and the absorbance was measured at 600 nm to confirm the relative invasion / migration relative to A549 cells.

As a result, it was confirmed that the infiltration and mobility in A549 cells irradiated with the split radiation were further increased as shown in Fig. 2A (Fig. 2A).

Cells irradiated with the split radiation were cultured for 7 days and then treated with the primary antibodies N-cadherin (santa cruz), E-cadherin (E-cadherin), etc. used as markers for epithelial to mesenchymal transition (EMT) Western blot experiments were performed using cadherin, β-actin (cell signaling), and Snail (abcam) and secondary antibody Rabbit (cell signaling).

As a result, as shown in FIG. 2B, the expression level of E-cadherin, which is an epithelial cell marker, decreased as a kind of EMT marker by dividing radiation, and N-cadherin and Snail, which are mesenchymal cell markers And the expression level was increased (FIG. 2B).

Therefore, it was confirmed that the split radiation has a close relationship with the EMT process, and that the infiltration and mobility of cells are increased by the split radiation.

< Example  3> Aldefluor Of lung cancer cell line ALDH1  Isolate active and inactive cells

&Lt; 3-1 > ALDH1  Isolate active and inactive cells

The present inventors carried out isolation of amyloid cells using fluorescence activated cell sorter (FACS) after Aldefluor staining to confirm the growth and TSPYL5 relationship of lung cancer stem cells through ALDH1 activity from A549 cells.

Specifically, Aldefluor product of stem cell company was used to isolate ALDH1 active cells and inactive cells from A549 cells. This product is based on the activity of aldehyde dehydrogenase (ALDH), and the level of ALDH expression in stem cells is higher than that of normal cells. When the enzyme activity is measured, the activity of the cell is not measured, It has the advantage that it is free of toxicity and easy to use by separating cells. 25 μl of DMSO (Dimethylsulphoxide) was added to the dried Aldefluor reagent, and reacted at room temperature for 1 minute. Then, the reagent, which was red orange, turned bright yellowish green. Then, 25 μl of 2 M hydrochloric acid (hydrocholoric acid) , And then 360 μl of Aldefluor assay buffer is added and the plate is refrigerated to prepare Aldefluor substrate. After dissolving the prepared A549 cells, the supernatant is separated using a centrifuge and 1 ml of Aldefluor assay buffer is added to prepare 1 × 10 ⁶ cells / ml. Prepare two empty tubes, add 5 μl of the activated Aldefluor substrate to the dissolved cells, add 500 μl to the control tube, and add 5 μl of DEAB (Diethylaminobenzaldehyde) solution only to the control group. After reacting at 37 ° C for 30 minutes, remove the supernatant through a centrifuge, add 500 μl of Aldefluor assay buffer, and incubate at 4 ° C for 24 hours. Subsequently, BAAA, which is a negatively charged ALDH-substrate, penetrates into living cells through passive diffusion and is converted into BAA- by the intracellular ALDH, which emits fluorescence light. The cells that emit fluorescence through the machine are called active cells. Were separated using FACS as inactive cells (Figure 3A).

&Lt; 3-2 > ALDH1  Of active and inactive cells Colony  Formation Analysis colony  formation assay )

The present inventors performed colony formation analysis with ALDH1 active cells and ALDH1 inactive cells isolated from A549 cells in order to compare the degree of growth of A549 cells and ALDH1 active cells and ALDH1 inactive cells isolated therefrom.

Specifically, ALDH1 active cells and inactive cells were inoculated on 1 × 10 ³ 35 mm plates, cultured for 8 days in a 37 ° C. carbon dioxide incubator, and then stained with crystal violet 0.5% reagent for 10 minutes, washed several times with PBS, Respectively.

As a result, as shown in FIG. 3B, it was confirmed that ALDH1-activated cells grew well but inactive cells did not grow (FIG. 3B).

<3-3> ALDH1  In active and inactive cells Cancer stem cell Marker TSPYL5 Confirmation of expression of

The present inventors confirmed the expression levels of CD44, CD133 and TSPYL5, which are other stem cell markers in ALDH1-activated and inactive cells, isolated from A549 cells through PCR and Western blotting.

Specifically, the separated ALDH1 active and inactive cells were mixed with 1 ml of trisole for 5 minutes, and then 200 μl of chloroform reagent was added. After 5 minutes of mixing, the mixture was centrifuged at 4 ° C for 10 minutes to obtain 200 Transfer the supernatant of ul into a new tube. The reaction mixture was incubated at room temperature for 10 minutes with 500 μl of isopropanol, and the supernatant was removed by centrifugation at 4 ° C. The precipitate was washed with diethylpyrocarbonate (DEPC) solution containing 75% ethanol Remove the supernatant using a centrifuge. The precipitate was dissolved in DEPC solution and quantified. 1 ug of RNA was synthesized by using Intron 's oligo dT cDNA kit at 45 ° C for 1 hour and 95 ° C for 5 minutes using an amplification machine. After 30 cycles of denaturation at 94 DEG C for 5 minutes, denaturation at 94 DEG C for 30 seconds, extension at 56 DEG C for 30 seconds, and extension at 72 DEG C for 30 seconds were repeated with the primer and I-taq polymerase (Intron) Lt; / RTI &gt; after 10 min. The post-yield was confirmed by loading on 1% agarose gel.

