Human breast cancer MDA-mb-231 cell proliferation inhibitor

Abstract

[Problem] The present invention provides an inhibitor such as an MDA-MB-231 cell proliferation inhibitor containing DHMBA as an active ingredient, the inhibitor affecting the proliferation, metastatic activity, and bone microenvironment of MDA-MB-231 cells in vitro, and making it possible to provide a useful therapeutic strategy for human breast cancer. [Solution] The present invention is characterized by having the effect of inhibiting the proliferation of human breast cancer MDA-MB-231 cells containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient.

Description

Growth inhibitor for human breast cancer MDA-MB-231 cells

[0001] The present invention relates to a growth inhibitor for metastatic human breast cancer cells.

[0002] Breast cancer is the most serious malignant tumor and the leading cause of cancer-related deaths in women. Breast cancer has a high tendency to metastasize to bone, which promotes bone turnover and leads to bone damage.

[0003] Approximately 70% to 80% of patients with advanced breast cancer suffer from bone metastasis. Bone metastasis results from interactions between metastatic tumor cells and cells of the bone microenvironment. The spine is the most common site of bone metastasis in breast cancer patients. Tumor cell invasion into bone tissue can cause bone loss, pain, pathological fractures, hypercalcemia, and spinal cord compression, significantly reducing patients' quality of life.

[0004] Breast cancer can be classified into four molecular subtypes based on the expression of hormone receptors (estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2). Triple-negative breast cancer (TNBC) lacks expression of all three hormone receptors and cannot be effectively targeted with hormonal therapy. Notably, TNBC is highly heterogeneous, with wide variation in molecular characteristics and response to treatment. TNBC is highly aggressive and metastatic, with a rapid risk of recurrence and a poor prognosis, including low survival rates.

[0005] Bone loss induced by bone metastasis of breast cancer cells is thought to be due to both activated osteoclastic bone resorption and suppressed osteoblastic bone formation. Bisphosphonates and anti-RANKL antibodies (denosumab) are the current standard treatments for patients with bone metastases.

[0006] Furthermore, chemotherapy drugs have been shown to be more effective against breast cancer. However, these treatments are associated with severe side effects. To date, the most recommended drug is the anthracycline doxorubicin, which is more effective against breast cancer but is associated with cardiotoxicity. Due to the limitations of chemotherapeutic drugs, more effective compounds and hormones with less or no adverse effects on normal tissues are needed as a preferred treatment option.

[0007] A novel phenolic antioxidant, 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), was discovered in the Pacific oyster Crassostrea gigas. DHMBA possesses dual properties, acting as a radical scavenger and preventing oxidative stress in various cells. In particular, DHMBA is an excellent peroxyl radical scavenger, approximately 15 times and four orders of magnitude more potent than Trolox in lipid and aqueous media, respectively. This compound reacts more rapidly with HOO(-) than other known antioxidants, such as resveratrol and ascorbic acid. As an antioxidant, DHMBA may be an important dietary factor in regulating cellular function. Recently, DHMBA has been shown to inhibit the growth of metastatic prostate cancer cells by targeting various signaling pathways. The present inventors have already filed and presented a patent application for a novel strategy for the treatment of prostate cancer using DHMBA. Thus, elucidating the pharmacological actions of DHMBA may be important for the prevention and treatment of various diseases.

[0008] Patent Publication No. 2012-153852

[0009] In this invention, we investigated whether DHMBA has an inhibitory effect on human bone metastatic breast cancer MDA-MB-231 cells in vitro. We confirmed that incubation with DHMBA affects MDA-MB-231 cell proliferation, metastatic activity, and the bone microenvironment in vitro. Mechanistically, DHMBA inhibits several molecules related to EGF signaling and blocks TNF-α signaling in MDA-MB-231 cells. This demonstrates that this invention can provide a useful therapeutic strategy for human breast cancer. Therefore, the present invention aims to provide inhibitors, such as MDA-MB-231 cell proliferation inhibitors, containing DHMBA as an active ingredient.

[0010] The present invention is characterized by: having an inhibitory effect on the proliferation of human breast cancer MDA-MB-231 cells, containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient; or characterized by: the inhibitory effect on the proliferation of human breast cancer MDA-MB-231 cells is exerted by blocking different cell signaling pathways; or characterized by: the inhibitory effect on the proliferation of human breast cancer MDA-MB-231 cells is exerted by blocking different cell signaling pathways and by regulating protein levels involved in different cell signaling pathways in MDA-MB-231 cells; or characterized by: having an effect of promoting apoptotic cell death in human breast cancer MDA-MB-231 cells, containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient; or characterized by: having an effect of suppressing the metastatic activity of human breast cancer MDA-MB-231 cells, containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient; or The present invention is characterized by: a composition comprising 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, which has the effect of inhibiting the reduction of osteoblastic MC3T3-E1 cells co-cultured with human breast cancer cells MDA-MB-231, and the effect of inhibiting the promotion of apoptosis of the osteoblastic MC3T3-E1 cells; or a composition comprising 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, which has the effect of inhibiting the reduction of macrophage RAW264.7 cells co-cultured with human breast cancer cells MDA-MB-231, and the effect of inhibiting the promotion of apoptosis of the macrophage RAW264.7 cells; or a composition comprising 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, which has the effect of inhibiting TNF-α signaling in human breast cancer MDA-MB-231 cells and reducing TNF-α production, thereby inhibiting TNF-α signaling in human breast cancer MDA-MB-231 cells and reducing TNF-α production.

[0011] According to the present invention, the inhibitory effect of DHMBA on human bone metastatic breast cancer MDA-MB-231 cells was examined in vitro, and it was confirmed that incubation with DHMBA affected the proliferation, metastatic activity, and bone microenvironment of MDA-MB-231 cells in vitro. Mechanistically, DHMBA inhibited several molecules related to EGF signaling and blocked TNF-α signaling in MDA-MB-231 cells, providing a useful therapeutic strategy for human breast cancer. In other words, the present invention has the excellent effect of providing inhibitors, such as inhibitors of MDA-MB-231 cell proliferation, containing DHMBA as an active ingredient.

