WO2013022157A1 - 피불린-3 단백질을 유효성분으로 함유하는 암 줄기세포 성장 억제용 약학적 조성물 - Google Patents
피불린-3 단백질을 유효성분으로 함유하는 암 줄기세포 성장 억제용 약학적 조성물 Download PDFInfo
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- WO2013022157A1 WO2013022157A1 PCT/KR2011/010001 KR2011010001W WO2013022157A1 WO 2013022157 A1 WO2013022157 A1 WO 2013022157A1 KR 2011010001 W KR2011010001 W KR 2011010001W WO 2013022157 A1 WO2013022157 A1 WO 2013022157A1
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- Prior art keywords
- fibulin
- cells
- cancer
- protein
- cancer stem
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- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Definitions
- composition for inhibiting cancer stem cell growth containing fibulin-3 protein as an active ingredient
- the present invention relates to a method for inhibiting the growth of cancer stem cells using fibulin-3 or a pharmaceutical composition for inhibiting cancer stem cell growth containing fibulin-3 protein as an active ingredient.
- cancer stem cell because it had.
- the paradigm of cancer stem cells is that tumors are caused by specific cell populations that show various potential potency and self renewal [BM Boman, MS Wicha, Cancer stem cells: a step toward the cure, J Clin. Oncol.
- cancer stem cells show resistance to anticancer drugs developed so far. 80% to 90% of cancer cells In fact, there is no recurrence, but the remaining 10 to 20% (tumor stem cells) do not die and remain relapsed when the anticancer drug is administered.
- the anticancer agent that removes cancer stem cells by targeting cancer stem cells having the characteristics of pleasant cells beyond the limitations of current cancer treatment methods that attack cancer cells only. Need development
- chemotherapy is mainly used for surgery, chemotherapy and radiotherapy.
- various agents and radiation are used, but their application is limited to the range that the human body can afford.
- the limited application of these chemotherapy and radiotherapy results in the fact that no matter how good they can be in animal studies, they have the disadvantage of offsetting the effect, limiting coverage and limiting efficacy in the clinic. This is due to the emergence of anticancer drug resistant cancer cells or side effects due to the cytotoxicity of each therapy.
- Cisplatin for example, is the most effective cancer treatment and is one of the 30 most effective anticancer drugs currently used in the clinic.
- radiation therapy is an anticancer therapy that treats various cancers occurring in the human body by irradiating the affected area with an appropriate amount of radiation.
- Such radiotherapy reduces the hematopoietic function of the human body and suppresses the immune system, thereby improving the effectiveness of cancer treatment.
- cancer cells become resistant due to repeated irradiation, the radiation therapy also has limitations in anticancer therapy.
- markers of such cancer stem cells CD133 (prominin-1 or AC133) and CD44 (hyaluronate receptor or P-glycoprotein 1) are used. Recently, acetaldehyde dehydrogenases (ALDH1) have been identified.
- ALDH Detoxifying enzymes that oxidize intracellular aldehydes, resulting in significant resistance to alkylating agents or oxidative stress [Blood 87 (1996) 1097-1103; Chem. Biol. Interact. 143-144 (2003) 5-22].
- ALDH converts retinol (Vit amine A) into retinoic acid, the most active form of retinoids important for the treatment and prevention of cancer, thereby affecting cell growth and proliferation.
- ALDH is generally known to have 19 genes and is divided into ALDH1, 2, and 3 classes.
- ALDH1 has retinal dehydrogenase activity that converts retinal to retinoic acid, and ALDH1A1 and ret inal dehyde dehydrogenase 3 are most efficient. Has activity [Biochim. Biophys. Acta 1790 (2009) 1660-1664. Therefore , the activity of ALDH1 is important to isolate the subpopulation of cancer stem cells in lung cancer cell lines [Mol. Cancer. Res. 7 (2009) 330-338. The winter t / (3-catenin) pathway has recently been reported to play an important role in the generation of cancer stem cells that cause cancer recurrence (Nature Reviews Cancer 5, 744-749 (September 2005)).
- wnt is well known to be active in cancer, resulting in glycogen synthase kinase 3: GSK3) activity is inhibited and beta-catenin, a substrate of GSK3, accumulates, and this abnormal wnt pathway induces neoplastic prol i ferat ion.
- GSK3 glycogen synthase kinase 3
- beta-catenin a substrate of GSK3
- this abnormal wnt pathway induces neoplastic prol i ferat ion.
- (Fibulin) protein family are extracellular matrix Elastic cells (extracel hilar matrix,
- ECM As a protein bound to ECM, it is a protein that contributes to cell-to-cell interactions or to cell-to-substrate interactions.
- Six types are known from Type 1 to Type 6.
- Such Fibulin proteins are proposed as new gene targets that suppress tumors in some species.
- fibulin-3 homologous to fibulin-5 acts on cell growth regulation and some tumors, and fibulin-5 inhibits tumor proliferation, invasion, vascular endothelial growth factor and tumor angiogenesis.
- fibulin-3 homologous to fibulin-5 acts on cell growth regulation and some tumors, and fibulin-5 inhibits tumor proliferation, invasion, vascular endothelial growth factor and tumor angiogenesis.
- Fibulin-3 is also known to reduce cell growth regulation, reduce tumor angiogenesis and some tumors [Cancer Res. 66 (2006) 2621-2629.
- the effect of fibulin 3 on cancer stem cells, which can cause tumors to develop or maintain their properties and resist chemotherapy is not yet known.
- the present inventors isolated cancer stem cells using ALDH1 activity as a marker in H460 and A549 cells, which are non-small cell lung cancer cells but with different sensitivity to cytotoxicity such as permeability and ionizing radiation, and isolated ALDH1 activity or inactivation. It was confirmed that there was a difference in the expression level of fibrin # 3 in the cells.