As a result, as shown in FIG. 3C, gene expression of the CDRs ALDH1A1, ALDH1A3, CD44 and CD133, and TSPYL5 was increased in the ALDH1-activated cells than in the inactive cells. On the contrary, PTEN was found in the ALDH1- (Fig. 3C).

50 μl of each solution was added to each cell and reacted at 4 ° C. for 30 minutes. Then, the supernatant was obtained by centrifugation at 4 ° C. and 40 μg of each protein was loaded onto SDS-gel using a protein quantification kit (sigma) Respectively. The cells were identified by using the primary antibodies ALDH1A1, ALDH1A3 (abcam), TSPYL, PTEN (santa cruz), CD44, β-actin (cell signaling) and CD133 (biorbyt) in the same manner as in Example <1-1>.

As a result, as shown in Fig. 3D, it was confirmed that the protein expression of ALDH1A1, ALDH1A3, CD44 and CD133, and TSPYL5 in the ALDH1 active cells was increased and PTEN was decreased in the same manner as in the polymerization chain reaction 3D).

Therefore, it was confirmed that TSPYL5 gene is present in ALDH1 activated cell, which is a cancer stem cell marker, and is associated with the growth of lung cancer cells.

purpose
gene Primer designation order direction SEQ ID NO: Tm / cycle ALDH1A1
ALDH1A1_F TGTTAGCTGATGCCGACTTG Forward SEQ ID NO: 3 58 ° C / 30
ALDH1A1_R TTCTTAGCCCGCTCAACACT Reverse SEQ ID NO: 4 ALDH1A3
ALDH1A3_F TCTCGACAAAGCCCTGAAGT Forward SEQ ID NO: 5 58 ° C / 30
ALDH1A3_R TATTCGGCCAAAGCGTATTC Reverse SEQ ID NO: 6 CD133
CD133_F ATGGCCCTCGTACTCGGCTCCCTGT Forward SEQ ID NO: 7 57 ° C / 30
CD133_R ATGTTGTGATGGGCTTGTCATAACA Reverse SEQ ID NO: 8 CD44
CD44_F ATGGACAAGTTTTGGTGGCACGCA Forward SEQ ID NO: 9 57 ° C / 30
CD44_R TCACCCCAATCTTCATGTCCACAT Reverse SEQ ID NO: 10 TSPYL5
TSPYL5_F TTCGGCTCTCCAGGAAGTTT Forward SEQ ID NO: 11 57 ° C / 30
TSPYL5_R GGGGATGGTTCTGAAATGCT Reverse SEQ ID NO: 12 PTEN
PTEN_F TGTGGTCTGCCAGCTAAAGG Forward SEQ ID NO: 13 57 ° C / 30
PTEN_R CACACAGGTAACGGCTGAGG Reverse SEQ ID NO: 14 GAPDH
GAPDH_F AAGGGTCATCATCTCTGCCC Forward SEQ ID NO: 15 56 ° C / 25
GAPDH_R AGGGGTGCTAAGCAGTTGGT Reverse SEQ ID NO: 16

< Example  4> TSPYL5 Wow Cancer stem cell Marker Inn ALDH1 The relationship of

The present inventors confirmed the effect of TSPYL5 overexpression and TSPYL5 inhibition to control the expression level of TSPYL5 in order to confirm the relationship with the amniotic stem cell marker ALDH1 according to the amount of TSPYL5 expression.

Specifically, in order to suppress the expression of TSPYL5 using TSPYL5 siRNA against A549 cells expressing TSPYL5 protein, the siRNA for TSPYL5 gene was sequenced using a forward primer 5'-AAAGGUAGAACUGCAAGGGAUUGGG-3 '(SEQ ID NO: 17) And the reverse primer 5'-CCCAAUCCCUUGCAGUUCUACCUUU-3 '(SEQ ID NO: 18) were used. Intracellular introduction of the siRNA was carried out using 100 nM of each RNAi per 2 × 10 ⁵ A549 cells and 100 nM of Scrambled Stealth ^™ RNA molecules (siControl) in which Scrambled Stealth ^™ RNA molecules were introduced The cells were transfected with Lipofectamine RNAi MAX (Invitrogen) in the absence of penicillin-streptomycin solution (Hyclone) and reacted for 4 to 6 hours. 100 units / ml penicillin-streptomycin And the cells were cultured for 72 hours. In contrast, overexpression clones of the TSPYL5 gene were transfected into H460 cells to artificially overexpress the TSPYL5 gene in H460 cells. CTTAAGCTTATGAGCGGCCGAAGTCGG-3 '(SEQ ID NO: 19) and reverse primer 5'-TGGAATTCGTGTTGGATTGGCTCACCCC (SEQ ID NO: 19) prepared so as to include a Hind III restriction enzyme recognition sequence at the 5'-end and an EcoR I restriction enzyme recognition sequence at the 3'- Denatured at 94 ° C for 1 minute, denatured at 94 ° C for 1 minute, attached at 56 ° C for 1 minute, and extended at 72 ° C for 1 minute and 30 seconds. , Followed by extension at 72 &lt; 0 &gt; C for 5 minutes. By carrying out polymerization chain reaction under such conditions, 1,253 bp TSPYL5 gene was obtained. The resulting polymerase chain reaction product and pcDNA3.1 (Invitrogen, USA) vector were treated with restriction enzymes, respectively, and ligated to ligase to obtain pcDNA3.1 for fibulin-3 overexpression / TSPYL5 vector. Then, 2 ug of the TSPYL5 overexpression vector was transfected in 2 × 10 ⁵ / ml of H460 cells in a medium without penicillin-streptomycin using Lipofecamine 2000, reacted for 4 to 6 hours, and then 100 units / ml penicillin-streptomycin, and cultured for 48 hours.

