WO2011093559A1 - Composition for the prevention or treatment of gastric cancer containing ursodeoxycholic acid as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of gastric cancer containing ursodeoxycholic acid (UDCA) or a salt thereof as an active ingredient, and more specifically the present invention relates to a composition for the prevention or treatment of gastric cancer comprising ursodeoxycholic acid or a salt thereof as an active ingredient, and relates to a method for the prevention or treatment of gastric cancer comprising the step of administering an apoptosis-inducing-agent composition and ursodeoxycholic acid or a salt thereof together with a pharmaceutically acceptable carrier to an individual in need thereof. The ursodeoxycholic acid or the salt thereof according to the present invention can be used to advantage in the prevention or treatment of gastric cancer because it has an outstanding activity in activating caspase, and ultimately in inducing apoptosis in gastric cancer cells, by inducing the activity of PKC and growth promotion of oxygen species (ROS) in a fashion which is lipid-raft dependent in gastric cancer cells, and adjusting the expression or activity of cell death receptors (TNF receptor super family).

Description

Composition for the prevention or treatment of gastric cancer, which contains urosodeoxycholic acid as an active ingredient

The present invention relates to a composition for preventing or treating gastric cancer, which contains ursodeoxycholic acid (UDCA) as an active ingredient.

Stomach cancer is the highest cancer morbidity and cancer mortality in the world and is the most common cause of death from cancer in Korea, Japan, China, Russia, Central Europe, South and Central America, Hong Kong and Scandinavia. More than 16% of Korean and Japanese men are dying from stomach cancer.

In particular, in Asia such as Korea and Japan, the incidence rate of gastric cancer is higher than that of other continental races. It is reported to come from a difference. In the case of Koreans, the daily salt intake is about 20g, which is twice as much as that of Westerners. In particular, the incidence of gastric cancer is reported in Korea, Japan, Finland, and Iceland, which have a habit of eating salted fish. In addition, as a cause of gastric cancer, genetic causes other than eating habits are involved, and it is known that the incidence of gastric cancer is high in the first generation offspring of gastric cancer patients.

In addition, the symptoms of gastric cancer show a variety of symptoms ranging from no symptoms to severe pain, the symptoms of gastric cancer does not have any characteristics but general digestive symptoms, most of the early symptoms of stomach cancer And, even if the symptoms are relatively mild to feel a slight indigestion or upper abdominal discomfort, most people are easy to overlook this, causing the death rate of stomach cancer.

Currently, the main treatments for various cancers, including gastric cancer, are used by one or a combination of surgical surgery, irradiation and chemotherapy, which includes removing most of the diseased tissue. Surgical surgery is effective for removing tumor tissue or surrounding tissue but cannot be used to treat tumors in difficult-to-operate areas such as the spine or to disperse diffuse tumors scattered throughout the body, such as leukemia. Chemotherapy can treat cancer by disrupting cell replication or cell metabolism, but can be used for the treatment of various tumors, but it also acts on normal cells, causing serious side effects. In particular, it acts on hematopoietic organs in which cell division and cell metabolism are active, causing serious side effects that weaken the patient's immune system. These side effects have a great impact on the patient's life and also have a main dose limiting toxicity (DLT) which should be taken into account when administering the drug. As side effects caused by chemotherapeutic agents and radiation therapy are a major problem in the clinical treatment of cancer patients, it is urgent to develop new anticancer drugs having excellent stability in the body that can reduce the side effects of chemotherapy treatments.

Meanwhile, ursodeoxycholic acid (3α, 7β-dihydroxy-5β-cholanoic acid, hereinafter referred to as 'UDCA') has been used for liver, biliary system diseases in Korea, China, and Japan for a long time. As a main component, it is a type of bile acid found in bile, has a molecular weight of 392.58, and a molecular formula of C ₂₄ H ₄₀ O ₄ . The UDCA has a function of cleaning the microbilitery in vivo, thus releasing wastes and toxic bile acids in the microbilitery, stabilizing and protecting hepatocytes, increasing hepatic blood flow, inhibiting cholesterol absorption and biosynthesis, It has the effect of dissolving gallstones, inhibiting production, and pharmacological activity to normalize immune activity. It is a drug used with major clinical indications for gallstones and biliary tract diseases, chronic liver disease and liver function improvement, indigestion after small intestine resection and fatty liver. . In particular, the mechanism of cholesterol gallstone dissolving effect of UDCA decreases the activity of HMG CoA reductase, an enzyme required for cholesterol synthesis in the liver, reduces cholesterol secretion into bile, increases 7α-hydroxylase activity, and absorbs cholesterol in the intestine. It is known to act to lower. In addition, administration of UDCA transforms cholesterol-saturated bile into unsaturated bile. Desaturation of bile resulting from the administration of UDCA increases the ability to transport cholesterol and is a highly soluble multilamellar liposome in bile. (multilamellar liposome (mesophase)) forms liquid crystals in bile saturated with cholesterol. Therefore, gallstones are dissolved in bile supersaturated with cholesterol after UDCA due to the formation of liquid crystals.

Therefore, UDCA is widely used as a therapeutic agent for liver-related diseases, and recently, it has been found that UDCA can effectively treat rectal cancer (Korean Patent Publication No. 2008-0061327), and is induced by environmental hormones including dioxin. It has also been reported that there is an effect that can suppress the toxicity (Republic of Korea Patent No. 0368936).

However, there have been no reports on the effects of UDCA on preventing or treating gastric cancer.

Accordingly, an object of the present invention is to provide a composition for preventing or treating gastric cancer, which comprises ursodeoxycholic acid (UDCA) or a salt thereof as an active ingredient.

It is another object of the present invention to provide an apoptosis inducer composition comprising urousodeoxycholic acid (UDCA) or a salt thereof as an active ingredient.

It is another object of the present invention to provide a method for preventing or treating gastric cancer, comprising administering urousodeoxycholic acid (UDCA) to a subject in need thereof except a human with a pharmaceutically acceptable carrier. It is.

In order to achieve the above object, the present invention provides a composition for the prevention or treatment of gastric cancer comprising ursodeoxycholic acid (UDCA) or a salt thereof as an active ingredient.

In one embodiment of the present invention, the urousodeoxycholic acid (UDCA) or salts thereof may have anticancer activity by inducing apoptosis of gastric cancer cells to induce cell death.

In one embodiment of the present invention, the apoptosis (apoptosis) is by the treatment of urosodeoxycholine acid (UDCA) or salts thereof, promoting the activity of caspase (gaspase) in gastric cancer cells; Promoting activity or expression of cell death receptors; Translocation of the protein kinase C (PKC) δ protein from the cytosol to the membrane; Or by the generation of reactive oxygen species (ROS).

In one embodiment of the present invention, the caspase may be selected from the group consisting of caspase-3, caspase-6, caspase-8, caspase-9, and combinations thereof.

In one embodiment of the present invention, the cell death receptor may be selected from the group consisting of DR3, DR4, DR5, DR6, FAS, TNFR and combinations thereof.

In one embodiment of the present invention, promoting the activity or expression of the cell death receptor may be to induce apoptosis in gastric cancer cells by promoting the activity of FADD or RIP1 protein.

In one embodiment of the present invention, the generation of reactive oxygen species (ROS) or the activity of PKC (protein kinase C) δ protein may be controlled by a lipid raft located in the cell membrane.

In one embodiment of the present invention, the urousodeoxycholic acid may be included in 50μM ~ 5,000μM relative to the total weight of the composition.

The present invention also provides an apoptosis inducer composition comprising urousodeoxycholic acid (UDCA) or a salt thereof as an active ingredient.

In one embodiment of the invention, the composition may induce apoptosis in gastric cancer cells.

The present invention also provides a method for the prevention or treatment of gastric cancer, comprising administering urousodeoxycholic acid (UDCA) or a salt thereof to a subject in need thereof, including a human, with a pharmaceutically acceptable carrier. to provide.

In one embodiment of the present invention, the carrier may be one or more selected from the group consisting of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives.

Urosodeoxycholine acid or a salt thereof according to the present invention promotes the activity of caspase in gastric cancer cells, promotes the activity or expression of apoptosis receptors, promotes the activity of PKC and the activity of reactive oxygen species (ROS). Since it promotes production and ultimately induces apoptosis of gastric cancer cells, it can be usefully used for preventing or treating gastric cancer.

Figure 1A shows the concentration of UDCA and DCA (0, 250, 500, 1000μM) for each cell line of gastric cancer cell lines snu-601, snu-638, snu-1 and snu-216, respectively, MTT assay It is a graph showing the comparison of the cell survival rate, apoptotic body percent and LDH activity percent through each, Figure 1B shows the photo of the colony formed on the plate after the treatment of UDCA at each concentration for the gastric cancer cell lines 1C is a graph showing counting the number of colonies formed.

