Inhibitors for the proliferation of human glioblastoma cells

Abstract

The present invention provides an inhibitor of human glioblastoma cell proliferation. [Solution] A human glioblastoma cell proliferation inhibitor containing 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as the active ingredient is used. DHMBA increases the levels of tumor suppressor factors p53, p21, and Rb, thereby suppressing the proliferation of cancer cells.

Description

[Technical Field]

[0001] This invention relates to an inhibitor of human glioblastoma cell proliferation. [Background technology]

[0002] Glioblastoma is the most common malignant brain tumor in adults. Glioblastoma is a malignant brain tumor that develops in the brain, usually in the cerebrum, and spreads to surrounding brain tissue. Specifically, it is a tumorous form of glial cells (astrocytes, glia) that support nerve cells in the brain. This tumor is highly invasive and has the highest mortality rate. Therefore, experiments and research to improve the prognosis of patients with this tumor are important. Currently, there is said to be no effective treatment for malignant glioma. Glioblastoma is characterized by the overexpression of ligand-independent epidermal growth factor (EGF) receptors. EGF receptor signaling promotes tumorigenesis by increasing cell proliferation and tissue invasion and inhibiting apoptosis of cancer cells. The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter referred to as DHMBA) has been shown to block oxidative stress by scavenging free radicals in various cell types. This invention confirms the effect of DHMBA on human glioblastoma cells in vitro. Glioblastoma cells were cultured in DMEM-low glucose containing 10% fetal bovine serum (FBS) in the presence of DHMBA (0.1-100 μM). Culture with DHMBA suppressed cell proliferation in the presence of FBS or EGF. Mechanistically, DHMBA treatment reduced the levels of cell proliferation promoters PI3 kinase 100α, Akt, MAPK, phosphor-MAPK, and mTOR, and increased the levels of tumor suppressors p53, p21, and Rb, leading to the suppression of cancer cell proliferation. DHMBA treatment stimulated glioblastoma cell death by increasing caspase-3 and cleavage caspase-3 levels. Furthermore, culture with DHMBA inhibited metastatic activity, including cancer cell adhesion and migration. Thus, DHMBA may have an inhibitory effect on the activity of human glioblastoma cells in vitro. This invention is expected to provide a novel strategy for the treatment of glioblastoma.

[0003] 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a phenolic antioxidant derived from the Pacific oyster (Crassostrea gigas), possesses two properties: it acts as a radical scavenger in various cells, blocking oxidative stress. In particular, DHMBA is approximately 15 times, and four orders of magnitude, more effective than Trolox as a peroxyl radical scavenger in lipid and aqueous media. DHMBA 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 and preventing various diseases. Recently, DHMBA has been shown in vitro to suppress adipogenesis, inflammatory macrophage activity, and related osteoclast formation, while stimulating osteoblastic osteogenesis. Furthermore, DHMBA has been shown to inhibit the proliferation of metastatic prostate cancer cells by targeting various signaling pathways, offering a new strategy for cancer treatment using DHMBA. Thus, elucidating the pharmacological role of DHMBA is considered important for the prevention and treatment of various diseases, including cancer. Glioblastoma is the most common malignant brain tumor in adults. It is highly aggressive and has the highest mortality rate. Currently, there is no effective treatment for malignant gliomas. Attempts to improve the prognosis of patients affected by this tumor remain unsuccessful. Glioblastoma is characterized by ligand-independent overexpression of the epidermal growth factor (EGF) receptor. EGF receptor signaling can promote tumorigenesis by increasing cell proliferation and tissue invasion and inhibiting apoptosis in cancer cells. The mitogen-activated protein kinase (MAPK) network has been studied and is significantly altered in glioblastoma cells. Mutations in the MAPK pathway most frequently affect ERK, c-Jun N-terminal kinase (JNK), and the RAS and B-RAF of the p38 pathway, leading to malignant transformation. Targeting the EGF receptor and its associated signaling pathways is crucial in the treatment of malignant gliomas. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2023-177458 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] This invention investigated whether DHMBA has anticancer activity against human glioblastoma cells in vitro. It was found that culturing with DHMBA inhibited cell proliferation by suppressing various pathways related to EGF signaling, thereby stimulating glioblastoma cell death and preventing cell proliferation and carcinogenesis. Furthermore, DHMBA increased the levels of tumor suppressor factors p53, p21, and Rb, consequently suppressing cancer cell proliferation. In particular, culturing with DHMBA inhibited metastatic activity, including cancer cell adhesion and migration. Thus, DHMBA exhibits anticancer activity against human glioblastoma cells in vitro, suggesting its potential to provide a new strategy for the treatment of glioblastoma. [Means for solving the problem]

[0006] The present invention provides a human glioblastoma cell proliferation inhibitory effect using 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient. It is characterized by the following: or A substance containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient has an inhibitory effect on the EGF-enhancing proliferation of glioblastoma cells. It is characterized by the following: or A glioblastoma cell death stimulating agent containing 3,5-dihydroxy-4-methoxybenzyl alcohol as the active ingredient. It is characterized by the following: or The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol, which has the effect of inhibiting the metastatic activity of glioblastoma cells. It is characterized by the following: [Effects of the Invention]