<4-1> TSPYL5  Using overexpression and inhibition Cancer stem cell Marker ALDH1 Confirmation of expression with

The present inventors performed TSPYL5 and ALDH1 primers in a polymerization chain reaction and TSPYL5 and ALDH1 antibodies in a Western blot experiment in the same manner as in the above Example <3-3>. Thus, TSPYL5 overexpression in H460 cells and TSPYL5 inhibition in A549 cells Respectively.

As a result, as shown in FIG. 4A, when TSPYL5 was overexpressed in H460 cells, the expression of the cancer stem cell marker ALDH1 was increased, and when the expression of TSPYL5 was inhibited in A549 cells, the expression of the cancer stem cell marker ALDH1 was decreased 4A).

Therefore, it was confirmed that the regulation of TSPYL5 expression is closely related to the genes and proteins of the cancer stem cell markers ALDH1A1 and ALDH1A3.

<4-2> TPSYL5  Over-expression and inhibition of cancer cell growth

The present inventors confirmed A549 cells inhibiting A549 cells and TSPYL5 expression by using the same method as Example <3-2>, and H460 cells expressing T4Y cells and H460 cells as a control.

As a result, as shown in FIG. 4B, TSPYL5 inhibition of A549 cells confirmed a decrease in cell growth, and more cell proliferation was confirmed through overexpression of TSPYL5 in H460 cells (FIG. 4B).

<4-3> TPSYL5  Overexpression and inhibition ADLH  Check the expression level

The present inventors used A549 cells and A549 cells inhibiting TSPYL5 expression and H460 cells and T4YL5-overexpressing H460 cells as a control group in the same manner as in Example <3-1>, and then subjected to Aldefluor staining followed by FACS.

As a result, as shown in FIG. 4C, TSPYL5 inhibition of A549 cells confirmed a decrease in cell growth, and cell proliferation was confirmed by overexpression of TSPYL5 in H460 cells (FIG. 4C).

<4-4> ADLH1  Active cell separation and TPSYL5  Inhibition ADLH  Change in expression level

The present inventors used TSPYL5 in the same manner as in Example <4-1> and Example <4-2> using ALDH (+, high) active cells, which were the cGMP cells isolated in Example <3-1> , Followed by polymerization chain reaction using TSPYL5 and ALDH1 primers and Western blotting and colony formation analysis using TSPYL5 and ALDH1 antibodies.

As a result, as shown in FIG. 4D, TSPYL5 and ALDH genes and proteins were inhibited in TSPYL5 inhibition in cancer stem cell ALDH activated cells, and cancer cell growth was also inhibited (FIG. 4D).

< Example  5> TSPYL5 Of cell metastasis using overexpression and inhibition

<5-1> TSPYL5 Overexpression and inhibition EMT Marker  Confirm

The present inventors used a Western blotting method and a matrigel kit to confirm the infiltration and mobility due to the TSPYL5 gene regulation using A549 cells inhibiting lung cancer cell line A549 cells, A549 cells inhibiting TSPYL5 expression, H460 cells and H460 cells overexpressing TSPYL5 as a control group .

Specifically, A549 cells and A549 cells inhibiting TSPYL5 expression and H460 cells and TSPYL5-overexpressing H460 cells were cultured in the same manner as in Example <4-1> (N-cadherin), Twist (santa cruz), E-cadherin, β-actin (cell signaling), Snail (abcam) and Vimentin (Thermo) and secondary antibodies Rabbit, Mouse Cell signaling was used to perform Western blot experiments.

As a result, as shown in FIGS. 5A and 5C, A549 cells inhibiting TSPYL5 expression increased expression of E-cadherin, an epithelial marker of EMT markers, and increased expression of N-cadherin, Vimentin, Twist and Snail (FIG. 5A). On the contrary, it was confirmed that expression of mesenchymal markers in EMT markers was increased in T4Y5-overexpressing H460 cells (FIG. 5C).

<5-2> TSPYL5 Of Cancer Cells Using Overexpression and Suppression of Cancer Cells

The present inventors performed invasion and mobility analysis using Transwell (Falcon, USA) in the same manner as in <Example 2> to measure the metastatic ability of cancer cells.

Specifically, A549 cells and A549 cells inhibiting TSPYL5 expression, and H460 cells and T4YL5-overexpressing H460 cells were cultured. Then, 5 × 10 ⁴ cells were placed in a Transwell upper chamber, and then 7% FBS (20 ug / well; BD GBiosciences) was preliminarily coated onto both chambers and a Transwell upper chamber by placing 500 μl of RPMI 1640 medium supplemented with 5% % CO ₂ The membrane of the Upper chamber was stained with a crystal violet solution and observed with a microscope to confirm the number of stained cells.

As a result, as shown in Figs. 5B and 5D, TSPYL5 gene inhibitory infiltration and mobility ability were decreased, while conversely, overexpression of TSPYL5 gene increased infiltration and mobility ability (Fig. 5B and 5D).

