KR101571098B1 - Pharmaceutical composition for preventing or treating cancer comprising tamoxifen and methyl sulfonyl methane - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer diseases comprising tamoxifen and methyl sulfonyl methane as active ingredients and, more specifically, to a composition for preventing and treating cancer diseases which inhibits proliferation, metastasis, infiltration and growth of cancer cells. It has effective effects for preventing or treating cancer diseases due to exhibiting a synergistic effect when compared to using individual compounds in the case of using the composition of the present invention and exhibiting an effective anti-cancer effect even with a drug of low concentration.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer diseases, which comprises tamoxifen and methylsulfonylmethane as an active ingredient. BACKGROUND ART [0002]

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer diseases comprising tamoxifen and methylsulfonylmethane as an active ingredient, and more particularly to a pharmaceutical composition for preventing or treating cancer diseases, which comprises an effective anticancer effect by synergistic action by the combined administration of tamoxifen and methylsulfonylmethane ≪ / RTI >

Cancer or neoplasm is a malignant neoplasm that occurs in any part of the body. It is characterized by uncontrolled proliferation of cells and is an important public health problem that is estimated to reach about 6 million people worldwide annually. In most countries, cancer is the second leading cause of death following heart disease. Each cancer spreads from one cell that divides at a particular stage, forming a family of infinitely proliferating cells that appear in the form of tumors.

Breast cancer is one of the leading causes of cancer death among western women and it is expected that it will be a major cause of cancer death among Oriental women in the future. The American Cancer Society estimates that one out of nine women is at risk for breast cancer and about one-quarter of patients with breast cancer have a fatal outcome.

A synthetic, non-steroidal selective estrogen receptor modulator, tamoxifen, has been used effectively for breast cancer care for the past 20 years. Tamoxifen is one of the most widely prescribed antineoplastic agents in the United States and the United Kingdom and is one of the best early hormone treatments available for both premenopausal and postmenopausal women with estrogen receptor positive metastatic disease. In addition, adjuvant therapy studies have shown a substantial reduction in the incidence of prevalent primary breast cancer among women receiving tamoxifen treatment, suggesting that tamoxifen can be used to prevent breast cancer.

Tamoxifen has a tissue-specific estrogenic effect and an antiestrogenic effect. Estrogen, an ovarian hormone, increases the risk of estrogen receptor-mediated breast and endometrial cell division, increasing the risk of breast cancer and endometrial cancer. Cell division is an essential factor in the complex process of human cancer development, as this cell division itself increases the risk of genetic errors, particularly genetic errors such as inactivation of tumor suppressor genes.

Methylsulfonylmethane (MSM) is a dietary sulfur compound that is a single substance with an effective ability to act on tumor cells.

The larger the dose of the drug, the greater the side effect. As a result, the combination therapy is often used in chemotherapy. Combination therapy can reduce the dose of each drug and increase the efficacy. Approach.

Korean Patent Publication No. 10-2013-0051359

The present invention solves the above-mentioned problems, and it is an object of the present invention, which is devised in view of the above-mentioned needs, and an object of the present invention is to provide a cancer agent which effectively inhibits the growth and metastasis of cancer cells by exhibiting a synergistic anticancer effect by the combined administration of tamoxifen and methylsulfonylmethane. And a method of preventing and treating cancer by using the composition.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer diseases, which comprises tamoxifen or a pharmaceutically acceptable salt thereof and methylsulfonylmethane as an active ingredient.

In one embodiment of the present invention the cancer is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, melanoma, pancreatic cancer, colorectal cancer, kidney cancer, leukemia, non- And it is more preferable that the cancer disease is breast cancer, but it is not limited thereto.

In another embodiment of the present invention, the composition ratio of tamoxifen or a pharmaceutically acceptable salt thereof and methylsulfonylmethane is preferably 1: 10000 to 3: 40000, but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition is preferably, but not limited to, inducing apoptosis of cancer cells or inhibiting proliferation or growth of cancer cells.

In another embodiment of the present invention, the pharmaceutical composition preferably inhibits the Jak2 / STAT5b mechanism but is not limited thereto.

The present invention also provides a pharmaceutical composition for suppressing metastasis or invasion of cancer cells containing tamoxifen or a pharmaceutically acceptable salt thereof and methylsulfonylmethane as an active ingredient.

