Compositions comprising telmisartan for enhancing activity of trail to prostatic cancer

Abstract

To provide a composition for enhancing the anticancer activity of TRAIL to prostatic cancer, that is, to selectively induce apoptosis of cancer cells without affecting normal cells, by overcoming the resistance to TRAIL.SOLUTION: A composition comprising telmisartan for enhancing the activity of TRAIL to prostatic cancer is provided.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a composition containing telmisartan for enhancing the activity of TRAIL against prostate cancer. [Background technology]

[0002] Cancer is one of the most serious health threats to humankind, and is caused by a series of mutations that cause cells to become immortal in an uncontrolled and unregulated manner. The causes of cancer include environmental or external factors such as chemicals, viruses, bacteria, and ionizing radiation, and internal factors such as congenital genetic mutations.

[0003] In the case of cancer that is discovered in the early stages, there are treatments such as surgery, radiation therapy, and chemotherapy, but the side effects of these treatments have also emerged as a major problem, and in the case of terminal cancer or cancer that has metastasized, there is no specific treatment, and the patient is given a prognosis of how long they have left to live, and their life ends. Furthermore, although various biochemical mechanisms related to cancer have been elucidated and corresponding therapeutic agents have been developed, a fundamental method of treating cancer has not yet been presented.

[0004] TRAIL (Tumor necrosis factor-related apoptosis inducing ligand) is a type of tumor necrosis factor (TNF) and is known as a ligand that induces autoapoptosis in tumor cells. TRAIL has four receptors, among which death receptors (DR4, DR5) are overexpressed in cancer cell lines and decoy receptors (DcR1, DcR2) are overexpressed in normal cell lines. TRAIL can bind to death receptors such as DR4 and DR5 on the surface of tumor cells and induce the death of tumor cells. In the case of the decoy receptors, the apoptosis signaling is not transmitted into the cells because there is no apoptosis domain at the c-terminal end. Therefore, TRAIL-based therapy has the characteristic of being harmless to normal cells and inducing toxicity only to various cancer cell lines, and is a very effective next-generation anti-cancer therapeutic agent.

[0005] However, it has been shown that when cancer cell lines are continuously treated with TRAIL, even cancer cell lines that were previously sensitive to TRAIL gradually become resistant to TRAIL. Specifically, many cancer cells resist TRAIL-induced apoptosis through various mechanisms, including a decrease in apoptosis receptors and an increase in anti-cell death proteins, such as cellular FLICE-like inhibitory protein (c-FLIP(L)), B-cell lymphoma-2, B-cell lymphoma-extra large (Bcl-xL), and myeloid cell leukemia-1 (Mcl-1).

[0006] Therefore, there is a need for research into a method for preventing cancer cell proliferation and promoting apoptosis by overcoming the resistance of TRAIL, which can selectively induce apoptosis in cancer cells.The present inventors have therefore researched a therapeutic agent that can overcome TRAIL resistance and prevent cancer cell proliferation, and have confirmed that telmisartan inhibits autophagy at the late stage, thereby upregulating DR5 and inducing TRAIL-mediated cell death, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a composition for promoting the activity of TRAIL against prostate cancer, containing telmisartan, which can overcome resistance to TRAIL, which selectively induces apoptosis in cancer cells without affecting normal cells, and effectively induce apoptosis in cancer cells, thereby promoting the anti-cancer effect. [Means for solving the problem]

[0008] In order to achieve the above object, the present invention can provide a composition for enhancing the activity of TRAIL against prostate cancer, comprising telmisartan. The present invention can provide a pharmaceutical composition for preventing or treating prostate cancer, comprising telmisartan and TRAIL. Effect of the Invention

[0009] Telmisartan of the present invention upregulates DR5 through autophagy inhibition at the late stage, thereby inducing TRAIL-mediated cell death, and has therapeutic effect on prostate cancer. [Brief description of the drawings]

