Pharmaceutical composition for treating cancer including 2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol as active ingredient

Abstract

The present disclosure relates to an anticancer use of a novel compound, i.e., 2-methoxy-4-(3-(4-methoxyphenyl)propyl-1-en-1-yl)phenol. The compound of the present disclosure effectively inhibits the growth of cancer cells and tumors in vitro and in a xenograft animal model. The compound of the present disclosure illustrates an anticancer activity by inhibiting a DNA binding activity of transcription factor STAT 3 in cancer cells, inducing apoptosis of cancer cells, and reducing the expression of a cell cycle regulatory protein. The compound of the present disclosure can be developed as an active ingredient of a strong anticancer drug.

Claims

1. A method for treating cancer, comprising administering to a subject in need thereof pharmaceutical composition comprising a pharmaceutically effective amount of a compound represented by the following formula 1; and a pharmaceutically acceptable carrier ##STR00002##

2. The method of claim 1, wherein the compound represented by the formula 1 increases the expression of a pro-apoptotic protein in cancer cells.

3. The method of claim 2, wherein the pro-apoptotic protein is cleaved caspase-3, cleaved caspase-8 or Bax protein.

4. The method of claim 1, wherein the compound represented by the formula 1 reduces the expression of an anti-apoptotic protein in cancer cells.

5. The method of claim 4, wherein the anti-apoptotic protein is a Bcl-2 protein.

6. The method of claim 1, wherein the compound represented by the formula 1 reduces the expression of a cell cycle regulatory protein.

7. The method of claim 6, wherein the cell cycle regulatory protein is Cdk (cyclin-dependent kinase) 2, Cdk (cyclin-dependent kinase) 4, Cdk (cyclin-dependent kinase) 6, cyclin B1, cyclin D1, or cyclin E1.

8. The method of claim 1, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colorectal cancer, colon cancer, tubal cancer, endometrial cancer, cervical cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer or bone marrow cancer.

9. A method for treating cancer by inhibiting DNA binding activity of transcription factor STAT3, comprising administering to a subject in need thereof pharmaceutical composition comprising a pharmaceutically effective amount of a compound represented by the following formula 1; and a pharmaceutically acceptable carrier ##STR00003##

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates the results of inhibition of the growth of SW480 cell line in dependence on the concentration of MMPP. The upper right panel illustrates cell photos exhibiting the results of inhibition of the growth of SW480 cells in dependence on the concentration of MMPP. The lower right panel illustrates the effect of MMPP on the colon cancer cell line SW480.

(2) FIG. 2 illustrates the results of changes in the expression of cell cycle regulatory proteins and apoptotic signal proteins by MMPP. The upper part of panel A of FIG. 2 illustrates the result that the expression of the cell cycle regulatory signal protein is reduced by MMPP. The lower part of panel A of FIG. 2 illustrates the results of an increase in expression of pro-apoptotic protein and a decrease in expression of anti-apoptotic protein. Panel B of FIG. 2 illustrates the results of inhibition of DNA binding activity of STAT3 by MMPP.

(3) FIG. 3 illustrates the results of MMPP inhibition of colon cancer growth in a xenograft model.

MODES OF THE INVENTION

EXAMPLE

(4) Experimental Materials and Methods

(5) 1. Cell Culture

(6) The human colon cancer cell line, SW480, was purchased from the American Type Culture Collection (Manassas, Va., USA) and cultured in an RPMI (Roswell Park Memorial Institute)-1640 medium (Gibco-BRL) to which 10% heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin at 37 C. humidified atmosphere of 5% CO.sub.2. The cells were cultured in a 100 mm culture dish at a concentration of 410.sup.5 cells for experiments.

(7) 2. Cell Viability Analysis Using MTT Assay

(8) The influence of 2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) on the viability of cells was confirmed by colorimetric and metabolic activity assay using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Put briefly, cells were inoculated on 96-well plates at 110.sup.4 cells/well and cultured for 24 hours. Cells were treated with 2.5 to 10 g/ml MMPP or DMSO (dimethyl sulfoxide), a solvent for MMPP. After culturing for 24 hours, the medium including MMPP was removed and replaced with 100 l of fresh medium. After culturing for 1.5 hours, medium including MTT was removed and 200 l DMSO was added to each well. The plate was then agitated at a weak intensity until the color reaction of the plate became uniform. Colorimetric evaluation was performed using a microplate reader at 540 nm.

(9) 3. Western Blot Analysis

(10) Lysates, cytosolic and nuclear extracts of whole cells were obtained. SDS-PAGE and western blot analysis were performed as described in published documents 1 and 2 [1, 2]. Put briefly, the cells of 510.sup.5 cells/well were inoculated in 6-well plates and cultured for 24 hours. Cells were then treated with MMPP or DMSO for 24 hours. The treated cells were washed twice with PBS and lysed. Proteins from the lysed cells were separated on 10 to 15% SDS-PAGE. Proteins were transferred to a PVDF membrane and the PVDF membrane was blocked with TBS/T-buffer including 5% defatted milk for 2.5 hours at room temperature. The membrane to which the proteins are transferred was analyzed by using a mouse monoclonal antibody for as a primary antibody. Protein expression was visualized using a chemiluminescence reagent (Amersham Pharmacia Biotech, Inc., Buckinghamshire, UK) and measured using a in which a CCD camera (Fusion-FX, Fisher BioTech, Ltd., Wembley, Australia) is installed.