2A shows Z-DEVD-FMK, Z-VEID-FMK, Z-IETD-FMK, Z-LEHD-FMK, or Z-VAD-FMK, inhibitors of caspase 3, 6, 7, and 8 for gastric cancer cell lines After the first treatment, and then treated with 1000μM UDCA, and shows the result of measuring the degree of cell death (nucleus fragmentation) by HO / PI double staining method, Figure 2B after treating 1000μM UDCA to gastric cancer cell line, The results of measuring the activation of caspase-8 using a FLICE / Caspase 8-colorimetric assay kit are shown, and FIG. 2C is first treated with Z-IETD-FMK in gastric cancer cell lines, followed by 1000 μM UDCA treatment. Western blot shows the results of confirming the amount of protein cut caspase 3, 6 and PARP.

FIG. 3A shows siRNA FAS, siRNA DR4, siRNA DR5, or siRNA Control for gastric cancer cell lines snu-601 and snu-638 after transduction through AMAXA method, followed by 1000 μM UDCA, followed by HO / PI double The result of measuring the degree of cell death (nucleus fragmentation) by staining method is shown, Figure 3B shows the result of confirming the protein amount of caspase 3, 6 and PARP cut through Western blot, Figure 3C is FLICE / The results of measuring the caspase-8 activation using the caspase 8-colorimetric assay kit are shown.

Figure 4A is transduced siRNA FADD, siRNA RIP1 or siRNA Control for the gastric cancer cell lines snu-601 and snu-638 through the AMAXA method, and then treated with 1000μM UDCA, and then killed cells by HO / PI double staining Figure 4B shows the result of measuring the degree (nucleus fragmentation or condensation of the nucleus), Figure 4B shows the result of confirming the amount of protein of caspase 3, 6 and PARP, FADD and RIP1 cut through Western blot, Figure 4C The results of measuring caspase-8 activation using the FLICE / Caspase 8-color assay kit are shown.

Figure 5A is treated with 1000μM UDCA for gastric cancer cell lines snu-601 and snu-638, followed by incubation with time, and then fractionated the cytosolic protein and organ / membrane protein fractions from the cells, respectively. Western blot shows the results of confirming the amount of PKC δ, PKC α, PKC θ, and PKC ε protein, Figure 5B is treated with a PKC inhibitor such as rottlerin (Rottlerin), and then HO / PI double staining method The degree of cell death (nucleus fragmentation or nuclear condensation) and Western blot showed the expression level of DR5 protein and the amount of cut caspase 3 and 6 protein, and FIG. 5C shows siRNA for each gastric cancer cell line. PKC δ or siRNA Control was transduced via AMAXA method, treated with 1000 μM UDCA, followed by HO / PI double staining to confirm the degree of cell death (nucleus fragmentation or nuclear condensation) and Western blot Via PKC The expression levels of the δ and DR5 proteins and the amounts of the caspase 3, 6 and PKC δ proteins that were truncated are shown.

FIG. 6A shows the results of measuring the production of ROS by treating 1000 μM UDCA against gastric cancer cell lines snu-601 and snu-638 and calculating the ratio of DCFH-DA / HO per cell after 3 hours or 5 hours. 6B shows treatment of ROS inhibitors, 10 mM NAC, 100 μM BHA, 10 mM Tiron, 2 μM DPI, or 1000 U catalase, respectively, followed by 1000 μM UDCA treatment and HO / PI double staining for gastric cancer cell lines. Degree (nucleus fragmentation or nuclear condensation), and FIG. 6C measures the amount of caspase-3, 6, and PARP protein truncated via Western blot for gastric cancer cell lines treated as in FIG. 6B above. The results are shown.

FIG. 7A shows that the snu-601 gastric cancer cell line was treated with 1000 μM UDCA in the presence or absence of 10 mM NAC or 100 μM BHA, respectively, and then the expression level of DR5 was confirmed by Western blotting, and FIG. 7B is the snu-638 gastric cancer cell line. After treatment with 1000 μM UDCA in the presence or absence of 10 mM NAC or 100 μM BHA, respectively, the expression level of DR5 was confirmed by Western blot.

FIG. 8A shows pretreatment of 10 mM NAC to snu-601 gastric cancer cell line followed by 1000 μM UDCA and fractionation of cytosolic and organ / membrane protein fractions from cells, followed by Western blot for PKC δ. Figure 8B shows the measurement of the expression level, Figure 8B shows the measurement of the expression level of PKC δ by performing the same experiment as in Figure 8A for the snu-638 gastric cancer cell line.

9 shows confocal microcsope after treatment with gastric cancer cell lines with vehicle or UDCA, staining and staining lipid rafts with choleratoxin-fluorosome (CTx-FITC) and DR5 antibody. As observed.

FIG. 10A shows that MBCD was previously treated in gastric cancer cell lines snu-601 and snu-638, and treated with 1000 μM UDCA, respectively, and cell viability was obtained through MTT assay, and the degree of cell death through HO / PI double staining (nucleus) Fragmentation or condensation of the nucleus), FIG. 10B shows FLICE / Caspase 8-coloration, including the results of measuring the expression level of DR5 and the amount of caspase-3, 6, and PARP protein truncated by Western blot. The results of measuring the caspase-8 activation using the assay kit are shown.

FIG. 11A shows that 1 mM MBCD was pretreated with gastric cancer cell lines snu-601 and snu-638, and treated with 1000 μM UDCA, respectively, followed by fractionation of cytosolic protein and organ / membrane protein fractions from cells. , Western blot confirmed the amount of PKC δ protein, Figure 11B is pre-treated with gastric cancer cell lines snu-601 and snu-638 MBM of 1mM or 2mM, and treated with 1000μM UDCA, respectively, DCFH per cell The result of ROS generation was measured by calculating the ratio of -DA / HO.

[Revisions under Rule 91 28.09.2010]
12A and 12B show subcutaneous injection of 5 × 10 ⁶ cells / 200 ul of snu-601 cells into the side of 6 week old Athymic balb / c nude mice, and the experimental group was treated with UDCA solution (150 mg / kg / day). , 6days / week), the control group to the control group (vehicle) by intraperitoneal injection as a result of analyzing the growth of xenograft tumor in vivo, Figure 12A shows the size of the tumor (volume) formed on the side of the nude mouse It is a photograph, FIG. 12B is a result graph which shows the size of the tumor of the experimental group and a control group from the 14th to 33rd day. Statistical significance was confirmed by paired T-test.

The present invention is characterized by providing a composition for preventing or treating gastric cancer comprising ursodeoxycholic acid (UDCA) or a salt thereof as an active ingredient.

In the present invention, the pharmacologically active ingredient of the composition for preventing or treating gastric cancer may be the ursodeoxycholic acid (UDCA) compound, and the urusdeoxycholic acid compound is a salt, preferably pharmaceutical It can also be used in the form of acceptable salts.

The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid may be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid may include, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

Urusodeoxycholic acid according to the present invention is a component present in the bile of the animal in general, it can be used that is isolated from the bile of the animal or prepared by chemical synthesis known in the art, commercially available urusode Any of oxycholic acid can be used.

When using urusodeoxycholic acid separated from the bile of the animal, the separation can be obtained from the bile using the method of extracting and separating conventional materials. At this time, the extraction can be extracted using a suitable solvent known in the art, that is, water or an organic solvent, preferably purified water, methanol (methanol), ethanol (ethanol), propanol, isopropanol ), Butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane Various solvents, such as), can be used alone or in combination.

In addition, separation and purification of urusodeoxycholic acid extracted using the extraction solvent is performed by column chromatography and high-speed liquid filled with various synthetic resins such as silica gel or activated alumina. Chromatography (HPLC; High Performance Liquid Chromatography) may be performed alone or in combination, and the method of extracting and separating and purifying the active ingredient is not necessarily limited to the above-mentioned methods, and any method used in the art may be used. It is possible.

In one embodiment of the present invention was used to purchase urosodeoxycholine acid sold by ICN Biomedical or Sigma.

On the other hand, the present invention is characterized by the first time that the urusodeoxycholic acid (UDCA) or salts thereof have the activity to prevent or treat gastric cancer.

In particular, urosodeoxycholine acid or a salt thereof of the present invention is characterized by having anticancer activity by inducing apoptosis in gastric cancer cells.

In the method of treating cancer, apoptosis control therapy is recently used to block the pathological growth of unregulated cells such as cancer cells. Existing extensive necrosis drug therapy for diseases such as cancer Cytotoxic enzymes (e.g., lysozyme) leaked by killing pathological cells and destroying cell membranes of pathological cells show cytotoxicity to surrounding normal cells, which inevitably leads to excessive inflammation, resulting in high side effects. There is this.

