KR101131675B1 - Composition comprising ursodeoxycholic acid for preventing or treating gastric cancer - Google Patents

Abstract

The present invention relates to a composition for preventing or treating gastric cancer containing ursodeoxycholic acid (UDCA) as an active ingredient, and more particularly, the present invention relates to ursodeoxycholic acid (UDCA). Or administering to the subject in need thereof a composition for preventing or treating gastric cancer, apoptosis inducing agent composition and urusdeoxycholine acid (UDCA) comprising a salt thereof as an active ingredient, together with a pharmaceutically acceptable carrier. It relates to a method for preventing or treating gastric cancer. Urusodeoxycholine acid according to the present invention promotes the activity of caspases in gastric cancer cells, promotes the activity or expression of apoptosis receptors, promotes the activity of PKC and the generation of reactive oxygen species (ROS) Ultimately, since the activity of inducing apoptosis of gastric cancer cells is excellent, it can be usefully used for preventing or treating gastric cancer.

Description

Composition comprising ursodeoxycholic acid for preventing or treating gastric cancer}

The present invention relates to a composition for the prevention or treatment of gastric cancer containing 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 continents, which is hardly regarded as a race or ethnic difference, and the most important difference in living environment, especially dietary life 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, a genetic cause other than eating habits is involved, and it is known that the incidence of gastric cancer is high in the first generation 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 little indigestion or upper abdominal discomfort, most people are easy to overlook this cause 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 waste and toxic bile acids in the microbilitery, stabilizing and protecting the hepatocytes, increasing hepatic blood flow, inhibiting cholesterol absorption and biosynthesis, It is a drug that has major clinical indications for gallstone disease, biliary tract disease, chronic liver disease and liver function improvement, indigestion after small bowel resection and fatty liver, as it has pharmacological activity that dissolves gallstones, inhibits production and normalizes immune activity. . In particular, the mechanism of cholesterol gallstone dissolution of UDCA decreases the activity of HMG CoA reductase, an enzyme necessary for cholesterol synthesis in the liver, which 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 bile cholesterol-saturated bile into unsaturated bile. Desaturation of bile resulting from 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, it is an object of the present invention to provide a composition for the prevention or treatment of gastric cancer comprising ursodeoxycholic acid (UDCA) or a salt thereof as an active ingredient.

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

Another object of the present invention is 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. .

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 urusodeoxycholine acid (UDCA) may have anticancer activity by inducing apoptosis (apoptosis) of gastric cancer cells to induce apoptosis.

In one embodiment of the present invention, the apoptosis (apoptosis) is by the treatment of urosodeoxycholine acid (UDCA), promoting the activity of caspase (gaspase) in gastric cancer cells; 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 one embodiment of the present invention, the caspase may be selected from the group consisting of caspase-3, caspase-6, caspase-8 and caspase-9.

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

In one embodiment of the present invention, promoting the activity or expression of the apoptosis 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) and 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 a 250μM ~ 1000μM relative to the total weight of the composition.

The present invention also provides an apoptosis inducer composition comprising urusodeoxycholic 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 preventing or treating gastric cancer, comprising administering urosodeoxycholic acid (UDCA) to a subject in need thereof except a human with a pharmaceutically acceptable carrier.

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.

Urusodeoxycholine acid according to the present invention promotes the activity of caspases in gastric cancer cells, promotes the activity or expression of apoptosis receptors, promotes the activity of PKC and the generation of reactive oxygen species (ROS) Ultimately, since the activity of inducing apoptosis of gastric cancer cells is excellent, it can be usefully used for preventing or treating gastric cancer.

