KR20160103648A - A composition comprising alpha or gamma mangostin for preventing, improving or treating pancreatic cancer - Google Patents

Abstract

The present invention relates to a composition comprising -mangostin or -mangostin as an active ingredient for preventing, alleviating or treating pancreatic cancer, and more specifically, to a composition comprising -mangostin or -mangostin as an active ingredient for preventing, alleviating or treating pancreatic cancer, which suppresses pancreatic cancer through autophagy activation. The composition according to the present invention contains 0.1-20 M of at least one selected among -mangostin and -mangostin, and has the effect of increasing the expression of LC3-II in MIA PaCa-2 or PANC-1 cell lines.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, ameliorating or treating pancreatic cancer, which contains alpha or gamma mangosteen as an active ingredient. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for preventing,

The present invention relates to a composition for the prevention, amelioration or treatment of pancreatic cancer which contains? -Mangostin or? -Mangostin as an active ingredient.

More particularly, the present invention relates to a composition for preventing, ameliorating, or treating pancreatic cancer containing, as an active ingredient,? -Mangostin or? -Mangostin, which inhibits pancreatic cancer through autophagy activation will be.

The composition according to the present invention contains 0.1-20 .mu.M of at least one selected from among .alpha.-mangostin and .gamma.-mangostin and increases the expression of LC3-II in MIA PaCa-2 or PANC-1 cell lines .

Pancreatic cancer has a survival rate of less than 6% within 5 years of diagnosis and is the fourth most deadly cancer in the world. This is because the early prognosis is difficult because the prognosis is poor, and when it is detected, the metastasis already occurs and many surgical procedures can not be attempted.

The only way to treat pancreatic cancer is with medication, unless early diagnosis has been developed and surgery is done.

The drug gemcitabine is mainly used for the treatment of pancreatic cancer, but its effect is minimal and it is the gemcitabine effect that increases the survival period up to 6 months.

Therefore, it is necessary to develop pancreatic cancer treatment drugs that are more effective than gemcitabine, adjuvants that increase the effect of gemcitabine, and health foods for pancreatic cancer patients.

To date, anticancer agents have been shown to reduce the survival rate of cancer cells through apoptosis, slow down the rate of cancer cell division through cell cycle arrest, increase antioxidant, anti-inflammatory, It was developed for the purpose of.

Autophagy was found by Christian de duve in 1963, and it has recently been shown that self-extinguishing affects the survival rate of cancer cells, and it is a new target of development of anticancer drugs.

While the apoptosis is the mechanism of cell death, the original concept of autophagy is to decompose damaged or useless organs in the cell during nutrient deficiency and supply the resulting energy to the cells, Has been known to increase survival rate.

From the cell membrane, vesicle nucleation begins, phagophore is formed, elongation occurs, and autophagosome is formed. When autophagosome and lysosome are fused, autolysosome is formed. Then energy is generated through the process of degradation.

Many proteins are involved in the autophagy mechanism, among which LC3 (microtubule-associated protein 1A / 1B-light chain 3) -II is used as a marker of autophagy.

Recently, autophagy has been reported as a tumor suppressor, and autophagy is called programmed cell death type 3 or autophagic cell death.

On the other hand, Mangosteen ( Garcinia mangostana L.) is a fruit tree originating in Malaysia. Its fruit is a flat ball with a diameter of 4-7 cm and ripened in purplish black.

Southeast Asia has been used as a traditional folk remedy for the treatment of abdominal pain, diarrhea, dysentery, wound infections, pus, pus and chronic ulcers. Antimicrobials, antibacterial, anti-inflammatory, anti-allergic, neuroprotective, cardiovascular protective effects and antioxidant effects of α and γ-mangostin, components extracted from the bark of mangosteen fruit, have been experimentally proven.

Mangosteen functional food compositions are disclosed in Korean Patent Publication No. 10-2005-0083860 as prior art related to the present invention.

The prior art is directed to a food composition having the functionality of an active ingredient contained in mangosteen by using mangosteen, wherein the xanthone contained in the mangosteen rind is selected from the group consisting of antidepressants, anti-tuberculosis, Inflammatory, cardiovascular, anti-leukemia, anti-tumor, anti-ulcer, anti-allergic, antiallergic and antirino virus activity.