However, cell apoptosis control therapy by apoptosis induces spontaneous killing of pathological cells or strongly inhibits the growth of these cells, which can reduce the inflammatory side effects induced by cell death of cancer cells compared to cell necrosis. There is this.

Apoptosis is genetically controlled programmed cell death, which refers to active cell death, and the apoptosis process requires energy and exhibits characteristic cell morphology. In other words, when apoptosis signal is transmitted, the cell death is determined within the cell, and the cell death process is performed. In the execution step, the cell shows characteristic biochemical and morphological changes, and when the cell death progresses, the cell contracts. The cell membrane becomes bubble-like (blebbing), and the nucleus is condensed, the DNA in the nucleus is cut into small oligonucleotide fragments, forming an apoptotic body. The apoptotic body thus formed undergoes a series of processes in which phagocytosis by macrophages causes cell death.

Apoptosis can also be broadly divided into two pathways, one is the mitochondrial regulatory endogenous pathway and the other is the exogenous pathway.

Exogenous pathways involve the assembly of a death inducing signaling complex (DISC) from activation of a death receptor (eg, FADD) induced by a ligand and thus the activation of the protease caspases. In particular, activation of caspase 8 and 10 activates cleavage of caspase-3 and proceeds downstream of the caspase chain reaction pathway (U. Sartorius et al., Chembiochem 2 (2001) 20-29).

In other cell types, signals from activated receptors do not produce caspase signal transduction cascades that are strong enough to cause cell death. In this case, the signal needs to be amplified via the mitochondrial dependent apoptosis pathway. In this pathway, caspase-8 activates Bid and translocates it to the mitochondria, which releases cytochrome c into the cytoplasm. Cytochrome c interacts with Apaf-1 and Apaf-1 activates caspase-9, which in turn activates caspase dependent pathways (EA Slee et al, J. Cell Biol. 144 (1999) 281-292) .

Mitochondria play a key role as modulators in the endogenous apoptosis pathway, where cellular damage is mediated by DNA damage, hypoxia, cellular stress or chemotherapeutic agents (A. Ashkenazi, Nature Reviews Cancer 2 (2002), 420-430). Disruption of the mitochondria leads to the release of SMAC / DIABLO and cytochrome c into the cytoplasm.

Therefore, the present inventors investigated whether urosodeoxycholic acid has an effect of inducing apoptosis on gastric cancer cells. According to one embodiment of the present invention, gastric cancer cell lines snu-601, snu-638, snu-1 and snu Induction of apoptosis by treatment of urosodeoxycholine acid (UDCA) with -216 cell lines, respectively, followed by MTT assay, apoptotic body percent, LDH activity percent, colony formation assay, HO / PI double staining, etc. As a result, it was confirmed that apoptosis was induced by UDCA in all of the gastric cancer cell lines, resulting in cell death (see FIGS. 1A to 1C), and it was shown that cell death was increased in a concentration-dependent manner of UDCA. In particular, it was confirmed that cell death by UDCA is caused by apoptosis (apoptosis) rather than cell necrosis (necrosis).

Therefore, the present inventors have found that gastric cancer cell death by urosodeoxycholine acid (UDCA) of the present invention is due to apoptosis mechanism, not cell necrosis.

In addition, in the case of cell death induced by activation of the death receptor in the apoptosis process, the initiation of the cell death mechanism is activated from the reaction of the death receptor and the ligand, and then the adapter associated with the cell death to the death receptor. Factors are recruited and involved in oligomerization (Krammer PH. CD95 (APO-1 / Fas) -mediated apoptosis: Live and let die. Adv Immunol . 71: 163-210,1999).

These death receptor proteins are mostly type I transmembrane proteins, such as TNFR-1, CD95 / Fas, TNF-associated apoptosis-inducing ligand (TRAIL) receptor-1 (DR4), TNF-related apoptosis-inducing ligand (TRAIL) receptor- 2 (DR5), belonging to the Tumor Necrosis Factor (TNF) receptor superfamily, including death receptors 3 and 6, and when TRAIL binds to DR4 or DR5 or Fas ligand binds to CD95 / Fas, it is Fas-associated as a receptor Recruit the death domain (FADD), FADD again attracts caspase-8 to the death receptor to form a death attracting signal complex (DISC). DISC formed activates caspase-8 and activated caspase-8 induces apoptosis in cells according to its underlying signaling system.

On the other hand, PKC (protein kinase C) is a protein present in many tissues or organs of animals such as the brain, spleen, and heart, and functions as signal transdution, cell growth, gene expression, and tumor promotor. It is known to have In many animal cells, changes in pH due to phosphorylation of PKC and concentration changes of Na ⁺ and H ⁺ are known to be involved in cell activity, and in some cells are also known to be proliferation signals.

PKC also increases the transcription of certain genes. In particular, the activity of PKC by DAG in secondary messengers produced by hydrolysis of phosphatidylinositol may be similarly expressed by the binding of monoacylglycerol or phorbol esters. . Phorbol esters have the function of a cancer promoter in animal cells and are known to induce the growth of cancer cells.

In addition, PKC is known to play an important role in the production of free radicals (ROS) in the cell, the production of free radicals such as superoxide anion in the cell is a flavoprotein-containing enzyme NADPH oxidase, It is known to occur through xanthine oxidase, NO synthase, and mitochondrial electron transport system.

In addition, in the inactivated form of PKC, most of the PKC is present in the cytosol, and when PKC is activated, the cytosol moves from the cytosol to the cytoskeleton associated with the cell membrane, nucleus and membrane. (translocation) is (Nishizuka Y. The molecular heterogeneity of protein kinase C and its implication for cellular regulation Nature .334:. 661-665,1998).

In addition, PKCs are subdivided into three different subfamily and there are more than 12 isomeric forms in mammalian cells. Among these isomeric forms, in particular the activity of PKC δ and migration into the cell membrane act to induce the initiation of apoptosis in cells, which is responsible for promoting the activity of caspase 3 and lower signal transduction to induction of apoptosis. Known (Brodie C and Blumberg PM.Regulation of cell apoptosis by protein kinase c δ. Apoptosis . 8: 19-27,2003).

Accordingly, the present inventors analyzed whether caspases, which are cell death factors, are involved in apoptosis induction by UDCA. According to one embodiment of the present invention, after treating a caspase inhibitor in a gastric cancer cell line, Treatment with UDCA and confirming the induction of apoptosis of cells showed that treatment with caspase inhibitors inhibited apoptosis by UDCA compared to the absence of treatment (see FIG. 2A). It has been shown that there is activity to increase (see FIG. 2B), and when the inhibitor of caspase 8 was treated, it was found that cleavage of caspase 3, 6 and PARP by activation of apoptosis did not occur (FIG. 2C). Reference).

Thus, the UDCA of the present invention can induce apoptosis by activating caspases in gastric cancer cells, which may be, but are not limited to, caspases-3, 6, 8 and 9, preferably Apoptosis can be induced by activating caspase-8 and cascaded caspases 3, 6 and 9 present downstream thereof.

In addition, the UDCA of the present invention is characterized by having an action of promoting the expression or activity of apoptosis receptors in gastric cancer cells, the apoptosis receptor is not limited thereto, DR3, DR4, DR5, DR6, FAS and TNFR It may include.

For example, according to one embodiment of the present invention, the expression of DR5 was increased when UDCA was treated to gastric cancer cells, whereas siRNA was used to suppress the expression of DR4, DR5 and FAS in gastric cancer cells. Subsequently, treatment with UDCA showed that apoptosis by UDCA was suppressed (see FIGS. 3A-3C).

According to another embodiment of the present invention, when apoptosis is induced by UDCA in gastric cancer cells, to investigate the effect of UDCA on the expression or activity of cell death factors, ie FADD and RIP1, using siRNA After suppressing the expression of the gene, treatment of gastric cancer cells with UDCA showed that apoptosis was reduced compared to the control (see FIGS. 4A and 4B).

Thus, the UDCA of the present invention promotes expression or activity of apoptosis receptors in gastric cancer cells, and activation of such apoptosis receptors promotes apoptosis by promoting the activity of adapter factors (eg, FADD or RIP1) associated with cell death. Can be derived.