Figure 1a is treated with each concentration of UDCA (0, 250, 500, 1000μM) for each cell line of gastric cancer cell lines SNU-601, SNU-638, SNU-1 and SNU-216, the cells through the MTT assay It is a graph showing a comparison of the survival rate, percentage of apoptotic nucleus and LDH release, respectively, Figure 1b shows the visual observation of the colonies 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.
Figures 2a and 2b is treated with UDCA (0, 250, 500, 1000μM) for each concentration for gastric cancer cell lines SNU-601 and SNU-638, the degree of cell death by HO / PI double staining method using a fluorescence microscope The number of observed and killed cells is graphed, and 2c shows the results of the LDH release analysis using the LDH-toxicity assay kit for gastric cancer cell lines treated with UDCA for each concentration. The amount of caspase 3, 6 and proteins of PARP and LC3 cut in UDCA treated gastric cancer cell lines was confirmed by Western blot.
Figure 3a is a graph showing the cell viability through MTT assay after the treatment of each concentration of DCA (0, 250, 500, 1000μM) for gastric cancer cell lines SNU-601 and SNU-638, 3b and 3c is HO The degree of cell death was observed by fluorescence microscopy through the / PI double staining method, and the number of cells killed was graphed respectively, and 3d is a graph showing the degree of LDH release using an LDH-toxic assay kit.
4A 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 treatment with 20μM first, 1000μM UDCA treatment, and then showed the result of measuring the degree of cell death (nucleus fragmentation) by HO / PI double staining method, 4b is treated with gastric cancer cell line 1000μM UDCA, The results of measuring the activation of caspase-8 using FLICE / Caspase 8-colorimetric assay kit, 4c was first treated with 20μM Z-IETD-FMK to gastric cancer cell line, followed by 1000μM UDCA treatment , Western blot shows the results of confirming the amount of protein cut caspase 3, 6 and PARP.
Figure 5a is a transduction of siRNA FAS, siRNA DR4, siRNA DR5 or siRNA control for the gastric cancer cell lines SNU-601 and SNU-638 through the AMAXA method, and then treated with 1000μM UDCA, HO / PI double The staining method shows the result of measuring the degree of cell death (nucleus fragmentation), 5b shows the result of confirming the protein amount of caspase 3, 6 and PARP cut through Western blot, 5c is FLICE / caspase The results of measuring the caspase-8 activation using the 8-color assay kit are shown.
Figure 6a 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 through HO / PI double staining The degree (nucleus fragmentation or nuclear condensation) is measured, and 6b shows the result of confirming the amount of the caspase 3, 6 and the protein of PARP, FADD and RIP1 cut through Western blot, and 6c is FLICE / The results of measuring the caspase-8 activation using the caspase 8-colorimetric assay kit are shown.
Figure 7a is treated with 1000μM UDCA for gastric cancer cell lines SNU-601 and SNU-638, followed by incubation by time, and then the cytosolic (cytosolic) protein fraction and organ / membrane protein fraction from the cells respectively , Western blot shows the results of confirming the amount of PKC δ, PKC α, PKC θ, and PKC ε protein, and 7b shows the degree of cell death (nucleus fragmentation or nuclear condensation) through HO / PI double staining 7c shows the result of confirming the amount of the caspase 3 and 6 protein cut through the Western blot, 7d is transduced siRNA PKC δ or siRNA Control by AMAXA method for each gastric cancer cell line, 1000μM UDCA After the treatment was confirmed by Western blot the cut amount of caspase 3, 6 and PKC δ protein is shown.
Figure 8a is a treatment for each concentration of rottlerin (rottlerin) for gastric cancer cell lines SNU-601 and SNU-638, and then treated with 1000μM UDCA and confirmed the expression level of DR5 protein by Western blot, 8b is gastric cancer cell line After transducing siRNA PKC δ or siRNA Control by AMAXA method, and then treated with 1000μM UDCA, the expression level of PKC δ and DR5 protein was confirmed by Western blot.
FIG. 9A shows the results of measuring the generation of ROS by treating 1000 μM UDCA for gastric cancer cell lines SNU-601 and SNU-638, calculating the ratio of DCFH-DA / HO per cell after 3 hours or 5 hours. 9b is treated with 10 mM NAC, 100BHA, 10 mM Tiron, 2DPI, or 1000 U catalase, respectively, for gastric cancer cell lines, and then treated with 1000 μM UDCA, and HO / PI double staining to determine the degree of cell death (nucleus fragmentation or Nucleus condensation), 9c shows the results of measuring the amount of caspase-3, 6, and PARP protein cut through Western blot for gastric cancer cell lines treated as 9b.
Figure 10a shows that the treatment of 1000μM UDCA in the presence or absence of 10mM NAC or 100BHA in the SNU-601 gastric cancer cell line, respectively, and confirmed the expression level of DR5 through Western blot, 10b is 10mM in the SNU-638 gastric cancer cell line After treatment with 1000 μM UDCA in the presence or absence of NAC or 100BHA, respectively, the expression level of DR5 was confirmed by Western blot.
FIG. 11A shows pretreatment of 10 mM NAC to SNU-601 gastric cancer cell line followed by 1000 μM UDCA and fractionation of cytosolic protein and organ / membrane protein fractions from cells, followed by PKC δ via Western blot. Figure 11b shows the measurement of the expression level, Figure 11b shows the measurement of the expression level of PKC δ by performing the same experiment as in FIG. 11a for the SNU-638 gastric cancer cell line.
12a is a graph showing cell viability through MTT assay after pretreatment of 1 mM MBCD to gastric cancer cell lines SNU-601 and SNU-638, respectively treated with 1000 μM UDCA, and 12b shows HO / PI double staining method. Cell death rate (nuclear fragmentation or nuclear condensation), 12c shows the amount of caspase-3, 6, and PARP protein cut through Western blot, and 12d shows FLICE / Kaspa. The results of measuring the caspase-8 activation using the eighth colorimetric assay kit are shown.
FIG. 13 shows that 1 mM MBCD was previously treated in gastric cancer cell lines SNU-601 and SNU-638, and treated with 1000 μM UDCA, respectively, and then confirmed the expression level of DR5 through western blot.
FIG. 14A shows that 1 mM MBCD was previously treated with gastric cancer cell lines SNU-601 and SNU-638, and treated with 1000 μM UDCA, respectively, and then the cytosolic protein fraction and organ / membrane protein fraction were respectively fractionated from the cells. Then, the amount of PKC δ protein was confirmed by Western blot, and 14b was pretreated with gastric cancer cell lines SNU-601 and SNU-638 with 1 mM or 2 mM MBCD, and 1000 μM UDCA, respectively, followed by DCFH per cell. It shows the result of measuring the generation of ROS by calculating the ratio of -DA / HO.