[0012] The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits the growth of human breast cancer MDA-MB-231 cells in vitro. (A) Effect on colony formation. MDA-MB-231 cells (1x10 per well of a 6-well plate) 3 (B) Photographs of plates stained with crystal violet are shown. (C) Colony counts are shown. Data are presented as mean ± SD of six wells using different cell preparations. (D) Effect on cell proliferation. Cells (1x10 per well in 24-well plates) were cultured for 9 days in DMEM containing 10% FBS and 1% P / S in the presence of vehicle (final concentration 1% ethanol) or DHMBA (1 or 10 μM). (E) Photographs of plates stained with crystal violet are shown. (F) Colony counts are shown. Data are presented as mean ± SD of six wells using different cell preparations. (F) Effect on cell proliferation. Cells (1x10 per well in 24-well plates) were cultured for 9 days in DMEM containing 10% FBS and 1% P / S in the presence of vehicle (final concentration 1% ethanol) or DHMBA (1 or 10 μM). 5 (1x10 cells / mL) were cultured in the presence of vehicle (1% ethanol as a final concentration) or DHMBA (10 μM) for 1, 2, 3, 4, and 5 days. The number of cells attached to the dish was counted. (D) Effect of increasing DHMBA concentrations. Cells (1x10 cells / mL) were cultured in DMEM containing DHMBA (0.1, 1, 10, 100, and 1000 μM) for 1, 2, 3, 4, and 5 days. The number of cells attached to the dish was counted. (E) Effect of increasing DHMBA concentrations. Cells (1x10 cells / mL) were cultured in DMEM containing DHMBA (0.1, 1, 10, 100, and 1000 μM) for 1, 2, 3, 4, and 5 days. The number of cells attached to the dish was counted. (F) Effect of increasing DHMBA concentrations. 5 (1x10 cells / mL) were cultured for 3 days. (E) Effect of epidermal growth factor (EGF). Cells (1x10 cells / mL) were cultured for 3 days. 5Cells (1x10 cells / mL) were cultured in DMEM containing EGF (50, 100, or 200 ng / mL) without FBS for 3 days in the presence of vehicle (1% ethanol) or DHMBA (10 μM), and the number of adherent cells was counted. Data are shown as the mean ± SD obtained from 8 wells of duplicate plates using different cell preparations. *: p<0.001 vs. the control group (gray bar). 1-way ANOVA, Tukey-Kramer post-test. Inhibitory effect of DHMBA on the proliferation of human breast cancer MDA-MB-231 cells in the presence of cell cycle inhibitors or inhibitors of intracellular signaling factors. (A) and (B) Cells (1x10 cells / well) 5 (C) and (D) Cells (1x10 cells / mL) were cultured for 3 days in the presence of vehicle (1% ethanol as a final concentration) or DHMBA (10 μM) with or without cell cycle inhibitors including roscovitine (10 or 100 nM), butyrate (10 or 100 μM), or sulforaphane (1 or 10 nM). 5 Cells (1x10 cells / mL) were cultured for 3 days in the presence of vehicle (1% ethanol) or DHMBA (10 μM), or in the presence or absence of intracellular signaling inhibitors, including wortmannin (10 or 100 nM), PD98059 (10 or 100 μM), or staurosporine (1 or 10 nM). After culture, the number of cells attached to the dish was counted. Data are shown as mean ± SD obtained from 8 wells of duplicate plates using different cell preparations. *: p<0.001 vs. the control group (gray bar). One-way ANOVA, Tukey-Kramer post-test. DHMBA regulates the levels of several proteins related to the proliferation of human breast cancer MDA-MB-231 cells in vitro. Cells (1x10 cells / 100 mm dish) were cultured for 3 days in the presence or absence of vehicle (1% ethanol) or DHMBA (10 μM), or in the presence or absence of intracellular signaling inhibitors, including wortmannin (10 or 100 nM), PD98059 (10 or 100 μM), or staurosporine (1 or 10 nM). 6Cells (10 mL / 10 mL medium) were cultured in DMEM containing 10% FBS and 1% P / S in the presence of vehicle (1% ethanol) or DHMBA (10 μM) for 3 days. After incubation, cells were removed from the dishes using a cell scraper in cell lysis buffer containing protease inhibitors. 40 micrograms of supernatant protein per lane was separated by SDS-PAGE (12%), transferred to a nylon membrane, and subjected to Western blotting using antibodies against various proteins. (A) Representative data are shown. (B) Bands are shown as fold increases relative to the control. Data are shown as means ± SD of values ​​obtained from four dishes using different cell preparations. *: p<0.001 vs. the control group. One-way ANOVA with Tukey-Kramer post-test. DHMBA promotes the death of MDA-MB-231 human breast cancer cells in vitro. Cells (1x10 cells / well in a 24-well plate) were cultured in vitro. 5 (100mm dish) 1x10 cells / mL were cultured in DMEM containing 10% FBS and 1% P / S for 3 days after reaching subconfluence and then further cultured in the presence of vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 μM) for 24 (A) or 48 (B) hours, respectively. (C) Subconfluent cells were further cultured in the presence of vehicle (1% ethanol) or DHMBA (1 μM or 10 μM) with or without caspase-3 inhibitor (10 μM) for 48 hours. The number of cells attached to the dish was counted. Data are shown as the mean ± SD from eight wells in duplicate plates using different cell preparations. (D) To measure the levels of caspase-3 or cleaved caspase-3, cells (1x10 cells / 100mm dish) were plated. 6Cells (10 ml / 10 ml medium) were cultured in DMEM containing 10% FBS and 1% P / S for 3 days in the presence of either vehicle (1% ethanol) or DHMBA (10 μM). After culture, cell lysates were obtained, and 40 micrograms of supernatant protein per lane was separated by SDS-PAGE (12%), transferred to a PVDF membrane, and subjected to Western blotting using antibodies against various proteins. Representative data are shown. Bands are expressed as fold increases relative to the control. Data are presented as the mean ± SD of values ​​obtained from four dishes using different cell preparations. *: p<0.01 vs. the control group (gray bar). One-way ANOVA with Tukey-Kramer post-test. DHMBA inhibits migration of human breast cancer MDA-MB-231 cells. An in vitro scratch assay was used to analyze cell migration. MDA-MB-231 cells (2 x 10 cells per well in a 24-well plate) were cultured. 5 Cells (1000 cells / mL) were cultured in DMEM containing 10% FBS and 1% P / S for 48 hours. Upon reaching confluence, a linear wound was made in the center of the cell monolayer. Cells were then cultured for 24 or 48 hours in the above medium supplemented with vehicle (1% ethanol) or DHMBA (0.1, 1, 10, 50, or 100 μM). After incubation, fixed cells were stained with crystal violet. To assess cell migration, images were taken of the migration distance, and representative photographs are shown (Figures A and B). The migration distance was expressed as a relative value (% of the cell-free area) between the start of incubation (control) and 24 or 48 hours after incubation (Figures C and D). Data are shown as the mean ± SD of values ​​obtained from a total of eight wells in two plates using different cell preparations. *: p<0.001 vs. control group (before incubation; white bar). #: p<0.001 vs. DHMBA-free group (gray bar). One-way ANOVA with Tukey-Kramer post-test. DHMBA inhibits the adhesion of human breast cancer MDA-MB-231 cells in vitro. Cancer cell adhesion was assessed using Matrigel. Cancer cells (10 per well in a 24-well plate) were cultured. 