Furthermore, the present inventors examined whether UDCA can activate PKC in a cell death mechanism of gastric cancer cells by UDCA, and according to an embodiment of the present invention, after treating UDCA to gastric cancer cell lines, cytoplasmic fraction and membrane of cells The fractions were extracted and Western blots were used to confirm the positional shifts of the PKC isomers in the cells. In the case of PKC δ, the migration from the cytosol to the membrane by UDCA was confirmed (see FIG. 5A). On the other hand, when PKC δ inhibitor or siRNA PKC δ was treated to inhibit the expression or activity of PKC δ, caspases not only inhibited the activity but also decreased the expression of the death receptor DR5, indicating that apoptosis was suppressed (FIG. 5B and 5C).

Therefore, the present inventors found that the activity of the death receptor DR5 and caspases in the apoptosis of cancer cells by UDCA is regulated by PKC δ, so that PKC δ plays a very important role in the initiation of apoptosis of cancer cells by UDCA. I could see that.

On the other hand, reactive oxygen species (ROS), that is, active oxygen, are also called oxygen-derived species because they are oxygen-derived materials, and some of them have a single electron at the outermost shell and are highly reactive. Also known as (radical). ROS is a singlet oxygen, hydrogen, as well as radicals such as superoxide anion radicals (O2-), hydroxyl radicals (OH.), Peroxyl radicals (ROO), and the like. It refers to all non-radical materials such as peroxide, etc. Its variety is very diverse, and the reaction time of ROS is very short while the reaction force is very large, so that it is possible to denature various proteins, lipid peroxidation and DNA in vivo. It is known to have harmful effects such as mutation (Li W, Hill HZ, Induced melanin reduces mutations and cell killing in mouse melanoma.Phytochem Photobiol . 65, pp.480-485, 1997), ultimately cell damage and cell death. (McCord JM. Human disease, free radicals and the oxidant / antioxidant balance. Clin Biochem . 26: 351-357,1993).

Therefore, the inventors confirmed that such ROS is involved in the anticancer activity of gastric cancer of UDCA. According to one embodiment of the present invention, when UDCA was treated to gastric cancer cells, the production of ROS increased and cell death was increased. On the other hand, treatment with ROS scavenger reduced ROS production and decreased cell death (see FIGS. 6A to 6C). Expression of DR5 by UDCA and PKC δ activity (from cytosol to membrane) It can be seen that the movement of is also regulated by ROS production (see FIGS. 7A, 7B, 8A, and 8B), and the fact that UDCA has an effect of inducing ROS production was first identified in the present invention.

On the other hand, cholesterol present in the cell membrane of eukaryotic cells is a component that plays a very important role in the composition, maintenance and fluidity of the membrane, such cholesterol is not formed in a uniform form in the cell membrane but is formed in a dense form at a specific site, This site in the stomach is referred to as "lipid raft".

Thus, these lipid raft domains contain relatively high amounts of cholesterol and glycosphingolipids, and include signal transduction proteins such as Src-family kinase, hetero-trimeric G protein subunits, and receptor tyrosine kinase to activate the phosphorylation chain reaction. (Simons K and Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol. 1: 31-9,2000; Dimanche-Boitrel MT, Meurette O, Rebillard) A and Lacour S. Role of early plasma membrane events in chemotherapy-induced cell death.Drug Resist Updat. 8: 5-14, 2005).

Accordingly, the present inventors have investigated MBCD, a reagent that removes cholesterol from lipid rafts and damages the structure of lipid rafts, in order to investigate whether lipid raft domains are involved in the death signal transmission process of cancer cells by UDCA, in particular, apoptosis. After treatment with cells, apoptosis by UDCA was observed, and MBCD treatment resulted in inhibition of caspases activity by UDCA, decreased expression of DR5 and ultimately suppressed apoptosis (FIG. 9, 10A and 10B).

In addition, through another embodiment of the present invention, as a result of damaging the structure of the rapid raft domain by treating MBCD, it was shown that the production of ROS by UDCA is partially inhibited and the activity of PKC δ is also inhibited (FIGS. 11A and 11B). Reference).

Thus, the present inventors have found that the structure of the lipid raft domain present in the cell membrane during the apoptosis process of gastric cancer cells by the UDCA of the present invention acts as an important regulator of cell death signal transduction.

Therefore, ursodeoxycholic acid (UDCA) of the present invention is effective in treating gastric cancer by inducing cell death in gastric cancer cells, and the gastric cancer cells in the present invention may include any gastric cancer cells. And for example snu-601, snu-638, snu-1 and snu-216.

Accordingly, the present invention can provide a composition for preventing or treating gastric cancer, which includes ursodeoxycholic acid (UDCA) or a salt thereof as an active ingredient.

In the present invention, the composition for preventing or treating gastric cancer may include a pharmaceutically effective amount of the urousodeoxycholic acid compound alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount in the above means an amount sufficient to prevent, ameliorate and treat gastric cancer symptoms.

The pharmaceutically effective amount of urosodeoxycholic acid according to the present invention is 0.1 to 1,500 mg / day / kg body weight, preferably 0.5 to 900 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the degree of gastric cancer symptoms, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

In addition, "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like when administered to a human.

Examples of such carriers, excipients and diluents include lactose, 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 and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

The composition for preventing or treating gastric cancer symptoms according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscle, and the dosage of the active ingredient is determined by the route of administration, age, sex, weight and It may be appropriately selected depending on various factors such as the severity of the patient. In addition, the composition for preventing or treating gastric cancer of the present invention can be administered in parallel with a known compound having the effect of preventing, improving or treating gastric cancer symptoms.

Therefore, the present invention may provide a method for preventing or treating gastric cancer, comprising administering urosodeoxycholic acid to a subject in need thereof except a human with a pharmaceutically acceptable carrier. May be used at least one selected from the group consisting of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives.

Furthermore, the present invention can provide an apoptosis inducer composition comprising ursodeoxycholic acid (UDCA) or a salt thereof as an active ingredient, and the apoptosis inducer composition according to the present invention can induce apoptosis in gastric cancer cells. have.

In general, cancer is caused mostly by an imbalance between cell death by apoptosis and cell proliferation by intracellular signal transduction mechanisms. In general, in cancer tissues, the signal transduction mechanisms involved in cell proliferation are activated, whereas cells related to apoptosis are activated. It is well known in the art that internal signal transduction is inhibited.

Thus, apoptosis inducers are effective in treating cancer by activating apoptosis in cancer cells and inducing planned cell death in cancer cells.

In the present invention, the apoptosis (apoptosis) can be induced through any apoptosis activity mechanism occurring in the cell, and preferably, by the treatment of urosodeoxycholine acid (UDCA), caspase in gastric cancer cells (caspase) Promotion of activity); Promoting activity or expression of apoptosis receptors; Translocation of the protein kinase C (PKC) δ protein from the cytosol to the membrane; Or by the generation of reactive oxygen species (ROS).

In addition, gastric cancer prevention or treatment composition and apoptosis inducer composition comprising the urusodeoxycholic acid (UDCA) or a salt thereof according to the present invention as an active ingredient in an amount of 250 μM to 1000 μM based on the total weight of the composition. May be included.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example>

(1) experimental material

The reagents and antibodies used in the experiments of the following examples are as follows. That is, Ursodeoxycholic acid (UDCA) was purchased from ICN Biomedical or Sigma, and Deoxycholic acid (DCA), Z-DEVD-FMK (caspase-3 inhibitor) ), Z-VEID-FMK (caspase-6 inhibitor), Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), Z-VAD-FMK (poly-caspase inhibitor) Ac-LEVD-CHO (caspase-4 inhibitor) was purchased from Calbiochem and used N-acetyl-L-cysteine (NAC), Butylated hydroxyanisole (BHA) and 1,2-dihydroxybenzene-3, 5-disulforic acid (Tiron) was purchased from Roche, Methyl-β-Cyclodextrin (MBCD), catalase and DPI were purchased from Sigma Aldrich, UO126, PD98059, Rottlerin, 109203x and The GO6976 reagent was used from AG Scientific.

In addition, for antibodies used, Cleaved caspase-3 (Asp175) antibody, Cleaved caspase-6 (Asp315) antibody, p-MEK1 / 2 antibody, t-MEK1 / 2 antibody, p-EGFR antibody and t-EGFR antibody were P-ERK1 / 2 antibody, t-ERK1 / 2 antibody, FADD antibody, Caveolin-1, Goat anti-mouse IgG-HRP antibody and Goat anti-rabbit IgG-HRP were used from Cell signaling. Antibodies were purchased from Santa Cruz Biotechnology Inc., and anti-PARP antibodies, RIP1 antibodies, PKC α, PKC δ, PKC θ and PKC ε were those purchased from BD pharmingen. DR4 antibody and DR5 antibody were used from ProSic, and Tubulin was used from Biogenex Laboratories.

