KR20220006364A - Composition for preventing, alleviating or treating liver cancer comprising alpha mangostin glycoside - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating liver cancer comprising alpha-mangostin glycoside. The alpha-mangostin glycoside of the present invention not only has excellent bioavailability, but also exhibits an excellent effect in the treatment of liver cancer such as an ability to kill liver cancer cells, inhibit cancer cell metastasis, inhibit angiogenesis, inhibit HIF-1α accumulation, inhibit tumor sphere formation, thereby being able to be usefully used in related medicines and health functional food fields.

Description

A composition for preventing, alleviating or treating liver cancer comprising alpha mangostin glycoside;

The present invention relates to a composition for preventing, improving or treating liver cancer comprising alpha mangosteen glycoside.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, accounting for 85% to 90% of all cases of liver cancer, and has been reported as the second leading cause of cancer mortality worldwide. HCC is a multifactorial disease caused by smoking, alcohol, mycotoxin, human hepatitis virus, fatigue and obesity. Current HCC treatments include surgery, radiofrequency ablation, and chemotherapy, but the treatment results are not satisfactory due to metastasis and high recurrence rates. .

On the other hand, alpha mangosteen is a polyphenolic xanthone isolated from the peel of a tropical fruit mangosteen (Garcinia mangostana Linn.). Phytochemicals have been reported to have various biological activities such as antioxidant, anti-inflammatory, anti-tumor, anti-diabetic, anti-bacterial, anti-fungal, anti-obesity, cardioprotective and neuroprotective effects. Various in vitro and in vivo studies have confirmed that alpha-mangosteen has chemopreventive and chemotherapy potential against a wide range of cancer cell types. However, the low oral bioavailability of alpha-mangosteen due to first-pass metabolism, poor absorption due to low water solubility and the effect of P-glycoprotein efflux limit further clinical applications.

Therefore, a novel alpha-mangosteen derivative design is required to improve bioavailability, but research on this is insufficient.

Korean Patent Registration Publication No. 10-1621162

An object of the present invention is alpha-mangosteen-3-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside) or a pharmaceutically acceptable To provide a pharmaceutical composition for preventing or treating liver cancer comprising a salt as an active ingredient.

An object of the present invention is alpha-mangosteen-6-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 6-O- β -D-2-deoxyglucopyranoside) or a pharmaceutically acceptable To provide a pharmaceutical composition for preventing or treating liver cancer comprising a salt as an active ingredient.

An object of the present invention is alpha-to β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside) or a salt thereof, an active ingredient-mangosteen-3-O It is to provide a food composition for preventing or improving liver cancer comprising.

An object of the present invention is alpha-to β -D-2- deoxy gluconic nose Llano side (α-mangostin- 6-O- β -D-2-deoxyglucopyranoside) or a salt thereof, an active ingredient-mangosteen-6-O It is to provide a food composition for preventing or improving liver cancer comprising.

The present invention to achieve the above object of the present invention is alpha-mangosteen-3-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside) Or it provides a pharmaceutical composition for preventing or treating liver cancer comprising a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention is alpha-mangosteen-6-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 6-O- β -D-2-deoxyglucopyranoside) or a pharmaceutically acceptable It provides a pharmaceutical composition for preventing or treating liver cancer comprising a salt as an active ingredient.

The present invention is alpha-to β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside) or a salt thereof, an active ingredient-mangosteen-3-O It provides a food composition for preventing or improving liver cancer, including.

The present invention is alpha-to β -D-2- deoxy gluconic nose Llano side (α-mangostin- 6-O- β -D-2-deoxyglucopyranoside) or a salt thereof, an active ingredient-mangosteen-6-O It provides a food composition for preventing or improving liver cancer, including.

In one embodiment of the present invention, the composition may further include an autophagy inhibitor.

In one embodiment of the present invention, the autophagy inhibitor may be 3-methyladenine.

In one embodiment of the present invention, the molar concentration ratio of the autophagy inhibitor and the active ingredient compound may be 1: 15 to 25.

In one embodiment of the present invention, the composition may inhibit the formation of tumor spheres.