5Cells (1000 cells / mL) were suspended in DMEM containing 10% FBS and 1% P / S in the presence of vehicle (1% ethanol) or DHMBA (0.1, 1, 10, or 100 μM). After 30 minutes of incubation at 37°C, nonadherent cells were removed by washing with PBS. Adherent cells were fixed and stained with crystal violet. To assess cell adhesion, one field per well was photographed and measured using Image J2 software. The number of adherent cells was counted under a light microscope (40x magnification) and averaged from five randomly selected fields. Figure A: Photographs taken using a crystal stick for adherent cells. Figure B: Results are shown as the mean ± SD of values ​​obtained from a total of six wells using two plates with different cell preparations. *: p<0.001 vs. cells incubated without DHMBA (white bar). 1-way ANOVA, Tukey-Kramer post-test. The in vitro effect of DHMBA on the proliferation and death of osteoblastic MC3T3-E1 cells co-cultured with human breast cancer MDA-MB-231 cells. The crosstalk between MDA-MB-231 and MC3T3-E1 cells was investigated using a transwell chamber. (A): To investigate the effect of co-culture with MDA-MB-231 cells on the proliferation of MC3T3-E1 cells, MC3T3-E1 cells (10 per well of a 24-well plate) were cultured in the lower chamber in the presence of DHMBA (0, 1, 10 μM). 5 The upper chamber was filled with 500 μL (5 x 10 cells / ml) of MDA-MB-231 cells. 4 After 3 days of culture, MC3T3-E1 cells attached to the dish were counted. (B): To examine the effect of conditioned medium obtained by culturing with MDA-MB-231 cells, MC3T3-E1 cells were cultured for 3 days in the presence of either Bay 11-7082 (10 nM) or DHMBA (10 μM) in conditioned medium (0.25 mL or 0.5 mL per 1 mL of total medium in the well) or conditioned medium (0.5 mL per well). After culturing, MC3T3-E1 cells attached to the dish were counted. In other experiments, to examine the effect of co-culture with MDA-MB-231 cells (C) or conditioned medium (D) on MC3T3-E1 cell death, cells (10 per well of a 24-well plate) were cultured. 5 (C): After the culture, 500 μL of MDA-MB-231 cells (5 x 10 cells / mL) were added to the upper compartment of the transwell chamber. 4(Cells / ml) were added to the wells and cultured for an additional 2 days. After incubation, the attached MC3T3-E1 cells were counted. (D): To examine the effect of conditioned medium obtained from culture with MDA-MB-231 cells on cell death, MC3T3-E1 cells were cultured for 3 days until they reached subconfluence. Then, the conditioned medium was replaced with 0.25 mL or 0.5 mL per mL of total medium in the well containing either Bay 11-7082 (10 nM) or DHMBA (10 μM) and cultured for an additional 2 days. After incubation, the attached MC3T3-E1 cells were counted. Results are shown as the mean ± SD of values ​​obtained from a total of 8 wells on two plates using different cell preparations. *: p<0.001 vs. control (light gray bar). #: p<0.001 vs. the Bay11-7082-free group (black bars with 0.5 mL of conditioned medium) or the DHMBA-free group (dark gray bars in Figures A and C). 1-way ANOVA, Tukey-Kramer post-test. In vitro effects of DHMBA on the proliferation and death of macrophages, RAW264.7 cells, co-cultured with human breast cancer cells, MDA-MB-231. The effects of crosstalk between MDA-MB-231 and RAW264.7 cells were investigated using a transwell chamber. (A): To examine the effect of co-culture with MDA-MB-231 cells on RAW264.7 cell proliferation, RAW264.7 cells (10 cells per well of a 24-well plate) were placed in the lower chamber in the presence of DHMBA (0, 1, or 10 μM). 5 The upper chamber was filled with 500 μL (5 x 10 cells / ml) of MDA-MB-231 cells. 4After 3 days of culture, RAW264.7 cells were counted. (B): To examine the effect of conditioned medium obtained by culturing with MDA-MB-231 cells, RAW264.7 cells were cultured for 3 days in the presence of either Bay 11-7082 (10 nM) or DHMBA (10 μM) in conditioned medium (0.25 mL or 0.5 mL per mL total medium in the well) or conditioned medium (0.5 mL per well). After culturing, RAW264.7 cells were counted. In other experiments, to examine the effect of co-culture with MDA-MB-231 cells (C) or conditioned medium (D) on RAW264.7 cell death, cells (10 per well of a 24-well plate) were cultured. 5 (5 x 10 cells / mL) were plated in the presence of DHMBA. After reaching subconfluence, the cells were cultured for 3 days. (C): After culture, 500 μL (5 x 10 cells / mL) of MDA-MB-231 cells were added to the upper compartment of the transwell chamber. 4 (Cells / mL medium) were added to the wells and cultured for an additional 2 days. After incubation, the attached RAW264.7 cells were counted. (D): To examine the effect of conditioned medium obtained from culture with MDA-MB-231 cells on cell death, RAW264.7 cells were cultured for 3 days and then replaced with conditioned medium (0.25 mL or 0.5 mL per mL of medium in the well) containing or without Bay 11-7082 (10 nM) or DHMBA (10 μM) and cultured for an additional 2 days. After incubation, the attached RAW264.7 cells were counted. Results are shown as the mean ± SD of values ​​obtained from a total of 8 wells on two plates using different cell preparations. *: p<0.001 vs. control (light gray bar). #: p<0.001 vs. the group without Bay11-7082 (black bars with 0.5 mL of conditioned medium) or the group without DHMBA (dark gray bars in panels A or C). 1-way ANOVA, Tukey-Kramer post-test. Incubation with DHMBA suppresses TNF-α production in human breast cancer MDA-MB-231 cells. MDA-MB-231 cells (1x10 per well) 5Cells (0.1, 1, 10, 100, 250 μM) were cultured in DMEM containing 10% FBS and 1% P / S in 24-well plates for 3 days until subconfluence was reached. (A) After culture, the medium was collected for TNF-α assay, and the TNF-α concentration in the medium was analyzed using a human TNF-α ELISA kit according to the manufacturer's instructions. TNF-α production was expressed as picograms (pg) secreted per mL of culture medium. Results are shown as the mean ± SD of values ​​obtained from eight wells in two plates using different cell preparations. *: p<0.001 vs. control (white bar). One-way ANOVA with Tukey-Kramer post-test. Hypothesis: Mechanism by which DHMBA suppresses human breast cancer MDA-MB-231 cell proliferation, metastatic activity, and the bone microenvironment. DHMBA suppresses proliferation and metastatic activity in breast cancer cells by regulating various signaling pathways, reducing the levels of Ras / PI3K / Akt / MAPK and mTOR, and increasing the levels of Rb, p53, and p21. In addition, DHMBA promotes cell death associated with caspase 3 activation, leading to cell growth inhibition. Furthermore, TNF-α produced by MDA-MB-231 cells suppresses osteoblasts and macrophages, which are involved in bone formation in the bone microenvironment, leading to impaired bone formation. This process is protected by DHMBA. Therefore, DHMBA may have a potential inhibitory effect on human breast cancer MDA-MB-231 cells metastasizing to bone.