(2) cell culture

In addition, the cells used in the following examples used human gastric cancer cell lines (snu-601, snu-638, snu-1 and snu-216 cells), and the cells were obtained from the Korea Cell Line Bank of Seoul National University (Korea) These cells were RPMI 1640 medium (Invitrogen, CA) supplemented with heat-inactivated 10% fetal bovine serum (FBS; Invitrogen, CA) and 1% penicillin-streptomycin (Welzen, Seoul, Korea). Incubated at 37 ° C. and at 5% carbon dioxide conditions.

The cells were also incubated overnight for drug exposure testing, then 5 × 10 ⁶ cells / 20 cm ² dish, 1.5 × 10 ⁶ cells / 10 cm ² dish, 5 × 10 ⁵ cells / 6 cm ² dish, 2 × 10 ^The cells were divided into ⁵ cells / 3.5 cm ² dishes, and in the case of 24 well plates, cells were divided and used to have a cell number of 5 × 10 ⁴ cells / well.

<Example 1>

MTT In an assay  Cell survival rate, Apoptotic body  percent, LDH  Percent and Colony  In human gastric cancer cells through formation analysis UDCA of Apoptosis  Induction measurement

[Revisions under Rule 91 28.09.2010]
The present inventors investigated whether urosodeoxycholine acid (UDCA) has activity to induce apoptosis of the cancer cells against gastric cancer cell lines. For this purpose, the cancer cell lines snu-601, snu-638, snu- 1 and snu-216 cell lines were used, and the cells were each dispensed into 24 well plates at 5 × 10 ⁴ cells / well, treated with UDCA at concentrations of 250, 500 and 1000 μM and then in FIGS. 1A-1C. All cells were incubated for 48 hours, and in subsequent experiments, SNU601 was incubated for 24 hours and SNU638 for 48 hours, respectively, and the survival rate of the cells was analyzed by MTT analysis.

In this case, the MTT assay is treated with 0.5μg / ml MTT to each of the cells cultured after treatment with UDCA and incubated for 4 hours, the cells of each plate collected and collected at room temperature for 5 minutes at 1000 rpm for 5 minutes Centrifuged. The medium was then removed, 750 ul of DMSO was added to dissolve the formazin crystal, and the absorbance was measured at 540 nm using an ELISA microplate reader (Pekin-Elmer). In this case, cells treated with nothing were used as a control group, and the cell viability of the cells was measured based on 100%.

In addition, to analyze the apoptosis form of gastric cancer cells by UDCA, a double staining method using Hoechst 33342 (HO) and PI (Propidium Iodide) was performed. HO stains the nuclei of cells with blue fluorescence, while PI It penetrates the cell membrane of damaged cells and is characterized by fluorescence. Thus, double staining with HO / PI shows normal round nuclei of blue when the cells are alive, condensed and cleaved blue nuclei in the early apoptotic phase, and late apoptotic phase (secondary necrotic). In the case of condensation and cleaved pink nucleus can be observed, in the case of cell necrotic (necrotic cell) it is observed as a pink nucleus that maintains a round shape without condensation and cleavage.

HO / PI double staining using this principle is performed by dispensing snu-601, snu-638, snu-1 and snu-216 cells into 2 × 10 ⁵ cells / 3.5 cm ² dishes, respectively, followed by the same concentration as in the MTT assay. UDCA and cells were stained by treating the cells with 1 μg / ml Hoechst 33342 and 5 μg / ml PI for each hour. Cells were then collected by treatment with trypsin, centrifuged at 1500 rpm for 10 minutes at a temperature of 4 ° C., and the cells collected at the bottom were washed with 1 × cold PBS solution and then centrifuged again as above conditions. Cells were then lysed with 500ul of 3.7% paraformaldehyde and allowed to react for 5 minutes, and then centrifuged in a cytospinner. The slides were then immediately fixed with mounted gels, washed with water, dried and covered with a glass cover slip. The slides were observed under a DM5000 fluorescence microscope (Hanil) under excitation / emission wavelengths of 340/425 nm (HO) and 580/630 nm (PI), respectively, to identify apoptosis-induced cells.

Furthermore, the present inventors performed lactate dehydrogenase (LDH) release assay to determine whether UDCA affects the process of inducing cell necrosis in the process of inducing apoptosis of gastric cancer cells. The assay is used to measure the degree of cytotoxicity caused by cell membrane damage. LDH is a relatively stable enzyme, present in all cell types and rapidly released into the cell medium when the cell membrane is damaged.

To this end, snu-601, snu-638, snu-1 and snu-216 cells are each dispensed into 5 × 10 ⁴ cells / well in a 24-well plate, incubated in 500 ul RPMI medium, and the method described above. UDCA was treated for each concentration in the same manner. Thereafter, 50ul of LDH lysis buffer was added 30 minutes before the end of constant culture time of each cell to lyse the cells. Lysed cells were centrifuged at 600 g for 10 minutes, each supernatant was placed in a 24-well micro-dispensing plate of 10 ul, followed by 100 ul of LDH reaction mixture (mix of 200 ul of WST substrate mixture and 10.5 ml of LDH assay buffer). Was added and mixed. Thereafter, the plate was stirred at room temperature for 30 minutes, and measured at a wavelength of 450 nm and a reference wavelength of 650 nm at a primary wavelength using a plate reader (Bio Rad, USA). In addition, the percent release of LDH released from each cell was calculated by the following formula.

Percent (%) Toxicity = [(Experimental LDH Release)-(Spontaneous LDH Release by Effector or Target) / (LDH Release Maximum)-(Voluntary LDH Release)] × 100%

Here, LDH activity spontaneously released from control cells was less than 2% LDH release maximum measured from fully lysed cells.

In addition, to confirm that UDCA can inhibit colony formation of gastric cancer cells, the UDCA was treated for 12 hours at concentrations of 250, 500, and 1000 μM for the gastric cancer cell lines, respectively, followed by 37 ° C./5% CO.₂Under conditions Incubate for 2 weeks and the colonies were stained with crystal violet to analyze the degree of colony formation through the colony formation assay. At this time, the measurement of the degree of colony formation was visually observed colonies formed on the plate (> 1mm).

As a result, as shown in FIG. 1A, when UDCA was treated in human gastric cancer cell lines, MTT analysis showed that survival rate of gastric cancer cells decreased, and the effect of reducing survival was that the higher the concentration of treated UDCA, As a result, the longer the treatment time, the lower the survival rate.

In addition, when gastric cancer cell lines were treated with UDCA and subjected to double staining using HO / PI, gastric cancer cells were confirmed to induce cell death by apoptosis, whereas cell necrosis in all gastric cancer cell lines was associated with apoptosis. Almost no induction was observed.

In addition, lactate dehydrogenase (LDH) release analysis showed that the activity of LDH was almost 1% or less in gastric cancer cell lines treated with UDCA. In general, when cell membrane damage occurs, the release of LDH occurs in the early stages of cell death, which is typical of necrotic cell death. On the other hand, cell death of gastric cancer cells by the UDCA of the present invention did not induce the release of LDH.

Therefore, it can be seen that cell death of gastric cancer cells by UDCA of the present invention is caused by apoptosis rather than cell necrosis.

Therefore, the present inventors have found that UDCA induces cell death by apoptosis in gastric cancer cells, but does not induce cell death by cell necrosis.

[Revisions under Rule 91 28.09.2010]
1A to 1C show the experimental results for DCA as well as the UDCA to prepare for the experimental results when the UDCA was treated.

Deoxycholine acid (DCA) has been disclosed to induce apoptosis in liver cells and colon cancer cells. However, there is no known content of inducing apoptosis in gastric cancer cells in the case of deoxycholic acid, and the present inventors treated deoxycholic acid with gastric cancer cell lines, respectively, followed by MTT assay, HO / PI double staining. And LDH release assays.

MTT analysis showed that, similarly to the treatment of gastric cancer cells with UDCA, DCA treatment also induced apoptosis of gastric cancer cells in a concentration-dependent manner, but HO / PI double staining showed 0.5 mM DCA. Abnormal treatment resulted in a significant decrease in apoptotic body percentage compared to UDCA treatment.In other words, DCA was shown to induce cell necrosis in gastric cancer cells, unlike UDCA. In the case of DCA treatment in terms of LDH activity, gastric cancer cells were found to increase the release of LDH out of the cell membrane in a concentration-dependent manner.

Through the above results, the present inventors found that DCA has an activity of inducing apoptosis of gastric cancer cells, but unlike UDCA, the mechanism of inducing cell death is achieved through cell necrosis, which is treated with UDCA in FIG. 1A. Apoptotic bis are formed without LDH release, which is supported by the presence of LDH release when treated with DCA.