The present invention is characterized by providing a composition for the prevention or treatment of 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 ursodeoxycholic acid compound may be a salt, preferably a 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 can be used as a component present in the bile of the animal is generally isolated from the bile of the animal or prepared by chemical synthesis methods known in the art, commercially available urusodeoxy Any cholic 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-performance liquid chromatography filled with various synthetic resins such as silica gel or activated alumina. It can be used alone or in combination with graphigraphy (HPLC), and the extraction and separation and purification method of the active ingredient is not necessarily limited to the above-described method, any method that is performed in the art can be used.

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 (ursodeoxycholic acid: UDCA) has the activity that can prevent or treat stomach cancer.

In particular, the urousodeoxycholic acid 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 pathological growth of unregulated cells such as cancer cells. Extensive necrosis pharmacotherapy is used 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, apoptosis-induced apoptosis control may induce spontaneous killing of pathological cells or strongly inhibit the growth of these cells, thereby reducing the inflammatory side effects induced by apoptosis of cancer cells compared to cell necrosis. have.

Apoptosis is genetically regulated programmed cell death, which means active cell death, and the apoptosis process requires energy and exhibits characteristic cell morphology. In other words, when apoptosis signal is transmitted, apoptosis is determined within the cell, and the cell death process is performed. In the execution step, the cell shows characteristic biochemical and morphological changes. 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 pagocytosis is carried out by macrophages, resulting in cell death.

In addition, apoptosis can be largely 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 the activation of the resulting 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 signaling cascades that are strong enough to kill apoptosis. 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 regulators 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) The disruption of the mitochondria leads to the release of SMAC / DIABLO and cytochrome c into the cytoplasm.

Therefore, the present inventors investigated whether urosodeoxycholine 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 The gastric cancer cell lines were treated with urosodeoxycholic acid (UDCA) for -216 cell lines, and MTT assay analysis, colony formation assay, HO / PI double staining method, and the like to confirm that apoptosis was induced. In all, it was confirmed that apoptosis was induced by UDCA and apoptosis occurred (see FIGS. 1A to 1C), and apoptosis was increased in a concentration-dependent manner of UDCA. It was confirmed that it is caused by apoptosis (apoptosis), not cell necrosis (necrosis) (see Fig. 2a and 2b).

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

In addition, in the case of apoptosis induced by activation of apoptosis receptor, the initiation of apoptosis mechanism is activated from the reaction of a death receptor and a ligand, and then an adapter associated with apoptosis in the apoptosis 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 the TRAIL binds to DR4 or DR5 or the 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 8 induces apoptosis intracellularly 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, the phosphorylation of PKC and the change of pH due to the change of Na + and H + concentrations are known to be involved in cell activity, and in some cells it is known to be a signal of proliferation. PKC also increases the transcription of certain genes. In particular, the activity of PKC by DAG in secondary messengers produced by the hydrolysis of phosphatidylinositol may be similarly expressed by the binding of monoacylglycerol or phorbol ester. 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 be made through xanthine oxidase, NO synthase, and mitochondrial electron transfer system.

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

Therefore, the present inventors analyzed whether caspases, which are apoptosis 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 confirmation of the induction of apoptosis of cells showed that treatment with caspase inhibitors inhibited apoptosis by UDCA compared to the absence of treatment (see FIG. 4A), whereas UDCA was associated with caspase 8 It has been shown that there is activity to increase the activity of (see Fig. 4b), and when the inhibitor of caspase 8 was treated, it was found that cleavage of caspase 3, 6 and PARP by the activation of apoptosis does not occur ( See FIG. 4C).

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, caspase-3, 6, 8 and 9, preferably caspase Apoptosis can be induced by activating Cas-8 to cascade the 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. 5A-5C).

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 apoptosis factors, namely FADD and RIP1, using siRNA After suppressing gene expression, treatment of gastric cancer cells with UDCA showed that apoptosis was reduced compared to the control (see FIGS. 6A and 6B).