[0007] According to the present invention, it was confirmed that culturing with DHMBA inhibits cell proliferation by suppressing various pathways related to EGF signaling, thereby stimulating glioblastoma cell death and preventing cell proliferation and carcinogenesis. Furthermore, DHMBA increased the levels of tumor suppressor factors p53, p21, and Rb, and as a result, suppressed the proliferation of cancer cells. In particular, culturing with DHMBA inhibited metastatic activity, including the adhesion and migration of cancer cells. Thus, DHMBA showed anticancer activity against human glioblastoma cells in vitro. Therefore, it has been pointed out that the present invention has the potential to provide a new strategy for the treatment of glioblastoma. [Brief explanation of the drawing]

[0008] [Figure 1](A) Effect on colony formation. Cells (1 x 10³ cells / 2 mL per well in a 6-well plate) were cultured for 10 days in DMEM containing 10% FBS in the presence of a medium (final concentration 1% ethanol) or DHMBA (10 μM). A photograph of the plate stained with crystal violet is shown. (B) OD570 nm of crystal violet solubilized with 100% methanol is shown. Data are shown as the mean ± SD of 6 wells using different cell preparations. (C) Changes in cell proliferation with increasing culture days. Cells (1 x 10⁵ cells / mL per well in a 24-well plate) were cultured for 1, 2, 3, 4, and 5 days in the presence of a medium (final concentration 1% ethanol) or DHMBA (10 μM). The number of cells attached to the dish was counted. (D) Effect of increasing DHMBA concentration. This is an explanatory diagram illustrating the state of cells (1 x 10⁵ cells / mL per well) cultured for 3 days in DMEM containing DHMBA (0.1, 1, 10, 100, 250 μM). [Figure 2]Culturing with DHMBA inhibits the proliferation of human glioblastoma cells enhanced in vitro in the presence of epidermal growth factor (EGF). Cells (1 x 10⁵ cells / mL per well) were cultured in DMEM (serum-free) for 2 days (Figure A) or 4 days (Figure B) in the presence of a medium (PBS) or EGF (100 or 300 ng / ml), with or without DHMBA (10 μM). After culturing, the number of cells attached to the dish was counted. Data are shown as mean ± SD obtained from 8 wells of 2 replicate plates using different cell preparations. *: p < 0.001 compared to control (Graber). One-way ANOVA, Tukey-Kramer post-test. To measure EGF receptor levels, cells (1 x 10⁶ cells / 10 ml medium in a 100 mm dish) were cultured in DMEM containing DHMBA (10 μM) for 3 days. After culturing, 40 micrograms of cell-lysed protein per lane were separated by SDS-PAGE (12%), and Western blotting was performed using a specific antibody. (C) Representative data is shown. (D) The bands are shown in multiples of the control. [Figure 3] Inhibitory effect of DHMBA on the proliferation of human glioblastoma cells in vitro in the presence of cell cycle inhibitors or intracellular signaling factors. Cells (A) and (B) (1 x 10⁵ cells / mL per well) were cultured for 3 days in the presence of DHMBA (10 μM), with or without the presence of various cell cycle inhibitors including roscovitine (10 or 100 nM), butyrate (10 or 100 μM), or sulforaphane (1 or 10 nM). Cells (C) and (D) (1 x 10⁵ cells / mL per well) were cultured for 3 days in the presence of DHMBA (10 μM), with or without the presence of intracellular signaling inhibitors including wortmannin (10 or 100 nM), PD98059 (1 or 10 μM), or staurosporine (1 or 10 nM). After culture, the number of cells attached to the dish was counted. The data are shown in an explanatory diagram as mean ± SD of 8 wells in 2 replicate plates using different cell preparations. [Figure 4] DHMBA modulates the levels of several proteins involved in signaling pathways related to the proliferation of human glioblastoma cells in vitro. Cells (1 x 10⁶ cells / 10 mL medium in a 100 mm dish) were cultured for 3 days in DMEM containing 10% FBS and 1% P / S in the presence of a medium (1% ethanol) or DHMBA (10 μM). After culturing, cells were removed from the dish using a cell scraper in cell lysis buffer containing a protease inhibitor. 40 micrograms of supernatant protein per lane were separated by SDS-PAGE (12%) and transferred to a nylon membrane for Western blotting using specific antibodies against various proteins. (A) Representative data are shown. (B) Bands are shown as multiples of the control. The data are shown in an explanatory diagram as the mean ± SD of values ​​obtained from four dishes using different cell preparations. [Figure 5]DHMBA-induced cell death in vitro was stimulated in human glioblastoma cells. (A) Cells (1 x 10⁵ cells / mL per well in a 24-well plate) were cultured for 3 days after reaching subconfluence in DMEM containing 10% FBS and 1% P / S, and then cultured for 48 hours each in the presence of DHMBA (0.1, 1, 10, 100, 250 μM). (B) Cells at subconfluence were cultured for an additional 48 hours in the presence of DHMBA (1 μM or 10 μM) with or without a caspase-3 inhibitor (10 μM). The number of cells adhering to the dish was counted. Data are shown as mean ± SD of 8 wells in two replicate plates using different