Therefore, we confirmed that the regulation of TSPYL5 gene changes the abundance, invasion and mobility of EMT markers.

< Example  6> TSPYL5  By modulation of expression Cancer stem cell  growth( sphere formation ) Confirm

Malignant cancer cells undergo anchorage-independent cell growth on an agar plate, and anchorage-independent cell growth is a major trait to determine whether a cell is malignantly transformed . Under conditional media, TSPYL5 was inhibited by shRNA expression and non-adherent cell proliferation was compared.

<6-1> TSPYL5  For inhibition shRNA Lentivirus  Transduction ( lentivirus transduction )

The present inventors have prepared for shRNA TSPYL5 inhibition to suppress the expression of TSPYL5 continuously in order to confirm the relationship with sphere formation, which is a main trait of malignant transformation of cancer cells by TSPYL5 inhibition.

Specifically, 1 × 10 ⁶ cells of 293T cells (Nuclear Medicine Institute, Seoul) were divided into 60 mm plates, and 1 ug / ul of virus early gene (pLP1, pLP2, pLP / VSVG; Invitrogen) 2 ug / ul of ShRNA (Table 2, SHCLNG-NM_033512; Sigma-Aldrich MISSION shRNA library) and lipofectamine 2000 were transduced in the medium without penicillin-streptomycin. After 12 hours, the medium was replaced with a medium supplemented with 100 units / ml of penicillin-streptomycin, and cultured for 24 hours. The culture solution containing viral particles was collected, and A549 cells isolated as a single cell Lt; / RTI &gt;

SHCLND-NM_033512 location order Average knockdown level SEQ ID NO: Cell line 3UTR CCGGAGCCAGTGCACATGGTATTAGCTCGAGCTAATACCATGTGCACTGGCTTTTTTG 0.76 SEQ ID NO: 21 A549 CDS CCGGGTCTCGTTCTACTCCAATCCACTCGAGTGGATTGGAGTAGAACGAGACTTTTTG 0.76 SEQ ID NO: 22 A549 CDS CCGGGCCCAATCCCTTGCAGTTCTACTCGAGTAGAACTGCAAGGGATTGGGCTTTTTG 0.82 SEQ ID NO: 23 A549 CDS CCGGCTTCGATCGCAACCCGTATTTCTCGAGAAATACGGGTTGCGATCGAAGTTTTTG 0.6 SEQ ID NO: 24 A549 CDS CCGGAGAGGTACTGAGCTACTTAAACTCGAGTTTAAGTAGCTCAGTACCTCTTTTTTG 0.67 SEQ ID NO: 25 A549

&Lt; 6-2 > TSPYL5  By inhibition of expression sphere formation  Experiment

The present inventors conducted experiments in cells inhibiting the expression of TSPYL5.

Specifically, for the sphere formation experiment, 2 × 10 ⁴ A549 cells were suspended in DMEM (Invitrogen) containing stem cell-permissive medium and suspended in DMEM-F12 medium (Invitrogen) ng / ml EGF, 20 ng / ml basic fibroblast growth factor (bFGf) and B27 serum-free supplement (50x; Invitrogen) were mixed and cultured on a 60 mm plate pretreated with 0.8% agar As a control, a culture solution containing virus particles was added to inhibit expression of TSPYL5 prepared in the same manner as in Example <6-1>. The cells were incubated at 37 ° C in a 5% CO ₂ And sphere formation was confirmed after 10 days of incubation.

In addition, the primary antibodies ALDH1A1, ALDH1A3 (Abcam), CD133 (biorbyt), Oct3 / 4 (millipore), CD44, Sox2, Nanog and β-actin Western blot experiments were performed using cell signaling.

As a result, as shown in FIG. 6A, when the expression of TSPYL5 was inhibited in lung cancer cell line A549 cells, the size of sphere, which is a main characteristic of malignant transformation, was relatively small and the expression level of major stem cell markers decreased, (Fig. 6A).

&Lt; 6-3 > TSPYL5  Over-expression sphere formation  Experiment

The present inventors conducted experiments in cells overexpressing TSPYL5.

Specifically, H 2 O 4 cells overexpressing TSPYL5 expression were suspended in DMEM (Invitrogen) containing stem cell-permissive medium in the same manner as in Example <4-1> above with 2 × 10 ⁴ H460 cells as a control, And formation of sphere formation was confirmed in the same manner as in Example <6-2>.

Further, the western blot experiment was carried out in the same manner as in Example <1-1>.

As a result, as shown in FIG. 6B, in the case of overexpression of TSPYL5 expression in lung cancer cell line H460 cells, the size of sphere, which is a main characteristic of malignant transformation, was increased, and the expression of major stem cell markers was increased, (Fig. 6B).

Therefore, it was confirmed that cell growth of malignant traits was inhibited by TSPYL5 inhibition.

< Example  7> Cancer stem cell Marker Inn ALDH1  Through modulation of expression TSPYL5 The relationship of

The present inventors have confirmed the relationship with TSPYL5 by controlling or overexpressing the expression of ALDH1, a cancer stem cell marker.