- fibulin-3 when the expression level of fibulin-3 is increased in the isolated cells, beta-catenin ( ⁇ -catenin) and infiltration related factors ⁇ P-2 and ⁇ P-7 which decreases the characteristics of cancer stem cells decrease, It was confirmed that the growth of cancer stem cells was reduced by fibulin-3, which was the same result even when treated with the medium overexpressing fibulin-3.
- a fibrin-3 purified vector was prepared to purify the fibulin-3 protein, and the purified fibulin-3 protein is A549 cells which are non-small cell lung cancer cells, MDA-MB231 cells which are breast cancer cells, and cancer enjoyment cells.
- the present invention was completed by identifying that the fiblin-3 may be usefully used as a composition for inhibiting growth of cancer stem cells by confirming that it inhibits cell growth of phosphorus ALDH1 activated cells.
- An object of the present invention is to provide a fibulin-3 protein having cancer stem cell growth inhibitory activity.
- another object of the present invention comprising the step of treating a fibulin ⁇ 3 overexpressing cell line or culture medium thereof produced by transforming an effective amount of fibulin-3 protein or fibulin-3 gene in cancer stem cells It is to provide a method for inhibiting growth of cancer stem cells.
- Another object of the present invention is to provide a method for inhibiting cancer stem cell growth comprising administering a pharmaceutically effective amount of fibrin-3 protein to a subject having cancer stem cells.
- Another object of the present invention comprising administering to a subject with cancer-loving cells a fibulin-3 overexpressing cell line or a culture thereof prepared by transforming a pharmaceutically effective amount of fibulin-3 gene It is to provide a method for inhibiting cancer stem cell growth.
- Still another object of the present invention is to provide a use of fibulin ⁇ 3 protein for use in the manufacture of a pharmaceutical composition for inhibiting cancer stem cell growth.
- Another object of the present invention to use in the manufacture of a pharmaceutical composition for inhibiting cancer stem cell growth, provides a use of a fibulin I overexpressing cell line prepared by transforming the fibulin-3 gene or a culture thereof It is to.
- the present invention provides a fibulin-3 protein having cancer stem cell growth inhibitory activity.
- the present invention is fibulin-3 (fibulin-3) having cancer stem cell growth inhibitory activity It provides a pharmaceutical composition for inhibiting cancer stem cell growth containing a protein as an active ingredient.
- the present invention provides a method for inhibiting growth of cancer stem cells comprising the step of treating an effective amount of fibulin-3 in cancer stem cells.
- the present invention also provides a pharmaceutical composition for inhibiting cancer stem cell growth, comprising a fibulin-3 overexpressing cell line prepared by transforming a fibulin-3 gene or a culture medium thereof as an active ingredient.
- the present invention also provides a method for inhibiting growth of cancer stem cells, comprising the step of treating a fibulin-3 overexpressing cell line or a culture medium thereof prepared by transforming the fibulin-3 gene.
- the present invention also provides a method for inhibiting cancer stem cell growth comprising administering a pharmaceutically effective amount of fibrin-3 protein to a subject having cancer stem cells.
- the present invention is a method for inhibiting cancer stem cell growth comprising administering to a subject with cancer stem cells a fibulin-3 overexpressing cell line or culture medium prepared by transforming a pharmaceutically effective amount of fibulin-3 gene To provide.
- the present invention also provides the use of a fibulin-3 protein for use in the manufacture of a pharmaceutical composition for inhibiting cancer stem cell growth.
- the present invention provides a use of a fibulin-3 overexpressing cell line prepared by transforming a fibulin-3 gene or a culture thereof for use in the manufacture of a pharmaceutical composition for inhibiting cancer stem cell growth.
- the fibulin-3 protein of the present invention is a beta-catenin ( ⁇ - catenin) and infiltration-related factors ( ⁇ P ⁇ 2 and — P— 7) can effectively inhibit the activated cell growth of cancer stem cells, inhibit the growth of non-small cell lung cancer cells, breast cancer cells and cancer stem cells, pharmaceuticals for inhibiting cancer stem cell growth It can be usefully used in a composition.
- ⁇ - catenin beta-catenin
- infiltration-related factors ⁇ P ⁇ 2 and — P— 7
- FIG. 1 is a diagram showing a protein purification vector by attaching a histidine protein sequence to the c-terminal region of the fibulin-3 gene.
- FIG. 2 is a diagram showing the result of separating ALDH1 active cells and ALDH1 inactive cells using an FACS machine after Aldefluor staining on A549 cells;
- AL (-) cells without ALDH1 activity.
- Figure 3 is the result of overexpressing the fibulin-3 gene with histidine attached to CH0-K1 cells and confirmed the expression through reverse transcriptase reaction and Western blot;
- RT-PCR reverse transcriptase Banung
- pcDNA CH0-K1 cells overexpressed void as control; And ,
- His-pibulin 3 ⁇ - ⁇ cells overexpressing a fibulin-3 vector with histidine attached.
- FIG. 4 shows the results of colony formation analysis of A549 cells, ALDH1 active cells isolated from A549, and ALDH1 inactive cells isolated from A549;
- A549 colony formation of A549 cells
- ALDH + colony formation of cells with ALDH1 activity
- ALDH- Colony formation in cells without ALDH1 activity.
- Figure 5 shows the results of ALDH1 activated cells and ALDH1 inactive cells isolated from A549, the expression level of ALDH1A1, betacatenin, and fibulin-3 using A549 cells as a control, through reverse transcriptase polymerase reaction;
- ALDH1A1 ALDH1.
- RT-PCR result of reverse transcriptase polymer reaction
- Western Result of Western Blot.