In one embodiment of the present invention, the composition can inhibit the expression of one or more cancer stem cell markers selected from the group consisting of Nanog, Oct4, Sox2, CD44 and CD133.

The alpha-mangosteen glycoside of the present invention has excellent bioavailability and exhibits excellent effects in the treatment of liver cancer, such as hepatocarcinoma cell killing ability, cancer cell metastasis inhibition ability, angiogenesis inhibition ability, HIF-1α accumulation inhibition ability, and tumor cell formation inhibition ability. And it can be usefully used in the field of health functional food.

1 shows the structures of alpha-mangosteen and alpha-mangosteen glycoside compounds.
Figure 2 shows the growth inhibitory effect of alpha-mangosteen glycoside on hepatocellular carcinoma (HCC).
3 shows the effect of alpha mangosteen glycoside on the inhibition of Hep3B cell migration.
Figure 4 shows the effect of alpha mangosteen glycoside on the cell cycle and apoptosis of cells.
5 shows the effect of alpha mangosteen glycoside on the expression of mitochondrial-mediated apoptosis regulators in Hep3B cells.
6 shows the effect of alpha mangosteen glycoside on Hep3B cell autophagy.
7 shows the effect of alpha mangosteen glycoside on the c-Met signaling pathway in Hep3B cells.
8 shows the effect of alpha mangosteen glycoside on Hep3B cell-induced angiogenesis.
9 shows the effect of alpha mangosteen glycoside on HIF-1α activity in Hep3B cells.
Figure 10 shows the effect of alpha mangosteen glycoside on tumorisphere formation and cancer stem cell marker expression.

The inventors of the present invention are alpha to the human hepatocyte-mangosteen-3-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside) and alpha- Mangosteen - 6-O - β -D- 2- deoxy gluconic nose Llano side (α-mangostin- 6-O- β -D-2-deoxyglucopyranoside) a result of intensive evaluation of the antitumor activity of the compounds of the invention The present invention was completed by demonstrating that HCC growth, migration, angiogenesis, and stemness of cancer cells were inhibited through inhibition of c-Met signal transduction and inhibition of HIF-1α expression.

Thus, the present invention is alpha-mangosteen-3-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside) or a pharmaceutically acceptable It provides a pharmaceutical composition for preventing or treating liver cancer comprising a salt as an active ingredient.

In another aspect, the present invention is alpha-mangosteen-6-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 6-O- β -D-2-deoxyglucopyranoside) or a pharmaceutically acceptable salt thereof It provides a pharmaceutical composition for preventing or treating liver cancer comprising as an active ingredient.

The alpha-mangosteen-3-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin-3 -O- β -D-2-deoxyglucopyranoside) is to have the structure of formula (I) for the HCC It has high anticancer activity.

[Formula 1]

The alpha-mangosteen-6-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 6-O- β -D-2-deoxyglucopyranoside) is to have the structure of formula (II) for the HCC It has high anticancer activity.

[Formula 2]

The composition of the present invention may be applied for treatment, prevention or improvement of liver cancer. The liver cancer includes both primary liver cancer or metastatic liver cancer, and includes both hepatocellular carcinoma and cholagiocarcinoma, but preferably can be applied to hepatocellular carcinoma. In addition, the liver cancer may be early stage liver cancer, advanced liver cancer, or end stage liver cancer.

In the present invention, "pharmaceutically acceptable salt" means a salt formed with any inorganic or organic acid or base that does not cause serious irritation to the administered organism and does not impair the biological activity and physical properties of the compound. As the salt, a salt commonly used in the art may be used, such as an acid addition salt formed with a pharmaceutically acceptable free acid.

As used herein, the term “prevention” refers to any act of inhibiting or delaying the onset of liver cancer by administering the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which the symptoms of liver cancer are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

A pharmaceutical composition according to the invention are alpha-mangosteen-3-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside), alpha-mango Stein- 6-O - β -D-2-deoxyglucopyranoside (α-mangostin- 6-O-β- D-2-deoxyglucopyranoside) or a salt thereof is included as an active ingredient, and is pharmaceutically acceptable possible carriers. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It does not, and may further include other conventional additives, such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection or oral ingestion.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously or subcutaneously) according to a desired method, and the dosage may vary depending on the patient's condition and weight, the degree of disease, drug form, Although it varies depending on the route and time of administration, it may be appropriately selected by those skilled in the art.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like.

