KR101458272B1 - Inhibitor of cancer cell growth comprising retinoid and C-Jun N-terminal kinase inhibitor - Google Patents

Abstract

The present invention relates to a cancer cell growth inhibitor comprising a retinoid and a p38 kinase inhibitor or a C-Jun N-terminal inhibitor, wherein the expression and activity of the metrix metalloproteinase is increased and the growth of cancer cells is inhibited And thus can be usefully used as a medicine for inhibiting the growth of cancer cells.

Description

Inhibitor of cancer cell growth comprising retinoid and C-Jun N-terminal kinase inhibitor comprising retinoid and C-Jun N-terminal inhibitor [

The present invention relates to a growth inhibitor of cancer cells comprising a retinoid and a p38 kinase inhibitor, a C-Jun N-terminal inhibitor. In addition, the present invention relates to a cancer cell growth inhibitor whose expression and activity of metrix metalloproteinase increases when the cancer cell growth inhibitor is administered.

Retinoids are structural analogs of vitamin A and include both natural and synthetic compounds. Retinoic acid, 'ATRA', 9-cis-retinoic acid, trans-3,4-didehydrolate acid, 4-oxo retinoic acid, 13-cis- Retinoid compounds, such as retinol, are multimeric expression modulating compounds that affect a number of inflammatory, immune, and structural cells.

Among the retinoids, retinoic acid is known to be an important regulator of cartilage and skeletal formation and is known to be a derivative of effective chemotherapeutic agents in the treatment of various types of cancer, including lung, breast, head and neck cancer, and hematologic cancer (Kakizuka A. et al. Cell, 1991, vol. 66, p663-671). In addition, retinoic acid has been reported to inhibit cell growth and proliferation in a variety of cancers, including lung cancer, breast cancer, leukemia, and rhabdomyosarcoma (Sun SY et al., Cancer Res, 1997, vol. 57, p4931-4939). However, as an anticancer agent, retinoids have lower therapeutic efficiency than other anticancer drugs, and therefore, anticancer agents having higher therapeutic efficiency are required.

Accordingly, an object of the present invention is to provide an agent for inhibiting growth of cancer cells, which is superior to an existing cancer cell growth inhibitor. The present invention provides a cancer cell growth inhibitor having a higher effect than a conventional retinoid alone by simultaneous administration of a retinoid and a mitogen-activator protein kinase modulator in cancer cells.

The present inventors have been studying cancer cell growth inhibitors with higher therapeutic efficiency than retinoids, and have found that when retinoids are administered to cancer cells, they act as modulators of metrix metalloproteinases and that the retinoids also act as mitotic-activated protein kinase signaling Lt; RTI ID = 0.0 > activity. ≪ / RTI > Mitogen-Activated Protein Kinase (MAPK) plays an important role in the growth of cancer cells as an important signaling protein that plays a key role in almost all physiological phenomena such as cell growth, differentiation and death. As a result, the concurrent administration of p38 kinase inhibitor and C-Jun N-terminal kinase inhibitor as a retinoid and a mitogen-activated protein kinase regulator whose anticancer efficacy has been proven results in a cancer cell with a higher efficiency than the administration of one of retinoids or the inhibitor And that it would be possible to curb growth. As a result of the above research, it was surprisingly found that simultaneous administration of retinoid and the inhibitor is more effective in inhibiting cancer cell growth than existing retinoids alone.

The cancer cells of the present invention are not particularly limited, but include cancer cells such as fibrosarcoma, lung cancer, breast cancer, leukemia, rhabdomyosarcoma, ovarian cancer, liver cancer, pancreatic cancer and prostate cancer. The fibrous sarcoma is a malignant tumor and may occur in any part of the body. Examples of fibrosarcoma include, but are not limited to, HT1080.

Retinoids are one of a group of compounds in which four isoprenoid units are linked in a head-to-tail fashion. Vitamin A is a generic term for retinoids that qualitatively indicate the biological activity of retinol. The retinoid of the present invention can increase the expression and activity of the matrix metalloproteinase in the growth process of cancer cells. The retinoid is not particularly limited and includes, for example, retinol, retinal, retinoic acid, retinol and fatty acid esters, retinoid derivatives such as esters of aliphatic alcohol and retinoic acid, and vitamin A Analogs can be used.

The term "p38 kinase inhibitor " as used herein relates to compounds that target, decrease or inhibit p38 kinase. p38 kinase is a mitogen-activated protein kinase that responds to stress stimuli such as cytokines, ultraviolet light, thermal shock, and osmotic shock, and is involved in cell division and apoptosis. Examples of the p38 kinase inhibitor are not particularly limited, but SB203580 represented by the following formula (1) is particularly preferable.