cell preparations. *p<0.001 vs control (Graber). One-way ANOVA, Tukey-Kramer post-test. To measure the levels of caspase-3 or cleavage caspase-3, cells (1 x 10⁶ cells / 10 ml medium in a 100 mm dish) were cultured for 3 days in DMEM containing either a medium (1% ethanol) or DHMBA (10 μM). After culturing, 40 micrograms of cell-lysed protein per lane were separated by SDS-PAGE (12%) and Western blotting was performed using specific antibodies. (C) Representative data are shown. (D) Bands are shown as multiples of the control. The data are shown as mean ± SD values ​​obtained from four dishes using different cell preparations. [Figure 6]The stimulating effect of DHMBA on the death of human glioblastoma cells is not enhanced in vitro by the presence of apoptotic cell death stimulants. Cells (1 x 10⁵ cells / mL per well in a 24-well plate) were cultured for 3 days in DMEM containing 10% FBS and 1% P / S until subconfluence was reached. The cells were then cultured for 48 hours in the presence of a signaling medium (PBS or 1% ethanol), TNF-α (0.1 or 1 ng / ml) (Figure A), NF-κB signaling inhibitor Bay 11-7062 (1 or 10 nM) (Figure B), Bay K 8644 (0.1 or 1 μM) (Figure C), or calcium-dependent protein kinase inhibitor staurosporine (1 or 10 nM) (Figure D), with or without DHMBA (10 μM). The number of cells adhering to the dish was counted. Data are presented as mean ± SD of 8 wells in two replicate plates using different cell preparations. The data is shown in an explanatory diagram as the mean ± SD of 8 wells in two replicate plates using different cell preparations. [Figure 7] The effect of DHMBA on glioblastoma cell proliferation and death is inhibited in vitro by the presence of an aryl hydrocarbon receptor inhibitor (CH223191). A: Cells (1 x 10⁵ cells / ml per well in a 24-well plate) were cultured for 3 days in DMEM containing 10% FBS, 1% P / S, and 1% fungizone in the presence or absence of CH223191 (1, 10, 25 μM) and DHMBA (10 μM), either in the presence or absence of these two. B: Cells (1 x 10⁵ cells / ml per well in a 24-well plate) were cultured for 3 days in DMEM containing 10% FBS and 1% P / S until subconfluence was reached, and then cultured for an additional 48 hours in the presence of CH223191 (1, 10, or 25 μM), with or without DHMBA (10 μM). The number of cells adhering to the dish was counted. The data is shown in an explanatory diagram as mean ± SD obtained from a total of 8 wells in two replicate plates using different cell preparations. [Figure 8]DHMBA inhibits the adhesion of human glioblastoma cells in vitro. The adhesion of cancer cells was evaluated using Matrigel. Cancer cells (105 cells / mL per well in a 24-well plate) were suspended in DMEM containing 10% FBS and 1% P / S in the presence of vehicle (1% ethanol) or DHMBA (0.1, 1, 10, 100, 250 μM). After incubation at 37 °C for 30 min, non-adherent cells were washed away with PBS. The adherent cells were fixed and stained with crystal violet. To evaluate cell adhesion, one field per well was photographed and measured using Image J2 software (Figure A). The number of adherent cells was counted randomly in 5 fields with an optical microscope (40x) and averaged (Figure B). The results are shown as the mean ± SD of values obtained from a total of 8 wells using two plates with different cell preparations in an explanatory diagram. [Figure 9] DHMBA inhibits the migration of human glioblastoma cells in vitro. Cells (2x105 cells / mL per well in a 24-well plate) were cultured in DMEM containing 10% FBS and 1% P / S for 48 h. When confluent, a linear scratch was made in the center of the cell monolayer. Then, the cells were cultured for 48 h in the above medium supplemented with vehicle (1% ethanol) or DHMBA (0.1, 1, 10, 100 μM). After culture, the fixed cells were stained with crystal violet. To evaluate cell migration, the migration distance was imaged and a representative photograph was presented (Figure A). The results of the migration distance were expressed as the relative value (% relative to the control) of the distance of the cell-free area at the start of culture (control) and after 48 h (Figure B). The data are shown as the mean ± SD of values obtained from a total of 8 wells using two plates with different cell preparations in an explanatory diagram. [Figure 10]This is an explanatory diagram of the hypothesized mechanism by which DHMBA exhibits an anti-cancer effect on human glioblastoma cells. DHMBA inhibited the stimulatory effect of EGF on cancer cell proliferation. This was independent of changes in EGF receptor levels. The inhibitory effect of DHMBA on cell proliferation and metastatic activity by controlling various signal transduction pathways. DHMBA decreased the levels of Ras / PI3K / Akt / MAPK and mTOR, which are involved in promoting cell proliferation. Furthermore, DHMBA increased the levels of the tumor suppressors Rb, p53, and p21 in cancer cells. Furthermore, this is an explanatory diagram showing that DHMBA stimulated cell death associated with increased levels of caspase-3 and cleaved caspase-3, leading to a decrease in the number of glioblastoma cells.