[0010] [Figure 1]Figure 1 shows that telmisartan induces TRAIL-mediated cell death in prostate cancer cell lines. (A) shows the results of treating PC3 with various concentrations of telmisartan (0, 25, 50, 100 μM) for 12 hours, followed by 100 ng / ml TRAIL for 3 hours, and then examining the cells with an optical microscope. (B) shows the results of staining the cells with crystal violet. (C) shows the results of quantitatively confirming the crystal violet staining by calculating the relative viability of the control cells, with the viability of the control cells set at 100%. (D) shows the results of measuring the LDH assay of the cells. (E) shows the results of treating PC3 with 100 μM telmisartan for various times (0, 6, 12, 24 hours), followed by 100 ng / ml TRAIL. (F) shows the results of observation under an optical microscope after 3 hours of TRAIL treatment; (G) shows the results of crystal violet staining of the above cells; (H) shows the results of quantitative confirmation by calculating the relative viability of the above cells, with the viability of the control cells set at 100%; and (H) shows the results of LDH assay of the above cells. [Diagram 2] FIG. 2 confirms that telmisartan upregulates DR5 expression and induces TRAIL-mediated cell death. (A) shows the results of treating PC3 with various concentrations of telmisartan (0, 25, 50, 100 μM) for 12 hours, followed by 100 ng / ml TRAIL for 3 hours, and then Western blotting the cells to confirm the expression levels of DR5 and DR4. (B) shows the results of treating PC3 with 100 μM telmisartan for various times (0, 6, 12, 24 hours), followed by 100 ng / ml TRAIL for 3 hours, followed by Western blotting the cells to confirm the expression levels of DR5 and DR4. (C) shows the results of immunocytochemistry of the cells to confirm that telmisartan upregulates DR5 in the cells. (D) shows the results of Western blotting the cells to confirm the expression of cleaved caspase-8 and cleaved caspase-3, which are intracellular apoptosis regulatory proteins. [Diagram 3]Figure 3 shows the results of confirming that telmisartan induces upregulation of DR5 through protein stabilization. (A) PC3 was treated with various concentrations of telmisartan (0, 25, 50, 100μM) for 12 hours, followed by 100ng / ml TRAIL for 3 hours, and the expression level of DR5 was confirmed by RT-PCR in the above cells. (B) The expression level of DR5 was confirmed by Western blotting after treating the above cells with 200μg / ml cycloheximide (CHX), a strong protein stabilizer that interferes with new protein synthesis, for various periods of time. [Figure 4] FIG. 4 shows the results of confirming that telmisartan inhibits the degradation of DR5 through inhibition of autophagic flux. (A) shows the results of treating PC3 with various concentrations of telmisartan (0, 25, 50, 100 μM) for 12 hours, followed by treatment with 100 ng / ml TRAIL for 3 hours, and then Western blotting the cells to confirm the transition from LC3-I to LC3-II and the level of p62. (B) shows the results of treating the cells with 100 μM telmisartan for various times (0, 6, 12, 24 hours), followed by treatment with 100 ng / ml TRAIL for 3 hours, followed by Western blotting the cells to confirm the conversion from LC3-I to LC3-II and the level of p62. (C) shows the results of confirming the DR5 and LC3 levels of telmisartan in the cells by immunocytochemistry. (D) shows the results of transmission electron microscopy (TEM) analysis to confirm that autophagy is inhibited in cells treated with telmisartan. [Diagram 5]FIG. 5 shows the results confirming that inhibition of autophagy by telmisartan enhances TRAIL-mediated cell death. (A) shows the cell morphology photographed after treating PC3 with 20 μM chloroquine for 12 hours and 100 μM telmisartan for 12 hours, followed by treatment with 100 ng / ml TRAIL for 3 hours. (B) shows the result of staining the above cells with crystal violet. (C) shows the quantitative confirmation of crystal violet staining by calculating the relative viability with the viability of control cells set at 100%. (D) shows the result of measuring the LDH assay of the above cells. [Figure 6] Figure 6 shows the effect of telmisartan on surface DR5. (A) shows the result of treating PC3 with various concentrations of telmisartan (0, 25, 50, 100 μM) for 12 hours, followed by measurement of the amount of surface DR5 by flow cytometry. (B) shows the result of treating PC3 with 20 μM chloroquine for 3 hours and 100 μM telmisartan for 12 hours, followed by measurement of the amount of surface DR5 by flow cytometry. (C) shows the result of isolating cell membranes from an untreated group, a group treated with 50 μM telmisartan for 12 hours, and a group treated with 20 μM chloroquine (Sigma-Aldrich) for 12 hours, and performing Western blotting on the above cells to confirm an increase in DR5 present in the cell membrane. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below. The present inventors have been researching therapeutic agents that can overcome TRAIL resistance and prevent the proliferation of cancer cells, and have confirmed that telmisartan inhibits autophagy at a late stage, thereby upregulating DR5 and inducing TRAIL-mediated cell death, thereby completing the present invention.