- A549 A549 cell
- A549 / V.C A control in which a vector was expressed in A549 cells
- A549 / pcFBLN3 expresses fibulin _3 overexpressing vector in A549 cells;
- H460 H460 cells
- H460 / siCtl a control in which sicon vector was expressed in H460 cells
- H460 / siFBLN3 expresses fibulin-3 expression inhibitory vector in H460 cells
- FBLN 3 fibulin-3
- RT-PCR result of reverse transcriptase polymer reaction
- FIG. 8 shows the results of Western blot analysis of microscopic photographs and beta-catenin expression of cells observed after treatment of fibulin-3 overexpression medium or H460 cell culture medium with overexpression of fibulin-3 in A549 cells. ;
- A549 A549 cell
- A549 / H460 M cells in which A549 cells were treated with H460 culture medium; And ,
- A549 / FBLN3 (+) M Cells treated with the culture medium of A549 cells overexpressing the fibulin-3 protein in A549 cells.
- Figure 9 is treated with fibulin-3 overexpression medium or H460 cell culture medium overexpressing fibrin-3 in A549 cells, or in H460 cell culture medium overexpressing fibulin-3 overexpression medium or fibulin-3 Western blot confirmed the change in the expression levels of betacatenin, P-2 and V-7 in cells treated with fibrin-3 antibody;
- A549 A549 cell
- A549 / H460 M A549 cells were treated with H460 culture medium
- A549 / FBLN3 (+) M treated with a culture medium of A549 cells overexpressing fibulin-3 protein in A549 cells;
- A549 / H460 M + FBLN3 Ab treating A549 cells with H460 culture medium and fibulin-3 antibody; And, A549 / FBLN3 (+) M + FBLN3 Ab: Treated culture medium and fibulin-3 antibody of A549 cells overexpressing fibulin-3 protein in A549 cells.
- FIG. 10 shows HLD cell culture medium in which ALDH1 activated cells are treated with fibulin-3 overexpression medium or H460 cell culture medium overexpressing fibulin-3, or fibulin-3 overexpression medium or fibulin-3 overexpression Western blots showed changes in the expression levels of betacatenin, MMP-2 and MMP-7 in cells treated with epiphyllin-3 antibody;
- ALDH + ALDH1 active cells
- ALDH + / H460 M ALDH1 activated cells were treated with culture medium of H460 cells
- ALDH + / H460 M + FBLN3 Ab ALDH1 activated cells were treated with culture medium of H460 cells and fibrin-3 antibodies
- FIG. 11 shows the results of colony formation of A549 cells after treatment with different concentrations of fibulin ⁇ 3 overexpression medium and H460 cell culture medium overexpressing fibulin-3 in A549 cells;
- Control group control group treated with growth medium of A549 cells
- Fibulin 3-% ratio of fibulin-3 overexpression medium in the culture medium of A549 cells overexpressing fibulin ⁇ 3 and the mixed medium of A549 growth medium; And ,
- H460-% ratio of H460 cell culture medium in the mixed medium of H460 cells and the mixed medium of A549 growth medium.
- FIG. 12 shows the result of confirming colony formation after treatment of the culture medium of CH0-K1 cells or 293T cells overexpressing the heath-pibulin-3 vector at A549 cells at a rate of 50%;
- CH0-K1 medium medium of CH0-K1 cells overexpressed Hispenfibulin-3 vector
- 293T medium medium of 293T cells overexpressed his-pibulin-3 vector
- b Graph of colony count results.
- Figure 13 shows the results of colony formation after treatment of purified fibulin-3 protein to A549 cells
- CH0- 1 24h-l A549 cells treated with 1 ug of fibulin protein purified from CH0-K1 cells overexpressing his-fibulin-3 vector after 24 hours;
- CH0-K1 24h—2 A549 cells treated with 1 ug after 24 hr of purified fibulin protein purified from CH0-K1 cells overexpressing his-pibulin-3 vector, and again after 1 ug after 24 hr;
- 293T 24h-l A549 cells treated with 1 ug of fibulin protein purified from 293T cells overexpressing hissingfibulin-3 vector after 24 hours;
- 293T 24h'2 A549 cells treated with 1 ug after 24 hours of purified fibulin protein purified from 293T cells overexpressing his-pibulin-3 vector and once again after 24 hours;
- CH0-K1 24h-l MDA-MB231 cells treated with 1 ug of fibulin protein purified from CH0-K1 cells overexpressing a histopipilin-3 vector after 24 hours;
- CH0-K1 24h-2 MDA-MB231 treated with 1 ug of purified fibulin protein in CH0-K1 cells overexpressing his-pibulin-3 vector after 24 hours, and then again 1 L1 g after 24 hours cell;
- 293T 24h-l MDA—MB231 cells treated with 1 ug of purified fibulin protein after 24 hours in 293T cells overexpressing a His-Goblin-3 vector;
- 293T 24h-2 MDA-MB231 cells treated with 1 ug after 24 hours of purified fibulin protein purified from 293T cells overexpressing his-pibulin ⁇ 3 vector and once again after 24 hours; pc-DNA: control group; And ,
- ALDH1 + ALDH1 active cells
- Control group control group treated with growth medium of ALDH1 cells
- Fibulin 3-% ratio of fibulin-3 overexpression medium in the mixed medium of A549 cells overexpressing fibulin ⁇ 3 and ALDH1 growth medium; And ,
- H460-% ratio of H460 cell culture medium in the mixed medium of H460 cells and ALDH1 growth medium.
- FIG. 16 shows the result of confirming colony formation after treatment of the culture medium of CH0-K1 cells or 293T cells overexpressing his-pibulin-3 vector with ALDH1 active cells at a rate of 50 3 ⁇ 4;
- ALDH + ALDH1 active cells
- CH0-K1 medium medium of CHO—K1 cells overexpressed his-pibulin-3 vector
- 293T medium medium of 293T cells overexpressed his-fibulin-3 vector
- b Graph of colony count results.
- ALDH + ALDH1 active cells
- CHO-Kl 24h-l ALDH1 activating cells treated with 1 ug of fibulin protein purified from ALDH1 activating cells overexpressing his-pibulin-3 vector after 24 hours;
- CHO-Kl 24h-2 ALDH1 activating cells treated with 1 ug after 24 hr of purified fibulin protein in ALDH1 activating cells overexpressing his-pibulin-3 vector and again after 1 ug after 24 hr;
- 293T 24h-l ALDH1 activating cells treated with 1 ug of fibulin protein purified from 293T cells overexpressing Hissopiblin-3 vector 24 hours later;
- the present invention provides fibulin-3 (fibL in-3) protein having cancer stem cell growth inhibitory activity.