Mode for Carrying Out the Invention

[0009] Hereinafter, experiments regarding each inhibitor according to the present invention were conducted, and the experimental methods and other details of the experiments will be described. (Experimental Materials and Methods) Reagents Dulbecco's Modified Eagle Medium - Low Glucose (DMEM; containing 1000 mg / L, L-glutamine, and sodium bicarbonate) was purchased from Sigma-Aldrich, Inc. (St. Louis, Missouri, USA). Antibiotics (100 units / mL penicillin and 100 μg / mL streptomycin; P / S) were purchased from Gibco Life Technologies Corporation (Grand Island, NY, USA). Fetal Bovine Serum (FBS) was purchased from Omega Scientific Inc. Roscovitine, sulforaphane, butyric acid, tumor necrosis factor α (TNF-α) were purchased from R&D Systems (Minneapolis, Minnesota, USA). PD98059, staurosporin, wortmannin, Bay K 8644, Bay 11-7089, crystal violet, human epidermal growth factor (EGF), caspase-3 inhibitor, and all other reagents were purchased from Sigma-Aldrich unless otherwise specified. (3,5-dihydroxy-4-methoxybenzyl alcohol) 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), an amphiphilic phenolic compound, was initially isolated from the Pacific oyster (Crassostrea gigas) and characterized as an antioxidant. The chemical structure of DHMBA is shown in the prior patent obtained by the present inventor.

[0010] In this invention, 100% pure synthetic DHMBA obtained from Watanabe Oyster Research Institute Co., Ltd. (Hachioji City, Tokyo) was used. The DHMBA was dissolved in 100% ethanol and stored at -20°C until use. (Human glioblastoma cells) Human glioblastoma cells, CVCL-12231, isolated from glioblastoma, were obtained from the Cell Bank-(RIKEN BRC) in Tokyo [RCB0763; GI-1]. This cell line was isolated from gliosarcoma in a 61-year-old Japanese male. The glioma cells were cultured in DMEM (low glucose, 1000 mg / L) containing 1% P / S and 10% FBS. (Colony formation and crystal violet staining) Glioblastoma cells (1x10 3 (cells / 2mL / 6-well plate) 1x10 3Cells were seeded at a density of / well in a 6-well dish and cultured for 10 days in DMEM containing 10% FBS and 1% P / S under conditions of 5% CO2 and 37°C. Cells adhering to the dish were washed with phosphate-buffered saline (PBS; 2 mL, 3 times), fixed with methanol (0.5 mL per well) at room temperature for 20 minutes, and then washed three times with PBS. Fixed cells were stained with 0.1% crystal violet (1 mL) at room temperature for 30 minutes. Stained cells were washed four times with PBS (2 mL). The plate was air-dried at room temperature for 2 hours. The plate was photographed and stained cells were confirmed under a microscope (Nikon TMS, Tokyo, Japan). To measure the stained cells, 1.0 mL of methanol (100%) was added to the dish, covered, and incubated with shaking at room temperature for 20 minutes. After extraction, the optical density of each well was measured at 520 nm using a plate reader.

[0011] (Assays on cell proliferation and cell death) Glioblastoma cells (1 x 10⁶ cells per well in a 24-well plate) 5 Cells ( / mL) were cultured for 1, 2, 3, 4, and 5 days in DMEM containing 10% FBS and 1% P / S in the presence of a medium (1% ethanol as the final concentration) or DHMBA (10 μM) (Figure 1C). Cells were also cultured for 3 days with increasing concentrations of DHMBA (0.1, 1, 10, 100, 250 μM) (Figure 1D). In another experiment, cells (1 x 10 per well) 5 Cells (1 x 10 / mL) were cultured in DMEM (serum-free) with EGF (100 or 300 ng / mL) added (Figure 2). In another experiment, cells (1 x 10 per well in a 24-well plate) were cultured. 5Cells were grown to subconfluence for 3 days in DMEM containing 10% FBS and 1% P / S in the presence of either a medium (1% ethanol as the final concentration) (Figure 3). Roscovitine (10 or 100 nM), butyrate (10 or 100 μM), sulforaphane (1 or 10 nM), wortmannin (10 or 100 nM), PD98059 (1 or 10 μM), or staurosporine (1 or 10 nM) were added at effective concentrations, with or without DHMBA (10 μM). In another experiment, cells were cultured in the presence of the aryl hydrocarbon receptor agonist CH223191 (1, 10, or 25 μM), with or without DHMBA (10 μM) (Figure 6A). After culturing, cells were detached from each well and counted as described below. To investigate the effect of DHMBA on cell death, glioma cells (1 x 10⁶ per well) were used. 5 Cells (1x10 / mL) were cultured subconfluently for 3 days in DMEM containing 10% FBS and 1% P / S in the presence of a medium (1% ethanol as final concentration) or DHMBA (0.1, 1, 10, 100, 250 μM), and then cultured for a further 24 or 48 hours in the presence of a medium (PBS or 1% ethanol as final concentration) or DHMBA (0.1, 1, 10, 100, 250 μM). In additional experiments, cells (1x10 per well) were cultured subconfluently for 3 days in DMEM containing 10% FBS and 1% P / S, in the presence of a medium (1% ethanol as final concentration) or DHMBA (0.1, 1, 10, 100, 250 μM). 5After reaching sub-confluence in a 24-well plate in DMEM (containing 10% FBS and 1% P / S) at ( / mL), the cells were cultured for 3 days, and then cultured for 48 hours in the presence of vehicle (1% ethanol as the final concentration) or DHMBA (1 μM or 10 μM), regardless of the presence or absence of caspase-3 inhibitor (10 μM). In another experiment, after culturing the cells at sub-confluence for 3 days, they were treated with TNF-α (0.1 or 1 ng / mL), Bay K 8644 (0.1 or 1 μM), Bay 11-7086 (1 or 10 nM), or staurosporine (1 or 10 nM), with or without DHMBA (10 μM) (Figure 5). In other experiments, the cells were cultured in the presence of CH223191 (1, 10, or 25 μM), with or without DHMBA (10 μM) (Figure 7B). After culture, Ca 2+ / Mg 2+ A sterile solution of 0.05% trypsin + EDTA in Ca / Mg-free PBS (Thermo Fisher Scientific, Waltham, MA, USA) (0.1 mL per well) was added while incubating at 37 °C for 2 minutes, and then DMEM containing 10% FBS (0.9 mL per well) was added to detach the cells. To measure the viable cell count, the medium containing floating cells (0.1 mL) was mixed with 0.1 mL of 0.5% trypan blue staining solution. Using a microscope (10×, Olympus MTV-3), a hemocytometer (Sigma-Aldrich, St. Louis, Missouri), and a cell counter (Line Precision Machinery H-102P, Tokyo, Japan), the viable cells were counted. The average of two counts was taken for each dish. The cell count was expressed as the number per well.