Thus, the UDCA of the present invention promotes the 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 apoptosis. Can be induced.

Furthermore, the present inventors examined whether UDCA can activate PKC in apoptosis mechanism of gastric cancer cells by UDCA. According to one embodiment of the present invention, after treating UDCA to gastric cancer cell lines, cytosolic fractions of cells and As a result of extracting each of the membrane fractions and confirming the positional movement of the PKC isomers in the cells through Western blot, it was confirmed that the PKC δ was moved from the cytosol to the membrane by UDCA (see FIG. 7A). On the other hand, treatment with PKC δ inhibitor or siRNA PKC δ inhibited the expression or activity of PKC δ, which not only inhibited the caspases activity but also decreased the expression of the death receptor DR5, indicating that apoptosis was inhibited ( 7C-7D and 8A-8B).

Therefore, the present inventors have found that the activity of the death receptor DR5 and caspases in the process of apoptosis of cancer cells by UDCA is regulated by PKC δ. I could see that.

Reactive oxygen species (ROS), or active oxygen, are also called oxygen-derived species because they are oxygen-derived substances. 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., and their types are very diverse, and the reaction time of ROS is very short, but the reaction power is very large, so that in vivo, denaturation of various proteins, lipid peroxidation, DNA It is known to have harmful effects, such as mutations and (Li W, Hill HZ, Induced melanin reduces mutations and cell killing in mouse melanoma. Phytochem Photobiol . 65, pp. 480-485, 1997), ultimately known to act to induce cell damage and apoptosis (McCord JM. Human disease, free radicals and the oxidant / antioxidant balance. Clin Biochem . 26: 351- 357,1993).

Therefore, the inventors confirmed whether such ROS is involved in the anticancer activity of gastric cancer of UDCA, and according to one embodiment of the present invention, when UDCA was treated to gastric cancer cells, the production of ROS increased and apoptosis appeared to be increased. On the other hand, when ROS scavenger was treated, ROS production was decreased, and apoptosis was also reduced (see FIGS. 9A to 9C). 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 generation (see FIGS. 10A to 10B and 11A to 11B), and it was first identified in the present invention that UDCA has an effect of inducing the production of ROS.

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". Therefore, 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. is inhibited by known which serves to control the signaling (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).

Therefore, in order to investigate whether lipid raft domains are involved in the death signal transmission process of cancer cells by UDCA, in particular, apoptosis, gastric cancer cells, MBCD, a reagent that removes cholesterol from lipid rafts and damages the structure of lipid rafts After treatment with, apoptosis by UDCA was observed, and MBCD treatment resulted in inhibition of caspase activity by UDCA, decreased expression of DR5 and ultimately suppressed apoptosis (FIGS. 12A-12D). And FIGS. 13A-13B).

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 and the activity of PKC δ are also inhibited (see FIGS. 14A and 14B).

Therefore, 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 transmission.

Therefore, urusodeoxycholic acid (ursodeoxycholic acid: UDCA) of the present invention is effective in treating gastric cancer by inducing apoptosis in gastric cancer cells, the gastric cancer cells in the present invention may include any gastric cancer cells and , Preferably, SNU-601, SNU-638, SNU-1 and SNU-216.

Accordingly, the present invention can provide a composition for preventing or treating gastric cancer, including 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 urusodeoxycholic acid according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 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, the pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorders, dizziness or the like when administered to humans. 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 so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine 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 most often caused by an imbalance between apoptosis-cell death and cell proliferation by intracellular signaling mechanisms. In general, in cancer tissues, signaling mechanisms related to cell proliferation are activated, whereas cells related to apoptosis are activated. It is well known in the art that internal signaling is inhibited. Thus, apoptosis inducers are effective in treating cancer by activating apoptosis in cancer cells and inducing planned apoptosis in cancer cells.

In the present invention, the apoptosis (apoptosis) can be induced through all the apoptosis activity mechanisms 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, the 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. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< 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), 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. In addition, the cells were cultured overnight for drug exposure ^{test, 5 x 10 6 cells / 20cm} 2 dish, 1.5x10 6 cells / 10cm 2 dish, 5 x 10 5 cells / 6 cm 2 dish, 2 x 10 5 The cells were divided into cells / 3.5 cm ² dish, and in the case of 24 well plates, the cells were divided into 5 x 10 ⁴ cells / well to divide the cells.