- the fibulin-3 protein preferably has one amino acid sequence selected from the amino acid sequences of 1) to 3), but is not limited thereto.
- the cancer is lung cancer or breast cancer, and the lung cancer is more preferably non-small cell lung cancer.
- the cancer is lung cancer or breast cancer, and the lung cancer is more preferably non-small cell lung cancer.
- the present inventors A fibulin ⁇ 3 vector with a histidine protein attached to the c-terminal site was prepared (see FIG. 1), and to confirm the expression of the vector, CH0-K1 cells (Biotechnology Research Institute, Daejeon) and 293T cells (Hanyang University) , Seoul), respectively, and overexpression of fibulin-3 protein was confirmed by polymerase chain reaction and Western blot (see FIG. 3).
- the medium was grown to A549 cells and cancer stem cells ALDH1 activated cells that are non-small cell lung cancer. This inhibition was confirmed (see FIGS. 11 and 16).
- using the transduced cell line to purify the fibulin-3 protein and confirmed the growth inhibitory effect of cancer cells of the purified fibulin-3 protein, A549 cells, non-small cell lung cancer cells, cancer stem cells It was confirmed that there is an effect of inhibiting cell growth in ALDH1 activated cells and MDA-MB231 cells which are breast cancer cells (see FIGS. 12, 17 and 13).
- the fibulin ⁇ 3 protein can be usefully used to inhibit the growth of cancer cells and cancer stem cells.
- the present invention provides a pharmaceutical composition for inhibiting cancer stem cell growth, which contains the fibrin-3 protein according to the present invention as an active ingredient.
- the fibulin-3 protein preferably has one amino acid sequence selected from the amino acid sequences of 1) to 3), but is not limited thereto.
- the cancer is lung cancer or breast cancer, and the lung cancer is more preferably non-small cell lung cancer.
- the cancer stem cell is a marker of cancer stem cells CD133 (prominin-l; AC 133),
- CD44 honeyironate receptor; P— glycoprotein 1
- ALDHl is preferably selected from any one selected from the group consisting of ALDH1 is more preferably selected, but not limited thereto.
- the present inventors are non-small cell lung cancer cells Cancer stem cells were distinguished and selected using ALDEFLUOR products (stem cells) based on ALDH1 activity in A549 cells (see FIG. 2).
- the present inventors analyzed the growth of ALDH1 activated cells and ALDH1 inactive cells isolated by colony formation experiments to identify the difference between non-small cell lung cancer and cancer stem cells.
- ALDH1 activated cells grew better than A549 cells, whereas ALDH1 inactive cells did not grow than A549 cell control, indicating that ALDH1 activated cancer stem cells are the driving force for non-small cell lung cancer growth.
- RT-PCR reverse transcriptase polymerase chain reaction
- Western blot Western blot to confirm the expression levels of ALDH1, fibulin3 and betacatenin in ALDH1 active and inactive cells.
- the present inventors overexpressed fibulin-3 in A549 cells with high expression of beta-catenin in order to confirm the expression level of betacatenin by fiblin-3 (fibulin-3).
- the vector was transduced, and H460 cells with little beta-catenin expression level were inhibited with fibrin-3 expression by siRNA for fibulin-3 inhibition.
- beta-catenin expression was reduced in A549 cells overexpressing fibulin-3, and in contrast, beta-catenin expression was increased in H460 cells in which fibulin-3 was inhibited (see FIGS. 6 and 7). ).
- overexpressed fibulin-3 inhibits betacatenin, which is important for cancer stem cell growth, and that fibulin-3 may be useful for inhibiting growth of cancer stem cells.
- fibulin-3 may be useful for inhibiting growth of cancer stem cells.
- the present inventors inhibit the expression of beta-catenin and the invasion factor ⁇ P-2,7, which are important for tumorigenesis and proliferation by fibulin-3 in non-small cell lung cancer cells A549.
- the A549 cells were treated with the culture medium of A549 cells overexpressing fibulin-3 and the culture medium of H460 cells with high expression of fibulin-3.
- the cells treated with the medium were killed. It was confirmed by a microscope, it was confirmed that the expression of beta-catenin and -2 P-2, 7 compared to the control (see FIGS. 8 and 9).
- the fibulin-3 antibody was used to further confirm that the result was due to fibulin-3.
- fibulin ⁇ 3 directly inhibits the expression of betacatenin and ⁇ P– 2,7.
- the inventors of the present invention to determine the expression of beta-catenin and ⁇ ?-2,7 by fibulin -3 in cancer enjoyment cells.
- the culture medium and the culture medium of H460 cells having high expression of fibulin-3 were treated with ALDH1 activated cells and Western blot was performed.
- the beta-catenin and MMP ⁇ 2,7 expression was reduced in the two medium-treated group.
- the fibulin-3 antibody was used to further confirm that the result was due to fibulin-3 (see FIG. 14).
- fibulin-3 inhibited the growth of non-small cell lung cancer cells.
- growth of A549 cells was inhibited in a concentration-dependent manner both in the culture medium of A549 cells overexpressing fibulin-3 and the culture medium overexpressing medium of H460 cells expressing high fibulin-3. It was confirmed (see FIG. 10).
- the present inventors conducted colony formation experiments to determine whether the growth of cancer-like cells is inhibited by fibulin-3, and as a result, the concentration of medium in both of the fibulin ⁇ 3 overexpressing medium was mixed. It was confirmed that the growth of ALDH1 activated cells was inhibited (see FIG. 10).