The term "C-Jun N-terminal kinase (JNK) inhibitor" as used herein relates to a compound that targets, decreases or inhibits C-Jun N-terminal kinase. JNK, a serine-specific protein kinase, is involved in the phosphorylation and activation of c-Jun and ATF2 and plays an important role in metabolism, growth, cell differentiation and apoptosis. Examples of the JNK kinase inhibitor are not particularly limited, but a pyrazolanthrone represented by the following formula 2 is particularly preferable.

As used herein, the term " Matrix Metalloproteinases "is a zinc-dependent family of enzymes that has a specific structure and catalytic properties and plays an important role in tumor invasion and metastasis. The Metrix metalloproteinase system participates in a variety of processes that regulate connective tissue remodeling in the body, including fetal development, organogenesis, neovascularization, bone growth, and cartilage remodeling. However, in pathological conditions such as tumors, the regulatory function of the matrix metalloproteinase system is destroyed, resulting in the degradation and reconstruction of a wide range of extracellular matrix. The matrix metalloproteinase is a matrix metalloproteinase-1 / Metrix metalloproteinase-8 / Metrix metalloproteinase-13 / Metrix metalloproteinase-18 interstitial collagenases, matrix metalloproteinase-2 / metrix metalloproteinase-9, matrix metalloproteinase-3 / metrix metalloproteinase-10, elastase elastases, and membrane-type matrix metalloproteinases (MMPs), among which gelatinases, Metrix metalloproteinase-9 and Metrix metalloproteinase-2 Is known to be a key regulator of tumor invasion and metastasis. Examples of the matrix metalloproteinase include, but are not limited to, Metrix metalloproteinase-2, Metrix metalloproteinase-9.

The term "cancer cell growth inhibitor " as used herein may be any drug that directly or indirectly inhibits the physical, chemical and / or physiological actions of cells involved in the growth of cancer. Among these drugs, anticancer agents are particularly preferable in terms of therapeutic effects and the like.

In accordance with the present invention, a combination of a p38 kinase inhibitor or a C-Jun N-terminal kinase inhibitor as a regulator of metrix metalloproteinase as a retinoid and a mitogen-activating protein kinase regulator, Cancer cell growth inhibitory effect.

FIG. 1 is a photograph of a fibrosarcoma HT1080 treated with various concentrations of retinoic acid at 0, 10, 20, 30, and 50 μM for 24 hours under a phase contrast microscope.
FIG. 2 is a graph showing the degree of cell proliferation by MTT assay in various concentrations of retinoic acid at 0, 10, 20, 30, and 50 μM for 24 h in fibrous sarcoma HT1080.
Figure 3 shows the results of treatment of fibrosarcoma HT1080 with 50 μM retinoic acid for 0, 10, 30, 1, 3, 6, 12, (MMP-2) and Metrix metalloproteinase-9 (MMP-9) through gelatin zymogens after 30, 50, and 50 μM, respectively.
4 is a graph obtained by quantifying the processing result of FIG. 3 through an image J program.
Figure 5 shows the results of treatment of fibrosarcoma HT1080 with 50 μM retinoic acid at 0, 10, 30, 1, 3, 6, 12 and 24 hours and fibrous sarcoma HT1080 at 0, 10 and 20 (MMP-2), Matrix metalloproteinase-9 (MMP-9), and Actin were determined by Western blot analysis after treatment with 30, 50, Results.
6 is a graph obtained by quantifying the processing result of FIG. 5 through the image J program.
FIG. 7 shows the results of Western blot analysis after treatment with 50 μM of retinoic acid in fibrosarcoma HT1080 at 0, 10, 30, 1, 3, 6, 12, and 24 hours, followed by p38 kinase and pp38 kinase, pJNK , And C-Jun N-terminal kinase (JNK).
Figure 8 shows the expression levels of p38 kinase, pp38 kinase, pJNK, and C-Jun N-terminal kinase (JNK) by Western blot analysis after treatment with fibrosarcoma HT1080 for 24 hours at 0, 10, 20, 30, .
Fig. 9 shows the results of treatment of fibrosarcoma HT1080 in a control group treated with 50 μM of retinoic acid only, a control group of 50 μM of retinoic acid and SB203580 (SB) of p38 kinase inhibitor, 50 μM of retinoic acid, - SP600125 (SP), an end-kinase inhibitor, was used to measure the activity of Metrix metalloproteinase-9 (MMP-9) and Metrix metalloproteinase-2 (MMP-2) The result is confirmed.
FIG. 10 is a graph obtained by quantifying the processing result of FIG. 9 through an image J program.
Figure 11 shows that fibrosarcoma HT1080 was treated with 50 μM of retinoic acid, 50 μM of retinoic acid and SB203580, a p38 kinase inhibitor, 50 μM of retinoic acid and C-Jun N-terminal kinase (MMP-9), Matrix metalloproteinase-2 (MMP-2), and Actin (MMP-9) by Western blot analysis using the inhibitor SP600125 And the amount of expression was confirmed.
12 is a graph obtained by quantifying the processing result of FIG. 10 through an image J program.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms.