[0013] The inventors of the present invention have conducted experiments on the present invention, the details of which are described below.

[0014] Materials and Methods: Reagents: Dulbecco's modified Eagle's medium (DMEM) was purchased from Mediatech (Herndon, VA, USA). Antibiotics (100 units / mL penicillin and 100 μg / mL streptomycin; P / S) were purchased from Gibco Life Technologies Corporation (Grand Island, NY, USA). α-Minimum Essential Medium (α-MEM) was purchased from Invitrogen Corp. (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from Omega Scientific Inc. Roscovitine, sulforaphane, butyrate, and tumor necrosis factor α (TNF-α) were purchased from R&D Systems (Minneapolis, MN, USA). PD98059, Staurosporin, Wortmannin, Bay 11-7089, crystal violet, human epidermal growth factor (EGF), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted.

[0015] 3,5-Dihydroxy-4-methoxybenzyl alcohol (DHMBA) is an amphiphilic phenolic compound originally isolated from the Pacific oyster (Crassostrea gigas) and recognized for its antioxidant properties. The chemical structure of DHMBA is shown in the specifications of the inventor's previously obtained patents.

[0016] In the present invention, 100% pure DHMBA was used, obtained from Watanabe Oyster Laboratory Co., Ltd. (Hachioji, Tokyo). The DHMBA was dissolved in 100% ethanol and stored at −20° C. until use.

[0017] (Human breast cancer cells) MDA-MB-231 human breast cancer cells were obtained from the American Type Culture Collection (ATCC CRL-1435™, ATCC; Rockville, MD, USA). MDA-MB-231 cells lack receptors for estrogen, progesterone, and human epidermal growth factor receptor 2, which are found in triple-negative breast cancers. These cells express high levels of epidermal growth factor receptor (EGFR), and activation of this receptor and its downstream signaling events promotes the migration, proliferation, invasion, and progression of the malignant phenotype of these cells. MDA-MB-231 cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

[0018] Mouse osteoblastic MC3T3-E1 cells were obtained from the American Type Culture Collection (HTB-77™, ATCC; Rockville, MD, USA). MC3T3-E1 cells were cultured on plastic dishes in α-MEM containing 10% FBS and 1% P / S at 37°C in a CO2 incubator, as previously described.

[0019] (Macrophage RAW264.7 Cells) Mouse RAW264.7 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). RAW264.7 cells are a monocyte / macrophage-like cell line derived from an Abelson leukemia virus-transformed cell line derived from BALB / c mice. RAW264.7 cells were cultured in CO 2 The cells were cultured on plastic dishes in α-MEM containing 10% FBS and 1% P / S in an incubator at 37°C.

[0020] (Colony formation assay) MDA-MB-231 cells (1x10 3 cells / 2mL / well, 6-well plate) 3Cells were seeded into 6-well dishes at a density of 1 / well and cultured in DMEM containing 10% FBS and 1% P / S at 5% CO2 and 37°C for 9 days until visible clones formed on the plate. The resulting colonies were washed three times with phosphate-buffered saline (PBS) (2 mL), fixed with methanol (0.5 mL per well) at room temperature for 20 minutes, and then washed three times with PBS. The colonies were then stained with 0.1% crystal violet (1 mL) at room temperature for 30 minutes. The stained cells were washed four times with PBS (2 mL). The plates were air-dried at room temperature for 2 hours. More than 50 colonies were counted under a microscope (Nikon TMS, Tokyo, Japan).

[0021] (Cell proliferation and cell death assay) MDA-MB-231 cells (1x10 per well in a 24-well plate) 5 / mL) were cultured in DMEM containing 10% FBS and 1% P / S for 1, 2, 3, 4, and 5 days in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 μM). In separate experiments, cells (1x10 per well of a 24-well plate) were cultured in DMEM containing 10% FBS and 1% P / S for 1, 2, 3, 4, and 5 days in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 μM). 5 In a separate experiment, cells (1x10 per well in a 24-well plate) were cultured in DMEM containing EGF (50, 100, or 200 ng / mL) without FBS in the presence of vehicle (final concentration 1% ethanol) or DHMBA (10 μM) for 3 days. 5 Cells (10 μM / mL) were grown to subconfluence for 3 days in DMEM containing 10% FBS and 1% P / S in the presence of a vehicle (final concentration of 1% ethanol) containing effective concentrations of roscovitine (10 or 100 nM), butyrate (10 or 100 μM), sulforaphane (1 or 10 nM), wortmannin (10 or 100 nM), PD98059 (10 or 100 μM), or staurosporin (1 or 10 nM), with or without DHMBA (10 μM). After incubation, cells were detached from each well and counted as described below.

[0022] To measure the effect of DHMBA on cell death, MDA-MB-231 cells (1x10 per well) were cultured in 100 wells of 1000 MDA-MB-231 cells. 5 Cells (1x10 per well) were cultured in DMEM containing 10% FBS and 1% P / S for 3 days at subconfluence in the presence of vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 μM), followed by an additional 24 or 48 hours in the presence of vehicle (PBS or 1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 μM). In additional experiments, cells (1x10 per well) were cultured in DMEM containing 10% FBS and 1% P / S for 3 days at subconfluence in the presence of vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 μM). 5 / mL) were cultured in 24-well plates in DMEM (containing 10% FBS and 1% P / S) for 3 days after reaching subconfluence, and then cultured for an additional 48 hours in the presence of vehicle (final concentration of 1% ethanol) or DHMBA (1 μM or 10 μM) with or without caspase-3 inhibitor (10 μM).

[0023] After culturing, Ca 2+ / Mg 2+ Cells were detached from each well by adding 0.1 mL of a sterile solution of 0.05% trypsin and EDTA in PBS (Thermo Fisher Scientific, Waltham, MA, USA) without HCl and incubating at 37°C for 2 minutes. Then, 0.9 mL of DMEM containing 10% FBS was added. To measure viable cell counts, 0.1 mL of medium containing suspended cells was mixed with 0.1 mL of 0.5% trypan blue staining solution. Viable cells were counted using a microscope (10x magnification, Olympus MTV-3) and a hemocytometer (Sigma-Aldrich, St. Louis, MO) using a cell counter (Rhein Seiki H-102P, Tokyo, Japan). Duplicate counts were averaged for each dish. Cell numbers were expressed as the number per well.