- the present inventors overexpressed the heat-fibulin-3 vector for the expression of fibulin-3 protein in CHCHQ cells and 293T cells, which are easy to purify proteins, and then cultured the culture medium for 48 hours to form non-small cell lung cancer cells.
- the cell treated with the medium was confirmed that the cell growth is inhibited compared to the control (see FIGS. 11 and 16).
- the inventors of the present invention in order to determine whether the growth of cancer cells and cancer stem cells by purified fibulin-3 protein is inhibited A549 cells, non-small cell lung cancer cells, breast cancer cells MDA-MB231 cells and cancer stem cells Colony formation experiments were carried out after treatment of ALDH1 activated cells with fibrin-3 purified protein obtained by transforming a fibulin-3 protein-expressing heath-fibulin3 vector into CH0-K1 cells or 293T cells.
- A549 cells which are non-small cell lung cancer cells
- MDA 'MB231 cells which are breast cancer cells
- ALDH1 activated cells which are cancer stem cells (see FIGS. 12, 13, and 17).
- the fibulin-3 protein may inhibit the cell growth and penetration factors of cancer-proliferating cells in inhibiting the growth of cancer stem cells. It can be usefully used as.
- the therapeutically effective amount of the composition of the present invention may vary depending on several factors, such as the method of administration, the target site, the condition of the patient, and the like. Therefore, when used in humans, the dosage should be determined in an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from an effective amount determined through animal testing. These considerations in determining the effective amounts are given in, for example, Hardman and Limbird, eds. , Goodman and Gi lman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Mart in ed. , Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
- compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents.
- Pharmaceutically acceptable carriers are not particularly limited so long as they are suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc.
- Compounds, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltocholine solution, glycerol, ethane and one or more of these ingredients can be used in combination.
- Other conventional additives such as bacteriostatic agents can be added.
- diluents may be additionally added to formulate main formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
- diluents such as aqueous solutions, suspensions, emulsions and the like.
- dispersants such as aqueous solutions, suspensions, emulsions and the like.
- surfactants such as aqueous solutions, suspensions, emulsions and the like.
- binders and lubricants may be additionally added to formulate main formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
- lubricants may be additionally added to formulate main formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
- Remington's Pharmaceutical Sc ience Mack Pub 1 ishing Company, East on PA, 18th, 1990
- composition of the present invention comprises 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.
- the composition of the present invention can be administered orally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally according to the desired method, and the dosage is based on the patient's weight, age, sex, health condition, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease.
- the daily dose of the composition according to the present invention is 0.0001 to 10 mg / m «, preferably 0.0001 to 5 mg / m «, more preferably administered once a day to dividing the water.
- the present invention also provides a method for inhibiting growth of cancer stem cells, comprising the step of treating an effective amount of fibulin-3 in cancer enjoyment cells.
- the present invention also provides a method for inhibiting cancer stem cell growth comprising administering a pharmaceutically effective amount of fibulin-3 protein to a subject having cancer stem cells.
- the cancer is lung cancer or breast cancer, and the lung cancer is more preferably non-small cell lung cancer.
- the inventors of the present invention provide a culture medium of ALDH1 active cells overexpressing fibulin-3 protein to determine whether cancer-bearing cells are inhibited by fibulin-3 and fibulin-3 protein.
- the culture medium of H460 cells having high expression of A460 was isolated from cancer stem cells in A549 cells and treated with ALDH1 activated cells, and then colony formation experiments were performed.
- the purified fibulin-3 protein was purified by transforming histofibrin-3 vector for purification of fibulin-3 protein into CH0-K1 cells or 293T cells, and the ADA cell, which is a non-small cell lung cancer cell, and the MDA ⁇ , a breast cancer cell.
- the fibulin-3 protein of the present invention can be usefully used for a method of inhibiting the growth of cancer cell.
- the present invention also provides a pharmaceutical composition for inhibiting cancer stem cell growth, comprising a fibulin-3 overexpressing cell line prepared by transforming a fibulin-3 gene or a culture medium thereof as an active ingredient.
- the cancer is lung cancer or breast cancer, and the lung cancer is more preferably non-small cell lung cancer.
- the present invention provides a method for inhibiting growth of cancer stem cells, comprising the step of treating a fibulin-3 overexpressing cell line or a culture medium prepared by transforming the fibulin-3 gene.
- the present invention is a method for inhibiting cancer stem cell growth comprising administering to a subject with cancer stem cells a fibulin-3 overexpressing cell line or culture medium prepared by transforming a pharmaceutically effective amount of fibulin-3 gene To provide.
- the cancer is lung cancer or breast cancer, and the lung cancer is more preferably non-small cell lung cancer.
- fibulin-3 The overexpressing vector was overexpressed in 5 x 10 5 ALDH1 active cells and treated with ALDH1 activated cells for 48 hours of culture and for 48 hours incubation of 5 ⁇ 10 5 ⁇ 460 cells for Western blot. As a result, it was confirmed that the expression of beta-catenin and ⁇ 2,7 related to the growth of cancer stem cells was reduced in the two medium-treated groups.
- the fibulin-3 antibody was used to further confirm that the result was due to fibulin-3. Through these results, the fibulin-3 overexpressing cell line prepared by transforming the fibulin-3 gene 11010001
- the present inventors conducted colony formation experiments to confirm whether the growth of cancer stem cells is inhibited by fibulin-3, and as a result, the concentration of medium in both of the fibulin ⁇ 3 overexpressing medium was mixed. It was confirmed that the growth of ALDH1 activated cells was inhibited.
- the inventors of the present invention transformed a culture medium by transforming a heath-fibulin-3 vector for the expression of a fibulin-3 protein into CH0-K1 cells or 293T cells, which are easy to purify proteins, of lung cancer cells, breast cancer cells and cancer stem cells. Colony formation experiments were conducted to see if they inhibited growth. As a result, it was confirmed that the growth of lung cancer cells, breast cancer cells and cancer stem cells by the culture medium.