< Example  1>

In human gastric cancer cells UDCA of Apoptosis  Induction activity

<1-1> MTT  Analysis and Colony  In gastric cancer cells through formation analysis UDCA of Apoptosis  Induction measurement

First of all, the present inventors investigated whether urosodeoxycholine acid (UDCA) has activity to induce apoptosis of the cancer cells against gastric cancer cell lines, and for this purpose, the cancer cell lines SNU-601, SNU-638, SNU -1 and SNU-216 cell lines were used, and the cells were dispensed into 24 well plates at 5 x 10 ⁴ cells / well, respectively, and treated with UDCA at concentrations of 250, 500 and 1000 μM, followed by 12, 24, The cells were incubated for 36 and 48 hours, respectively, and analyzed for the degree of apoptosis of cells by MTT analysis. In addition, in order to confirm that UDCA can inhibit colony formation of gastric cancer cells, the UDCA was treated with each of the gastric cancer cell lines at concentrations of 250, 500, and 1000 μM for 12 hours, and then at 37 ° C. for 15 hours. The degree of colony formation was analyzed by colony formation assay. In this case, the MTT assay is treated with 0.5ug / ml MTT to each of the cells cultured after treatment with UDCA and incubated for 4 hours, the cells of each plate were collected at room temperature for 5 minutes at 1000 rpm for 5 minutes Centrifugation was performed. 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, the measurement of the degree of colony formation was visually observed colonies formed on the plate.

As a result, as shown in Figures 1a to 1c, when UDCA treatment of human gastric cancer cell lines, MTT analysis showed that the survival rate of gastric cancer cells is reduced, the survival reduction effect is that the concentration of the treated UDCA The higher, the longer treatment time was shown to decrease the survival rate (FIG. 1A). In addition, colony formation also showed that the number of colonies formed was significantly decreased as the treatment concentration of UDCA was increased, and in particular, when treated with 1000 μM of UDCA, almost no colonies were formed (see FIGS. 1B and 1C).

<1-2> Hoechst  33342 and PI ( Propidium Iodide Gastric cancer cells through analysis UDCA Induction of apoptosis

To analyze the apoptosis form of gastric cancer cells by UDCA, double staining was performed using Hoechst 33342 (HO) and PI (Propidium Iodide), where HO stains the nucleus of cells with blue fluorescence, while PI is damaged cells. Penetrates the cell membrane of the fluorescence is characterized. Thus, dual staining with HO / PI is blue when the cells are alive, condensed and cleaved in the early apoptotic phase to observe the stained nuclei, and in the late apoptotic phase (secondary necrotic). And the cut pink nucleus can be observed, in the case of necrotic cells, the whole nucleus is observed in pink. HO / PI double staining using this principle is to divide the SNU-601, SNU-638, SNU-1 and SNU-216 cells with 2 x 10 ⁵ cells / 3.5cm ² dish, respectively, and then the above Example <1-1 Treated with UDCA at a concentration as described above, the cells were treated with Hogst 33342 and 5 ug / ml of PI to the cells at each time and incubated for 15 minutes under conditions of 5% carbon dioxide at a temperature of 37 ° C. The cells were stained. Then, the cells were collected by treatment with trypsin, centrifuged at 1500 rmp 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 described above. Cells were then lysed with 500ul of 3.7% paraformaldehyde and incubated for 5 minutes, followed by winch separation in cytospinner. The slides were then immediately fixed with mounted gels, washed with water, dried and covered with a glass cover slip. The slices were observed under a DM5000 fluorescence microscope (Hanil) under excitation / emission wavelengths of 340/425 nm (HO) and 580/425 nm (PI), respectively. In addition, the degree of Cleaved caspase-3, Cleaved caspase-6, Cleaved PARP, LC3 and tubulin protein in the cells treated with UDCA was observed by Western blotting method. Identifying antibodies were used according to Western blot methods known in the art.

Furthermore, the present inventors performed lactate dehydrogenase (LDH) release analysis to determine whether UDCA affects the process of inducing apoptosis 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 aliquoted to 5 x 10 ⁴ cells / well in a 24-well plate and then cultured 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 completion of the constant culture time of each cell to lyse the cells. The 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 (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.

As a result, as shown in Figures 2a and 2b, when treated with UDCA and double staining using HO / PI to the gastric cancer cell lines, it was confirmed that all of the cancer cells were induced by apoptosis, whereas In all gastric cancer cell lines, cell necrosis was not induced in comparison with apoptosis.

In addition, lactate dehydrogenase (LDH) release analysis, as shown in Figure 2c, the UDCA-treated gastric cancer cell lines, LDH activity was found to be almost 1% or less. Generally, 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, it could be seen that apoptosis of gastric cancer cells by UDCA of the present invention did not induce the release of LDH. Therefore, it could be seen that the apoptosis of gastric cancer cells by UDCA of the present invention was caused by apoptosis rather than cell necrosis, and these results can be clearly confirmed through the Western blot results of FIGS. 2D and 2E. That is, when apoptosis occurs, caspase is activated, and PARP (poly (ADP-ribose) polymerase) is characterized in that the cleavage. As a result of treating the gastric cancer cell line with UDCA according to the present invention, caspase-3, Cleaved fragments of caspase-6 and PARP were identified, further confirming that no transition from LC-I to LC-II was induced, indicating that UDCA does not induce autophagy in gastric cancer cells. there was.