Specifically, in order to inhibit the expression of ALDH1A1 and ALDH1A3 in A549 cells expressing ALDH1A1 and ALDH1A3 proteins, the ALDH1A1 forward primer 5'-GAGAGUACGGUUUCCAUGA-3 '(SEQ ID NO: (SEQ ID NO: 27) and the reverse primer 5'-UACUGACGUUGUCAUCUGUG-3 ' ) Stealth-RNAi. Intracellular introduction of the siRNA was carried out using 100 nM of each RNAi per 2 × 10 ⁵ A549 cells and 100 nM of Scrambled Stealth ^™ RNA molecules (siControl) in which Scrambled Stealth ^™ RNA molecules were introduced The cells were transfected with Lipofectamine RNAi MAX (Invitrogen) in the absence of penicillin-streptomycin solution (Hyclone) and reacted for 4 to 6 hours. 100 units / ml penicillin-streptomycin And the cells were cultured for 72 hours.

In contrast, overexpression clones of ALDH1A1 and ALDH1A3 genes were transfected into H460 cells to artificially overexpress the ALDH1A1 and ALDH1A3 genes in H460 cells. (SEQ ID NO: 30) and reverse primer 5'-ATATAAGCTTATGTCATCCTCAGGCACGCC-3 '(SEQ ID NO: 30) prepared to include a Hind III restriction enzyme recognition sequence at the 5'-end and an EcoR I restriction enzyme recognition sequence at the 3'- ATATGAATTCTTATGAGTTCTTCTGAGAGATTTTC-3 '(SEQ ID NO: 31) and ALDH1A3 forward primer 5'-ATATAAGCTTATGGCCACCGCTAACGGGGC-3' (SEQ ID NO: 32) and the reverse primer 5'-ATATGAATTCTCAGGGGTTCTTGTCGCCAAG-3 '(SEQ ID NO: Deg.] C for 5 minutes, followed by 30 cycles of denaturation at 94 DEG C for 1 minute, extension at 56 DEG C for 1 minute and extension at 72 DEG C for 1 minute and 30 seconds, followed by extension at 72 DEG C for 5 minutes Respectively. The polymerization chain reaction was carried out under these conditions to obtain ALDH1A1 and ALDH1A3 genes. The resulting polymerase chain reaction product and pcDNA3.1 (Invitrogen, USA) vector were treated with restriction enzymes, respectively, and ligated to ligase to obtain pcDNA3.1 for fibulin-3 overexpression / ALDH1A1 and ALDH1A3 vectors were constructed. Subsequently, 2 ug of the ALDH1A1 and ALDH1A3 overexpressing vectors were transfected in 2 × 10 ⁵ / ml of H460 cells using Lipofecamine 2000 in a medium without penicillin-streptomycin, reacted for 4 to 6 hours, 100 units / ml penicillin-streptomycin, and cultured for 48 hours.

<7-1> ALDH1A1 , ALDH1A3 Using inhibition of TSPYL5  Confirm expression relationship

The present inventors conducted experiments by inhibiting ALDH1A1 and ALDH1A3 gene expression in A549 cells.

In the same manner as in Example 1-1, ALDH1A1, ALDH1A3 (Abcam), TSPYL5, PTEN, β-catenin, Jagged1, Notch1, Notch2, Notch3 (santa cruz), Oct4 (millipore), and Cd133 biorbyt) and CD44, Sox2, Nanog and β-actin (cell signaling) antibodies.

As shown in FIG. 7A, when the expression of ALDH1A1 and ALDH1A3 was inhibited, the amount of expression of Sox2, Oct4 and β-catenin was decreased, and the expression amounts of CD44, Cd133, Nanog, Notch and TSPYL5 were not changed Confirming that TSPYL5 is regulated at higher levels of ALDH1 (Fig. 7A).

In addition, A549 cells and A549 cells inhibiting ALDH1A1 and ALDH1A3 expression were assayed for colony formation using the same method as Example <3-2> above.

As a result, as shown in Fig. 7B, it was confirmed that when the expression of ALDH1A1 and ALDH1A3 was inhibited, cell growth was remarkably reduced (Fig. 7B).

<7-2> ALDH1A1 , ALDH1A3 of Overexpression  Used TSPYL5  Confirm expression relationship

The present inventors performed experiments by overexpressing ALDH1A1 and ALDH1A3 genes in H460 cells.

In the same manner as in Example 1-1, ALDH1A1, ALDH1A3 (Abcam), TSPYL5, PTEN, β-catenin, Jagged1, Notch1, Notch2, Notch3 (santa cruz), Oct3 / Cd133 (biorbyt) and CD44, Sox2, Nanog and β-actin (cell signaling) antibodies.

As shown in FIG. 7C, when ALDH1A1 and ALDH1A3 were overexpressed, the amount of expression of Sox2, Oct4 and β-catenin was increased and the expression level of TSPYL5 was not changed (FIG. 7C).

In addition, H460 cells and ALDH1A1 and ALDH1A3-overexpressed H460 cells were colony formed using the same method as in Example <3-2>.

As a result, as shown in Fig. 7D, it was confirmed that cell growth was increased upon overexpression of ALDH1A1 and ALDH1A3 (Fig. 7D).

< Example  8> ALDH1  Analysis of Cell Transition Capacity Using Suppression

The present inventors used a Western blotting method and a matrigel kit to confirm the invasion and mobility of ALDH1A1 and ALDH1A3 gene-regulated A549 cells and ALDH1A1 and ALDH1A3-overexpressed H460 cells in lung cancer cell lines, .