(11) 4. Analysis of DNA Binding Activity of STAT3 Using EMSA

(12) The DNA binding activity of STAT3 was measured using an electrophoretic mobility shift assay (EMSA) as described in document 1. Put briefly, cells were cultured in a 100 mm culture dish at 37 C. for 24 hours and then treated with MMPP or DMSO. After incubation for 24 hours, the cells were washed three times with cold PBS and nuclear extracts for EMSA experiments were prepared. Relative thickness of the DNA-protein binding band was quantified using Lab Works 4.0 software (UVP Inc., Upland, Calif., USA) after scanning with densitometry.

(13) 5. Anticancer Activity Research of a Xenograft Animal Model

(14) 6-week-old BALB/c athymic nude mouse was purchased from Japan SLC. The mice were bred and maintained in accordance with the Aseptic Environment certified by the American Association for Accreditation of Laboratory Animal Care and the standards and regulations established by the Ministry of Food and Drug Safety. Human colon cancer cell line SW620 was injected subcutaneously (110.sup.7 cancer cells/0.1 ml PBS/animal) into the lower flank of host mice using a 27-gauge needle. When the mean tumor volume was 300 to 400 mm.sup.3 after 20 days, MMPP dissolved in 0.01% DMSO were injected intraperitoneally (2.5 mg/kg and 5 mg/Kg) into tumor bearing mice twice a week for 3 weeks. The group treated with 0.01 mol/L of DMSO was used as a negative control group.

(15) The weight of the mice and the tumor volume were observed twice a week. The volume of the tumor was calculated and computed by the equation: (AB.sup.2)/2 after measuring with vernier calipers. In the above equation, A and B mean the long and short lengths of the tumor, respectively. At the end of the experiment, mice were sacrificed by cervical dislocation. The tumor was surgically removed and separated from the muscles and skin surrounding the tumor, and then the weight was measure.

(16) Experiment Results

(17) 1. Effect of MMPP on Growth of Human Colon Cancer Cells

(18) The inhibitory effect of MMPP on the growth of human colon cancer cells was examined using SW480 cells. As illustrated in FIG. 1, when MMPP (2.5 to 10 g/ml) was treated for 24 hours, MMPP inhibited the growth of SW480 cells in a concentration-dependent manner, and the IC.sub.50 value was 5.9 g/ml. In addition, it was confirmed that MMPP significantly reduced cell density as compared to the control group of SW480 cells. These results indicate that MMPP has a strong inhibitory effect on the growth of colon cancer cells.

(19) 2. Effect of MMPP on Cell Cycle Regulatory Signal and Apoptosis Signal In order to investigate the basic mechanism of the growth inhibitory effect of colon cancer cells of MMPP, it was examined whether MMPP treatment affects cell cycle regulation and apoptosis signals in SW480 cells. As illustrated in panel A of FIG. 2, the treatment of MMPP effectively reduced the expression of cell cycle regulatory signal proteins. In addition, MMPP increased the expression of cleaved caspase-3, cleaved caspase-8 and Bax, which are pro-apoptotic proteins, but reduced the expression of the anti-apoptotic proteins Bcl-2 (Panel A of FIG. 2).

(20) 3. Effect of MMPP on STAT3 DNA Binding Activity

(21) In order to determine whether MMPP reacted with STAT3 to inhibit DNA binding activity of STAT3, MMPP was exposed to SW480 cells for 1 hour and the DNA binding activity of STAT3 was measured. As a result of the experiment, it was confirmed that MMPP effectively inhibited DNA binding activity of STAT3 in SW480 cells (panel B in FIG. 2).

(22) 4. Effect of MMPP on Growth of Colon Cancer in a Xenograft Model

(23) In order to confirm the anticancer effect in vivo, nude mice with xenografted colon cancer were treated with MMPP and tumor growth was investigated. In a SW480 xenografted mouse study, mice with a tumor volume of 100 to 300 mm.sup.3 were injected via intraperitoneal injection of MMPP twice a week for 3 weeks. Tumor volumes of mice treated with 2.5 mg/kg and 5 mg/kg of MMPP were 65% and 35%, respectively, compared to the control group (FIG. 3). In the same manner as an in vitro experimental result, these results suggest that MMPP inhibits the growth of colon cancer in vivo.

(24) The specific embodiments described herein are representative of preferred embodiments or examples of the present disclosure, and thus the scope of the present disclosure is not limited thereto. It will be apparent to those skilled in the art that modifications and other uses of the present disclosure do not depart from the scope of the invention described in the claims.