- the fibulin ⁇ 3 overexpressing cell line prepared by transforming the fibulin-3 gene or its culture medium inhibited the growth and penetration factor of cancer stem cells.
- Fibulin-3 overexpressed cell lines or cultures thereof may be usefully used in the pharmaceutical composition for inhibiting the growth of cancer-loving cells and methods for inhibiting cancer stem cell growth.
- the present invention also provides the use of fibulin-3 protein for use in the manufacture of a pharmaceutical composition for inhibiting cancer stem cell growth.
- the present invention provides a fibulin-3 overexpressing cell line prepared by transforming a fibulin-3 gene or a culture solution thereof for use in the manufacture of a pharmaceutical composition for inhibiting cancer stem cell growth.
- the fibulin-3 protein is characterized by having an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto.
- the cancer may be lung cancer or breast cancer, but is not limited thereto.
- the inventors purified the fibulin-3 protein confirmed the cancer cell growth inhibitory effect of the purified fibulin ⁇ 3 protein, A549 cells that are non-small cell lung cancer cells, cancer stem cells ALDH1 activated cells and breast cancer cells It has been confirmed that MDA-MB231 cells have an effect of inhibiting cell growth, and thus the fibulin-3 protein can be usefully used to inhibit the growth of cancer cells and cancer stem cells. It was confirmed that the expression of beta-catenin and VII 2,7 related to the growth of cancer stem cells was directly suppressed by Boolean 3 overexpressing cell line or culture medium thereof.
- the fibulin-3 produced by transforming the fibulin-3 gene in inhibiting the growth of cancer stem cells. It was confirmed that the overexpressing cell line or its culture medium inhibited the cell growth and penetration factor of cancer stem cells, through which the fibulin-3 ' overexpressing cell line or its culture medium was useful for the preparation of pharmaceutical compositions for inhibiting the growth of cancer stem cells. It can be seen that it can be used.
- the present invention will be described in detail by Examples, Experimental Examples and Preparation Examples.
- the inventors constructed an overexpressed clone of the fibulin-3 gene.
- non-small cell lung cancer cells H46C ATCC no. HTB-177) was used to extract the whole RNA using the High Pure RNA Isolation Kit (Roche, Germany), and then reverse RNA 1 "polymerase chains to total RNA 1 ig using Transcriptor First Strand cDNA Synthesis Ki Roche, Germany).
- Reverse transcrition polymerase chain reaction (RT-PCR) was performed, PCR conditions were 30 minutes at 55 ° C., and 85 0 C.
- Hinc restriction enzyme recognition sequence at 5'_end and restriction enzyme recognition sequence at 3'-end After 5 min pre-denature at 94 ° C using a primer pair of forward primer 5'-ATATMGCTTATGTTGAAAGCCC— 3 '(SEQ ID NO: 2) and reverse primer 5'-ATATCTCGAGCTAAMTG AATGGCCCC-3' (SEQ ID NO: 3) prepared to include , 1 min degeneration at 94 ° C ,
- the inventors produced a fibulin-3 clone with histidine protein attached to the c-terminal site for protein purification.
- PCR was performed by preparing primers such that histidine protein was expressed in the back of fibulin-3 using DNA /0.1 of the overexpression of the fibulin-3 clone of ⁇ Example 1-1> as a template.
- Forward primer 5'-CGGMTTCATGTO ⁇ GCCCTmCCTMCTATG— 3 '(SEQ ID NO: 14) prepared by attaching the histidine (his) protein sequence with the c Y restriction enzyme recognition sequence at the 5' end and the Xhol restriction enzyme recognition sequence at the 3 'end.
- the present inventors prepared a culture medium in which cells overexpressing fibulin-3 were used as the fibulin-3 overexpressing vector of ⁇ Example 1-1>.
- the penicillin-streptomycin solut ion (Hyclone) of the fibulin-3 overexpression vector 2 of ⁇ Example 1-1> was used for Lipofecamine 2000 on 5 ⁇ 10 5 cells / m A549 cells. °] was transduced in medium not added. After 4-6 hours of reaction, the cells were replaced with medium containing 100 units / mi of penicillin-straptomycin and cultured for 48 hours to obtain the medium.
- the present inventors prepared a medium in which the cells transformed with the purified fibulin-3 protein vector (his-fibulin-3) of ⁇ Example 1_2> were cultured.
- CH0—K1 cells Life Science Research Institute, Daejeon
- 293T cells Haanyang University, Seoul
- Fibulin-3 vector 4 / ig and lipofect amine 2000 were used to transduce in DMEM: F12 (Gibco) medium and DMEM (Hyclone) medium without penicillin ⁇ streptomycin (penici 11 in— streptomycin solution, Hyclone).
- DMEM Fetrachloride
- penicillin ⁇ streptomycin penici 11 in— streptomycin solution, Hyclone
- the inventors of the present invention purified his-pibulin-3 protein in the fibulin ⁇ 3 protein purification medium of ⁇ Example 2-2>.
- the inventors of the stem cell company based on Aldehyde dehydrogenase (ALDH) activity based on Aldehyde dehydrogenase (ALDH) activity
- ALDEFLU0R was used to differentiate and select cancer stem cells from A549 cells, which are non-small cell lung cancer cells.
- the dried ALDEFLU0R reagent was added with 25 ⁇ DMSO (dimethylsulphoxide) to react at room temperature for 1 minute, and the reagent was activated from red orange to light yellow. Add 25 M of hydrochloric acid (0 ⁇ 0101 " acid) to the activated reagent
- ALDEFLU0R substrate was made. Thereafter, the supernatant was isolated ⁇ ⁇ ⁇ 6 of A549 cells by centrifugation. Discard the supernatant and mix 1 ml of ALDEFLU0R assay buffer. Prepare two empty tubes, each containing 500 ⁇ sample containing the cells and ALDEFLU0R assay buffer, add 5 activated ALDEFLU0R substrates to one tube, and add DEAB (Di ethyl ami nobenzaldehyde) solution to another tube. f, mix and mix.