However, the above-mentioned prior art has an unknown effect on the inhibitory effect of? And? -Mangostin on pancreatic cancer.

In addition, the prior art documents related to the present invention have revealed that? -Mangostin inhibits pancreatic tumors in a xenograft mouse model (Non-Patent Document 1), suggesting that? -Mangostin inhibits pancreatic cancer cell survival and epithelial transition , epithelial-mesenchymal transition) (Non-Patent Document 2).

Non-Patent Document 1 describes that apoptosis of pancreatic cancer cell line is induced and suppresses pancreatic cancer. Non-Patent Document 2 discloses that pancreatic cancer is suppressed by inhibiting P13K / Akt pathway, All of the patent documents have a problem that can not be known about the mechanism of inhibiting pancreatic cancer through activation of autophagy.

Also, as a prior art document related to the present invention, there is a thesis that α-mangostin inhibits tumor growth through autophagy activation (Non-Patent Document 3).

However, the above non-patent document 3 confirms the effect on the tumor associated with the ER (endoplasmic reticulum) stress, and the test using the mouse colon carcinoma CT26 and human Her-2 / neu expressing CT26 (Her-2 / CT26) Respectively.

Tumors are classified as benign or malignant. In the case of malignant tumors, the tumor can be divided into an adnexal tumor, epithelial tumor, mixed tumor, mixed tumor, and teratoma.

Tumors are classified according to the primary organs in which tumors such as stomach cancer, thyroid cancer, colorectal cancer, lung cancer, liver cancer, breast cancer, prostate cancer, gallbladder, other bile duct cancer, pancreatic cancer, bladder cancer, kidney cancer, ovarian cancer and cervical cancer occur. The mechanism is also known to be progressed by several stages, and studies should be conducted according to the type of each tumor.

Korean Patent Publication No. 10-2005-0083860 (2005.08.26)

 Hafeez BB, Mustafa A, Fischer JW, Singha, Zhong W, Shekhani MO, Meske L, Havighurst T, Kim K, Verma AK. α-Mangostin: a dietary antioxidant derived from the pericarp of Garcinia mangostana L. inhibits pancreatic tumor growth in xenograft mouse model. Antioxid Redox Signal. 2014 Aug 10; 21 (5): 682-99.  Xu Q, Ma J, Lei J, Duan W, Sheng L, Chen X, Hu A, cells by downregulating the PI3K / Akt pathway. Biomed Res Int. 2014; 2014: 546353.  Kim, SJ, Hong, EH, Lee, BR, Park, MH, Kim, JH, Pyun AR, Kim YJ, Chang SY, Chin YW, Ko HJ. α-Mangostin Reduced ER Stress-mediated Tumor Growth through Autophagy Activation. Immune Netw. Dec 12 (6): 253-60.

A problem to be solved by the present invention is to provide a composition for preventing, ameliorating or treating pancreatic cancer containing? -Mangostin or? -Mangostin as an active ingredient.

A problem to be solved by the present invention is to provide a method for preventing, ameliorating or treating pancreatic cancer comprising, as an active ingredient,? -Mangostin or? -Mangostin which inhibits pancreatic cancer through autophagy activation To provide a composition.

The problem to be solved by the present invention is to provide a method for the production of LC3-Ⅱ in an MIA PaCa-2 or PANC-1 cell line, which comprises 0.1-20 μM of at least one selected from α-mangostin and γ-mangostin Ameliorating or treating pancreatic cancer, comprising an alpha or gamma mangosteen having an efficacy of increasing the amount of alpha or gamma mangosteen.

The present invention aims at solving the technical problem by providing a composition for prevention, improvement or treatment of pancreatic cancer which contains? -Mangostin or? -Mangostin as an active ingredient.

The present invention provides a composition for preventing, ameliorating or treating pancreatic cancer containing, as an active ingredient,? -Mangostin or? -Mangostin, which inhibits pancreatic cancer through autophagy activation, I want to solve the problem.