Specifically, A549 cells inhibited the expression of ALDH1A1 and ALDH1A3, A449 cells inhibited the expression of ALDH1A1 and ALDH1A3, and H460 cells, ALDH1A1 and ALDH1A3-overexpressed H460 cells were used as a control, The western blot experiment was carried out in the same manner as in Example <1-1>.

As a result, as shown in FIGS. 8A and 8C, inhibition of ALDH1A1 and ALDH1A3 expression inhibited metastasis. As EMT markers, N-cadherin, Vimentin, Twist, Snail1, and Slug, which are protein markers of mesenchymal cells, And the expression of E-cadherin, which is an epithelial cell marker, was increased, and it was confirmed that the invasion and mobility of A549 cells inhibiting the expression of ALDH1A1 and ALDH1A3 were markedly reduced (Fig. 8A ). In contrast, when ALDH1 overexpression of EMT markers through H460 cells overexpressing ALDH1A1 and ALDH1A3, expression of N-cadherin, Vimentin, Twist, Snail1 and Slug, which are protein markers of mesenchymal cells, is increased and epithelial cells ) Of the E-cadherin marker (Fig. 8C).

In addition, invasion / mobility analysis was performed using Transwell (Falcon, USA) in the same manner as in Example 2 to measure the transfer ability of cancer cells.

Specifically, A549 cells, A549 cells inhibiting the expression of ALDH1A1 and ALDH1A3, and H460 cells and ALDH1A1 and ALDH1A3-overexpressing H460 cells as control groups were cultured, and 5 × 10 ⁴ cells were added to the transwell upper chamber. , 500 μl of RPMI 1640 medium supplemented with 7% FBS was added and 100 μl of Matrigel (20 μg / well) was previously coated on both chambers and a Transwell upper chamber. The cells were incubated for about 40 hours in a 37 ° C. incubator % CO ₂ The membrane of the Upper chamber was stained with a crystal violet solution and observed with a microscope to confirm the number of stained cells.

As a result, as shown in FIGS. 8B and 8D, the ALDH1A1 and ALDH1A3 gene inhibitory infiltration / mobility abilities were decreased, and conversely, the overexpression of ALDH1A1 and ALDH1A3 genes increased infiltration / mobility ability (FIGS. 8B and 8D) .

Therefore, it was confirmed that the expression level, invasion and mobility of EMT markers were altered through the regulation of ALDH1A1 and ALDH1A3 genes, and it was confirmed that the ability of TPSYL5 to modulate ALDH1 could change the cell metastasis ability.

< Example  9> TSPYL5  Growth in Various Cancer Cells by Regulation of Expression sphere  formation inhibition confirmation

The present inventors have found that Calu3 cells (adenocarcinoma), hepatocellular HepG2 cells and pancreatic cancer cells, such as A549 (adenocarcinoma) cells which are non-small cell lung cancer cells, are expressed in the same manner as in Example <6-2> The expression of TSPYL5 was inhibited and sphere formation was confirmed.

Specifically, for the sphere formation experiment, 2 × 10 ⁴ cells were suspended in DMEM (Invitrogen) containing stem cell-permissive medium and cultured in DMEM-F12 medium with 20 ng / ml EGF , 20 ng / ml basic fibroblast growth factor (bFGf) and B27 serum-free supplement (50x) were mixed and cultured on a 60 mm plate pretreated with 0.8% agar. As a control, In order to inhibit the expression of TSPYL5 produced in the same manner as in < 6-1 &gt;, a culture solution containing virus particles was added. The cells were incubated at 37 ° C in a 5% CO ₂ And sphere formation was confirmed after 10 days of incubation.

Western blotting experiments were also performed using the primary antibody ALDH1A1 or ALDH1A3 and β-actin (cell signaling) used as a cancer stem cell marker in the same manner as in Example <1-1>.

As shown in FIG. 9, when the expression of TSPYL5 was suppressed not only in lung cancer cell line A549 cells but also in lung cancer cell line Calu3 cell, liver cancer cell line HepG2 cell and pancreatic cancer cell line Panc1 cell, which had a high expression of TSPYL5, Was relatively small, and the expression of ALDH1, which is a major cancer stem cell marker, was decreased, and it was confirmed that cancer stem cell growth was inhibited (FIG. 9).