In one embodiment of the present invention, the transition is preferably lung metastasis, but is not limited thereto.

Pharmaceutically acceptable salts possess the biological effectiveness and properties of the compounds of the invention and represent biologically or undesirable salts. In many cases, the compounds of the invention may form acid and / or base salts by the presence of amino and / or carboxyl groups or the like groups.

Pharmaceutically acceptable base addition salts may be formed from inorganic and organic bases.

Examples of salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium and magnesium salts and the like, which are given as examples.

Examples of salts derived from organic bases include primary, secondary and tertiary amines such as alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di (substituted alkyl) amines, tri (substituted alkyl) amines , Tri (substituted alkenyl) amines, cycloalkylamines, di (cycloalkyl) amines, tri (substituted alkyl) amines, (Cycloalkyl) amines, substituted cycloalkylamines, disubstituted cycloalkylamines, triple substituted cycloalkylamines, cycloalkenylamines, di (cycloalkenyl) amines, tri (cycloalkenyl) But are not limited to, alkenylamines, double or triple substituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, Triheterocyclic amine, mixed Amines and triamines wherein two or more of the substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, Aryl, heteroaryl, heterocyclic, and the like), but is not limited thereto. Also included are amines in which two or three substituents together with the amino nitrogen form a heterocyclic or heteroaryl group. Suitable examples of amines include isopropylamine, trimethylamine, diethylamine, tri (isopropyl) amine, tri (n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, Such as caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamine, theobromine, purine, piperazine, piperidine, morpholine, N- ethylpiperidine, As an example.

Other carboxylic acid derivatives, such as carboxylic acid amides including carboxamides, lower alkyl carboxamides. Dialkylcarboxamides, and the like, may be useful in the practice of the present invention.

Pharmaceutically acceptable acid addition salts may be formed from inorganic and organic acids. Examples of salts derived from inorganic acids include hydrochloride, hydrogen bromide, sulfate, nitrate, phosphate and the like. Examples of salts derived from organic acids include salts derived from organic acids such as acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, p-toluenesulfonic acid, salicylic acid, and the like.

According to one embodiment, the pharmaceutical compositions of the present invention may be formulated in admixture with one or more pharmaceutically acceptable carriers. Such agents may be administered by standard routes. In general, the mixture can be administered by topical, transdermal, oral, rectal or parenteral (e.g. intravenous, subcutaneous or intramuscular) routes.

When the compound of the present invention is orally administered, the compound is administered as a solution, powder, tablet, capsule or lozenge. The compounds may be used in admixture with one or more conventional pharmaceutical additives or excipients used in tablets, capsules, lozenges, and other orally administrable forms.

When the compound of the present invention is parenterally administered, it is injected by subcutaneous injection, intravenous injection, intramuscular injection, or injection into the chest. At this time, in order to formulate the preparation for parenteral administration, the herbal extract or its fraction may be mixed with water or a stabilizer or buffer to prepare a solution or suspension, which may be prepared into a unit dosage form of an ampule or vial.

It is to be understood that the compositions of the present invention may include solubilizing agents, inert fillers, diluents, excipients and flavoring agents.

The preferred dose of the pharmaceutical composition for preventing or treating cancer diseases comprising tamoxifen and methylsulfonylmethane of the present invention as the active ingredient varies depending on the condition of the patient, the body weight, the degree of disease, the form of the drug, And may be suitably selected by those skilled in the art. However, an amount of 0.001 to 100 mg / kg is preferably administered once to several times per day. In the overall pharmaceutical composition, tamoxifen or a pharmaceutically acceptable salt thereof and methylsulfonylmethane of the present invention should be contained in an amount of 0.0001 to 10% by weight, preferably 0.001 to 1% by weight.

Hereinafter, the present invention will be described in detail.