Example 1-1

Human fibrosarcoma HT1080 used in this example was purchased from the American Type Culture Collection (Manassas, Va.). The human fibrosarcoma HT1080 contains 10% fetal bovine serum (purchased from Invitrogen, Burlington, CA), 50 μg / ml streptomycin and 50 units / ml penicillin (Purchased from incitrogen, Burlington, CA) medium in a 5% carbon dioxide (CO ₂ ) incubator maintained at 37 ° C and the density was cultured at a density of 1.2 × 10 ⁵ cells / dish .

Examples 1-2

The morphology of the cells was observed by phase contrast microscope by treating the human fibrosarcoma HT1080 cultured in Example 1-1 with different concentrations of retinoic acid for 24 hours, and MTT (3- (4,5-dimethylthiazol-2-yl ) -2,5-diphenyltetrazolium bromide) assay (FIG. 1, FIG. 2). The MTT assay proceeded in the following manner.

Examples 1-1 The fibrosarcoma HT1080 cultured in 96-well-seeded at a density of 1.2 × 10 ⁵ cells per well in the plate (well-plate) and cultured in 5% carbon dioxide incubator that is kept 37 ℃. When cells were grown at 70 ~ 80%, retinoic acid was treated and cultured for 24 hours. After culturing, MTT reagent I (10 mg / ml) was treated with 10 쨉 l per well and cultured for 4 hours. After that, MTT reagent II was treated with 100 μl per well and cultured for 12 hours, and the absorbance value at 550 to 600 nm was measured using an ELISA reader. The MMT reagent II is a solution prepared by mixing 10% SDS (sodium dodecyl sulfate), 0.01 N HCl, and dimethylsulfoxide (DMSO).

The fibrous sarcoma HT1080 was treated with various concentrations of retinoic acid at concentrations of 0, 10, 20, 30, and 50 μM for 24 hours, and it was observed as a phase contrast microscope that cell proliferation was inhibited depending on the concentration of retinoic acid ). As a result, the cell proliferation was inhibited dependently on retinoic acid treatment concentration, and cell proliferation was specifically inhibited by 50% at 50 μM of retinoic acid (FIG. 2).

Example 1-3

The activity of the matrix metalloproteinase was confirmed by gelatin zymography after treating the fibrous sarcoma HT1080 cultured in Example 1-1 with various concentrations of time and concentration of retinoic acid. The gelatin zymograph proceeds in the following manner.

The gelatin zymogram was purchased from RPMI-1640 (purchased from incitrogen, Burlington, Canada) containing 10% fetal bovine serum (purchased from Invitrogen, Burlington, Canada) Fibrosarcoma HT1080 was stained with RPMI-1640 supplemented with 2% fetal bovine serum and cultured for 12 hours. When cells grew 70-80%, retinoic acid was treated with RPMI-1640 containing 2% fetal bovine serum. After 24 hours, only the supernatant was collected separately and electrophoresed on SDS-polyacrylamide gel containing 0.1% of gelatin to separate the proteins by size. After washing with 2.5% Triton X-100 three times for 30 minutes, the cells were washed with distilled water to remove the SDS. Then, 5 mM calcium chloride (CaCl ₂ ), 0.2 M sodium chloride (NaCl), and 50 mM tris (hydroxymethyl) tris- (hydroxymethyl) -aminomethane) in a gelatin incubation buffer (pH 7.5) and shaking at 70 rpm in a 37 ° C incubator for 20 to 24 hours. After the reaction, the gel was stained with Coomassie blue solution.

 Western blot analysis was also performed on the amount of expression of matrix metalloproteinase in the fibrous sarcoma HT1080. First, a proteinase inhibitor (10 μg / ml) was added to a cell lysis buffer (cell lysis buffer) containing 50 mM Tris-HCl pH 7.4, 150 mM sodium chloride, and 1% SDS for quantitative analysis of protein. (1mM sodium orthovanadate, 1mM sodium dithiothreitol, 10μg / ml leupeptin, 10μg / ml pepstatin, 1mM 4- (2-aminoethyl) benzenesulfonyl fluoride) Fluorosodium) was added to extract the cell protein. The extracted proteins were separated by size by electrophoresis on SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Then, for the analysis of each protein expression, each protein-specific antibody, Metrix Metaloproteinase-9 (purchased from Santa Cruz Biotechnology, Santa Cruz, CA), Metrix Metaloproteinase-2 (Purchased from Santa Cruz Biotechnology, Santa Cruz, CA), pp38 kinase (purchased from Cell Signaling Technologies, Danvers, Mass.), P38 kinase (purchased from Santa Cruz Biotechnology, Santa Cruz, CA), pJNK kinase Purchased from C-Jun N-terminal kinase (purchased from Santa Cruz Biotechnology, Santa Cruz, CA), and Actin (purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA) Respectively.