[0024] (Cell migration test) To analyze cell migration in vitro, an in vitro scratch assay was used. MDA-MB-231 cells (2 x 10 cells per well in a 24-well plate) were cultured. 5Cells (100 cells / mL) were cultured in DMEM containing 10% FBS and 1% P / S for 48 hours. At confluence, a linear scratch was made in the center of the cell monolayer using a sterile 200 μL pipette tip. The wells were then washed twice with phosphate-buffered saline (PBS, 1 mL) to remove dead cells and cultured for 24 or 48 hours in the presence of vehicle (1% ethanol to a final concentration) or DHMBA (0.1, 1, 10, 50, or 100 μM). The cells were fixed in ice-cold 70% ethanol (0.5 mL / well) for 60 minutes at 4°C, stained with crystal violet (0.5 mL, 0.5% in 20% methanol) for 30 minutes, and then washed three times with PBS (1 mL). After drying overnight, migration distances were imaged. To assess cell migration, one field per well was photographed at baseline and after 24 or 48 hours of culture, and the distance between the cell-free zones between the two fronts was measured using ImageJ software. The migration distance was expressed as a relative value (% of the control) of the cell-free zone distance at baseline, 24 hours, and 48 hours of culture.

[0025] The effect of DHMBA on cancer cell adhesion was measured using Matrigel. Briefly, 200 μL of Matrigel matrix (diluted 1:1 with sterile PBS, Corning) was coated onto a 24-well plate and incubated at 37°C for 2 hours, after which excess Matrigel was removed. 1x10 cells were cultured in DMEM containing 10% FBS and 1% P / S in the presence of vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100 μM). 5A suspension of cancer cells (1 mL) at a density of 1 / mL was added to the coated wells. After incubation for 30 minutes at 37°C under 5% CO2 and 95% air, non-adherent cells were removed by washing three times with 500 μL of PBS. The cells were fixed with ice-cold 95% ethanol (0.5 mL / well) for 20 minutes, stained with crystal violet stain (0.2 mL, 0.5% in 20% methanol per well) for 30 minutes at room temperature, and then washed five times with PBS (1 mL). After drying overnight, one field per well was photographed and measured using Image J software to assess cell adhesion. The number of adherent cells was counted randomly in five fields under a light microscope (40x magnification) and the average was calculated.

[0026] Conditioned medium was prepared by culturing MDA-MB-231 cells according to a previous method. Briefly, MDA-MB-231 cells (1 x 10 cells per well in a 24-well plate) were cultured in 100 mL of PBS. 5 Cells (2000 cells / mL) were cultured in DMEM containing 10% FBS and 1% P / S for 3 days after confluence. The culture medium (conditioned medium) was collected and stored at -20°C until use in culture experiments.

[0027] (Crosstalk between MDA-MB-231 cells and bone microenvironment cells) The effects of DHMBA on the proliferation and death of bone microenvironment cells, including osteoblastic MC3T3-E1 cells and macrophage RAW264.7 cells, were investigated along with the crosstalk between human breast cancer MDA-MB-231 cells. Cells were co-cultured using a filter-equipped transwell chamber (Corning, Life Sciences, Cat. no. CLS3464-48EA, USA). MC3T3-E1 or RAW264.7 cells (10 per well of a 24-well plate) were placed in the lower compartment of the chamber. 5 The upper chamber contained 500 μL (5 x 10 cells / mL) of MDA-MB-231 cells and DHMBA (0, 1, or 10 μM). 4After incubation, the filters were removed and the cells attached to the dishes (wells) were counted as described in the "Cell proliferation and death assay" section.

[0028] First, to examine the effect of co-culture with MDA-MB-231 cells on the proliferation of MC3T3-E1 or RAW264.7 cells, cells (10 per well of a 24-well plate) were cultured in the lower compartment of the chamber with or without DHMBA (1 or 10 μM). 5 cells / mL) were co-cultured for 3 days with MDA-MB-231 cells in the upper chamber.

[0029] To examine the effect of conditioned medium obtained from MDA-MB-231 cells on the proliferation of MC3T3-E1 or RAW264.7 cells, cells were cultured for 3 days in the presence or absence of the TNF-α signaling inhibitors Bay 11-7082 (1 or 10 nM) or DHMBA (10 μM) in the conditioned medium (0.25 or 0.5 mL / well per mL of total medium in the well). After incubation, the number of attached MC3T3-E1 or RAW264.7 cells was counted.

[0030] Next, to examine the effect of co-culture with MDA-MB-231 cells on the death of MC3T3-E1 or RAW264.7 cells, cells (10 per well of a 24-well plate) were cultured. 5 The cells were cultured for 3 days until they became subconfluent, and then the culture medium was replaced. 500 μL of MDA-MB-231 cells (5 x 10 cells / mL) were placed in the upper compartment of the transwell. 4 The filters were filled with MC3T3-E1 cells or RAW264.7 cells (10 cells / mL) and then cultured for 2 days with DHMBA (0, 1, or 10 μM). After the culture was completed, the filters were removed and the MC3T3-E1 cells or RAW264.7 cells attached to the dishes were counted. Furthermore, to examine the effect of the conditioned medium on cell death, MC3T3-E1 cells or RAW264.7 cells (10 cells / mL) were added to the wells. 5After culturing the cells in the culture medium (0.25 or 0.5 mL per mL of culture medium) for 3 days, the medium was replaced with medium containing conditioned medium from MDA-MB-231 cells (0.25 mL or 0.5 mL per mL of culture medium) with or without Bay 11-7082 (1 and 10 nM) or DHMBA (10 μM). The cells were cultured for an additional 2 days. After culturing, the cells were counted.

[0031] (TNF-α production assay) MDA-MB-231 cells (1x10 per well) 5 Cells (100 μg / mL) were cultured in DMEM containing 10% FBS and 1% P / S with or without DHMBA (0.1, 1, 10, 100, 250 μM) in 24-well plates for 3 days until subconfluence was reached. After incubation, the medium was collected and TNF-α was measured. The TNF-α concentration in the medium was quantified using a human TNF-α ELISA kit (catalog number KHC301) from ThermoFisher Scientific (Waltham, MA, USA) according to the assay instructions. The amount of TNF-α produced was expressed as picograms (pg) secreted into the medium (mL).