[0012] (Western blot analysis) Human glioblastoma cells (100x21 mm dish, 1x10 per 10 mL 6Cells (dish) were cultured for 3 days at 37°C and 5% CO2 in DMEM containing 10% FBS and 1% P / S, in the presence of a medium (PBS) or DHMBA (10 μM). After culturing, the cells were washed three times with ice-cold PBS and the recovered lysates were centrifuged at 17,000 g for 10 minutes at 4°C in cell lysis buffer (Cell Signaling Technology, Inc.) supplemented with protease inhibitors and protein phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA) to obtain fractions containing the cytoplasm and endoplasmic reticulum of the cells. Protein concentrations in the supernatant were measured using Bio-Rad Protein Assay Dye (Bio-Rad Laboratories, Inc.) and bovine serum albumin (Bio-Rad Laboratories, Inc.) as standards. The cell lysate supernatant was stored at -80°C until use in the Western blot assay. 40 micrograms of cell lysate protein were applied to each lane and separated by SDS-PAGE (12%). After electrophoresis, the gel was transferred to a PVDF membrane and immunoblotting was performed using specific antibodies. After blocking with SuperBlock® T20 blocking buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA), the transferred membrane was immunoblotted with specific antibodies against various proteins, including Akt (catalog no. 9272, rabbit), Ras (catalog no. 3339, rabbit), PI3-kinase p1100α (catalog no. 4255, rabbit), mitogen-activated protein kinase (MAPK; catalog no. 4695, rabbit), phosphorylated-MAPK (catalog no. 4370, rabbit), and mechanistic-MAPK (catalog no. 4370, rabbit), obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).The antibodies include rapamycin's mechanical target (mTOR, catalog no. 4517, mouse), Rb (catalog no. 9309, mouse), p21 (catalog no. 2947, rabbit), caspase-3 (catalog no. 9662, rabbit), cleaved caspase-3 (catalog no. 9661, rabbit), EGF receptor (catalog no. 4267, rabbit), and β-actin (catalog no. 3700, mouse) and p53 (catalog no. sc-126, mouse) from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA). In the immunoblotting method using the above-mentioned specific antibodies, the membrane was incubated overnight at 4°C with each primary antibody, and then incubated with horseradish peroxidase-labeled secondary antibodies (catalog no. 7076P2 or 7074S for mouse and rabbit, respectively; Cell Signaling Technology, Inc.) at 4°C for 60 minutes. A total of four blots obtained from independent experiments were scanned using an Epson Perfection 1660 Photo scanner, and the bands were quantified using Image J2 software (National Institutes of Health, Bethesda, MD, USA).

[0013] (Cell adhesion test) The effect of DHMBA on cancer cell adhesion was measured using Matrigel. Briefly, 200 μL of Matrigel matrix (diluted 1:2 with sterile PBS, Corning) was coated onto a 24-well plate, incubated at 37°C for 2 hours, and then excess Matrigel was removed. In the presence of either a mediating solution (final concentration 1% ethanol) or DHMBA (0.1, 1, 10, 100, 250 μM), 1x10⁶ samples were placed in DMEM containing 10% FBS and 1% P / S. 5A suspension of glioma cells (1 mL) at a density of / mL was added to coated wells. After incubation for 30 minutes under conditions of 5% CO2, 95% air, and 37°C, non-adherent cells were removed by washing three times with 500 μL of PBS. Cells on the plate 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) at room temperature for 30 minutes per well, and then washed five times with PBS (1 mL). After drying overnight, one field per well was photographed and cell adhesion was evaluated using ImageJ software. The number of adherent cells was calculated by randomly counting 5 fields under a light microscope (40x magnification) and calculating the mean.