The present invention provides a method of increasing the expression of LC3-II in an MIA PaCa-2 or PANC-1 cell line, comprising 0.1-20 .mu.M of at least one selected from among .alpha.-mangostin and .gamma.-mangostin The present invention aims at solving the technical problem by providing a composition for prevention, improvement or treatment of pancreatic cancer containing alpha or gamma mangosteen as an active ingredient.

The composition according to the present invention has an effect of inhibiting pancreatic cancer through autophagy activation.

The composition according to the present invention has the effect of increasing the expression of LC3-II in MIA PaCa-2 or PANC-1 cell lines.

FIG. 1 is a graph showing survival rates of MIA PaCa-2 and PANC-1 pancreatic cancer cell lines after α-mangostin or γ-mangostin treatment at various concentrations.
FIG. 2 shows the results of TUNEL assay in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines after treatment with α-mangostin or γ-mangostin.
FIG. 3 is a Western blot experiment result in which α-mangostin or γ-mangostin was treated at different concentrations and the expression pattern of apoptosis-related protein was confirmed.
FIG. 4 is a Western blot experiment result in which α-mangostin or γ-mangostin was treated at different concentrations and the expression pattern of autophagy-related proteins was confirmed.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may properly define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

Therefore, the experimental examples and the reference examples described in the present specification are merely the most preferred embodiments of the present invention and are not intended to represent all of the technical ideas of the present invention. Therefore, various equivalents and variations Examples should be understood.

Experimental Example  1. Identification of survival rate of pancreatic cancer cells

1-1. Preparation for experiment

1) Cell culture

MIA PaCa-2 and PANC-1 pancreatic cancer cell lines. Both cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclon) + 10% Fetal bovine serum (FBS) + 0.1% Penicillin / Streptomycin and incubated in an incubator maintained at 5% CO ₂ and 37 ° C.

2) α and γ-mangostin

Samples were dissolved in DMSO and 20 mM stock was used. DMSO concentration was kept below 0.1% during drug treatment and 0.1% DMSO was always added to the control (0 μM). The drug was stored at 4 ° C.

1-2. Experimental course

Cells were treated with α and γ-mangostin (0, 0.1, 0.5, 1, 5, 10, 20 μM) after culturing in a 96-well plate at 5000 cells / well (100 μL) for 24 hours. Forty-eight hours after the drug treatment, 10 μL of WST-1 reagent was added to each well. After 2 hours, absorbance at 450 nm was measured. Cell viability was plotted against the control.

1-3. Experiment result

FIG. 1 is a graph showing survival rates of MIA PaCa-2 and PANC-1 pancreatic cancer cell lines after α-mangostin or γ-mangostin treatment at various concentrations.

As a result, the survival rate of MIA PaCa-2 and PANC-1 pancreatic cancer cell lines was decreased in α-mangostin or γ-mangostin-treated experimental group, and the inhibitory effect of pancreatic cancer cells was shown in a concentration-dependent manner.

Experimental Example  2. Self-destruction of pancreatic cancer cells apoptosis ) Confirm

2-1. Preparation for experiment

The experiment was carried out in the same manner as in Experimental Example 1.

2-2. Experimental course

TUNEL assay was performed to confirm the apoptosis of pancreatic cancer cells.

Cells were cultured in 15,000 cells / well (200 μL) for 24 hours in 8-well chamber slides. The cells were incubated for 24 hours with 0.1% DMSO in the control and IC ₅₀ (MIA PaCa-2 αM 8 μM and γ M 16 μM ; PANC-1 αM 13 μM, γM 20 μM).

HS-345, which was reported to cause auto-apoptosis in pancreatic cancer cells, was used as a positive control. After 48 hours of incubation, the cells were subjected to in situ cell death detection kit and AP (Roche) protocol.

2-3. Experiment result

FIG. 2 is a micrograph showing the results of TUNEL assay in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines after treatment with α-mangostin or γ-mangostin by concentration.

As a result, in the positive control group treated with HS-345, apoptosis of pancreatic cancer cells was observed. However, in the group treated with α-mangostin or γ-mangostin, Respectively.

Experimental Example  3. Self-destruction of pancreatic cancer cells apoptosis ) And self-extinguishing ( autophagy ) Related protein expression confirmation

3-1. Preparation for experiment

1) Protein extraction

Cells were cultured for 24 h at 2 × 10 ⁵ cells / well in 6-well plates, treated with α, γ-mangostin (0, 1, 5, 10, 20 μM) Proteins were extracted after further incubation.