[0032] Western blot analysis: MDA-MB-231 cells (100x21 mm dish, 1x10 per 10 mL) 6 Cells (cells / dish) were cultured in DMEM containing 10% FBS and 1% P / S in the presence of PBS or DHMBA (10 μM) at 37°C and 5% CO2 for 3 days. After incubation, cells were washed three times with ice-cold PBS and lysed in cell lysis buffer (Cell Signaling Technology, Inc.) supplemented with protease inhibitors and protein phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA). The collected lysates were centrifuged at 17,000 xg for 10 minutes at 4°C to obtain fractions containing the cytoplasm and endoplasmic reticulum. Protein concentrations in the supernatants were measured using Bio-Rad Protein Assay Dye (Bio-Rad Laboratories, Inc.). The cell lysates were stored at -80°C until use in Western blot assays.

[0033] Forty micrograms of supernatant protein was applied to each lane and separated by SDS-PAGE (12%). After electrophoresis, the gel was transferred to a PVDF membrane and immunoblotted using specific antibodies. Membranes were blocked with SuperBlock® T20 blocking buffer (Thermo Fisher Scientific, Inc.). The transferred membranes were obtained from Cell Signaling Technology (Danvers, MA, USA), and were stained with antibodies against Akt (Cat. no. 9272, rabbit), Ras (Cat. no. 3339, rabbit), PI3-kinase p1100α (Cat. no. 4255, rabbit), mitogen-activated protein kinase (MAPK; Cat. no. 4695, rabbit), phospho-MAPK (Cat. no. 4370, rabbit), mechanistic target of rapamycin (mTOR; Cat. no. 4517, mouse), Rb (Cat. no. 9309, mouse), p21 (Cat. no. 2947, rabbit), and β-actin (Cat. no. 3700, mouse), and p53 (sc-126, mouse). For immunoblotting using the specific antibodies listed above, membranes were incubated with each primary antibody overnight at 4°C and then analyzed by immunoblotting. Horseradish peroxidase-conjugated secondary antibodies (cat. no. 7076P2 or 7074S for mouse and rabbit antibodies, respectively; Cell Signaling Technology, Inc.; dilution 1:2,000) were incubated for 60 min at 4°C. A total of three blots from independent experiments were scanned with an Epson Perfection 1660 Photo scanner, and bands were quantified using Image J2 software (National Institutes of Health, Bethesda, MD, USA).

[0034] (Statistical Analysis) Statistical analysis of data was performed using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc. La Jolla, CA). Data are presented as mean ± standard deviation (SD). Multiple comparison tests were performed on parametric data using one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparison post-hoc test. Significant differences were considered to exist at a risk level of 0.05% or less.

[0035] (The experimental results of the above experiment are shown below.) 1. It was confirmed that the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits the proliferation of human breast cancer MDA-MB-231 cells in vitro.

[0036] First, we investigated whether DHMBA affects colony formation in vitro in human breast cancer MDA-MB-231 cells. MDA-MB-231 cells (1x10 per well) were cultured in vitro. 3 MDA-MB-231 cells (1x10 cells per well) were cultured in DMEM in the presence of a vehicle (1% ethanol as a final concentration) or DHMBA (1 or 10 μM) for 9 days. Colony formation was suppressed by culturing with DHMBA (Fig. 1A and B). To further examine the effect of DHMBA on MDA-MB-231 cell proliferation, cells (1x10 cells per well) were cultured in DMEM in the presence of a vehicle (1% ethanol as a final concentration) or DHMBA (1 or 10 μM) for 9 days. Colony formation was suppressed by culturing with DHMBA (Fig. 1A and B). 5 Cells (1000 cells / ml) were cultured in the presence of vehicle (1% ethanol) or DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. Incubation with DHMBA reduced the number of cells attached to the dish (Fig. 1C).

[0037] This effect was also confirmed by increasing the concentration of DHMBA (1, 10, 100, or 1000 μM). Notably, incubation with DHMBA (10 μM) blocked the stimulatory effect of epidermal growth factor (EGF; 50, 100, or 200 ng / ml) on MDA-MB-231 cell proliferation (Fig. 1E). Thus, DHMBA suppressed the proliferation of human breast cancer MDA-MB-231 cells in vitro.

[0038] 2. The inhibitory effect of DHMBA on breast cancer cell proliferation was confirmed to be related to the blockade of multiple signaling pathways.

[0039] The inhibitory effect of DHMBA on the proliferation of MDA-MB-231 breast cancer cells was examined using various inhibitors of intracellular signaling pathways (Fig. 2). When cancer cells were cultured with cell cycle inhibitors such as roscovitine (10 or 100 nM), butyrate (10 or 100 μM), or sulforaphane (1 or 10 nM) (Fig. 2A), or with intracellular signaling factor inhibitors such as wortmannin (10 or 100 nM), PD98059 (10 or 100 μM), or staurosporin (1 or 10 nM) (Fig. 2C), breast cancer cell proliferation was suppressed. These inhibitory effects were not further enhanced in the presence of DHMBA (10 μM) (Fig. 2C and D). These results suggest that the inhibitory effect of DHMBA on breast cancer cell proliferation is exerted by blocking different signaling pathways.

[0040] 3. DHMBA was confirmed to regulate the expression levels of proteins related to breast cancer cell proliferation.

[0041] We investigated whether DHMBA mechanistically regulates the levels of various proteins involved in signaling pathways in breast cancer MDA-MB-231 cell proliferation (Fig. 3). The levels of Ras, PI3K, Akt, MAP kinase (MAPK), phosphor-MAPK, and mTOR in MDA-MB-231 cells were reduced by incubation with DHMBA (Fig. 3A and B). These results support the notion that the inhibitory effect of DHMBA on cell proliferation is exerted by regulating the levels of various proteins involved in different signaling pathways in MDA-MB-231 cells.

[0042] 4. DHMBA was confirmed to promote breast cancer cell death.

[0043] To elucidate the effect of DHMBA on cell death in MDA-MB-231 breast cancer cells, subconfluent cells were further cultured in the presence of DHMBA (0.1, 1, 10, 100, or 1000 μM) for 24 or 48 hours (Figure 4A and B). After incubation, the number of cells attached to the dish was counted. A decrease in cell number was induced by incubation with DHMBA (1, 10, 100, or 1000 μM), indicating that DHMBA treatment induces cell death. Mechanistically, after the cell cultures reached subconfluence, they were further cultured in the presence of DHMBA (1 or 10 μM) for 48 hours, with or without a caspase-3 inhibitor (10 μM) (Figure 4C). The DHMBA-promoting effect on cell death was suppressed by the presence of the caspase-3 inhibitor. Furthermore, the levels of caspase-3 and cleaved caspase-3 in MDA-MB-231 cells were increased by treatment with DHMBA (10 μM) (Fig. 4D). These results suggest that incubation with DHMBA promotes apoptotic cell death in breast cancer MDA-MB-231 cells.