<110> Korea Atomic Energy Research Institute <120> Composition for cancer stem cell growth and anti-cancer          metastasis containing TSPYL5 expression or activation inhibitor <130> 2014P-04-005 <160> 33 <170> Kopatentin 2.0 <210> 1 <211> 418 <212> PRT <213> Homo sapiens <400> 1 Met Ser Gly Arg Ser Ser Arg Gly Arg Lys Ser Ser Arg Ala Lys Asn Arg   1 5 10 15 Gly Lys Gly Arg Ala Lys Ala Arg Val Arg Pro Ala Pro Asp Asp Ala              20 25 30 Pro Arg Asp Pro Asp Pro Ser Gln Tyr Gln Ser Leu Gly Glu Asp Thr          35 40 45 Gln Ala Ala Gln Val Gln Ala Gly Ala Gly Trp Gly Gly Leu Glu Ala      50 55 60 Ala Ala Ser Ala Gln Leu Leu Arg Leu Gly Glu Glu Ala Ala Cys Arg  65 70 75 80 Leu Pro Leu Asp Cys Gly Leu Ala Leu Arg Ala Arg Ala Ala Gly Asp                  85 90 95 His Gly Gln Ala Ala Ala Arg Pro Gly Pro Gly Lys Ala Ala Ser Leu             100 105 110 Ser Glu Arg Leu Ala Ala Asp Thr Val Phe Val Gly Thr Ala Gly Thr         115 120 125 Val Gly Arg Pro Lys Asn Ala Pro Arg Val Gly Asn Arg Arg Gly Pro     130 135 140 Ala Gly Lys Lys Ala Pro Glu Thr Cys Ser Thr Ala Gly Arg Gly Pro 145 150 155 160 Gln Val Ile Ala Gly Gly Arg Gln Lys Lys Gly Ala Ala Gly Glu Asn                 165 170 175 Thr Ser Val Ser Ala Gly Glu Glu Lys Lys Glu Glu Arg Asp Ala Gly             180 185 190 Ser Gly Pro Pro Ala Thr Glu Gly Ser Met Asp Thr Leu Glu Asn Val         195 200 205 Gln Leu Lys Leu Glu Asn Met Asn Ala Gln Ala Asp Arg Ala Tyr Leu     210 215 220 Arg Leu Ser Arg Lys Phe Gly Gln Leu Arg Leu Gln His Leu Glu Arg 225 230 235 240 Arg Asn His Leu Ile Gln Asn Ile Pro Gly Phe Trp Gly Gln Ala Phe                 245 250 255 Gln Asn His Pro Gln Leu Ala Ser Phe Leu Asn Ser Gln Glu Lys Glu             260 265 270 Val Leu Ser Tyr Leu Asn Ser Leu Glu Val Glu Glu Leu Gly Leu Ala         275 280 285 Arg Leu Gly Tyr Lys Ile Lys Phe Tyr Phe Asp Arg Asn Pro Tyr Phe     290 295 300 Gln Asn Lys Val Leu Ile Lys Glu Tyr Gly Cys Gly Pro Ser Gly Gln 305 310 315 320 Val Val Ser Ser Ser Thr Pro Ile Gln Trp Leu Pro Gly His Asp Leu                 325 330 335 Gln Ser Leu Ser Gln Gly Asn Pro Glu Asn Asn Arg Ser Phe Phe Gly             340 345 350 Trp Phe Ser Asn His Ser Ser Ile Glu Ser Asp Lys Ile Val Glu Ile         355 360 365 Ile Asn Glu Glu Leu Trp Pro Asn Pro Leu Gln Phe Tyr Leu Leu Ser     370 375 380 Glu Gly Ala Arg Val Glu Lys Gly Lys Glu Lys Glu Gly Arg Gln Gly 385 390 395 400 Pro Gly Lys Gln Pro Met Glu Thr Thr Gln Pro Gly Val Ser Gln Ser                 405 410 415 Asn ***         <210> 2 <211> 1254 <212> DNA <213> Homo sapiens <400> 2 atgagcggcc gaagtcgggg tcgaaagtcc tcccgcgcca aaaaccgggg caaaggccgc 60 gccaaagccc gagtccgccc tgctccggac gacgccccgc gcgacccgga cccttcacag 120 taccagagtc tcggggaaga cacccaggcg gcacaggtgc aggctggcgc ggggtggggt 180 ggcctggaag ccgctgcgtc cgcgcagctc ctccggctcg gggaggaggc cgcctgccgg 240 ctccccctgg actgtggcct cgcgctgcgg gcccgagctg cgggggacca cgggcaggcc 300 gcggccaggc ccggcccggg gaaggccgca tctctctcgg agcgcctggc cgcagacact 360 gtcttcgtgg gaacagcggg aaccgtggga aggccgaaaa atgccccccg cgttggaaac 420 cggcgtggcc ctgccgggaa gaaggcccca gaaacctgta gcaccgcggg gagggggcct 480 caggtcatag ctggtgggag gcagaagaaa ggggcggcag gggagaatac ctcggtgtca 540 gctggggagg aaaagaagga agagagggat gcagggtcgg ggcccccagc gacggaaggc 600 agcatggata cgctggagaa cgtgcagctg aagctggaga acatgaacgc ccaggcggac 660 agggcctacc ttcggctctc caggaagttt gggcagttgc gactgcagca cttggagcgc 720 aggaaccacc tcatccaaaa tatcccgggc ttctgggggc aagcatttca gaaccatccc 780 cagctagcat cctttctgaa tagccaagag aaagaggtac tgagctactt aaacagcttg 840 gaagtggaag agctcggcct tgccagattg ggctacaaaa tcaagttcta cttcgatcgc 900 aacccgtatt tccaaaataa ggtgctcatc aaggaatatg ggtgtggtcc ttctggccag 960 gtggtgtctc gttctactcc aatccagtgg ctcccagggc atgatctcca gtccctaagc 1020 cagggaaacc cagaaaacaa ccgtagtttc tttgggtggt tttcaaacca cagctccatt 1080 gagtctgaca agattgtgga gataatcaac gaagaattgt ggcccaatcc cttgcagttc 1140 taccttttga gtgaaggggc tcgtgtagag aaaggaaagg aaaaagaagg caggcaaggt 1200 ccaggaaagc agccaatgga gactactcag cctggggtga gccaatccaa ctga 1254 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALDH1A1_F <400> 3 tgttagctga tgccgacttg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALDH1A1_R <400> 4 ttcttagccc gctcaacact 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALDH1A3_F <400> 5 tctcgacaaa gccctgaagt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALDH1A3_R <400> 6 tattcggcca aagcgtattc 20 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CD133_F <400> 7 atggccctcg tactcggctc cctgt 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CD133_R <400> 8 atgttgtgat gggcttgtca taaca 25 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CD44_F <400> 9 atggacaagt tttggtggca cgca 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CD44_R <400> 10 tcaccccaat cttcatgtcc acat 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TSPYL5_F <400> 11 ttcggctctc caggaagttt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TSPYL5_R <400> 12 ggggatggtt ctgaaatgct 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTEN_F <400> 13 tgtggtctgc cagctaaagg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTEN_R <400> 14 cacacaggta acggctgagg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_F <400> 15 aagggtcatc atctctgccc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_R <400> 16 aggggtgcta agcagttggt 20 <210> 17 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siTSPYL5_F <400> 17 aaagguagaa cugcaaggga uuggg 25 <210> 18 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siTSPYL5_R <400> 18 cccaaucccu ugcaguucua ccuuu 25 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> THindlll_F <400> 19 cttaagctta tgagcggccg aagtcgg 27 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> TEcoRl_R <400> 20 tggaattcgt gttggattgg ctcacccc 28 <210> 21 <211> 58 <212> DNA <213> Homo sapiens <400> 21 ccggagccag tgcacatggt attagctcga gctaatacca tgtgcactgg cttttttg 58 <210> 22 <211> 58 <212> DNA <213> Homo sapiens <400> 22 ccgggtctcg ttctactcca atccactcga gtggattgga gtagaacgag actttttg 58 <210> 23 <211> 58 <212> DNA <213> Homo sapiens <400> 23 ccgggcccaa tcccttgcag ttctactcga gtagaactgc aagggattgg gctttttg 58 <210> 24 <211> 58 <212> DNA <213> Homo sapiens <400> 24 ccggcttcga tcgcaacccg tatttctcga gaaatacggg ttgcgatcga agtttttg 58 <210> 25 <211> 58 <212> DNA <213> Homo sapiens <400> 25 ccggagaggt actgagctac ttaaactcga gtttaagtag ctcagtacct cttttttg 58 <210> 26 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siALDH1A1_F <400> 26 gagaguacgg uuuccauga 19 <210> 27 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siALDH1A1_R <400> 27 ucauggaaac cguacucuc 19 <210> 28 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siALDH1A3_F <400> 28 cacagaugac aacgucgua 19 <210> 29 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siALDH1A3_R <400> 29 uacgacguug ucaucugug 19 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 1Hindlll_F <400> 30 atataagctt atgtcatcct caggcacgcc 30 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 1EcoRl_R <400> 31 atatgaattc ttatgagttc ttctgagaga ttttc 35 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 3Hindlll_F <400> 32 atataagctt atggccaccg ctaacggggc 30 <210> 33 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> 3EcoRl_R <400> 33 atatgaattc tcaggggttc ttgtcgccaa g 31