- DEAB Di ethyl ami nobenzaldehyde
- the inventors overexpressed the vector in order to confirm whether the fibulin-3 protein was overexpressed by a hiss-fibulin-3 vector for protein purification, and then performed polymerase chain reaction and western blot. .
- CH 10 -K1 cells Life Science Research Institute, Daejeon
- 293T cells Haanyang University, Seo-eul
- RNA 1 was dissolved at 45 0 C using Intron's oligo dT cDNA kit.
- CDNA was synthesized using an amplifier system (Takara, Japan) for 1 hour at 95 0 C for 5 minutes. Then, with the primer of Table 1 below, I-taq polymerase (Intron) 94 0 C 5 minutes
- vector-derived CH0-K1 cells were collected, and 50 ⁇ of lysis solution was added to each cell, followed by reaction at 4 ° C for 30 minutes, followed by 4 ° C. Centrifugation was performed using a centrifuge, supernatant was obtained, and then 40 g of each protein was loaded into the SDS-gel using a protein sigma kit.
- the protein loaded on the gel is then transferred to nitrocell membranes and reacted with BSA buffer for 30 minutes at room temperature to prevent other antibodies from binding.
- the primary antibody anti-betacatenin antibody (santa cruz) and anti- Beta-actin antibody (sigma) was reacted in PBS buffer diluted 1: 1000 for 4 hours and then secondary antibody anti-mouse (cell signaHng) was reacted in PBS buffer diluted 1: 10000 for 1 hour. .
- the nitrocelrose membrane was washed 5 times with PBS, and the film was then exposed to a film using a detection solution.
- the inventors confirmed the expression levels of fibulin-3 and betacatenin in ALDH1 activated cells and inactive cells isolated therefrom. Specifically, in order to extract RNA from A549 cells and ALDH1 active and inactive cells isolated therefrom, first, each cell was mixed with 1 m of trizol for 5 minutes, and then 200 chloroform reagent was added for 5 minutes. In a 4 ° C centrifuge
- RNA 1 / g was measured at 45 0 C for 1 hour and 95 minutes at 95 0 C using Intron oligo dT cDNA kit. Takara, Japan) was used to synthesize cDNA. Subsequently, with the primers of Table 1 below, I-taq polymerase (Intron) was used for 94 5 minutes.
- the present inventors confirmed the amount of nucleic acid and protein expression of betacatenin in A549 cells and H460 cells, which are non-small cell lung cancer cells.
- Each 1 I ⁇ was extracted and subjected to reverse transcription polymerase chain reaction (RT-PCR) using cDNA Synthesis Kit (Intron Biotechnology, Gyungki-do, Korea) to synthesize cDNA.
- Western Blot analysis (Western Blot) is first centrifuged to each 50 ⁇ put by using a banong a rear 4 0 C centrifuge for 30 min at 4 0 C was dissolved solution in each cell, to obtain a supernatant, and then, Each protein 40 / was loaded into SDS-gel using a protein sigma kit (sigma).
- the protein loaded on the SDS gel was transferred to the nitrocell membrane and reacted with BSA buffer for 30 minutes at room temperature to prevent other antibodies from binding.
- the primary antibody anti-betacatenin antibody (santa cruz) and anti- Beta actin The antibody (sigma) was reacted with PBS buffer solution diluted 1: 1000 for 4 hours, and then the secondary antibody anti-mouse (cell signaling) was reacted with PBS complete solution diluted 1: 10000 for 1 hour. Thereafter, the nitro salrose membrane was washed five times with PBS, and then exposed to a film with a detection solution.
- beta catenin is expressed more in A549 cells than in H460 cells (FIG. 6).
- the present inventors confirmed whether fibulin-3 affects the expression levels of nucleic acids and proteins of betacatenin in non-small cell lung cancer cells.
- the fibulin-3 gene overexpression clone prepared in ⁇ Example 1-1> was transduced into A549 cells having high beta-catenin expression.
- Fibulin-3 stealth-RNAi each of 25 mers of -3 '(SEQ ID NO: 13) was fabricated and ordered from Invitrogen. Introduction of the siRNA into the cell
- fibulin-3 was overexpressed by a fibulin-3 gene overexpression clone in A549 cells and fibulin-3 was inhibited by fibulin-3 siRNA in H460 cells. Confirmed. In addition, it was confirmed that betacatenin expression was decreased in A549 cells overexpressing fibulin-3 in each sample, and betacatenin expression was increased in H460 cells in which fibulin-3 expression was inhibited (FIG.
- betacatenin expression was inhibited by fibrin-3 in non-small cell lung cancer cell line.
- the present inventors confirmed the inhibitory effect of betacatenin expression by fibulin ⁇ 3 in A549 cells, which are non-small cell lung cancer cell lines.
- the medium of the fibulin-3 overexpression medium prepared in ⁇ Example 2-1> -A549 cell medium was mixed at a ratio of 1: 4, and the expression of fibulin-3 was higher than that of the A549 cell.
- the present inventors confirmed the growth inhibitory effect of A549 cells, which are non-small cell lung cancer cells by fibulin-3.
- the fibulin-3 overexpression medium prepared in Example 2-1 and the A549 cell medium were mixed at a ratio of 1: 4, and the expression of fibulin-3 was higher in H460 cells 5 than in A549 cells.
- A549 cells were treated with a mixture of ⁇ 460 cell culture medium and A549 cell medium in which ⁇ 10 5 was incubated for 48 hours at a ratio of 1: 4.
- the present inventors treated the His-fibulin-3 overexpression medium obtained by transforming the hiss-fibulin-3 vector for protein purification of ⁇ Example 2-2> to A549 cells to treat the cells.
- the growth inhibition effect of was confirmed by colony formation experiment.