[0044] 5. DHMBA was confirmed to suppress the metastatic activity of breast cancer cells.

[0045] To analyze the effect of DHMBA on cell migration, MDA-MB-231 cells were cultured with or without DHMBA (0.1, 1, 10, 50, or 100 μM) for 24 or 48 hours after cell scratching and allowed to reach subconfluence (Figure 5). Measurement of migration distance revealed that incubation with DHMBA inhibited MDA-MB-231 cell migration (Figure 5). Furthermore, incubation with DHMBA (1, 10, or 100 μM) inhibited MDA-MB-231 cell adhesion in vitro (Figure 6). These results suggest that incubation with DHMBA suppresses the metastatic activity of breast cancer cells.

[0046] 6. The in vitro effects of DHMBA on the proliferation and death of osteoblastic MC3T3-E1 cells co-cultured with human breast cancer cells MDA-MB-231 were confirmed.

[0047] The effect of crosstalk between MDA-MB-231 and MC3T3-E1 cells was investigated using a transwell chamber (Fig. 7). To examine the effect of co-culture with MDA-MB-231 cells on the proliferation of MC3T3-E1 cells, MC3T3-E1 cells (10 per well) were cultured in the lower chamber in the presence of DHMBA (0, 1, 10 μM). 5 The upper chamber was filled with 500 μL of MDA-MB-231 cells (5 x 10 cells / mL). 4 After 3 days of culture, MC3T3-E1 cells attached to the dishes were counted (Fig. 7A). The proliferation of MC3T3-E1 cells was suppressed by coculture with MDA-MB-231 cells, and this suppression was blocked by the presence of DHMBA (1 or 10 μM) (Fig. 7A). To further examine the effects of the conditioned medium obtained by coculture with MDA-MB-231 cells, MC3T3-E1 cells were cultured for 3 days in the presence of Bay 11-7082 (10 nM), an inhibitor of the TNF-α signaling pathway, or DHMBA (10 μM), with the conditioned medium (0.25 mL or 0.5 mL per 1 mL of medium in the well) (Fig. 7B). The proliferation of MC3T3-E1 cells attached to the dishes was suppressed by culturing them in the conditioned medium (Fig. 7B). These results suggest that the suppression of MC3T3-E1 cell proliferation by co-culture with MDA-MB-231 cells is mediated by TNF-α signaling, and that this pathway is also inhibited by DHMBA.

[0048] In other experiments, to examine the effect of co-culture with MDA-MB-231 cells (Fig. 7C) or conditioned medium (Fig. 7D) on the killing of MC3T3-E1 cells, cells (10 per well) were cultured. 5MC3T3-E1 cells (cells / ml) were cultured for 3 days after reaching subconfluence. After culture, MDA-MB-231 cells were seeded in the upper compartment of the transwell chamber and cultured for an additional 2 days. The proliferation of MC3T3-E1 cells was suppressed by coculture with MDA-MB-231 cells, and this suppression was blocked by the presence of DHMBA (1 or 10 μM) (Fig. 7C). To further examine the effect of conditioned medium obtained from coculture with MDA-MB-231 cells on cell death, MC3T3-E1 cells were cultured after reaching subconfluence for 3 days, replaced with conditioned medium (0.25 mL or 0.5 mL per mL of medium in the well) containing or without Bay 11-7082 (10 nM) or DHMBA (10 μM), and then cultured for an additional 2 days (Fig. 7D). The number of MC3T3-E1 cells attached to the dish was reduced by culturing in conditioned medium. This reduction was blocked by the presence of Bay 11-7082 or DHMBA (Fig. 7D). These results suggest that the suppression of MC3T3-E1 cells by coculture with MDA-MB-231 cells was mediated by TNF-α signaling and that this pathway was also blocked by DHMBA.

[0049] 7. The effect of DHMBA on the proliferation and death of macrophage RAW264.7 cells co-cultured with breast cancer cells was confirmed.

[0050] Macrophages play a regulatory role in the bone microenvironment. Therefore, we used a transwell chamber assay to examine whether coculture with breast cancer MDA-MB-231 cells altered the proliferation and death of macrophages in RAW264.7 cells (Figure 8). To examine the effect of coculture with MDA-MB-231 cells on RAW264.7 cell proliferation, RAW264.7 cells were placed in the lower chamber in the presence of DHMBA (0, 1, or 10 μM). MDA-MB-231 cells were seeded in the upper chamber. After 3 days of culture, the attached RAW264.7 cells were counted (Figure 8A). The proliferation of RAW264.7 cells was suppressed by coculture with MDA-MB-231 cells. This reduction was blocked by the presence of DHMBA (1 or 10 μM). The proliferation of RAW264.7 cells was suppressed when cultured in conditioned medium prepared from MDA-MB-231 cells (Figure 8B). This inhibition was blocked by treatment with Bay 11-7082 (10 nM) or DHMBA (10 μM).

[0051] We further examined the effect of coculture with MDA-MB-231 cells (Figure 8C) or conditioned medium (Figure 8D) on RAW264.7 cell death. RAW264.7 cell death was enhanced by coculture with MDA-MB-231 cells. This enhancement was blocked by the presence of DHMBA (1 or 10 μM) (Figure 8C). RAW264.7 cell death was enhanced by culturing in conditioned medium prepared from MDA-MB-231 cells (Figure 8D). This enhancement was blocked by treatment with Bay 11-7082 (10 nM) or DHMBA (10 μM) (Figure 8D). These results suggest that the suppression of RAW264.7 macrophages by coculture with MDA-MB-231 cells is mediated by TNF-α signaling, and that this pathway is also blocked by DHMBA.

[0052] 8. It was confirmed that incubation with DHMBA reduced TNF-α production in breast cancer cells.

[0053] TNF-α levels are elevated in the blood of breast cancer patients. The present inventors investigated whether incubation with DHMBA affects TNF-α production in MDA-MB-231 cells in vitro. The results showed that TNF-α production in MDA-MB-231 cells was reduced by incubation with DHMBA (1, 10, 100, and 250 μM). These results suggest that incubation with DHMBA suppresses TNF-α signaling in MDA-MB-231 cells.

[0054] (Discussion) Human breast cancer is a leading cause of cancer-related death in women, resulting in bone fractures and osteolysis accompanied by severe pain. Human breast cancer MDA-MB-231 cells, which cause bone metastasis, lack receptors for estrogen, progesterone, and epidermal growth factor receptor 2 and are triple-negative. The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has been shown to scavenge intracellular free radicals and prevent oxidative stress. However, its pharmacological role remains highly unknown.