Synergistic inhibition of cell proliferation by combination of tamoxifen and methylsulfonylmethane

The present inventors evaluated the proliferation inhibitory effect of human breast adenocarcinoma cell lines according to the combination of methylsulfonylmethane, tamoxifen and their concentrations. The number of cells treated at the greatest stage was compared to the number of untreated control cells. After 24 hours of treatment, cell growth was inhibited by 40% in 25 μM tamoxifen and by 45% in 300 mM methylsulfonylmethane (FIG. 1A). This concentration change is for further experiments. In order to obtain a combined dose showing a synergistic effect, tamoxifen and methylsulfonylmethane in different ratios (1: 10000 to 3: 40000) were used arbitrarily. Analysis of the proliferation inhibition data using Compusyn showed that the combination of tamoxifen and methylsulfonylmethane in a ratio of 3: 40000 showed a synergistic effect with Fa = 0.68 (Table 1). IC _{50 values} obtained by Compusyn Was a combination of 198.619 mM methylsulfonylmethane and 14.9 μM tamoxifen. This combination exhibits a synergistic action with a CI value of 0.52. Thus, the present inventors applied 200 mM methylsulfonylmethane and 15 μM tamoxifen in a further experiment (FIG. 1B)

[Table 1]

Table 1 shows the effects of tamoxifen, methylsulfonylmethane and their combination in the dose-dependent manner to inhibit the growth of MCF-7 breast cancer adenoma cells.

Induction of apoptosis by the combined administration of tamoxifen and methylsulfonylmethane in MCF-7 cells

The proliferation inhibition assay showed that co-administration induced growth inhibition. The next goal of the present inventors was to analyze whether co-administration can induce apoptosis, which is the most desirable characteristic of anticancer drugs. In order to detect and quantify cells that undergo apoptosis, we performed cell flow analysis of FITC-coated fluorescent Annexin-V-FITC cells (Fig. 1c). , And stained with propidium iodide (PI). 10 [mu] M of camptothecin was used as a positive control. The obtained results showed that the stronger apoptosis inducing effect (46%) than the individual administration of tamoxifen (30%) and methylsulfonylmethane alone (38%), ).

Synergistic Jak2 / STAT5b Inhibition by Combined Use of Methylsulfonylmethane and Tamoxifen

Expression levels of proteins related to the Jak2 / STAT mechanism were analyzed by Western blot analysis. As shown in FIG. 2A, the combination administration synergistically inhibits Jak2 expression and phosphorylation as compared to individual administration. STAT5b and its phosphorylation are inhibited by the action of tamoxifen and less inhibited by methylsulfonylmethane. When administered concomitantly, the degree of inhibition is much more noticeable than when administering the individual drugs. In the case of STAT3 and phosphorylated STAT3, the drug combination showed a higher degree of inhibition than the individual administration. Finally, IGF-1Rβ and its phosphorylation form are the strongest proofs to our hypothesis. This was down-regulated by co-administration with steady expression of the housekeeping gene, [beta] -actin. From these results, it can be seen that tamoxifen inhibits Jak2, STAT5b and IGF-1Rβ molecules, while methylsulfonylmethane down regulates STAT3, STAT5b, and Jak2. However, all of these signaling molecules were synergistically inhibited by concomitant administration, even though the concentration of the individual drugs was lower when administered concomitantly.

Inhibition of DNA Binding Activity of STAT5b by Combined Methylsulfonylmethane and Tamoxifen

The phosphorylated STAT3 and STAT5b translocate to the nucleus to perform their transcriptional control functions. Nuclear translocation was confirmed by nuclear extracts isolated from MCF-7 cells pretreated with the drug combination and individual administration, respectively. Western blot analysis of nuclear extracts reduced total and phosphorylated levels of STAT5 and STAT3 (FIG. 2c), as well as IGF-1Rβ and its phosphorylation pattern were further reduced in the combination treatment groups compared to the individual drug-treated groups Respectively. DNA binding activity was analyzed using EMSA analysis, which was confirmed by ChIP analysis (Fig. 2d, 2e). These results indicate that coadministration can play an important role in inhibiting binding activity.

Synergistic inhibition of STAT5b-mediated down-regulation by combination of methylsulfonylmethane and tamoxifen

EMSA data showed that the STAT5-DNA binding activity was further down-regulated in the drug-treated group than in the methylsulfonylmethane alone treatment group. This suggests that the downstream target of the STAT5b molecule is synergistically reduced in the co-administered cells. To confirm this hypothesis, the expression of the STAT5b downstream target was studied at both transcription and translation levels. Figure 3a showed mRNA levels in STAT5b and STAT3 downstream targets. Based on these results, the present inventors confirmed reduction of expression of cyclin D1, VEGF, IGF-1 and IGF-1Rβ in a co-administered sample. The level of loading control 18s RNA remained steady in all samples. Similarly, an assessment of the protein level of the STAT5b downstream target showed a significant inhibition of cyclin D1, VEGF, and VEGF-R2. On the other hand, Bax is a marker for an increase in the level of apoptosis, which is upregulated (Figure 3c)