[0014] (Cell migration test) For the analysis of cell migration in vitro, the in vitro scratch assay method was used. Glioblastoma cells (2 x 10⁶ cells per well in a 24-well plate) 5 Cells (cells / mL) were cultured for 48 hours in DMEM containing 10% FBS and 1% P / S. At confluence, a straight 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 48 hours in the presence of a medium (1% ethanol to final concentration) or DHMBA (0.1, 1, 10, 100 μM). Cells in the plates were fixed at 4°C for 60 minutes in ice-cold 70% ethanol (0.5 mL / well), 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 evaluate cell migration, one field per well was imaged at baseline and at 24 or 48 hours after culture, and the distance of the cell-free area was measured using ImageJ software. The migration distance results were expressed as a relative value (percentage of control) of the distance to the cell-free region at baseline and 24 or 48 hours after culture.

[0015] (statistical analysis) Statistical significance was shown using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc.) data as mean ± standard deviation (SD). Multiple comparisons were performed on parametric data using one-way analysis of variance (ANOVA) with Tukey-Kramer post-multiple comparison test. A p < 0.05 value was considered statistically significant. (Experimental results) The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has been shown to suppress the proliferation of human glioblastoma cells in vitro. The effect of DHMBA on glioblastoma cell colony formation in vitro was investigated. (1 x 10 cells per well) 3 Cells were cultured in DMEM for 10 days in the presence of a medium (1% ethanol as the final concentration) or DHMBA (10 μM). In in vitro culture of glioblastoma cells, cell proliferation increased after 10 days of culture, but no colonies were formed (Figure 1A and B). Cell proliferation was suppressed when cultured with 10 μM DHMBA (Figure 1A). To investigate the effect of DHMBA on glioblastoma cell proliferation, cells (1 x 10⁶ per well) were cultured. 5 Cells (cells / ml) were cultured for 1, 2, 3, 4, and 5 days in a medium (1% ethanol) or in the presence of DHMBA (10 μM). Culturing with DHMBA reduced the number of cells adhering to the dish (Figure 1C). This effect was also confirmed by increasing the concentration of DHMBA (1, 10, 100, or 250 μM) (Figure 1D).

[0016] (DHMBA inhibits EGF-enhanced proliferation of glioblastoma cells) Adding EGF (100 or 300 ng / mL) to serum-free medium increased glioblastoma cell proliferation (Figure 2A). Notably, cultures supplemented with DHMBA (10 μM) blocked the stimulating effect of EGF (100 or 300 ng / mL) on glioblastoma cell proliferation (Figure 2B). In addition, EGF receptor levels were not altered by culture with DHMBA (10 μM) (Figures 2C and D). Thus, DHMBA was found to inhibit the stimulating effect of EGF on glioblastoma cell proliferation, independently of changes in EGF receptor levels in in vitro. (The inhibitory effect of DHMBA on glioblastoma cell proliferation is related to the blockade of multiple signaling pathways.) The inhibitory effect of DHMBA on glioblastoma cell proliferation was investigated using inhibitors of various intracellular signaling pathways (Figure 3). Glioblastoma cell proliferation was suppressed by culturing with cell cycle inhibitors including roscovitine (10 or 100 nM), butyrate (10 or 100 μM), or sulforaphane (1 or 10 nM) (Figure 3A), or with intracellular signaling factors including wortmannin (10 or 100 nM), PD98059 (1 or 10 μM), or staurosporine (1 or 10 nM) (Figure 3C). These inhibitory effects were not further enhanced in the presence of DHMBA (10 μM) (Figures 3C and D). These results suggest that the inhibitory effect of DHMBA on glioblastoma cell proliferation is caused by the blockade of different signaling pathways. (The inhibitory effect of DHMBA on glioblastoma cell proliferation is involved in changes at multiple protein levels.) Mechanistically, it was determined whether DHMBA modulates the levels of various proteins involved in the signaling pathways of glioblastoma cell proliferation (Figure 4). Levels of Ras, PI3K, Akt, MAP kinase (MAPK), phosphor-MAPK, or mTOR in glioblastoma cells were decreased by culturing with DHMBA (Figures 4A and B). Notably, levels of the cell proliferation inhibitory proteins p53, p21, and Rb were increased by culturing with DHMBA (10 μM) (Figures 4A and B). These results suggest that the inhibitory effect of DHMBA on cell proliferation is caused by the modulation of different protein levels involved in different signaling pathways in glioblastoma cells.