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

<1-3> Deoxycholic acid  Caused by gastric cancer cells Apoptosis  Inductive analysis

Deoxycholine acid (DCA) has been disclosed to induce apoptosis in hepatocytes and colon cancer cells. However, in the case of deoxycholic acid, there is no known content of inducing apoptosis in gastric cancer cells, and the present inventors treated deoxycholic acid with gastric cancer cell lines, respectively, followed by MTT assay, HO / PI double staining method and LDH release assays were performed, and the experiments were performed in the same manner except that DCA was used instead of UDCA in Examples <1-1> to <1-2>.

As a result, DCA also induced apoptosis of gastric cancer cells in a concentration-dependent manner as in the case where UDCA was treated to gastric cancer cells (see FIG. 3A). As a result of analysis by HO / PI heavy staining, unlike UDCA, DCA has been shown to induce cell necrosis in gastric cancer cells (see FIGS. 3B and 3C). In addition, LDH release assays showed that DCA increases the release of LDH out of the cell membrane in a concentration-dependent manner in gastric cancer cells.

Therefore, the above results show that the present inventors have an activity of inducing apoptosis of gastric cancer cells in the case of DCA, but unlike UDCA, the mechanism of inducing apoptosis is achieved through cell necrosis.

< Example  2>

In stomach cancer cells UDCA On by Apoptosis  In judo Caspases  Impact Analysis

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 through apoptosis through Example 1. That is, 20 μM of caspase inhibitors (Z-DEVD-FMK, Z-VEID-FMK, Z-IETD-FMK, Z-LEHD-FMK or Z) in SNU-601 and SNU-638 cells, which are gastric cancer cell lines used in the above examples. -VAD-FMK) was treated for 1 hour, 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. The experiment was performed according to the method of use of the kit using (Biovision). That is, 5 x 10 ⁵ gastric cancer cells were treated with 1000 μM UDCA, and then cultured for 12, 18, 27, and 48 hours, respectively, and the cells were collected by centrifugation. The cells were then lysed with 50ul cold cell lysis buffer, incubated on ice for 10 minutes and centrifuged at 4 ° C. for 1 minute at 10,000 g. The supernatant was transferred to a new tube, quantified using the Biorad Protein Assay Kit, diluted with a volume of 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 with 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 Confirmed by performing blots.

As a result, as shown in Figure 4a, apoptosis induction of gastric cancer cells by UDCA was shown to be inhibited when treated with caspase inhibitors, the activity of caspase 8 is shown to increase the activity by the treatment of UDCA Appeared (see FIG. 4B). 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. 4C, caspase 3, 6 , And sections with PARP cleaved were not observed.

Thus, through the above results, the present inventors found that UDCA increases the activity of caspase 8 in the process of inducing apoptosis of gastric cancer cells by UDCA, and thus apoptosis of cells is induced by a signal transduction system related to the 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.

< Example  3>

UDCA On by DR5 Induction Analysis of

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, expression of these receptors in cells was removed using siRNAs of the cell surface killing receptors described in Table 1 below, i.e., siRNAs (8 ug) of the respective receptors were injected into the 1 × 10 ⁶ cells of gastric cancer cells by AMAXA method. The cells transduced and siRNA introduced were incubated for 40 hours at 37 ℃ temperature and 5% of carbon dioxide, and then treated with 1000μM UDCA to the cells, respectively, and cultured for a period of time (24 hours or 36 hours) Next, caspase-8 activity, MTT assay, HO / PI double staining and ROS assay were performed in the same manner as described in the above examples. At this time, the ROS analysis was performed by dispensing gastric cancer cells of 5x10 ⁴ cells in a 24-well plate, incubated with 500ul of RPMI medium, and then treated with UDCA, and incubated the cells under the same conditions. Then, 50 μM of 2'7'-dichlorofluorescein diacetate (DCFH-DA, Molecular probe) and 0.5 ug of HO were added to each cell to confirm the generation of reactive oxygen species (ROS) and incubated for 1 hour. . After washing, DCFH-DA was measured using a fluorocount model MQM200 plate reader (Packard instrument, USA) at 490 nm excitation wavelength and 530 nm emission wavelength, and measurement of HO was performed using excitation wavelength of 340 nm and It was measured at an emission wavelength of 425 nm. The amount of ROS produced was calculated by measuring the ratio of DCFH-DA / HO per well.