Claims (11)

A pharmaceutical composition for preventing and inhibiting cancer metastasis comprising an expression or activity inhibitor of TSPYL5 (testis-specific protein, Y-encoded-like 5) as an active ingredient.
2. The method according to claim 1, wherein the TSPYL5 expression inhibitor is an antisense nucleotide complementary to the mRNA of the TSPYL5 gene, a small interfering RNA or shRNA (short haripin RNA) A pharmaceutical composition.
The pharmaceutical composition for preventing and inhibiting cancer metastasis according to claim 1, wherein the activity inhibitor of TSPYL5 is any one selected from the group consisting of compounds, peptides, peptide mimetics and antibodies that complementarily bind to TSPYL5 protein .
The pharmaceutical composition for preventing and inhibiting cancer metastasis according to claim 1, wherein the TSPYL5 expression or activity inhibitor inhibits the growth and metastasis of cancer stem cells.
The composition for preventing and inhibiting cancer metastasis according to claim 1, wherein the cancer is selected from the group consisting of skin cancer, breast cancer, cervical cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer and stomach cancer.
A kit for inhibiting cancer stem cell growth comprising an expression or activity inhibitor of TSPYL5.
A method for inhibiting cancer stem cell growth comprising treating cancer cells with the expression or activity inhibitor of TSPYL5.
1) preparing a TSPYL5 expressing cell line;
2) treating the cell line of step 1) with the test substance;
3) measuring the expression or activity of ALDH (aldehyde dehydrogenase) of the cell line; And
4) screening a test substance whose ALDH expression or activity level is lower than that of a control group not treated with the test substance.
1) preparing a TSPYL5 expressing cell line;
2) treating the cell line of step 1) with the test substance;
3) measuring the expression or activity of ALDH in the cell line; And
4) selecting a test substance whose ALDH expression or activity level is lower than that of a control group in which the test substance is not treated; and screening the candidate substance for a cancer stem cell growth inhibitor.
10. The method according to claim 8 or 9, wherein the TSPYL5 expressing cell line of step 1) is prepared by irradiating a cancer cell line with radiation.
11. The method of claim 10, wherein the irradiation is a single irradiation of 2 Gy or 4 Gy, or 2 to 5 divided irradiation of 2 Gy.

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