After removing the medium, RIPA buffer was added, and incubated on ice for 5 minutes. Cells were collected with a scraper. After vortexing for 30 seconds, the supernatant was centrifuged at 14000 g for 15 minutes. Only the supernatant without pellet was transferred to a new tube and stored at -20 ° C. Phosphatase inhibitor (Roche) and protease inhibitor (Roche) were added to RIPA buffer.

3-2. Experimental course

Expression patterns of proteins were confirmed by Western blotting experiments.

Proteins were quantitated using the Pierce ™ BCA Protein Assay Kit (Thermo Scientific) according to the protocol and 30 μg protein per sample was used. 4X sample buffer and mercaptoethanol were mixed with protein, incubated at 95 ° C for 5 minutes, and placed on ice. SDS-polyacrylamide gel was prepared using 10% or 15% of the protein, and the protein was electrophoresed. After transferring to the nitrocellulose membrane, the cells were blocked with TBST buffer containing 5% milk for 1 hour, and the primary antibody was reacted overnight at 4 ° C. After washing with TBST buffer the next day, the secondary antibody was attached. After washing with TBST buffer, the band was confirmed using ECL solution. Actin was used as a loading control in this experiment.

3-3. Experiment result

FIG. 3 is a Western blot experiment result in which α-mangostin or γ-mangostin was treated at different concentrations and the expression pattern of apoptosis-related protein was confirmed.

PARP (poly (ADP-ribose) polymerase) is known to be associated with DNA repair function and programmed cell death.

The activity of PARP is involved in Caspase3. PARP is cleaved by cleaved caspase3 (c-caspase3), an activated form of caspase3.

Therefore, when PARP is cleavaged and becomes c-PARP, the function of PARP is lost and eventually apoptosis is caused.

Bax (Bcl-2-associated X protein) is a protein known to modulate mitochondrial apoptosis pathways and activates auto-apoptosis.

The results showed that the expression of Bax, c-PARP, and c-caspase3 did not change or decreased in the experimental group treated with α-mangostin or γ-mangostin.

FIG. 4 is a Western blot experiment result in which α-mangostin or γ-mangostin was treated at different concentrations and the expression pattern of autophagy-related proteins was confirmed.

LC3 (Light Chain 3) is a protein whose amount increases during self-digestion and is originally identified as a subunit of microtubule-associated proteins 1A and 1B (named MAP1LC3) [Mannm SS and Hammarback JA (1994) J Biol. Chem. 269, 11492-11497], followed by analogues to the yeast protein Apg8 / Aut7 / Cvt5, which plays an important role in self-digestion [Lang, T et al. (1998) EMBO J. 17,3597-3607].

In humans, LC3A, LC3B, and LC3C are known as three isoforms of LC3, and they undergo post-translational modification during cell self-destructing.

LC3 produces cytoplasmic form LC3-I through first cleavage of the carboxy terminal immediately following its synthesis and LC3-I is converted to LC3-II due to the ubiquitin-like system involving Apg7 and Apg3 during autolysis, This leads to autophagic vesicles (Kabeya, Y. et al. (2000) Embo J. 19, 5720-5728; He, H. et al. (2003) J. Biol. Chem. 278, 29278-29287; Tanida, I. et al. (2004) J. Biol. Chem. 279, 47704-47710; Wu, J. et al. (2006) Biochem. Biophys. Res. Commun. 339, 437-442; Ichimura, Y. et al. (2000) Nature 408, 488-492.

Thus, the presence of LC3 in autopagos and the conversion of LC3 to LC3-II, which is a lower mode of transport of LC3, is used as an index of self-extinguishing.

As a result, the expression of LC3-Ⅱ was increased in the group treated with α-mangostin or γ-mangostin compared to the control group. In particular, the expression of LC3-Ⅱ was significantly increased in the group treated with 20 μM .

Taken together, these results suggest that α-mangostin or γ-mangostin inhibits pancreatic cancer in the MIA PaCa-2 or PANC-1 cell line, which is inhibited by autophagy activation .