In another aspect the present invention is alpha-mangosteen-3-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside) or a salt thereof of the active ingredient It provides a food composition for preventing or improving liver cancer comprising a.

In another aspect, the present invention is alpha-mangosteen-6-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 6-O- β -D-2-deoxyglucopyranoside) or a salt thereof as an active ingredient comprising a It provides a food composition for preventing or improving liver cancer.

The food composition is a concept including health functional food.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated, for example, the severity of symptoms.

Food compositions of the present invention are alpha-mangosteen-3-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside), alpha-mangosteen - 6-O - β -D-2-deoxyglucopyranoside (α-mangostin- 6-O-β -D-2-deoxyglucopyranoside) or a salt thereof is added to food as it is or combined with other food or food ingredients. may be used, and may be appropriately used according to a conventional method. Alpha-mangosteen-3-O-β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside), alpha-mangosteen-6-O-β- D-2-deoxyglucopyranoside (α-mangostin- 6-O-β- D-2-deoxyglucopyranoside) or a salt thereof may be appropriately determined depending on the purpose of its use (for prevention or improvement). In general, in the production of food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term ingestion for health and hygiene purposes or for health control purposes, the amount may be less than or equal to the above range.

The health functional food composition of the present invention is not particularly limited in other ingredients other than containing the above ingredients as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like may be contained. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.

In the present invention, the pharmaceutical composition may further include an autophagy inhibitor, and the autophagy inhibitor may preferably be 3-methyladenine, but is not limited thereto.

In the present invention, the molar concentration ratio of the autophagy inhibitor and the active ingredient may be 1: 15 to 25, preferably 1: 20, but is not limited thereto.

In the present invention, the composition may inhibit the formation of tumor spheres.

In the present invention, the composition can inhibit the expression of one or more cancer stem cell markers selected from the group consisting of Nanog, Oct4, Sox2, CD44 and CD133.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example 1: Materials and Preparation>

Example 1-1. material

Alpha mangosteen, dimethyl sulfoxide (DMSO), 3-methyladenine (3-MA), dimethyloxalylglycine (DMOG), Z-DEVD-FMK, crystal violet, gelatin and 3 -(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) is Sigma - It was purchased from Aldrich (St. Louis, Missouri, USA). CellTiter-Glo® Luminescent Cell Viability Assay Kit was purchased from Promega (Madison, Wis.). Alpha Mangostin Glycoside, Alpha Mangostin-3-O-β-D-2-deoxyglucopyranoside (α-mangostin-3-O-β-D-2-deoxyglucopyranoside, Man-3DG) and Alpha Mango Steen-6-O-β-D-2-deoxyglucopyranoside (α-mangostin 6-O-β-D-2-deoxyglucopyranoside, Man-6DG) was obtained by applying a biocatalytic glycosylation reaction. Alpha mangosteen, Man-3DG and Man-6DG were prepared using DMSO at a concentration of 50 mM. Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM) and minimum essential medium (MEM) were purchased from Gibco (Grand Island, New York, USA). .

Endothelial growth medium-2 (EGM-2) and penicillin-streptomycin-amphotericin B were obtained from Lonza (Walkersville, MD, USA). Matrigel and transwell chamber systems were obtained from BD Biosciences (San Jose, CA) and Corning Costar (Acton, MA). Anti-cleaved caspase-3, anti-cleaved caspase-9, anti-Bcl-XL, anti-Bax, anti-Atg5, anti-Beclin-1, anti-LC3B, anti-p62, anti-phospho-Met, anti- Met, anti-phospho-AKT, anti-AKT, anti-phospho-ERK1/2, anti-ERK1/2, anti-HIF-1α, anti-Nanog, anti-Oct4, anti-Sox2, anti-CD44, anti- CD133, and anti-β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Example 1-2. Cell culture and hypoxia

Human liver cancer cells (HepG2, Huh7 and Hep3B) and human umbilical vein endothelial cells (HUVEC) were obtained from the Korean Cell Line Bank (Seoul, Korea) and the American Type Culture Collection (Manassas, VA, USA), respectively. . The HepG2 and Huh7 cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotics. Hep3B cells and HUVECs were grown in MEM and EGM-2 supplemented with 10% FBS and 1% antibiotics, respectively. Cells were maintained at 37 °C in a humidified 5% CO ₂ incubator (Thermo Fisher Scientific, Vantaa, Finland).