In addition, in FIG. 1B and 1C, the number of colonies formed was also markedly reduced as the concentration of UDCA was increased. In particular, when treated with 1000 μM of UDCA, almost no colonies were formed.

< EXAMPLE  2>

Analysis of the effects of caspases on induction of apoptosis by UDCA in gastric cancer cells

The present inventors performed the following experiment to confirm the effect of caspases on the apoptosis action by UDCA as UDCA confirms that the UDCA induces apoptosis in gastric cancer cells, resulting in cell death.

That is, 10 μM of caspase inhibitors (Z-DEVD-FMK, Z-VEID-FMK, Z-IETD-FMK, Z-LEHD-FMK or Z) in the gastric cancer cell lines snu-601 and snu-638 cells used in the above examples. -VAD-FMK) was treated for 30 minutes, and then treated with 1000 μM of UDCA, and then cultured for 24 hours for snu-601 cells and 36 hours for snu-638 cells. The cells were then identified by apoptosis-induced cells using the HO / PI double staining method performed in the above example, and the degree of nucleation aggregation or fragmentation was measured.

In addition, the degree of activation of caspase 8 in gastric cancer cells by UDCA treatment was observed through IETD-pNA substrate cleavage assay, which assay was performed using FADD-like IL-1β-converting enzyme (FLICE) / Caspase-8 colorimetric assay kit. Experiments were performed according to the method of use of the kit using (Biovision).

In other words, 5 × 10 ⁵ gastric cancer cells were treated with 1000 μM of UDCA, and then cultured for 12, 18, 24 and 48 hours, respectively, and the cells were collected by centrifugation. The cells were then lysed with 50 ul of cold cell lysis buffer, incubated on ice for 10 minutes and centrifuged at 4 ° C. for 1 minute at 10,000 g. Transfer the supernatant to a new tube, quantify using the Biorend Protein Assay Kit, dilute to 150 ul to 50 ul with cell lysis buffer, 50 ul of 2x reaction buffer (containing 10 mM DTT) and 5 ul of 4 mM IETD- pNA substrate was added to a final concentration of 200 μΜ. The cells were incubated at 37 ° C. for 2 hours, and the absorbance was measured at 405 nm using a plate reader. As a control, untreated cells were used.

In addition, the present inventors treated Z-IETD-FMK (20 μM), an inhibitor of caspase 8, with the gastric cancer cell line for 1 hour in advance in order to confirm the importance of caspase 8 in apoptosis of gastric cancer cells by UDCA, 1000 μM of UDCA was treated to snu-601 (for 24 hours) and snu-638 cells (for 36 hours), respectively, and the amount of protein of cleaved caspase 3, cleaved caspase 6, cleaved PARP and tubulin was measured. Confirmed by performing blots.

That is, when apoptosis occurs, caspase is activated, and PARP (poly (ADP-ribose) polymerase) is cleaved, which is characterized by Z-IETD, an inhibitor of caspase 8, before treating UDCA according to the present invention. Treatment of gastric cancer cell lines with -FMK (20 μM) confirmed the amount of cleaved sections of caspase-3, caspase-6 and PARP.

As a result, as shown in Figure 2A, apoptosis induction of gastric cancer cells by UDCA was shown to be inhibited when treated with caspase inhibitors, more specifically caspase-3 inhibitors (Z-DEVD-FMK) , Caspase-6 inhibitor (Z-VEID-FMK), caspase-7 inhibitor (Z-IETD-FMK), caspase-8 inhibitor (Z-LEHD-FMK), or pan-caspase inhibitor Z-VAD-FMK Induction of atopocytosis of gastric cancer cells by UDCA is almost completely inhibited by treatment of atopossis of gastric cancer cells by treatment of caspase-9 inhibitor (Z-LEHD-FMK). It was shown to be less suppressed than in the case of treatment, and the treatment of caspase-4 inhibitor (Z-LEVD-FMK) showed little inhibition of atopocsis of gastric cancer cells by UDCA. Apoptosis of cells is mainly regulated by exogenous pathways. C.

The activity of caspase 8 was shown to increase in activity by treatment of UDCA (see FIG. 2B). In addition, in the case of apoptosis of gastric cancer cells by UDCA, western blot after treatment with caspase-8 inhibitor to confirm whether caspase 8 plays an important role, as shown in FIG. 2C, caspase 3, 6 , And sections with PARP cleaved were not observed. This is more evident compared to the Western blot results on the right side of FIG. 2A.

Thus, through the above results, we found that UDCA increases caspase 8 activity in the process of inducing apoptosis of gastric cancer cells by UDCA, thus inducing apoptosis of cells by a signal transduction system related to caspase 8 downstream. In addition, it was found that caspase 8 and caspase 3, 6, and 9 were related to apoptosis of gastric cancer cells by UDCA.

On the other hand, in FIG. 2B, the maximum activity of caspase-8 is observed at 24 hours in snu-601 but is delayed at this time in snu-638. Thus, the time point for examining the apoptosis mechanism in snu-601 cells is 24 hours. 36 hours were selected for snu-536.

<Example 3>

Expression Induction Analysis of DR5 by UDCA

It is well known that cell surface killing receptors such as DR4, DR5 and FAS play an important role in the induction of cell death in cancer cells. The present inventors investigated whether cell surface killing receptors such as DR4, DR5 and FAS are involved in apoptosis induction of gastric cancer cells by UDCA.

To this end, the expression of these receptors was eliminated in cells using siRNAs of cell surface killing receptors described in Table 1 below. That is, siRNA (8ug) of each receptor was transferred to 1 × 10 ⁶ cells of gastric cancer cells. Cells transfected with siRNA were incubated for 40 hours at 37 ° C. and 5% carbon dioxide, and then treated with 1000 μM of UDCA, respectively, and cultured for a period of time (24 or 36 hours). Then, caspase-8 activity, MTT assay and HO / PI double staining assay were performed in the same manner as described in the above examples.

Table 1 siRNA sequence

Cell surface death receptor	siRNA sequence	SEQ ID NO:
siRNA FAS (S)	5'-GCUUAUACAUAGCAAUGGU (dtdt) -3 '	One
siRNA FAS (AS)	5'-ACCAUUGCUAUGUAUAAGC (dtdt) -3 '	2
siRNA RIP1 (S)	5'-CACACAGUCUCAGAUUGAU (dtdt) -3 '	3
siRNA RIP1 (AS)	5'-AUCAAUCUGAGACUGUGUG (dtdt) -3 '	4
siRNA FADD (S)	5'-CCAAGAUCGACAGCAUCGA (dtdt) -3 '	5
siRNA FADD (AS)	5'-UCGAUGCUGUCGAUCUUGG (dtdt) -3 '	6
siRNA DR4 (S)	5'-CUGGAAAGUUCAUCUACUU (dtdt) -3 '	7
siRNA DR4 (AS)	5'-AAGUAGAUGAACUUUCCAG (dtdt) -3 '	8
siRNA DR5 (S)	5'-CAGACUUGGUGCCCUUUG (dtdt) -3 '	9
siRNA DR5 (AS)	5'-UCAAAGGGCACCAAGUCUG (dtdt) -3 '	10
siRNA c-Jun (S)	5'-ACUGUAGAUUGCUUCUGUA (dtdt) -3 '	11
siRNA c-Jun (AS)	5'-UACAGAAGCAAUCUACAGU (dtdt) -3 '	12
siRNA NF-kB (p65) (S)	5'-CCUGAGCACCAUCAACUAU (dtdt) -3 '	13
siRNA NF-kB (p65) (AS)	5'-AUAGUUGAUGGUGCUCAGG (dtdt) -3 '	14
siRNA ERK1 (S)	5'-CUCUCUAACCGGCCCAUCU (dtdt) -3 '	15
siRNA ERK1 (AS)	5'-AGAUGGGCCGGUUAGAGAG (dtdt) -3 '	16
siRNA PKCd (S)	5'-CUCAUGGUACUUCCUCUGU (dtdt) -3 '	17
siRNA PKCd (AS)	5'-ACAGAGGAAGUACCAUGAG (dtdt) -3 '	18
siRNA control group (S)	5'-CCUACGCCACCAAUUUCGU (dtdt) -3 '	19
siRNA control group (AS)	5'-ACGAAAUUGGUGGCGUAGG (dtdt) -3 '	20

            

As a result, as shown in FIG. 3A, apoptosis of cells by UDCA was found to decrease apoptosis when siRNA DR5 or siRNA FAS was introduced in snu-601 cells, whereas apoptosis was reduced by siRNA DR4. Turned out not to be. In addition, the introduction of siRNA DR4 or siRNA DR5 in snu-638 cells has been shown to reduce apoptosis, while siRNA FAS has not been shown to reduce apoptosis. , 6 and PARP activity was the same result (see Fig. 3B).