The fibrous sarcoma HT1080 cultured in Example 1-1 was treated with 50 μM retinoic acid at 0, 10, 30, 1, 3, 6, 12, and 24 hours to determine the metrix metalloproteinase activity As a result, the activity of Metrix metalloproteinase-9 and Metrix metalloproteinase-2 was increased in a time-dependent manner. The concentration of retinoic acid was found to be 0, 10, 20, 30 and 50 μM for 24 hours, And the expression level of Metrix metalloproteinase-9 and Metrix metalloproteinase-2 was increased (FIG. 3). Figure 3 was quantified as a graph using the image J program (Figure 4). The degree of expression of Metrix metalloproteinase-2 and Metrix metalloproteinase-9 was examined by Western blot analysis. As a result, it was found that the concentration of Metrix metalloproteinase-2 and Metrix metalloproteinase-9 in a time- And the expression level of Metrix metalloproteinase-2 and Metrix metalloproteinase-9 was increased in a concentration-dependent manner (FIG. 5). The results obtained by performing the Western blot analysis were quantified through the image J program (Fig. 6). As a result, it was found that retinoic acid increases the activity and expression of Metrix metalloproteinase-2, Metrix metalloproteinase-9 in time and concentration-dependent manner in fibrosarcoma HT1080.

Example 2-1

Western blot analysis using the fibrous sarcoma HT1080 cultured in Example 1-1 with 50 μM of retinoic acid for various time periods was carried out. As a result, the activity of p38 kinase and C-Jun N-terminal kinase (Fig. 7). When the concentration of retinoic acid was gradually increased to 0, 10, 20, 30, and 50 μM for 24 hours, it was confirmed that the activity of p38 kinase and C-Jun N-terminal kinase decreased in a concentration-dependent manner ). On the other hand, the activity and concentration of ERK were independent.

As a result of the above experiment, the following was conducted with the hypothesis that the activity of matrix metalloproteinase would be further increased when retinoic acid and p38 kinase and C-Jun N-terminal kinase inhibitor were used together.

SB203580 (SB), an activity inhibitor of retinoic acid and p38 kinase, and SP600125 (SP), an activity inhibitor of C-Jun N-terminal kinase, were treated to determine the activity of Metrix metalloproteinase-2 and Metrix metalloproteinase- And degree of expression were confirmed by gelatin zymography and western blot analysis as used in Examples 1-3. As a result of gelatin zymography, the activity of matrix metalloproteinase-9 and matrix metalloproteinase-2 in fibrosarcoma HT1080 treated with 50 μM of retinoic acid alone was found to be twice as high . When SP600125 and SB203580 were treated together to inhibit p38 kinase and C-Jun N-terminal kinase, about 1.6 times more amount of metrix metalloproteinase-9 and metrix metalloproteinase- 2 was increased (Fig. 9). Western blot analysis also showed that the amount of expression of Metrix metalloproteinase-9 and Metrix metalloproteinase-2 in the treatment of fibrosarcoma HT1080 alone with 50 μM of retinoic acid was 2 times , And when SB203580 and SP600125 were treated together to inhibit the activity of p38 kinase and C-Jun N-terminal kinase, about 1.6 times more than the treatment with retinoic acid alone, Metrix metalloproteinase-9 and Metrix metalloproteinase -2 expression level was increased (FIG. 11). The results of the gelatin zymograph and Western blot analysis were plotted using the image J program (Figs. 10 and 12).

As described above, when retinoic acid and SB or SP are administered to cancer cells, it shows higher anticancer activity than retinoic acid alone treatment.

Claims (8)

A fibrosarcoma cancer cell growth inhibitor comprising retinoic acid and a compound represented by the following formula:

(2) delete delete delete The fibrosarcoma cancer cell growth inhibitor according to claim 1, wherein expression or activity of matrix metalloproteinases is increased upon administration of the cancer cell growth inhibitor. 6. The agent according to claim 5, wherein the matrix metalloproteinase is Metrix metalloproteinase-2 or Metrix metalloproteinase-9. delete delete

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