For hypoxic conditions, Hep3B cells were cultured in a hypoxic chamber (Forma Scientific, Marietta, Ohio) balanced with N _{2 under 5% CO 2} and 1% O _{2 .}

Examples 1-3. cell growth assay

HepG2, Huh7 and Hep3B cells (2×10 ³ cells/well) were seeded in 96-well culture plates. Cells were treated with various concentrations (0-100 μM) of alpha mangosteen, Man-3DG and Man-6DG and incubated for 72 hours. After that, 50 μl of MTT solution (2 mg/mL) was added to each well and incubated for 3 h. After incubation, formazan crystals were dissolved in 100 μL of DMSO for each well. The absorbance of each well was measured at a wavelength of 540 nm using a microplate reader (Thermo Fisher Scientific).

Examples 1-4. Confirm colony formation

HepG2, Huh7 and Hep3B cells (5×10 ² cells/well) were seeded in 6-well culture plates and treated with 10 μM of alpha mangosteen, Man-3DG and Man-6DG. After 7 days, cells were stained with crystal violet and colonies were counted for each well.

Examples 1-5. wound healing analysis

Hep3B cells (3 x 10 ⁵ cells/well) were seeded in 24-well culture plates. The fused monolayer cells were scraped using a tip, and then each well was washed with phosphate buffered saline (PBS) to remove floating cells. Cells were treated with 10 μM of alpha mangosteen, Man-3DG and Man-6DG and then cultured for 48 hours. Cell images were captured at 100x magnification at different times (0, 24 and 48 hours) under an optical microscope (Olympus, Center Valley, PA).

Example 1-6. cell cycle analysis

^{Hep3B cells (3 x 10 5} cells/well) treated with 10 µM of alpha-mangosteen, Man-3DG and Man-6DG were trypsinized, washed with cold PBS, fixed with cold 70% ethanol, and fixed at -20 °C. was stored overnight in Cells were washed with PBS and incubated in 100 μg/mL propidium iodide (PI) solution (Muse Cell Cycle Assay Reagent; Millipore, Hayward, CA, USA) for 30 min in the dark at room temperature. And the cellular DNA content was analyzed using flow cytometry (Muse Cell Analyzer; Millipore).

Example 1-7. apoptosis assay

^{Hep3B cells (3 × 10 5} cells/well) treated with 40 μM alpha-mangosteen, Man-3DG and Man-6DG were harvested, washed with PBS, and then 100 μl of annexin V-FITC and PI solution ( After staining with Muse Annexin V & Dead Cell Reagent; Millipore) at room temperature for 20 minutes, 100 μl of the medium was used to suspend. The stained cells were analyzed by flow cytometry (Muse Cell Analyzer; Millipore).

Examples 1-8. Western blot analysis

After treatment with α-mangosteen, Man-3DG, and Man-6DG (10 and 20 μM) for 24 h, Hep3B cells were harvested and lysed in 2x Laemmli sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA). made it Cell lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the isolated proteins were subjected to polyvinylidene dilution using standard electroblotting procedures. It was transferred to a fluoride (polyvinylidene difluoride, PVDF) membrane (Millipore). Blots were blocked using 5% or 1% skim milk for 1 h at room temperature, and cleaved caspase-3, cleaved caspase-9, Bax, Bcl-XL, Atg5, Beclin-1, LC3B, p62, phospho-Met (Tyr1234/Tyr1234/ 1235), Met, phospho-AKT (Ser473), AKT, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, HIF-1α, Nanog, Oct4, Sox2, CD44, CD133 and β-actin (dilution 1: 2000), and labeled overnight at 4°C. After washing 3 times with tris buffered saline with Tween 20 (TBST) for a total of 2 hours, secondary antibody (dilution 1:3000) to rabbit or mouse was added and incubated for 1 hour at room temperature. Immunolabeling was detected using an Enhanced Chemiluminescence (ECL) kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions.