In addition, as a result of measuring the activity of caspase 8, it was shown that the activity of caspase 8 by UDCA was inhibited by siRNA DR5 or siRNA FAS in snu-601 cells, but not by siRNA DR4. In the case of snu-638 cells, the activity of caspase 8 was shown to be reduced by siRNA DR4 or siRNA DR5, while not by siRNA FAS (see FIG. 3C).

Thus, the results indicate that the apoptosis induction process of gastric cancer cells by UDCA is regulated by DR5 and FAS in snu-601 cells and by DR4 or DR5 in snu-638 cells. In particular, it was found that DR5 induces a regulatory action, ie the onset of apoptosis by UDCA, in both gastric cancer cells.

<Example 4>

Induction of apoptosis activity of UDCA via FADD and RIP in gastric cancer cells

When apoptosis is induced in cells, TRAIL generally binds to the death receptors DR4 and DR5, and FAS ligands are characterized by inducing FADD to bind to FAS through their killing effect domains. Caspase 8 is induced to bind, resulting in the assembly of apoptosis inducing signal complexes to the receptor.

In order to confirm whether apoptosis of gastric cancer cells by UDCA is mediated through cell death factors, the present inventors transduced siRNA FADD and siRNA RIP1 described in Example 3 into gastric cancer cells, and then treated and cultured with 1000 μM of UDCA. Then, the HO / PI double staining method, the Western blot and the caspase 8 activity measuring method described in the above examples were performed, respectively.

As a result, as shown in FIGS. 4A and 4B, the apoptosis level of the cells by UDCA was reduced by siRNA FADD and siRNA RIP1 in both the cells of snu-601 and snu-638. As shown in 4C, the activity of caspase 3, 6 and PARP was also reduced by siRNA FADD and siRNA RIP1. In addition, the activity of caspase 8 was also shown to be reduced by siRNA FADD and siRNA RIP1.

Therefore, the above results showed that the apoptosis of gastric cancer cells by UDCA according to the present invention was carried out through apoptosis factors of FADD and RIP1, and they showed an effect of inducing caspase 8 as a death receptor. Was found to play an important role.

Example 5

Role of PKCδ in Apoptosis Action by DR5 Induced by UDCA

PKCδ is a type of PKC super family that is known to be involved in cell apoptosis, and when PKCδ is transported to the cell membrane, mitochondria and nucleus, it activates specific mechanisms to activate caspase 3 and ultimately induce apoptosis, thus, The activity of PKC is reported to be due to redistribution of kinase at cytosol and other intracellular locations.

In order to confirm whether the UDCA activates the intracellularly placed PKCδ, the present inventors treated 1000 μM of UDCA in gastric cancer cells, and then cultured each hour (0, 1, 2, 4, 8 and 12 hours). Cellular proteins (fraction 1) and organ / membrane proteins (fraction 2) were extracted using the Proteoextract subcellular proteome extraction kit (Calbiochem), and PKCδ, PKCα, PKCθ, and PKCε cytosolic membranes were extracted by Western blot. The shift was analyzed.

As a result, as shown in Fig. 5A, the treatment of UDCA had the effect of shifting PKCδ from the cytosol to the membrane, and the migration of PKCδ to the cell membrane was performed prior to the apoptosis of cells by UDCA (snu-601, UDCA). 2-4 hours after treatment, and 4-8 hours after UDCA treatment in snu-638). In PKCα, PKCε and PKCθ, no significant shift to the cell membrane was observed.

In addition, the present inventors treated 5 μM rottlerin, ie, PKCδ inhibitors, and 1000 μM UDCA for gastric cancer cell lines, respectively, in order to confirm the degree of change in apoptosis by UDCA by treating the inhibitor of PKCδ. Then, Western blot was performed using double staining of HO / PI and antibodies of caspases. Furthermore, after transduction into gastric cancer cells using siRNA PKCδ, the expression level of caspases in the cells was confirmed by Western blot. It was. In addition, in addition to the above-described inhibitor of PKCδ, roletrin, other PKC inhibitors, GF109203X (1 μM), and PKCα inhibitor GO6979 (1 μM) were also tested.

As a result, as shown in Fig. 5B, when treated with rotlerin, an inhibitor of PKCδ, apoptosis of cells by UDCA was suppressed, and the degree of inhibition was increased in a concentration-dependent manner (GF109203X and GO6979). When treated, apoptosis of cells by UDCA was hardly inhibited). In addition, when treated with rotlerin, the activity of caspases, a cell death factor, was shown to be inhibited.

According to FIG. 5C, when inhibiting the expression of PKCδ in gastric cancer cells by siRNA PKCδ, apoptosis of the cells by UDCA was decreased, and the activity of caspase 3, 6 and PARP was also inhibited and no cleaved forms thereof were observed. .

Therefore, the present inventors have found that when UDCA is treated in gastric cancer cells, PKCδ is induced by UDCA to the cell membrane and induces apoptosis through activation of cell death factors.

In addition, the present inventors confirmed the fact that UDCA induces the expression of DR5 in gastric cancer cells through the above-described examples, to analyze the relationship between DR5 and PKCδ in apoptosis by UDCA, 5 μM of rottlerin in gastric cancer cell lines. After pretreatment for 1 hour, and then treated with 1000μM UDCA, Western blot was performed to investigate the expression level of DR5, and also after transducing siRNA PKCδ into gastric cancer cell lines, treated with UDCA and Western blot Was performed to investigate the expression level of DR5 and PKCδ.

As a result, as shown in FIG. 5B, the treatment of gastric cancer cell line with rottlerin, an inhibitor of PKCδ, showed that the expression of DR5 by UDCA was decreased, and siRNA PKCδ was used to express PKCδ in cells. Inhibition also reduced the expression of DR5 by UDCA (see FIG. 5C).

Therefore, the above results showed that the inventors found that the expression of DR5 in the apoptosis process of gastric cancer cells by UDCA is regulated by PKCδ. Thus, PKCδ is the expression of DR5 by UDCA and initiation of apoptosis of gastric cancer cells. It was found to play a very important role in the process.

<Example 6>

Analysis of association with ROS generation in apoptosis action by expression of DR5 by UDCA and migration of PKCδ

Reactive oxygen species (ROS) are known to be the major determinants of cell death mode in the process of cell death, including apoptosis and cell necrosis.

Therefore, the present inventors treated 1000 μM of UDCA in gastric cancer cell lines to confirm whether ROS are involved in apoptosis of gastric cancer cells by UDCA, and then analyzed the degree of ROS generation after 3 hours or 5 hours of incubation. Double staining of PI was performed, and the expression level of caspases was confirmed by Western blot. In addition, scavengers of ROS, NAC (10 mM), BHA (100 μM), Trion (10 mM), DPI (2 μM), and catalase (1000U), respectively, were added to the gastric cancer cells through the same method as described above. The degree of apoptosis was measured.

At this time, the ROS assay was divided into 5 × 10 ⁴ cell number of gastric cancer cells in a 24-well plate, incubated with 500ul RPMI medium, and then treated with UDCA and the cells were again cultured under the same conditions. Then, 50 μM of 2 ', 7'-dichlorofluorescein diacetate (DCFDA, Molecular probe) and 0.5 μg / ml of HO were added to each cell to confirm the generation of reactive oxygen species (ROS) and incubated for 1 hour. It was. After washing, DCFDA was measured using a fluorocount model MQM200 plate reader (Packard instrument, USA) at an excitation wavelength of 490 nm and an emission wavelength of 530 nm, and the measurement of HO was performed at an excitation wavelength of 340 nm and of 425 nm. Measured at the emission wavelength. The amount of ROS produced was calculated by measuring the ratio of DCFDA / HO per well.

[Revisions under Rule 91 28.09.2010]
As a result, as shown in Figs. 6A to 6C, when UDCA was treated in gastric cancer cells, the production of ROS was increased in the cells (see Fig. 6A), and various kinds of ROS scavengers were treated. In one case, in particular, NAC and BHA have been shown to inhibit cell death through apoptosis by UDCA (see FIGS. 6B and 6C).

Thus, the results showed that apoptosis of gastric cancer cells by UDCA is regulated through the production of ROS.

In addition, the inventors added NAC or BHA, which is a scavenger of UDCA and ROS, to the gastric cancer cells, respectively, and examined DR5 through Western blot to investigate how the expression of ROS is induced by UDCA. The expression level of was investigated.

As a result, as shown in FIGS. 7A and 7B, DR5 induced by UDCA in gastric cancer cells was found to have decreased expression by NAC and BHA.