[0017] (DHMBA stimulates glioblastoma cell death) Next, we investigated whether DHMBA induces glioblastoma cell death. Subconfluenced cells were further cultured for 48 hours in the presence of DHMBA (0.1, 1, 10, 100, or 250 μM) (Figure 5A and B). After culturing, the number of cells attached to the dish was counted. The decrease in cell number was caused by culturing with DHMBA (1, 10, 100, or 250 μM), indicating that DHMBA treatment induces cell death. Mechanistically, after reaching subconfluence, cells were further cultured for 48 hours in the presence of DHMBA (1 or 10 μM), with or without a caspase-3 inhibitor (10 μM) (Figure 5B). The stimulating effect of DHMBA on cell death was inhibited by the presence of the caspase-3 inhibitor. Furthermore, the levels of caspase-3 and cleaved caspase-3 in glioblastoma cells were increased by DHMBA (10 μM) treatment (Figure 5C and D). These results suggest that culturing with DHMBA stimulates apoptotic cell death in glioblastoma cells.

[0018] Furthermore, we evaluated whether the stimulating effect of DHMBA on glioblastoma cell death is regulated by the presence of apoptotic cell death stimulants in vitro (Figure 6). Cells were cultured for 3 days after reaching subconfluence, and then TNF-α (0.1 or 1 ng / mL) (Figure 6A), the NF-κB inhibitor Bay 11-7062 (1 or 10 nM) (Figure 6B), Bay K 8644 (0.1 or 1 μM) (Figure 6C), or the calcium-dependent protein kinase inhibitor staurosporine (1 or 10 nM) (Figure 6D) were added, with or without DHMBA (10 μM) (Figure 6D). Glioblastoma cell death was stimulated by the presence of TNF-α (0.1 or 1 ng / mL) (Figure 6A) or Bay K 8644 (0.1 or 1 μM) (Figure 6C). These effects were not altered by treatment with DHMBA (10 μM). The stimulating effect of DHMBA on cell death was also induced by the presence of Bay 11-7062 (1 or 10 nM) (Figure 6B) or staurosporine (1 or 10 nM) (Figure 6D). These results suggest that the stimulating effect of DHMBA on glioblastoma cell death is involved in the signaling pathways of NF-κB and calcium-dependent protein kinases.

[0019] (Aryl hydrocarbon receptor antagonists inhibit the effects of DHMBA on glioblastoma cell proliferation and death.) The aryl hydrocarbon receptor (AHR) forms heterodimers with AHR nuclear localization factors and binds to heterologous responsive elements. CH223191 is a specific antagonist of AHR signaling that binds to this receptor. We investigated the effects of DHMBA on the proliferation and death of glioblastoma cells cultured in the presence of CH223191. Culture with CH223191 (1, 10, 25 μM) did not affect cell proliferation (Figure 7A) or cell death (Figure 7B). The effects of DHMBA (10 μM) on cell proliferation and cell death were blocked by the presence of CH223191 (1, 10, 25 μM) (Figures 7A and B). These results suggest that the effects of DHMBA on glioblastoma cell proliferation and death are partially mediated through processes related to AHR signaling that regulates transcriptional activity in the nucleus. (DHMBA inhibits the metastatic activity of glioblastoma cells) We investigated whether DHMBA affects the metastatic activity of glioblastoma cells. Cancer cell adhesion was evaluated in vitro using the Matrigel method. The presence of DHMBA (1, 10, 100, 250 μM) inhibited the adhesion of glioblastoma cells to the Matrigel phase of the dish (Figure 8). Furthermore, we determined whether DHMBA suppresses glioblastoma cell migration in vitro. Cell migration was determined using a scratch assay. Glioblastoma cell migration with wound healing 48 hours after scratching was suppressed by the presence of DHMBA (1, 10, 100 μM) (Figure 9). These results suggest that culturing with DHMBA inhibits the metastatic activity of glioblastoma cells.