[0012] In this regard, the present invention can provide a composition for enhancing the activity of TRAIL against prostate cancer, comprising Telmisartan. In the present invention, it was confirmed through light microscopy, crystal violet, and LDH assays that telmisartan induced TRAIL-mediated cell death in prostate cancer cell lines (PC3) treated with TRAIL after pretreatment with telmisartan for 12 hours (Figure 1A-Figure 1H). This confirmed that telmisartan upregulates DR5 expression to induce TRAIL-mediated cell death (Figure 2A-Figure 2D). This confirmed that the upregulation of DR5 is related to a post-translational process rather than a transcriptional process, and that telmisartan induces DR5 upregulation through protein stabilization (Figure 3). In addition, it was confirmed that telmisartan inhibits DR5 degradation through inhibition of autophagic flux, and inhibition of autophagic activity by telmisartan improved TRAIL-mediated cell death (Figures 4 and 5). Furthermore, the cell membrane of cells treated with telmisartan was isolated to confirm the expression of DR5, and it was confirmed that its expression was increased (Figure 6).

[0013] The above-mentioned "telmisartan" is commonly known by CAS Number 144701-48-4 and may have the structure of the following chemical formula 1, including a salt thereof or a pharma- ceutically acceptable salt thereof:

[0014] [ka]

[0015] In the present invention, the term "salt" refers to a salt prepared using a particular compound according to one embodiment and a relatively non-toxic acid or base. In the present invention, the term "pharmaceutically acceptable salt" refers to a salt prepared by utilizing a relatively non-toxic acid or base with a particular compound according to an embodiment. In cases where the compounds contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral forms of these compounds with a sufficient amount of base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. In cases where the compounds contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral forms of these compounds with a sufficient amount of acid in pure solution or in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include salts of inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate ion, phosphoric acid, monohydrogen phosphate ion, dihydrogen phosphate ion, sulfuric acid, hydrogen sulfate ion, hydroiodic acid or phosphoric acid, and salts of organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, stannic acid, methanesulfonic acid, and further include salts of amino acids (e.g., arginine, etc.) and salts of organic acids such as glucuronic acid.

[0016] The above pharma-ceutically acceptable salts can be synthesized by conventional chemical methods from parent compounds containing acidic or basic moieties. In general, these salts are prepared by reacting the free acid or base form of these compounds with a stoichiometrically appropriate amount of base or acid in water or an organic solvent, or in a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0017] In the present invention, the term "TRAIL (Tumor necrosis factor-Related Apoptosis-Inducing Ligand) protein" refers to a member of the cytokine proteins belonging to the Tumor Necrosis Factor (TNF) superfamily, and the sequence thereof can be obtained from a publicly known database such as NCBI's GenBank. Preferably, the TRAIL protein has the amino acid sequence of SEQ ID NO: 1 below: VRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG In the present invention, the above-mentioned Telmisartan can promote the sensitivity of prostate cancer to TRAIL.

[0018] The term "sensitivity" as used in the present invention means whether or not a particular drug is effective against cancer. In the present invention, when prostate cancer cell lines were treated with telmisartan, the effect of TRAIL was enhanced, and thus it was confirmed that telmisartan can enhance sensitivity to TRAIL.

[0019] "Prostate cancer", which is a disease targeted by the present invention, can include all cancers that can occur in the prostate, and the prostate cancer in the present invention can be TRAIL-resistant prostate cancer. The term "resistance" as used in the present invention refers to the phenomenon in which the efficacy of a drug is reduced by its use, and in the present invention, resistance refers to resistance in prostate cancer in which the efficacy of TRAIL is reduced.

[0020] In the present invention, the above-mentioned Telmisartan can increase the expression level of DR5 in TRAIL-resistant prostate cancer. In the present invention, said telmisartan can inhibit lysosomal degradation in TRAIL-resistant prostate cancer.

[0021] The pharmaceutical composition according to the invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions. Carriers, excipients or diluents that can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

[0022] The above composition can be administered orally or parenterally in clinical use. In the case of parenteral administration, it can be administered by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dura injection, cerebrovascular injection or intrathoracic injection, and can be used in the form of a general pharmaceutical formulation.

[0023] The compositions may be used alone or in combination with procedures using surgery, radiation therapy, hormonal therapy, chemotherapy and biological response modifiers. The daily dosage of the composition is about 0.0001 to 100 mg / kg, and preferably 0.001 to 10 mg / kg, and is preferably administered once or in several divided doses per day, but the range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

[0024] The composition of the present invention can be administered in various parenteral formulations during clinical administration, and when formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, tween 61, cacao butter, lauric butter, and glycerogelatin.