Synergistic inhibition of migration and invasion by concomitant administration of methylsulfonylmethane and tamoxifen

Inhibition of cell migration was confirmed by in vitro wound healing assay using MCF-7 cells. Plates containing scratch wounds were exposed for 24 hours to the drug combination and each drug, and pictures were taken to identify wound anastomosis areas (Fig. 4a). The wound anastomosis area was quantified by Image-J and the relative migration inhibition Respectively. Results showed that migration inhibition was significantly greater in the drug-treated group than in the individual drug treatment groups (Figure 4b, P <0.05 = *, and P <0.01 = **). Substrate metalloproteinases (MMPs) Plays an important role in cell infiltration through the extracellular matrix. FIG. 4E shows the effect of the drug combination in controlling transcription and translation levels in the expression of MMP2, MMP3, and MMP9. Inhibition of MMP expression inhibits the invasion of cancer cells. Infiltration inhibition was confirmed by matrigel infiltration assay (Figure 4c). Matrigel infiltration analysis showed a relatively high level of inhibition in the combination treatment group than in the individual treatment group of drugs (Fig. 4d). Statistical analysis was performed using t-test (P <0.01 = ** And P < 0.001 = ***)

Inhibitory effect of tamoxifen and methylsulfonylmethane on tumor growth

In vivo tumor suppressive activity of the drug combination was evaluated using breast cancer Balb / c nude mice induced by MCF-7 cells. After formation of tumors that could be touched by the hand, the mice were divided into four separate groups and drug administration started. The diameter of tumor mass was measured every 5 days using a digital caliper. Changes in tumor volume were plotted versus days of drug treatment daily. As shown in FIG. 5C, the present inventors observed a tumor volume inhibitory effect on a statistically significant level. Concomitant administration of the drug inhibited tumor growth relatively more. The toxicity of the drug to the total body weight of the mouse was always checked. Comprehensive data on mouse body weight showed no or slight side effects in mice given methylsulfonylmethane and drug combination without weight change. In tamoxifen treated mice, however, there was a slight change in mouse body weight, indicating a toxic effect (Fig. 5b). At the end of the drug treatment schedule, mice were sacrificed and xenotransplantation was cut for further analysis. Morphological analysis by hematoxylin-eosin (H & E) staining showed a relatively high degree of necrosis in the vehicle-treated group (control group) (Figure 5a)

Down-regulation of the STAT5b / IGF-1Rβ signaling mechanism by combined methylsulfonylmethane and tamoxifen

To understand the molecular mechanism by which drug administration inhibits tumor growth, analysis at the molecular level was performed for xenotransplantation. Drug conjugation has the ability to inhibit phosphorylation and activation of STAT5b, thereby also inhibiting the expression of IGF-1R [beta]. Expression of these molecules was analyzed by immunofluorescence microscopy. These results showed that the combined treatment of tamoxifen, methylsulfonylmethane and drug reduced the expression of STAT5b and IGF-1Rβ in the MCF-7 xenograft model without changes in nuclear levels (FIG. 6a). Jak2 and STAT3 were also analyzed to check for the association of this mechanism in inhibiting tumor growth. The present inventors confirmed that drug administration significantly inhibited the expression and phosphorylation of Jak2, STAT5b, STAT3 and IGF-1Rβ (FIG. 6b)

Inhibition of lung metastasis of cancer cells by combination of tamoxifene and methylsulfonylmethane in in vivo

In vitro analysis of inhibition of migration and invasion showed that drug combination inhibited the expression of MMPs and thus inhibited the migration and invasion of breast cancer cells. Thus, the efficacy of the drug combination therapy for pulmonary metastasis inhibition was analyzed through a transgenic animal model. The lungs were cut after sacrificing orthocraft mice injected with MCF-7 cells. The lungs were inserted into paraffin and analyzed for metastasis sites by hematoxylin-eosin (H & E) staining (Fig. 7a). The relative sites of metastasis were quantified. The high level of metastasis was observed in the control group and the metastatic site was decreased in the drug treatment group. Relative metastasis sites were plotted in terms of percentage of metastasis in the control group (Figure 7b). Statistical analysis was performed using t-test (P <0.01 = ** and P <0.001 = ***) (Fig. 7c). This shows that the combined administration of the drug inhibits VEGF and VEGF-R2 which play an important role in angiogenesis and is important for infiltration MMP2, MMP3, and MMP9, which play a role.