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. 5A, 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 decreased by siRNA DR4. It did not appear to be reduced. In addition, in SNU-638 cells, apoptosis was shown to be reduced when siRNA DR4 or siRNA DR5 was introduced, whereas apoptosis was not reduced by siRNA FAS, and the result was caspase-3. , 6 and PARP activity was the same result (see Fig. 5b). In addition, as a result of measuring the caspase 8 activity, caspase 8 activity by UDCA was found to be inhibited by siRNA DR5 or siRNA FAS in SNU-601 cells, while not by siRNA DR4. In the case of SNU-638 cells, caspase 8 activity was shown to be reduced by siRNA DR4 or siRNA DR5, while not by siRNA FAS (see FIG. 5C).

Therefore, 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 regulatory action, ie, initiation of apoptosis by UDCA, in both gastric cancer cells.

< Example  4>

In stomach cancer cells FADD  And RIP through UDCA of Apoptosis  Active induction

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 apoptosis factors, the present inventors transduced the siRNA FADD and siRNA RIP1 described in Example 3 to 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. 6A and 6B, the apoptosis of cells by UDCA was reduced by siRNA FADD and siRNA RIP1 in both SNU-601 and SNU-638 cells, and siRNA was also reduced compared to the control group. FADD and siRNA RIP1 have also been shown to reduce caspase 3, 6 and PARP activity. In addition, the activity of caspase 8 was also shown to be reduced by siRNA FADD and siRNA RIP1.

Therefore, the present inventors have found that the apoptosis of gastric cancer cells by UDCA according to the present invention is through cell death factors of FADD and RIP1, and they act to induce caspase 8 as a death receptor. It was found to play an important role.

< Example  5>

UDCA Expression induced by DR5 On by Apoptosis  In action PKC Role of δ

PKCδ is a type of PKC superfamily 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 PKC Has been reported to be due to redistribution of kinase at cytosol and other intracellular locations. In order to confirm whether the UDCA activates 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). The cytosolic protein (fraction 1) and organ / membrane protein (fraction 2) were extracted using the Proteoextract subcellular proteome extraction kit, and Western blots were used for the cytosolic migration of PKCδ, PKCα, PKCθ, and PKCε to the membrane. Analyzed.

As a result, as shown in Figure 7a, the treatment of UDCA had the effect of moving the PKCδ from the cytosol to the membrane, it can be seen that the migration of the PKCδ to the cell membrane is induced before the apoptosis of the cells by the UDCA .

In addition, the present inventors treated with 1, 2 and 5 μM of rottlerin, ie, PKCδ inhibitors, and 1000 μM for gastric cancer cell lines, respectively, in order to confirm the degree of change in apoptosis by UDCA by treating the inhibitor of PKCδ. After UDCA treatment, Western blot was performed using HO / PI double staining and caspase antibodies. Furthermore, after transduction into gastric cancer cells using siRNA PKCδ, the expression level of caspases was expressed in cells. Confirmed by Western blot.

As a result, as shown in Fig. 7b, when treated with rottlerin, an inhibitor of PKCδ, it was shown that apoptosis of cells by UDCA was suppressed, and the degree of inhibition was increased in a concentration-dependent manner. In addition, as shown in 7c, the treatment of rotlerin was shown to inhibit the activity of caspases, the cell death factors, and to inhibit the expression of PKCδ in gastric cancer cells by siRNA PKCδ, the cells by UDCA Of apoptosis was reduced, which also inhibited the activities of caspase 3, 6 and PARP so that their cleaved forms were not observed (see FIG. 7D).

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

Furthermore, 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. After pretreatment with rinrin for 1 hour, and then treated with 1000 μM of UDCA, Western blot was performed to investigate the expression level of DR5, and siRNA PKCδ was transduced into gastric cancer cell lines, respectively, and then UDCA Treatment and Western blot were performed to investigate the expression level of DR5 and PKCδ.

As a result, as shown in Fig. 8a, the treatment of gastric cancer cell line with rottlerin, an inhibitor of PKCδ, showed that the expression of DR5 by UDCA was reduced, and siRNA PKCδ was used to suppress the expression of PKCδ in cells. Inhibition also reduced the expression of DR5 by UDCA (see FIG. 8B).

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. It was found to play a very important role in the process.

< Example  6>

UDCA On by DR5 Expression and PKC by shift of δ Apoptosis  In action ROS  Analysis of association with occurrence

Reactive oxygen species (ROS) are known as key factors in determining cell death mode in apoptosis processes 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 cultured for 3 hours or 5 hours, followed by ROS as in the method described in Example 3. The degree of production was analyzed, double staining of HO / 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.

As a result, as shown in FIG. 9, when UDCA was treated in gastric cancer cells, ROS production was increased in the cells (see FIG. 9A), and when various types of ROS scavengers were treated, In particular NAC and BHA have been shown to inhibit apoptosis through apoptosis by UDCA (see FIGS. 9B and 9C).