Example  1. α- Mangostin  Preparation of a composition for prevention, improvement or treatment of pancreatic cancer containing active ingredient

The composition according to this embodiment can contain 0.1 to 20 μM of α-mangostin, based on the results of the above-described experimental examples.

In addition, the composition according to the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent, such as a-mangostin, to prevent, ameliorate, or treat pancreatic cancer formulated into a pharmaceutical unit dosage form. Can be prepared.

Examples of the carrier, excipient and diluent include tosse, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like can be used.

The pharmaceutical dosage forms may also be used in the form of pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

When the composition containing the active ingredient is formulated, it may be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant and the like which are usually used.

In addition, each of the above pharmaceutical dosage forms may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories, .

In the solid preparation for oral administration, at least one excipient such as starch may be prepared by mixing calcium carbonate, sucrose, lactose, gelatin or the like in the extract. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

The preparation for parenteral administration may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, and a suppository.

Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In addition, the present invention can produce a health functional food composition for preventing or ameliorating pancreatic cancer by adding a food supplementary additive to the composition containing the active ingredient.

Examples of foods to which the extract can be added include various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

The health functional beverage composition of the present invention is not particularly limited to the components other than those containing the above extract, and may contain various flavors or natural carbohydrates as additional components such as ordinary beverages.

Examples of the above-mentioned natural carbohydrates are monosaccharides such as glucose, fructose; Disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above, natural flavoring agents (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavorings.

In addition to the above, the extract of the present invention can be used as a nutritional supplement, a vitamin, a mineral (electrolyte), a flavor such as a synthetic flavor and a natural flavor, a coloring agent and an aggravating agent (cheese, chocolate etc.), a pectic acid and its salt, Colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may be used independently or in combination.

Example  2. γ- Mangostin  Preparation of a composition for prevention, improvement or treatment of pancreatic cancer containing active ingredient

The composition according to the present embodiment can contain 0.1 to 20 μM of γ-mangostin based on the experimental results of the above experimental examples.

In addition, the composition according to the present invention may further comprise at least one of gamma-mangostin and a pharmaceutically acceptable carrier, excipient or diluent to prevent, ameliorate, or treat pancreatic cancer formulated into a pharmaceutical unit dosage form. And food-acceptable food supplementary additives may be added to produce a health functional food composition for the prevention or amelioration of pancreatic cancer.

The types of pharmaceutically or pharmacologically acceptable additives are as described in Example 1, and commonly used additives may be used.

Example  3. α- Mangosteen ( mangosteen ) And γ- Mangostin  Preparation of a composition for prevention, improvement or treatment of pancreatic cancer containing active ingredient

Both a-mangostin and y-mangostin have potent efficacy in pancreatic cancer cell lines, and a synergistic effect can be expected by mixing them.

The composition according to the present embodiment can contain 0.1 to 20 μM of mangostin and γ-mangostin based on the experimental results of the above experimental examples.

In addition, the composition according to the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent, such as a-mangostin and y-mangostin, to formulate a pharmaceutical unit dosage form A composition for preventing, ameliorating or treating pancreatic cancer may be prepared, and a health functional food composition for preventing or ameliorating pancreatic cancer may be prepared by adding a food auxiliary additive.

The types of pharmaceutically or pharmacologically acceptable additives are as described in Example 1, and commonly used additives may be used.

Claims (5)

A composition for the prevention, amelioration or treatment of pancreatic cancer comprising as an active ingredient at least one selected from? -mangostin and? -mangostin.
The method according to claim 1,
Wherein said composition inhibits pancreatic cancer through autophagy activation. ≪ RTI ID = 0.0 > 11. < / RTI >
The method according to claim 1,
Wherein said composition contains 0.1-20 [mu] M of at least one selected from [alpha] -mangostin and [gamma] -mangostin.
The method according to claim 1,
The composition is useful for the prevention, amelioration or treatment of pancreatic cancer, characterized in that the expression of LC3 (microtubule-associated protein 1A / 1B-light chain 3) -II is increased in the human pancreatic cancer cell line MIA PaCa-2 or PANC- / RTI >
A health functional food composition for preventing or ameliorating pancreatic cancer containing? or? -mangostin as an active ingredient.

Images