As can be seen from the present invention, it was found that the combination of tamoxifen and methylsulfonylmethane inhibited the spread, metastasis, invasion and growth of cancer cells. The combination of tamoxifen and methylsulfonylmethane exhibits a synergistic effect as compared with the case where the individual compounds are used, so that they exhibit an effective anticancer effect even with a low concentration of the drug, so that they can be applied as a pharmaceutical composition.

FIG. 1 is a graph showing a synergistic effect on inhibition of cell proliferation due to the combined administration of tamoxifene and methylsulfonylmethane. FIG. a) represents the MTT analysis result. b) shows the result of analysis by Compusyn. c) shows the results of quantitative analysis using cell flow analysis.
FIG. 2 is a graph showing a synergistic effect on Jak2 / STAT5b inhibition due to the combined administration of tamoxifen and methylsulfonylmethane. FIG. a) shows Western blot analysis results for cytoplasmic protein levels. b) shows a graph of expression levels of various cytoplasmic proteins. c) shows the Western blot analysis results for the nuclear protein level. d) shows the result of gel shift analysis for the inhibition of DNA binding activity of STAT5b; e) shows the result of ChIP analysis.
FIG. 3 is a graph showing the synergistic effect of the STAT5b-mediated down-regulation target inhibition by the combination administration of tamoxifene and methylsulfonylmethane. a) shows the RT-PCR analysis of the RNA level of the down-regulated target. b) graphs the degree of inhibition of RNT levels by drug treatment. c) shows the Western blot results for cytoplasmic protein levels. d) is a figure showing the results in each experimental group for cytoplasmic protein expression.
FIG. 4 is a graph showing the synergistic effect of immobilization of tamoxifen and methylsulfonylmethane on inhibition of migration and invasion. FIG. a) represents the results of wound healing analysis. b) is a graphical representation of the results of the wound healing assay. c) shows the results of the infiltration analysis. d) is a graphical representation of the infiltration analysis results. e) shows the Western blot results showing the level of substrate metal protein.
FIG. 5 shows that the combination administration of tamoxifene and methylsulfonylmethane inhibited tumor growth. a) shows tumor size and hematoxylin-eosin (H & E) staining results in each experimental and control group. b) is the result of mouse weight analysis in each experimental group and control group. c) is a graphical representation of the tumor size analysis in each experimental and control group.
Figure 6 shows the result of the combined administration of tamoxifen and methylsulfonylmethane to down-regulate the STAT5b / IGF-IRβ signaling mechanism. a) shows immunofluorescence analysis results. b) shows the Western blot analysis result. c) is a graphical representation of the results for tissue protein levels.
Fig. 7 shows the result of the combined administration of tamoxifene and methylsulfonylmethane in Inhibivo inhibiting lung metastasis. a) is the result of analysis of the degree of metastasis in each experimental and control group. b) graphs lung metastasis results in each experimental and control group. c) represents tissue protein analysis results. d) is a graphical representation of tissue protein expression in each experimental and control group.

The present invention will now be described in more detail by way of non-limiting examples. It will be apparent to those skilled in the art that the following examples are illustrative of the present invention and that the scope of the present invention is not limited by the following examples.