[0055] In this study, we demonstrated that incubation with DHMBA inhibited colony formation and proliferation of MDA-MB-231 cells in vitro, promoting cell death and leading to the suppression of cancer cell proliferation. Furthermore, incubation with DHMBA was found to inhibit the migration and adhesion of MDA-MB-231 cells, suggesting the suppression of metastatic activity. These results demonstrate that DHMBA has an inhibitory effect on metastatic breast cancer cells in vitro.

[0056] In particular, the epidermal growth factor (EGF)-stimulatory effect on MDA-MB-231 cell proliferation was suppressed in the presence of DHMBA. Furthermore, we analyzed the underlying mechanism by which DHMBA inhibits MDA-MB-231 cell proliferation using various inhibitors of intracellular signaling pathways. The inhibitory effect of DHMBA on MDA-MB-231 cell proliferation was not enhanced in the presence of inhibitors of cell cycle and intracellular signaling pathways, suggesting that DHMBA inhibits diverse signaling processes. Furthermore, DHMBA treatment reduced the levels of PI3-kinase 100α, Akt, MAPK, phosphor-MAPK, or mTOR, which are associated with EGF-stimulated cell proliferation, and increased the levels of p53, p21, and Rb, which inhibit cell proliferation. Additionally, DHMBA suppressed the activation of caspase-3, which is associated with the induction of apoptotic cell death, resulting in a decrease in cell number. These findings suggest that DHMBA regulates the expression levels of various proteins associated with promoting cell proliferation, leading to a decrease in cell number. DHMBA is thought to regulate the gene expression of various proteins involved in cell proliferation signaling.

[0057] Interestingly, incubation with DHMBA was found to suppress the migration and adhesion of MDA-MB-231 cells, suggesting an inhibitory effect on metastatic activity. We previously showed that incubation with DHMBA inhibits the migration and invasion of human prostate cancer cells in vitro by reducing the levels of related proteins, such as NF-κB 65, caveolin-1, and integrins. DHMBA was also found to suppress the metastatic activity of human bone metastatic cancer cells, including prostate and breast cancer cells, in vitro.

[0058] Osteoblasts promote bone formation. Activated macrophages are the most likely candidates for promoting bone formation and are also involved in tissue repair processes in other tissues. Furthermore, we investigated whether coculture with MDA-MB-231 cells affects osteoblasts and macrophages involved in the bone microenvironment in vitro using a transwell chamber method with cancer cells and osteoblasts or macrophage RAW264.7 cells. We found that coculture with MDA-MB-231 cells reduced the proliferation and enhanced cell death of osteoblastic MC3T3-E1 cells or macrophage RAW264.7 cells, resulting in a decrease in the number of these cells. These findings suggest that MDA-MB-231 cells suppress osteoblasts and macrophages involved in bone formation in the bone microenvironment.

[0059] In particular, incubation with conditioned medium obtained from MDA-MB-231 cells reduced the proliferation and promoted the death of osteoblastic MC3T3-E1 cells and macrophage RAW264.7 cells. The effects of conditioned medium on the proliferation and death of MC3T3-E1 and RAW264.7 cells were suppressed in the presence of the signaling inhibitor TNF-α, suggesting that TNF-α is involved in the suppressive effect of MDA-MB-231 cells on bone microenvironment cells. TNF-α concentrations have been reported to increase in the serum of breast cancer patients depending on the stage of the disease. TNF-α was produced by MDA-MB-231 cells in vitro, and this production was reduced by incubation with DHMBA. Osteoblast function is suppressed by TNF-α signaling, resulting in impaired osteoblast formation and bone loss. Incubation with DHMBA may inhibit the effects of TNF-α on osteoblasts or macrophages. Presumably, breast cancer MDA-MB-231 cells produce TNF-α, which acts on osteoblasts and macrophages in the bone microenvironment, leading to impaired bone formation, a process that is inhibited by DHMBA.

[0060] (Conclusion) The present invention demonstrates that DHMBA suppresses the proliferation and metastatic activity of human breast cancer MDA-MB-231 cells and protects against cancer cell-induced impairment of the proliferation of bone microenvironment cells involved in bone formation.

[0061] The mechanism of action is based on the fact that DHMBA inhibits EGF-stimulated cell proliferation by controlling various signaling pathways in human breast cancer cells, thereby suppressing cell proliferation and metastatic activity. Furthermore, breast cancer cells suppress osteoblasts and macrophages, which promote bone formation, leading to bone loss. DHMBA may protect against this process. Therefore, DHMBA is useful as a means to suppress metastatic human breast cancer cells. Furthermore, this invention provides findings that could lead to a novel therapeutic agent for breast cancer treatment.

Claims

1. A growth inhibitor of human breast cancer MDA-MB-231 cells, characterized by having an inhibitory effect on the growth of human breast cancer MDA-MB-231 cells, containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient.

2. The growth inhibitor of human breast cancer MDA-MB-231 cells according to claim 1, wherein the growth inhibitory effect on human breast cancer MDA-MB-231 cells is exerted by blocking different cell signaling pathways.

3. The growth inhibitor of human breast cancer MDA-MB-231 cells according to claim 1, wherein the growth inhibitory effect on human breast cancer MDA-MB-231 cells is exerted by blocking different cell signaling pathways and by regulating the levels of proteins involved in different cell signaling pathways in MDA-MB-231 cells.

4. A cell death promoter for human breast cancer MDA-MB-231 cells, characterized in that it contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of promoting apoptotic cell death in human breast cancer MDA-MB-231 cells.

5. An agent for inhibiting the metastatic activity of human breast cancer MDA-MB-231 cells, which contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of inhibiting the metastatic activity of human breast cancer MDA-MB-231 cells.

6. An inhibitor of the decrease in MC3T3-E1 osteoblast cells co-cultured with human breast cancer cells MDA-MB-231 and an inhibitor of the promotion of apoptosis of MC3T3-E1 osteoblast cells, characterized in that the active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol, and the inhibitor has the effect of inhibiting the decrease in MC3T3-E1 osteoblast cells co-cultured with human breast cancer cells MDA-MB-231 and the effect of inhibiting the promotion of apoptosis of the MC3T3-E1 osteoblast cells.

7. An inhibitor of the reduction of macrophage RAW264.7 cells and an inhibitor of the promotion of apoptosis of macrophage RAW264.7 cells, which contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of suppressing the reduction of macrophage RAW264.7 cells co-cultured with human breast cancer cells MDA-MB-231 and the effect of suppressing the promotion of apoptosis of said macrophage RAW264.7 cells.

8. An agent for reducing TNF-α production in human breast cancer MDA-MB-231 cells, which contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and has the effect of suppressing TNF-α signaling in human breast cancer MDA-MB-231 cells and reducing TNF-α production, characterized in that it reduces TNF-α production by suppressing TNF-α signaling in human breast cancer MDA-MB-231 cells.

Images