Examples 1-9. Tumor cell-induced angiogenesis assay

Tumor cell-induced chemical invasion assays were performed using an in vitro co-culture system based on chemical invasion assays. Hep3B cells were seeded in the lower chamber and treated with 10 and 20 μM of alpha-mangosteen, Man-3DG and Man-6DG for 24 h. The medium in the lower chamber was replaced with fresh medium, and serum-depleted HUVECs (8×10 ⁴ cells/well) were added to the upper chamber. The chamber was incubated at 37 °C for 18 h. HUVECs that invaded the lower chamber were fixed using 70% methanol and stained with hematoxylin and eosin (H&E) for 5 min at room temperature. Totally invaded cells were photographed and counted at 100 magnification using an optical microscope (Olympus).

Examples 1-10. VEGF measurement using ELISA

The VEGF concentration in the medium generated from Hep3B cells treated with 10 and 20 μM of alpha-mangosteen, Man-3DG and Man-6DG was measured using a VEGF immunoassay kit (Koma Biotech, Seoul, Korea) according to the manufacturer's instructions. analyzed. Results were expressed as the concentration of VEGF relative to the total amount of protein from each well.

Examples 1-11. 3D cell culture

Hep3B cells were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12; Gibco) containing 1x B-27 serum-free supplement (Gibco), 5 µg/mL heparin (Sigma-Aldrich), 2 mM, Cultured in L-glutamine (Gibco), 20 ng/mL epidermal growth factor (EGF; Gibco), 20 ng/mL basic fibroblast growth factor (bFGF; Koma Biotech) and 1% penicillin/streptomycin (Gibco).

Examples 1-12. CellTiter-Glo® Luminescent Cell Viability Assay

Hep3B cells (3 × 10 ³ cells/well) were seeded in 96-white-well culture plates. Cells were treated with various concentrations (0-25 μM) of alpha-mangosteen, Man-3DG and Man-6DG and cultured in serum-free suspension spheroid culture conditions for 7 days. Then, 20 μl of the substrate solution was added to each well, the culture plate was shaken for 2 minutes and incubated in the dark for 8 minutes. Luminescence was detected using a multimode microplate reader (BioTek, Inc., Winooski, VT, USA).

Examples 1-13. Oncosphere formation assay

Hep3B cells (3 x 10 ³ cells/well) were seeded in 96-well culture plates and treated with 3.13 μM of alpha mangosteen, Man-3DG and Man-6DG. After culturing in a 3D culture environment for 7 days, the size and number of tumor spheres were checked with a 100 x optical microscope (Olympus).

Examples 1-14. statistical analysis

All experiments were repeated at least 3 times. Results were expressed as mean ± standard deviation (SD) and differences between groups were analyzed using analysis of variance (ANOVA) using the SPSS statistics package (SPSS 9.0; SPSS Inc.). Post hoc analysis was performed by Tukey's test and a p-value of <0.05 was considered to indicate a statistically significant difference.

<Example 2: Analysis of the effect of alpha mangosteen glycoside on HCC cell growth>

The effect of alpha mangosteen glycoside, Man-3DG and Man-6DG on the growth of three different HCC cell lines (HepG2, Huh7 and Hep3B) was investigated. The cells were treated with alpha-mangosteen, Man-3DG and Man-6DG (0-100 μM) for 72 hours and analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Cell growth was measured through

As a result, as shown in Fig. 2A, alpha mangosteen glycoside strongly inhibited the growth of HepG2, Huh7 and Hep3B cells (alpha mangosteen IC ₅₀ = 7.3, 15.9 and 13.1 μM; Man-3DG was IC ₅₀ = 12.6, 22.3 and 25.0 μM, _{respectively; Man-6DG has IC 50} = 7.0, 14.7 and 12.5 μM, respectively). In particular, Man-6DG exhibited a growth inhibitory effect on HCC cells to a degree similar to that of alpha mangosteen.