- the transformed CH0-K1 cells of ⁇ Example 2-2> and his-fibulin- 3 overexpression medium of 293T cells were mixed at a ratio of 1: 1 and treated with lxlO 3 A549 cells, 37 Incubated for 7 days in a carbon dioxide incubator. After staining with 0.5% reagent violet crystals for 10 minutes and washed several times with PBS, it was confirmed that A549 cells treated with heath-fibulin-3 overexpression medium inhibited cell growth compared to the control group (Fig. 11).
- the present inventors confirmed the cancer cell growth inhibitory effect by the purified fibulin-3 protein.
- lxlO 3 non-small cell lung cancer cells A549 cells and breast cancer cells
- the histopipibulin-3 purified protein of Example 2-3 was applied to lung cancer cells and breast cancer cells once or twice (once treated, again for 24 hours). One more time). Thereafter, the cells were incubated in a 37 ° C. incubator for 7 days, stained with Crystal Vial 0.5% reagent for 10 minutes, and washed several times with PBS.
- the present inventors confirmed the effect of inhibiting the expression of MMP-2,7 and betacatenin by fibulin-3 in ALDH1 activated cells, which are cancer stem cells.
- the fibulin-3 overexpression medium prepared in Example 2-1 and the ALDH1 active cell medium were mixed in a ratio of 1: 4, and H460 cells with higher expression of fibrin-3 compared to A549 cells.
- 5 ⁇ 10 5 dogs 48 hours incubation ⁇ 460 cell culture medium and ALDH1 active cell media 1: create a combined common culture medium in a ratio of 4, was treated with the medium in each 2 ⁇ 10 5 cells / of ALDH1 active cells.
- protein expression was confirmed by the Western blot method of ⁇ Experimental Example 2-1> using P2 (cell signaling), MMP-7CR & D system) and betacatenin (santa cruz) antibody.
- fibulin-3 antibody was diluted 500: 1 in two mediums mixed above and treated with ALDH1 activated cells 48 hours later. Protein expression was confirmed by Western blot using a beta-catenin antibody.
- beta-catenin protein, H460 cell culture medium and ALDH1 active cell medium were mixed at a ratio of 1: 4, fibulin-3 overexpression medium and ALDH1 active cell medium were 1: 4. It was confirmed that the protein expression of ⁇ P-2, 7 and betacatenin decreased in the cells treated with the mixed medium at the ratio of.
- Western blot results of treatment with ALDH1 activated cells by diluting the fibulin-3 antibody with 500: 1 in the medium showed that the protein expression of ⁇ P-2,7 and betacatenin was not different from that of the control group. It was confirmed that the expression inhibitory effect of -2,7 and betacatenin is due to the fibulin-3 protein (FIG. 14).
- the present inventors confirmed the growth inhibitory effect of ALDH1 activated cells, which are cancer stem cells by fibulin-3.
- the fibulin-3 overexpression medium prepared in Example 2-1 and ALDH1 activated cell medium were mixed in a ratio of 1: 4, and the expression of fibulin-3 was higher than that of A549 cells.
- the ALDH1 activated cells were treated with a mixture of ⁇ 460 cell culture medium and ALDH1 activated cell medium in which a large number of H460 cells 5 ⁇ 10 5 were incubated for 48 hours at a ratio of 1: 4. priority,
- One ⁇ 460 cell culture medium and ALDH1 active cell medium were mixed at a ratio of 1: 1 (50%), and the ⁇ 460 cell culture medium and ALDH1 activated cell medium were incubated for 48 hours with 5 ⁇ 10 5 cells of ⁇ 460 cells 5: 4. And treated with ⁇ 460 cell culture medium (100 3 ⁇ 4>), which were incubated for 48 hours in a medium mixed at 80 3 ⁇ 4 and 5 ⁇ 10 5 cells for 48 hours.
- the medium was treated in a 37 ° C. carbon dioxide incubator. After 8 days of incubation, 10 minutes of staining with crystal violet 0.5 3/4 reagent It confirmed cases washed with PBS.
- the present inventors confirmed the growth inhibitory effect of ALDH1 activated cells, which are cancer stem cells, by the heath-fibulin-3 overexpression medium prepared in Example 2-1.
- Example 2-2> A transgenic heath of CH0-K1 cells and 293T cells-over-expressing the -3 medium called P 1: a mixture in a ratio of 1 ⁇ ⁇ ⁇ treated in three ALDH1 activated cell After incubating for 7 days in a 37 ° C carbon dioxide incubator,
- the above ingredients were mixed and layered in an airtight cloth to prepare a powder.
- tablets were prepared by tableting according to a conventional method for producing tablets.
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US14/116,981 US20140147423A1 (en) | 2011-08-09 | 2011-12-22 | Pharmaceutical composition containing fibulin-3 protein as an active ingredient for inhibiting the growth of cancer stem cells |
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KR101635568B1 (ko) * | 2014-12-26 | 2016-07-20 | 한국원자력연구원 | 피불린-4 단백질 또는 이를 암호화하는 폴리뉴클레오티드를 유효성분으로 함유하는 폐암 예방 및 치료용 약학적 조성물 |
KR101746143B1 (ko) | 2015-07-08 | 2017-06-13 | 한국원자력연구원 | Fbln-3 단백질을 유효성분으로 함유하는 췌장암 예방 및 치료용 약학적 조성물 |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2016504422A (ja) * | 2013-04-02 | 2016-02-12 | イーホン・ジョウYihong ZHOU | フィビュリンタンパク質変異体および対応する核酸配列 |
Also Published As
Publication number | Publication date |
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KR101440487B1 (ko) | 2014-09-18 |
EP2742950B1 (en) | 2017-03-08 |
KR20130019341A (ko) | 2013-02-26 |
EP2742950A1 (en) | 2014-06-18 |
JP2014516053A (ja) | 2014-07-07 |
JP5717920B2 (ja) | 2015-05-13 |
US20140147423A1 (en) | 2014-05-29 |
EP2742950A4 (en) | 2015-04-08 |
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