Example 1 The antibody and reagent used in the present invention

RPMI-1640 was purchased from Sigma Chemical (St. Louis, MO). Penicillin-streptomycin solution and fetal bovine serum (FBS) were purchased from Hyclone (South of Logan, Utah). 0.05% trypsin-EDTA was purchased from Gibco-BRL (Grand Island, NY). The antibodies to STAT5b, VEGF, VEGF-R2, IGF-1Rβ, MMP2, MMP3, MMP9 and secondary antibodies (goat anti-mouse and rabbit IgG-Horseradish peroxidase) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Jak2 was obtained from Millipore (Bollerica, MA). Phosphorylated Jak2 was purchased from Cell Signaling Technology (Beverly, MA) and phosphorylated STAT5 was purchased from Upstate Biotechnology (Lake Placid, NY). beta -actin was purchased from Sigma Chemical Co. (St. Louis, Mo.). Enhanced chemiluminescence (ECL plus) assay kits were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Restore ™ western blot stripping buffer and NE-PER kit were purchased from Pierce (Rockford, Ill.). RNeasy mini kit and Qiaprep spin miniprep kit were purchased from Qiagen (Hilden, Germany). RT-PCR primers and 18s primers for VEGF, IGF-1, IGF-1Rβ, cyclin D1, MMP2, MMP3, MMP9 and RT-PCR were synthesized by Bioneer (Daejeon, Korea). Electrophoretic Mobility Shift Assay (EMSA) kit, oligonucleotide prove (STAT 5b), Luciferese Assay substrate, reporter lysis buffer were obtained from Promega Corp. (Madison, WI). Paraformaldehyde and mounting solutions for immunohistochemistry (IHC) were obtained from Dae Jung Metals Chemical (Korea) and Life Science (Mukilteo, Washington). Triton X-100 was obtained from Sigma Chmical Co. (St. Louis). Methylsulfonylmethane (MSM) was purchased from Fluka / Sigma Co. (St. Louis, MO). Tamoxifen for in vitro analysis was purchased from Sigma Chemicals (St. Louis, Mo.). 17β-estradiol pellet (0.72 mg, 60 days release) and tamoxifen tablet (0.72 mg, 60 days release) were purchased from Innovative Research of America (Sarasota, FL).

Example 2. Cell culture and treatment

Human breast adenocarcinoma, MCF-7 cell line, was maintained in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / ml penicillin and streptomycin in a 5% CO 2 environment at 37 ° C. The cells were placed in a closed compartment (Nu Aire, Plymouth, MN). At the beginning of each experiment, the cells were resuspended in the medium at a density of 2.5 x ¹⁰ cells / ml. Cells were treated with 25 μM tamoxifen, 300 mM methylsulfonylmethane and / or a combination thereof (tamoxifen 15 μM and methylsulfonylmethane 200 mM).

Example 3. Analysis of cell proliferation inhibitory activity

The cell viability was measured by the presence of a blue-forming (3-D, 5-dimethylthiazol-2-yl) -2,5-diphenyl tetra-zolium bromide (MTT) metabolite from mitochondrial dehidrogenase And analyzed by measuring formazan. Cells were resuspended in media for 1 day before drug treatment at a density of ³ x ¹⁰ cells per well in 96-well culture plates. The liquid medium was replaced with fresh medium containing DMSO as a vehicle. Cells were incubated with various concentrations of tamoxifen, methylsulfonylmethane, and combinations thereof (1: 10,000, 3: 40000). MTT (5 mg / ml) was added to each well and incubated at 37 占 폚 for 4 hours. Formazan products were formed and dissolved by adding 200 μl of dimethicyl sulphoxide (DMSO) to each well and absorbance was measured at 550 nm using an Ultra Multifunctional Microplate Reader (TECAN, Durham, NC). All measurements were repeated 3 times and at least 3 times.

Example 4. Analysis of apoptosis

Fluorescein-conjugated Annexin V-FITC was used to quantitatively determine the percentage of cells that underwent apoptosis. The drug-treated cells were washed and resuspended at a concentration of 1x10 ⁶ cells / ml in binding buffer solution. Cells that undergo apoptosis are stained with annexin V-FITC and propidium iodide. After incubation for 15 min at room temperature in darkness, the percentage of cell death was analyzed using cell flow analysis (Becton-Dickinson FACScan, San Jose, Calif.). 10 [mu] M camptothecin was used as a positive control for analysis.

Example 5. Western blot analysis

The MCF-7 cell line was treated with tamoxifen, methylsulfonylmethane and combinations thereof for a defined period of time. All cells were lysed on ice with radioimmunoprecipitation assay (RIPA) lysis buffer containing phosphatase and protease inhibitors. Cells were disrupted by aspiration through a 23-gauge needle and centrifuged at 15,000 rpm for 10 min at 4 ° C to remove cellular debris. Protein concentration was measured by the Bradford method. Protein equivalent was solubilized on SDS-PAGE and transferred to the nitrocellulose membrane. The blots were blocked with 5% skim milk powder for 1 hour. The membranes were detected as primary antibodies at 4 ° C overnight and then detected as HRP-conjugated secondary antibodies. Analysis was performed using an ECLplus assay kit and a LAS-4000 imaging device (Fujifilm, Japan).