Next, the effects of Man-3DG and Man-6DG on the clonal growth of HCC cells were investigated. As shown in Fig. 2B, colony formation of HepG2, Huh7 and Hep3B cells was inhibited after treatment with alpha mangosteen, Man-3DG and Man-6DG using a concentration of 10 μM. In addition, an alpha mangosteen glycoside analog was observed to have an antiproliferative effect on HCC cells.

<Example 3: Analysis of Effect of Alpha Mangosteen Glycoside on Hep3B Cell Migration>

To evaluate the effect of Man-3DG and Man-6DG on the migration of HCC cells, a wound-healing assay was performed using Hep3B cells. As a result, it was shown that Man-6DG significantly reduced the migration of Hep3B cells 48 hours after treatment compared to control cells, as observed in alpha mangosteen ( FIG. 3 ). This means that Man-6DG can inhibit the metastasis of HCC cells.

<Example 4: Analysis of the effect of alpha-mangosteen glycoside on apoptosis of Hep3B cells>

Abnormal cell cycle progression and avoidance of apoptosis are common features of cancer. Therefore, induction of cell cycle arrest and apoptosis in cancer cells is considered to be a major cellular mechanism of anticancer action.

To determine whether alpha-mangosteen glycoside inhibits HCC cell growth by inducing arrest at certain stages of the cell cycle, we analyzed the cell cycle distribution of Man-3DG and Man in Hep3B cells by flow cytometry analysis. The effect of -6DG was investigated. As a result, as shown in Fig. 4A, alpha mangosteen, Man-3DG and Man-6DG showed a significant decrease in the G2/M stage cell population compared to the untreated control cells, along with a G0/G1 stage cell population. induced a significant increase in These data demonstrate that alpha mangosteen and its glycosides inhibit the growth of Hep3B cells through cell cycle arrest in the G0/G1 phase.

We further investigated whether the antiproliferative effects of Man-3DG and Man-6DG on HCC cells were associated with apoptosis by flow cytometry after Annexin V-FITC/PI double staining. As a result, treatment with alpha-mangosteen, Man-3DG, and Man-6DG showed significant apoptosis in Hep3B cells at a rate of 3.15% in the control group, 65.2% in alpha-mangosteen, 10.25% in Man-3DG, and 28.55% in Man-6DG. It was shown that it was significantly improved (FIG. 4B).

Alpha mangosteen has been reported to induce mitochondrial-dependent apoptosis in HCC cells. Therefore, we evaluated the effect of Man-3DG and Man-6DG on the expression of important regulators of mitochondrial-mediated apoptosis in Hep3B cells. As a result, as shown in FIG. 5A , Man-3DG and Man-6DG increased cleaved caspase-3, cleaved caspase-9 and Bax protein levels, while decreasing the protein level of Bcl-XL. In particular, Man-6DG more effectively regulated the expression of apoptosis-related proteins than Man-3DG. In addition, the caspase-3 inhibitor Z-DEVD-FMK partially restored the viability of Hep3B cells inhibited by alpha mangosteen glycoside ( FIG. 5B ).

Collectively, these results suggest that Man-3DG and Man-6DG have excellent anticancer activity against HCC cells by G0/G1 cell cycle arrest and mitochondrial caspase-dependent apoptosis induction.

<Example 5: Analysis of the effect of alpha-mangosteen glycoside on autophagy of Hep3B cells>

To determine whether the activation of autophagy is related to the anticancer effect of alpha-mangosteen glycoside in HCC cells, we analyzed Man-3DG and Man-6DG for the expression of autophagy-related proteins in Hep3B cells by western blotting. was investigated for the modulating effect of As a result, as shown in FIG. 6A , Atg5, Beclin-1, LC3B-II and p62 protein levels were upregulated in Hep3B cells after treatment with alpha mangosteen glycoside, which is Man-3DG and Man-6DG means that it can induce autophagy activation and apoptosis in HCC cells.