Therefore, the results indicate that the expression of DR5 by UDCA is regulated by the production of ROS, and furthermore, ROS acts as another important regulator in the expression of DR5 by UDCA.

On the other hand, PKCδ is known as a factor present downstream of the ROS generation mechanism. Therefore, the present inventors performed an experiment to determine whether the production of ROS regulates the trasnlocation of PKCδ during apoptosis of gastric cancer cells by UDCA, that is, 1 hour treatment of 10 mM of NAC for gastric cancer cell lines for 1 hour. Cellular protein (fraction 1: C) and organ / membrane protein (fraction 2: M), respectively, were obtained from the cells at (2, 4, and 8 hours) and then the amount of PKCδ was determined via western blot.

As a result, the migration of PKCδ cytosol to membrane by UDCA was found to be reduced when the NAC, a scavenger scavenger, was treated (see FIGS. 8A and 8B).

Thus, the results indicate that the migration of PKCδ by UDCA is regulated by the production of ROS, and PKCδ is present downstream of the ROS production mechanism in the process of apoptosis by UDCA in gastric cancer cells. It was confirmed that the factor.

<Example 7>

Lipid Raft in Apoptosis Action by Expression of DR5 Induced by UDCA of lipid raft  role

Cholesterol has been known to play a very important role in the composition, maintenance and fluidity of the membrane. In particular, a cholesterol-rich microdomain, also known as a lipid raft, plays an important role in signal transduction of apoptosis and is associated with plasma cholesterol associated with the formation of FAS-FADD, DR4 or DR5-FADD complexes and activation of caspase 8 It is known to play an important role in the change.

The present inventors used MBCD, a lipid raft deficient reagent, to investigate the relationship between apoptosis and lipid raft of gastric cancer cells by UDCA. First, the gastric cancer cell line was treated with a vehicle or UDCA for 8 hours. , Fixed and anti-DR5 / FITC-CTxB (10μg / ml) / HO (1μg / ml) staining for 30 minutes and observed by confocal microscopy (confocal microcsope, FV1000, Olympus).

In addition, the gastric cancer cell line treated with 0.5 or 1 mM MBCD for 1 hour, and then treated with 1000 μM of UDCA, followed by incubation for 24 and 36 hours, respectively, MTT assay, HO / PI double staining method and caspase 8 activity The degree of death of the cells was measured through the measurement. In addition, the expression level of DR5 was measured by Western blot.

As a result, as shown in Fig. 9, treatment of gastric cancer cells with UDCA showed that the formation of lipid rafts was clearly increased.

In addition, cell death by apoptosis of gastric cancer cells by UDCA was shown to be inhibited when MBCD was treated (see FIG. 10A). In addition, HO / PI staining showed that, when MBCD treated, condensation and fragmentation of the nucleus showing apoptosis in gastric cancer cells was reduced compared to the group not treated with MBCD (see FIG. 10A), and caspa by UDCA Eighth activity was also shown to be inhibited by the treatment of MBCD (see Figure 10B).

In addition, in the case of DR5 induced expression by UDCA, it was confirmed that the expression level was changed by the treatment of MBCD. As a result, the expression of DR5 by UDCA was suppressed by MBCD (see FIG. 10B).

Accordingly, the results showed that the apoptosis of gastric cancer cells by UDCA was regulated by lipid rafts.

<Example 8>

Role of Lipid Rafts in ROSCA-activated ROS / PKCδ

As a result of Example 7 confirmed that the lipid raft plays a very important role in the cell death mechanism of gastric cancer cells by UDCA, whether the lipid raft is also involved in the activity of the ROS / PKCδ confirmed through the previous embodiment Investigated.

To this end, after treating 1 or 2 mM MBCD for gastric cancer cell line for 1 hour, 1000 μM of UDCA, followed by incubation for 24 hours and 36 hours, respectively, and extracting the cytoplasmic catalase / membrane fraction from the cells, respectively. The expression level of PKCδ was measured by Western blot, and the degree of ROS generation was measured by the same method as described in the above example.

[Revisions under Rule 91 28.09.2010]
As a result, as shown in FIGS. 11A-11B, the cytosolic migration of PKCδ by UDCA to membranes in gastric cancer cells was shown to be inhibited due to the treatment of MBCD (see FIG. 11A), and through these results PKCδ It can be seen that is a downstream event of lipid raft during apoptosis of cells. In addition, ROS production assays showed that ROS production by UDCA in gastric cancer cells was reduced due to the treatment of MBCD (see FIG. 11B).

Therefore, the above results indicate that the activity of ROS / PKCδ by UDCA is regulated by lipid raft. Thus, lipid raft is a function of DR5 by UDCA production of ROS and movement of PKCδ from cytosol to cell membrane. It was found that it acts as a very important regulator of apoptosis in gastric cancer cells.

Example 9

Analysis of the effect of UDCA administration on tumor growth in snu-601 xenograft mice in vivo

To analyze the growth of xenograft tumors in vivo, 6-week-old Athymic balb / c nude mice were purchased (Orien Bios, Korea) and snu-601 cells in the flank region of these mice. was injected ssikeul 5 × 10 ⁶ gae / 200ul (subcutaneous injection). From the day after injecting gastric cancer cells (day 1), 10 mice per group were administered intraperitoneally with UDCA solution (150mg / kg / day, 6days / week) or carrier (volume) formed from day 14 Was measured and the experiment was continued until day 33. Tumor size was expressed as relative value of mean tumor size formed on day 14. The body weight of the mice was also measured during the experiment, and no significant difference in body weight was observed between the UDCA treated group and the control group. Statistical significance was confirmed by paired T-test.

As a result, as shown in Figs. 12A and 12B, compared with the control group administered the carrier 300ul after snu-601 cell injection (upper line in Fig. 12A and the line in Fig. 12B), 300ul of UDCA after snu-601 cell injection. It can be seen that the tumor size of mice (bottom of FIG. 12A and line ▲ of FIG. 12B) of the group to which 300 ul of the solution was administered decreased noticeably.

In other words, it could be seen that the growth of tumors was statistically significantly suppressed in the UDCA-administered group.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

The present invention is a result supported by the following Korean national R & D projects:

(Project unique number) R13-2003-009

Ministry of Education, Science and Technology / Korea Research Foundation

(Research Project) Leading Research Center Development Project

(Name of Project) 1 Overall 2 Details: A Study on the Identification of Resistance Mechanisms by Endogenous Factors and Development of Overcoming Strategies

(Organizer) Resistant Cell Research Center

(Study period) September 1, 2003-August 31, 2012.

Claims (10)

1. A composition for preventing or treating gastric cancer, comprising ursodeoxycholic acid (UDCA) or a salt thereof as an active ingredient.
2. The method of claim 1,

   The urosodeoxycholine acid (UDCA) or a salt thereof is a composition for preventing or treating gastric cancer, characterized by having anti-cancer activity by inducing apoptosis (apoptosis) of gastric cancer cells to induce cell death.
3. The method of claim 2,

   The apoptosis (apoptosis) may be promoted by the treatment of urosodeoxycholine acid (UDCA) or salts thereof to promote the activity of caspase in gastric cancer cells; Promoting activity or expression of cell death receptors; Translocation of the protein kinase C (PKC) δ protein from the cytosol to the membrane; Or a composition for preventing or treating gastric cancer, which is induced by the generation of reactive oxygen species (ROS).
4. The method of claim 3,

   The caspase (caspase) is a composition for preventing or treating gastric cancer, characterized in that selected from the group consisting of caspase-3, caspase-6, caspase-8, caspase-9 and combinations thereof.
5. The method of claim 3,

   The cell death receptor is a composition for preventing or treating gastric cancer, characterized in that selected from the group consisting of DR3, DR4, DR5, DR6, FAS, TNFR and combinations thereof.
6. The method of claim 3,

   Promoting the activity or expression of the cell death receptor is a composition for preventing or treating gastric cancer, characterized in that to promote the activity of the protein of FADD, RIP1 or both to induce apoptosis in gastric cancer cells.
7. The method of claim 3,

   The production of reactive oxygen species (ROS), the activity of protein kinase C (PKC) δ protein or both are controlled by lipid raft (lipid raft) located in the cell membrane, characterized in that the composition for preventing or treating gastric cancer.
8. The method of claim 1,

   The urousodeoxycholic acid or its salt is a composition for preventing or treating gastric cancer, characterized in that it comprises 250μM ~ 1000μM relative to the total weight of the composition.
9. An apoptosis inducer composition comprising urousodeoxycholic acid (UDCA) or a salt thereof as an active ingredient.
10. The method of claim 9,

    Wherein said composition induces apoptosis in gastric cancer cells.

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