Example 6. RT-PCR

Total RNA was extracted using RNeasy Mini Kit (Qiagen) and quantitated by spectroscopy at 260 nm. RT-PCR analysis was performed on IGF-1, IGF-1R, cyclin D1, VEGF and 18s RNA. The cDNA was synthesized from total RNA by reverse transcription and treated with the first strand cDNA synthesis kit (Daejeon, Korea) for 1 hour at 42 ° C and 15 minutes at 80 ° C. PCR was performed using cDNA. The PCR conditions were denaturation at 30-95 ° C for 1 min at 94-95 ° C, annealing at 30-55 ° C for 1 min, and extension at 72 ° C for 30 min. The PCR products were analyzed by 1% agarose gel stained with ethidium bromide.

Example 7. Electrophoretic mobility shift assay (EMSA)

The DNA binding activity of STAT3 and STAT5b was detected using EMSA, where labeled double-stranded DNA is used as a DNA probe, which binds to activated STAT5b and STAT3 proteins in nuclear extracts. Nuclear protein extracts were prepared by Nuclear Extract Kit (Panomics, AY2002). EMSA experiments were performed by culturing progeny treated with nuclear protein and untreated biotin-labeled transcription factor (TF-STAT3). Proteins were digested with non-denaturing 6% PAGE-gel (Bio-Rad, Korea). Proteins in the gel were transferred to nylon membranes and then detected using streptavidin-HRP and a chemiluminescent substrate.

Example 8. Wound healing analysis

MCF-7 cells were cultured in 6-well plates at a concentration of 1 x ¹⁰ cells / well in RPMI-1640 medium and incubated in a wet chamber for 24 hours. After becoming a confluent monolayer, the cell layer was scratched with a pipette tip. Thereafter, it was washed with PBS to remove cell debris. Cells were treated with the required concentrations of drug (tamoxifen, methylsulfonylmethane, and combinations thereof). Control cells remained untreated. The corners of the wound were photographed using a microscope at different time intervals. The relative area of the wound suture was measured using Image J (Schneider CA, 2012).

Example 9. Matrigel Invasion Assay (Matrigel Invasion Assay)

Transwell invasion assays were performed with the aid of a Martrigel pre-coating, which immediately enabled the use of an invasion chamber (BD Biocoat, MA). The cells were made up of a suspension of 5x10 ⁴ and added to the insert. The medium containing the drug was transferred to a donor plate and the insert was then placed thereon. After incubation in a humidified environment at 37 [deg.] C for 24 hours, cells infiltrated into the surface apex were degraded into crystal violet. Cells on the upper surface were removed by scraping and infiltrated cells were observed under a microscope. Four distinct locations were focused and the number of cells counted.

Example 10. Tumorigenicity test

All animal experiments were approved by the Animal Use Protection Committee and conducted in accordance with the Commission's guidelines. For the establishment of estrogen receptor-positive MCF-7 xenografts, mouse ovaries were harvested and incubated with 17 [beta] -estradiol pellet (0.72 mg, 60 days release; Innovative Research of America, Sarasota, FL) And then transplanted under the neck. Xenotransplantation was initiated by injecting MCF-7 cells (1x107) into the subcutaneous tissue on the right hindlim lateral side. When the tumors reached 6-8 mm in diameter, the mice were randomly divided into 4 groups (control, tamoxifen, methylsulfonylmethane, combinations thereof) and 6 mice per group. In the methylsulfonylmethane administration group, 100 μl of 3% methylsulfonylmethane with 3 times distilled water was administered into the stomach. In the tamoxifen treatment group, tamoxifen pellets (0.72 mg, 60%, Innovative Research of America) were planted under the neck and methylsulfonylmethane was injected into the stomach. The injection was repeated once a day. Tumor growth was measured periodically with a digital caliper. The diameter of the tumor grew to 2 cm, and the animals were sacrificed 30 days after treatment. In the present invention, no experimental mice were observed to die from tumors. All possible breast cancer specimens obtained from human breast cancer xenografted mice were included in the present invention. The mice were anesthetized and the tumor was removed. Tumors were fixed with 4% formaldehyde, then paraffin was inserted and then divided into 5 mm. The sections were stained with hematoxylin and eosin (H &amp; E).