To further understand the role of autophagy and apoptosis induced by alpha-mangosteen glycosides in HCC cells, Hep3B cells were treated with alpha-mangosteen, Man-3DG and Man in the presence or absence of the autophagy inhibitor 3-MA. Treated with -6DG. As a result, as shown in FIG. 6B , compared to treatment with alpha mangosteen and Man-3DG and Man-6DG alone, treatment with alpha mangosteen and its glycosides and 3-MA more strongly enhanced the viability of Hep3B cells. decreased. These results suggest that autophagy activation induced by alpha-mangosteen, Man-3DG and Man-6DG may have a protective role in apoptosis induced in HCC cells.

<Example 6: Confirmation of the effect of alpha-mangosteen glycoside on the c-Met signaling pathway in Hep3B cells>

Abnormal c-Met signaling is one of the important factors in HCC progression. Several downstream signaling pathways, including MAPK and PI3K/AKT, can be activated by c-Met to upregulate the growth and metastasis of HCC cells. Therefore, we determined whether alpha-mangosteen glycoside affects the activation of c-Met and its downstream effectors, p44/42 MAPK (ERK1/2) and AKT in Hep3B cells.

As a result, as shown in Figure 7, Man-3DG and Man-6DG effectively inhibited phosphorylation of c-Met, AKT and ERK1/2 without inhibiting total protein levels, resulting in a c-Met mediated signaling cascade. It was confirmed that alpha-mangosteen glycoside can exhibit anticancer activity against HCC cells through down-regulation of .

<Example 7: Confirmation of Effect of Alpha Mangosteen Glycoside on Hep3B Cell-induced Angiogenesis>

To evaluate whether alpha-mangosteen glycoside inhibits HCC cell-induced angiogenesis, alpha-mangosteen, Man-3DG and Man on tubing and invasion of human umbilical vein endothelial cells (HUVECs) stimulated by Hep3B cells. The effect of -6DG was investigated. As a result, as shown in FIG. 8A , the conditioned medium (CM) from Hep3B cells induced tube formation of HUVECs compared to the control medium. However, CMs treated with alpha-mangosteen, Man-3DG and Man-6DG significantly blocked tube formation of HUVECs.

A co-culture assay was used to investigate the effects of alpha mangosteen, Man-3DG and Man-6DG on invasion of HUVECs induced by Hep3B cells. HUVECs co-cultured with Hep3B cells showed significant invasion compared to controls ( FIG. 8B ). However, Hep3B cells treated with alpha-mangosteen, Man-3DG and Man-6DG effectively prevented the increased invasion of HUVECs, confirming that alpha-mangosteen glycoside could inhibit HCC cell-induced angiogenesis. .

<Example 8: Confirmation of the effect of alpha-mangosteen glycoside on hypoxia-induced HIF-1α protein accumulation>

To evaluate the role of HIF-1α in mediating the tumor angiogenesis inhibitory effect of alpha-mangosteen glycoside, the effects of alpha-mangosteen, Man-3DG and Man-6DG on HIF-1α expression in Hep3B cells were westernized. Analyzed by lot. As a result, as shown in FIG. 9A , all of them significantly inhibited the hypoxia-induced accumulation of HIF-1α protein in Hep3B cells. In addition, the effect of alpha mangosteen glycoside on the expression of VEGF was confirmed through ELISA. As a result, it was confirmed that the VEGF production of Hep3B cells in hypoxia was significantly reduced by treatment with α-mangosteen, Man-3DG and Man-6DG ( FIG. 9B ). These data suggest that alpha mangosteen glycoside can inhibit HCC cell-induced angiogenesis by inhibiting the expression of HIF-1α and its downstream target VEGF.

In addition, it was evaluated whether the inhibitory effect of HIF-1α by alpha mangosteen glycoside was related to prolyl-hydroxylase (PHD) activity. As a result, as shown in FIG. 9C , treatment with dimethyloxalylglycine (DMOG), a PHD inhibitor, inhibited the degradation of HIF-1α protein by alpha mangosteen, Man-3DG and Man-6DG. This suggests that the compound may decrease the protein stability of HIF-1α through upregulation of PHD activity.