[0025] The present invention can also provide a pharmaceutical composition for preventing or treating prostate cancer, comprising Telmisartan and TRAIL. In the present invention, the above "telmisartan", "TRAIL" and "prostate cancer" may be within the aforementioned ranges.

[0026] The term "prevention" as used herein may refer to any act of inhibiting or delaying the onset of prostate cancer in an individual by administration of a pharmaceutical composition according to one embodiment. In the present invention, the term "treatment" can refer to any action in which an individual's symptoms for prostate cancer are improved or beneficially altered by administration of a pharmaceutical composition according to one embodiment.

[0027] The pharmaceutical composition according to the invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions. Carriers, excipients or diluents that can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

[0028] The compositions may be used alone or in combination with procedures using surgery, radiation therapy, hormonal therapy, chemotherapy and biological response modifiers. The pharmaceutical composition can be administered in parallel with TRAIL, which is used as an existing therapeutic agent for prostate cancer, and can be administered simultaneously, separately, or sequentially, and can be administered singly or in multiple doses. Taking into consideration all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0029] When the pharmaceutical composition is administered in combination with TRAIL, a synergistic effect may be observed in which the anti-cancer effects, i.e., tumor growth inhibition, immune activation, enhanced sensitivity of prostate cancer to TRAIL, and prevention or treatment of TRAIL-resistant prostate cancer, are more pronounced than when the pharmaceutical composition or TRAIL is administered alone.

[0030] In the above pharmaceutical composition, Telmisartan can be contained at 0.1 μM to 200 μM and TRAIL can be contained at 1 mg / ml to 200 mg / ml, preferably, Telmisartan can be contained at 50 μM to 150 μM and TRAIL can be contained at 50 mg / ml to 150 mg / ml, and more preferably, Telmisartan can be contained at 100 μM and TRAIL can be contained at 100 mg / ml.

[0031] In the present invention, the prostate cancer may be TRAIL-resistant prostate cancer. In the present invention, the term "administration" means introducing a predetermined substance into an individual by an appropriate method, and the term "individual" means all living organisms, including humans, such as rats, mice, livestock, etc., that may have prostate cancer. Specific examples include mammals, including humans.

[0032] In one embodiment, the route of administration of the pharmaceutical composition includes, but is not limited to, topical, oral, intravenous, intramuscular, intrathoracic, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal.

[0033] The pharmaceutical composition is administered in a pharma- tically effective amount. The term "pharma-tically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to any medical treatment, and an effective dose level can be determined based on factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. Specifically, the pharmaceutical composition can be administered at 0.001-1000 mg / kg / day, more specifically 0.1-100 mg / kg / day. The administration may be once a day or in multiple divided doses. For example, it may be administered every other day or once a week.

[0034] The present invention provides a health functional food containing telmisartan and TRAIL for preventing or ameliorating prostate cancer. In the present invention, the above-mentioned "prostate cancer", "prevention", etc. may be within the above-mentioned range, and the "pharmaceutical acceptable salt" may be a "food-based acceptable salt".

[0035] In the present invention, the term "food-based acceptable" means that the compound exhibits the property of being non-toxic to cells or humans exposed to the compound. In the present invention, the term "food-based acceptable salt" refers to a salt prepared using a specific compound according to one embodiment and a relatively non-toxic acid or base, and may be within the above-mentioned range for "salt".

[0036] In the present invention, the term "improvement" can mean any action that at least reduces a parameter related to the condition being treated, for example, the degree of symptoms. In this case, the above-mentioned health functional food can be used for the prevention or improvement of prostate cancer, either before or after the onset of the disease, simultaneously or separately with a drug for treatment.

[0037] In the above-mentioned health functional food, the active ingredient can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately according to the usual method. The amount of the active ingredient to be mixed can be appropriately determined according to the purpose of use (for prevention or improvement). In general, when producing a food or beverage, the above-mentioned health functional food can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health regulation, the amount may be less than the above range.

[0038] The health functional food may further include one or more of a carrier, a diluent, an excipient, and an additive, and may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid preparations. Foods to which the compound according to one embodiment may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, health functional foods, and the like.