Example 11. Transition animal model experiment

The orthotopic transgenic animal model was derived by tail vein injection of MCF-7 cells into 5-week-old BALB / c nude mice (Orient Bio, Seongnam City, Korea). To induce tumors in the MCF-7 model, the mice were ovariectomized and 17β-estradiol pellets were implanted under the neck. Mice were randomly divided into 4 groups and treated with the drug as a xenograft model. Tamoxifen pellets were subcutaneously injected into the neck along with 17 [beta] -estradiol. The control group was treated with vehicle and the methylsulfonylmethane-treated group was injected with 100 μl of 300 mM methylsulfonylmethane in the stomach. The combination administration group was treated with 300 mM methylsulfonylmethane and tamoxifen pellet. Treatment was continued for 30 days, and the mice were sacrificed and then the lungs were removed. They were fixed with 10% formalin and then paraffin was inserted. Tissue analysis was performed by hematoxylin-eosin (H & E) staining. After counting the number of metastatic tumors, the relative inhibition of metastasis was confirmed.

Example 12. Hematoxylin-eosin (H & E) staining assay

The xenotransplantation fixed with formalin and the lungs embedded in paraffin were successively cut into 5 mu m thick sections. Hematoxylin-eosin (H & E) staining was then performed with paraffin removal, rehydration with xylene, washing with an ethanol gradient (100%, 95%, 90%, 80%, 70% (Sigma-Aldrich) The slides were observed under a microscope and photographed.

Example 13. Immunofluorescence (IF) analysis

After formalin-fixed, paraffin-embedded breast carcinoma xenografts were removed by paraffin removal with 100% xylenes, rehydrated by decreasing ethanol concentration, and then passed through 0.1% Triton X-100 and fixed with 10% NGS (Normal Goat Serum, Normal goat serum in PBS). The cells were then incubated with STAT5b, IGF-1Rβ and then incubated with Alexa Fluor 488 (rabbit) and Alexa Fluor 594 (mouse) (Invitrogen) as secondary antibodies. For detection of nuclei, tissue sections were incubated with DAPI for 1 min and washed with PBS. The samples were observed under a fluorescence microscope and photographed.

EXAMPLE 14 Synergistic Quantitative and Statistical Analysis

The synergistic effects induced by the combined administration of the drugs were analyzed using Compusyn software. Combined index (CI) values were calculated based on the Chou and Talalay method. The CI calculation value can be interpreted as an additive effect when CI > 1 and a synergy when CI < 1. All experiments were repeated 3 times, and the results were expressed as mean ± standard deviation. Statistical analysis was performed by Student's t-test of the SAS program.

Claims (10)

A pharmaceutical composition for preventing or treating cancer diseases, which comprises tamoxifen or a pharmaceutically acceptable salt thereof and methylsulfonylmethane as an active ingredient. The cancer according to claim 1, wherein the cancer is cancer selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, melanoma, pancreatic cancer, colorectal cancer, kidney cancer, leukemia, non- Or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 1, wherein the composition ratio of tamoxifen or a pharmaceutically acceptable salt thereof and methylsulfonylmethane is 1: 10000 to 3: 40000. The pharmaceutical composition according to claim 2, wherein the cancer disease is breast cancer. The pharmaceutical composition for preventing or treating cancer diseases according to any one of claims 1 to 4, wherein the pharmaceutical composition inhibits proliferation of cancer cells. The pharmaceutical composition for preventing or treating cancer diseases according to any one of claims 1 to 4, wherein the pharmaceutical composition induces apoptosis of cancer cells. The pharmaceutical composition for preventing or treating cancer diseases according to any one of claims 1 to 4, wherein the pharmaceutical composition inhibits the growth of cancer cells. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition inhibits the Jak2 / STAT5b mechanism.  A pharmaceutical composition for suppressing metastasis or invasion of cancer cells containing tamoxifen or a pharmaceutically acceptable salt thereof and methylsulfonylmethane as an active ingredient. 10. The pharmaceutical composition according to claim 9, wherein the metastasis is metastasis or invasion inhibition.

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