<Example 9: Confirmation of Effect of Alpha Mangosteen Glycoside on Hep3B Cell Tumor Formation and Cancer Stem Function>

To further confirm the anticancer effect of alpha-mangosteen glycoside in a 3D culture environment, Hep3B cells were grown in serum-free suspension spheroid culture conditions. As a result, as shown in FIG. 10A, alpha mangosteen, Man-3DG and Man-6DG strongly inhibited the growth of 3D cultured Hep3B cells (IC ₅₀ = 4.17, 9.99 and 2.98 μM, respectively).

In particular, Man-6DG showed a better growth inhibitory effect in 3D cultured HCC cells compared to alpha mangosteen. In addition, it was confirmed that the tumorisphere formation ability of Hep3B cells was significantly inhibited when treated with 3.13 μM Man-6DG ( FIGS. 10B and 10C ). Man-6DG effectively reduced the size and number of tumor cells compared to alpha mangosteen and Man-3DG at the doses indicated.

We also investigated whether alpha-mangosteen glycoside regulates the expression of key stem-related markers for HCC CSCs in 3D tumorocyte culture conditions of Hep3B cells. As a result, treatment with alpha-mangosteen, Man-3DG and Man-6DG significantly reduced the expression levels of cancer stem regulators including Nanog, Oct4, Sox2, CD44 and CD133 in 3D tumorocyte cultured Hep3B cells (Fig. 10D). . In particular, the inhibitory effect of Man-6DG on the expression of cancer stem markers was stronger than that of alpha mangosteen and Man-3DG. These data suggest that Man-6DG may have superior therapeutic potential to clear HCC CSCs as well as better bioavailability than alpha-mangosteen in the in vivo environment.

Claims (14)

Alpha-by β -D-2- deoxy gluconic nose Llano side (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside) or a pharmaceutically acceptable salt thereof of the active ingredient-mangosteen-3-O A pharmaceutical composition for preventing or treating liver cancer, comprising.
Alpha-by β -D-2- deoxy gluconic nose Llano side (α-mangostin- 6-O- β -D-2-deoxyglucopyranoside) or a pharmaceutically acceptable salt thereof of the active ingredient-mangosteen-6-O A pharmaceutical composition for preventing or treating liver cancer, comprising.
The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition further comprises an autophagy inhibitor.
The pharmaceutical composition of claim 3, wherein the autophagy inhibitor is 3-methyladenine.
The pharmaceutical composition according to claim 3, wherein the molar concentration ratio of the autophagy inhibitor and the active ingredient compound is 1: 15 to 25.
The pharmaceutical composition according to claim 1 or 2, wherein the composition inhibits the formation of tumorspheres.
The pharmaceutical composition according to claim 1 or 2, wherein the composition inhibits expression of one or more cancer stem cell markers selected from the group consisting of Nanog, Oct4, Sox2, CD44 and CD133.
Alpha-mangosteen-3-O-β -D-2- deoxy gluconic nose Llano side liver cancer or prevention comprising the (α-mangostin- 3-O- β -D-2-deoxyglucopyranoside) or a salt thereof as an active ingredient A food composition for improvement.
Alpha-mangosteen-6-O-β -D-2- deoxy gluconic nose Llano side liver cancer or prevention comprising the (α-mangostin- 6-O- β -D-2-deoxyglucopyranoside) or a salt thereof as an active ingredient A food composition for improvement.
The food composition of claim 8 or 9, wherein the food composition further comprises an autophagy inhibitor.
The food composition of claim 10, wherein the autophagy inhibitor is 3-methyladenine.
The food composition according to claim 10, wherein the molar concentration ratio of the autophagy inhibitor and the active ingredient compound is 1: 15 to 25.
The food composition of claim 8 or 9, wherein the composition inhibits the formation of tumorspheres.
The food composition of claim 8 or 9, wherein the composition inhibits expression of one or more cancer stem cell markers selected from the group consisting of Nanog, Oct4, Sox2, CD44 and CD133.

Images