[0020] (discussion) Glioblastoma is an aggressive and deadly tumor, and the most common malignant brain tumor in adults. Effective treatments for malignant gliomas are needed. Glioblastoma is characterized by ligand-independent overexpression of the EGF receptor. EGF receptor signaling may promote tumorigenesis by increasing cell proliferation and tissue invasion and inhibiting apoptosis in cancer cells. Targeting the EGF receptor and its associated signaling pathways may be important in the treatment of malignant gliomas. We investigated in vitro whether DHMBA has anticancer effects on human glioblastoma cells. We found that culturing with DHMBA suppressed cell proliferation by inhibiting various signaling pathways related to EGF signaling. Furthermore, DHMBA stimulated glioblastoma cell death, leading to an inhibition of cell proliferation development. Interestingly, DHMBA increased the levels of tumor suppressor factors p53, p21, and Rb, consequently suppressing cancer cell proliferation. In particular, culturing with DHMBA inhibited metastatic activity, including cancer cell adhesion and migration. Thus, this invention demonstrates that DHMBA has anticancer activity against human glioblastoma cells in vitro, providing a new strategy for the treatment of glioblastoma tumors. Culturing with DHMBA inhibited the proliferation of human glioblastoma cells in vitro. The presence of DHMBA suppressed the stimulating effect of EGF on glioblastoma cell proliferation. This effect was independent of changes in EGF receptor levels. DHMBA may not affect the binding of EGF to the receptor on glioblastoma cells. Furthermore, the underlying mechanism by which DHMBA inhibits glioblastoma cell proliferation was evaluated using various inhibitors of intracellular signaling pathways. The inhibitory effect of DHMBA on glioblastoma cell proliferation was not enhanced by inhibitors of the cell cycle and intracellular signaling pathways, suggesting that DHMBA acts on various signaling processes, including the cell cycle. In particular, DHMBA administration reduced levels of PI3-kinase 100α, Akt, MAPK, phosphor-MAPK, and mTOR, which are associated with enhanced EGF-stimulated cell proliferation. This result suggests that DHMBA inhibits various signaling pathways involved in EGF receptor signaling. Culture with DHMBA may suppress EGF receptor-related signaling pathways in glioblastoma cells. In addition, culture with DHMBA increased levels of p53, p21, and Rb, which suppress cell proliferation. The inhibitory effect of DHMBA on glioblastoma cell proliferation may be related to the blockade of various processes associated with EGF signaling and the enhancement of inhibitory proteins associated with cell proliferation. DHMBA may also regulate the gene expression of several proteins in the nucleus. AHR forms heterodimers with AHR nuclear localization factors and binds to heterologous responsive elements. CH223191 is a specific antagonist of AHR signaling and inhibits the effects of various chemical compounds, including DHMBA, on the proliferation and death of various types of human cancer cells. The presence of AHR inhibited the effects of DHMBA on the proliferation and death of glioblastoma cells. This result suggests that the effects of DHMBA on glioblastoma cells may be partially involved in the activation of AHR signaling, which regulates transcriptional activity. DHMBA may also regulate the nuclear transcriptional activity of human glioblastoma cells, but this has not yet been clarified. Culture with DHMBA stimulated glioblastoma cell death in vitro, reducing cell number. The stimulating effect of DHMBA on glioblastoma cell death was inhibited by the presence of a caspase-3 inhibitor, suggesting that DHMBA-induced cell death is related to the activation of caspase-3, which is associated with the induction of apoptotic cell death. Mechanistically, culturing with DHMBA increased the levels of caspase-3 and cleavage caspase-3 in glioblastoma cells. The effect of DHMBA on cell death was characterized in comparison to the effects of TNF-α and Bay K 8674, which induce apoptotic cell death. Culture with TNF-α or Bay K 8674 stimulated glioblastoma cell death. Since these effects were not enhanced by the presence of DHMBA, it is suggested that DHMBA affects NF-κB or calcium-dependent signaling pathways. Furthermore, the stimulating effect of DHMBA on cell death was not enhanced in the presence of Bay-11-7062, an NF-κB signaling inhibitor, or staurosporine, a calcium-dependent protein kinase inhibitor. It has been reported that inhibition of NF-κB signaling induces apoptosis in primary exudative lymphoma cells infected with KSHV. Increased intracellular calcium may lead to apoptotic cell death. The stimulating effect of DHMBA on cell death is likely involved in its effects on these signaling processes. EGF receptor signaling may promote tumorigenesis by increasing cell proliferation and tissue metastasis and inhibiting apoptosis in cancer cells. Our study showed that culturing glioblastoma cells with DHMBA suppressed adhesion and migration, suggesting an inhibitory effect on metastatic activity. Previously, we reported that culturing with DHMBA inhibited migration and invasion of human prostate cancer cells in vitro by reducing the levels of related proteins such as NF-κB p65, caveolin-1, and integrins. Thus, DHMBA may inhibit the metastatic activity of various types of human cancer cells in vitro.

[0021] In conclusion, this invention demonstrates that the marine factor DHMBA suppresses cell proliferation enhanced by FBS or EGF and stimulates glioblastoma cell death by regulating various signaling processes. As summarized in Figure 10, mechanistically, DHMBA treatment reduced the levels of cell proliferation factors PI3-kinase 100α, Akt, MAPK, phosphor-MAPK, and mTOR, and increased the levels of tumor suppressors p53, p21, and Rb, leading to the suppression of cancer cell proliferation. Furthermore, culture with DHMBA inhibited metastatic activity such as adhesion and migration of cancer cells. Thus, DHMBA may have anticancer effects against human glioblastoma cells in vitro. This study provides a new strategy for the treatment of glioblastoma tumors.

Claims

1. A human glioblastoma cell proliferation inhibitor containing 3,5-dihydroxy-4-methoxybenzyl alcohol as the active ingredient, A growth inhibitor for human glioblastoma cells characterized by the following features.

2. A substance containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient has an inhibitory effect on the EGF-enhancing proliferation of glioblastoma cells. An EGF-enhancing and growth-inhibiting agent for glioblastoma cells, characterized by the following features.

3. A glioblastoma cell death stimulating agent containing 3,5-dihydroxy-4-methoxybenzyl alcohol as the active ingredient. A glioblastoma cell death stimulant characterized by the following:

4. The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol, which has the effect of inhibiting the metastatic activity of glioblastoma cells. An inhibitor of glioblastoma cell metastatic activity characterized by the following properties.

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