Therefore, 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. 10A and 10B, 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. Cytozoric protein (fraction 1: C) and organ / membrane protein (fraction 2: M) were obtained from the cells at (2, 4, and 8 hours), respectively, and then the amount of PKCδ was determined via western blot.

As a result, the migration of PKCδ from the cytosol to the membrane by UDCA was shown to be reduced by the treatment of NAC, a scavenging agent of ROS (see FIGS. 11A and 11B). Therefore, the above results indicate that the migration of PKCδ by UDCA is regulated by the production of ROS, and PKCδ is a factor present downstream of the ROS generation mechanism in the process of apoptosis by UDCA in gastric cancer cells. It was confirmed that.

< Example  7>

UDCA Induced to DR5 By expression of Apoptosis  In action Lipid Of the raft  role

Cholesterol has been known to play a very important role in the composition, maintenance and fluidity of the membrane. In particular, a domain rich in cholesterol-rich microdomains, aka lipid rafts, plays an important role in the signal transduction of apoptosis and is associated with the formation of FAS-FADD, DR4 or DR5-FADD complexes and the activation of caspase 8 plasma cholesterol. It is known to play an important role in the change. Therefore, the present inventors used MBCD, which is a lipid raft deficient reagent, to clarify the relationship between apoptosis and lipid raft of gastric cancer cells by UDCA, that is, gastric cancer cell lines treated with 1 or 2 mM MBCD for 1 hour. Thereafter, 1000 μM of UDCA was treated, and then cultured for 24 hours and 36 hours, respectively, and cell death was measured by MTT assay, HO / PI double staining method, and caspase 8 activity measurement. In addition, the expression level of DR5 was measured by Western blot.

As a result, as shown in FIG. 12, cell death by apoptosis of gastric cancer cells by UDCA was found to be suppressed when MBCD was treated (see FIG. 12A). 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. 12B). It was shown that it was inhibited by the treatment of MBCD (see FIG. 12C), and the activity of caspase 8 by UDCA was also inhibited by the treatment of MBCD (see FIG. 12D).

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 FIGS. 13A and 13B). .

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

< Example  8>

UDCA Activated by ROS Of PKC δ in Lipid Of the raft  role

As a result of Example 7 confirmed that the lipid raft plays a very important role in the apoptosis mechanism of gastric cancer cells by UDCA, whether the lipid raft is also involved in the activity of ROS / PKCδ confirmed through the previous embodiment Investigated. To this end, the gastric cancer cell line was treated with 1 or 2 mM MBCD for 1 hour, and then treated with 1000 μM of UDCA, followed by incubation for 24 hours and 36 hours, respectively. After extraction, 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.

As a result, as shown in FIG. 14, the cytosolic migration of PKCδ to gastric membrane by UDCA in gastric cancer cells was shown to be inhibited due to the treatment of MBCD (see FIG. 14A). It was found that this is a downstream event of lipid raft during apoptosis. 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. 14B).

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.

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 an illustrative rather than a restrictive sense. 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.

Attach an electronic file to a sequence list

Claims (10)

Gastric cancer prevention or treatment composition comprising ursodeoxycholic acid (UDCA) or salts thereof as an active ingredient. The method of claim 1,
The urosodeoxycholine acid (UDCA) is a gastric cancer prevention or treatment composition, characterized in that it has anti-cancer activity by inducing apoptosis (apoptosis) of gastric cancer cells to induce cell death. The method of claim 2,
The apoptosis (apoptosis) by the treatment of urosodeoxycholine acid (UDCA), promote the activity of caspase (gaspase) in gastric cancer cells; Promoting activity or expression of apoptosis 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). 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 and caspase-9. The method of claim 3,
The cell death receptors are DR4 (TNF-associated apoptosis-inducing ligand (TRAIL) receptor-1), DR5 (TNF-related apoptosis-inducing ligand (TRAIL) receptor-2), Tumor necrosis factor receptor superfamily (member 6) And TNFR (Tumor Necrosis Factor Receptor) composition for preventing or treating gastric cancer, characterized in that selected from the group consisting of. The method of claim 3,
Gastric cancer characterized by inducing apoptosis in gastric cancer cells by promoting the activity or expression of the apoptosis receptor Fas Associated protein with Death Domain (FADD) or receptor interacting protein 1 (RIP1) protein Prophylactic or therapeutic composition. The method of claim 3,
The generation of reactive oxygen species (ROS) and the activity of PKC (protein kinase C) δ protein is a composition for preventing or treating gastric cancer, characterized in that controlled by the lipid raft (lipid raft) located in the cell membrane. The method of claim 1,
The urosodeoxycholine acid is a composition for preventing or treating gastric cancer, characterized in that it comprises 250μM ~ 1000μM relative to the total weight of the composition. An apoptosis inducer composition comprising urosodeoxycholic 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.

Images