[0039] Specific examples of the carrier, excipient, diluent and additive may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl methylhydroxybenzoate, propyl propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0040] The health functional food may contain other ingredients as essential ingredients, without particular limitation, in addition to the above-mentioned active ingredients. For example, like ordinary beverages, it may contain various flavorings and natural carbohydrates as additional ingredients. Examples of the above-mentioned natural carbohydrates include monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; and polysaccharides, such as dextrin, cyclodextrin, and other ordinary sugars, and sugar alcohols, such as xylitol, sorbitol, and erythritol. As flavorings other than those mentioned above, natural flavorings (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavorings (saccharin, aspartame, etc.) can be advantageously used. The proportion of the above-mentioned natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

[0041] In addition to the above, the dietary supplement according to one embodiment may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and thickeners (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0042] The present invention will be described in more detail below through examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0043] [Example] Example 1. Confirmation of the enhancement of TRAIL-mediated apoptotic effect of telmisartan in prostate cells We confirmed the enhancement of TRAIL-mediated apoptosis by telmisartan in prostate cancer cells (PC3). PC3 cells were purchased from the Korea Cell Line Bank. PC3 cells were cultured in RPMI-1640 medium (Gibco BRL; Thermo Fisher Scientific, Inc.) containing 10% (v / v) fetal bovine serum (Sigma-Aldrich; Merck KGaA) and antibiotics (100 μg / ml) at 37°C and 5% CO2.

[0044] The two cells above are 1.0 × 10 4 After seeding in 12-well plates, the cells were cultured at 37°C for 24 hours, pretreated with various concentrations (0, 25, 50, 100 μM) of telmisartan (Cayman Chemical) for 12 hours, then treated with recombinant TRAIL protein (100 ng / ml) and cultured for another 3 hours. Cell morphology was observed under an optical microscope to confirm viable cells. As a result of confirmation under an optical microscope, JCFLRNS with TRAIL alone had little effect on cell viability, with only slight changes in morphology compared to the control group. This confirmed that PC3 cells were resistant to TRAIL. However, as the concentration of telmisartan co-treated with TRAIL increased, it had a significant effect on cell viability and morphology (Figure 1A and Figure 1E).

[0045] To confirm the effect of telmisartan on TRAIL-mediated cell death, cell viability was measured using MTT and crystal violet in cells treated under the same conditions as above. For the MTT assay, 50 μl of 5 mg / ml methyl-thiazolyl-tetrazolium (MTT) was added per well and incubated at 37°C for 2 hours. After discarding the medium, 500 μl of dimethyl sulfoxide was added and the absorbance was measured at 570 nm using a Bio-Rad spectrophotometer. The cells were stained with 0.5% crystal violet solution dissolved in 30% ethanol and 3% formaldehyde for 10 minutes, washed four times with PBS, and air-dried. Cell viability was measured by comparing the relative staining intensity with the control group. Results of crystal violet and MTT analysis showed that co-treatment of telmisartan with TRAIL significantly increased TRAIL-induced cell death and reduced the percentage of viable prostate cancer cells in a dose-dependent manner (Figure 1B, Figure 1C, Figure 1F, Figure 1G).

[0046] The LDH cytotoxicity detection kit (Takara Bio, Inc., Tokyo, Japan) was performed according to the manufacturer's instructions. LDH activity was measured by absorbance at 490 nm using a microplate reader (Spectra Max M2, Molecular Devices, Sunnyvale, CA, USA). Measurement and analysis of lactate dehydrogenase (LDH) in prostate cancer cells confirmed that combined treatment with telmisartan and TRAIL significantly enhanced TRAIL-mediated cell death in a dose-dependent manner, whereas telmisartan or TRAIL alone had minimal effects on cell death (Figure 1D and Figure 1H).

[0047] The combined use of telmisartan and TRAIL significantly enhanced TRAIL-mediated cell death and reduced cell viability in TRAIL-resistant prostate cancer cells, demonstrating its efficacy against prostate cancer.

[0048] Example 2. Confirmation of gene expression related to TRAIL-mediated apoptosis of telmisartan in prostate cancer cells To confirm the underlying mechanism of cell death induction in prostate cancer cells associated with the synergistic effect of telmisartan and TRAIL, the expression of death receptors (DRs) associated with TRAIL-induced cell death was confirmed using Western blot analysis and real-time PCR. Death receptors (DRs) are important cell death-regulating surface proteins that contain death-inducing domains and play a functional role in TAIL-induced cell death, and increased expression of DR5 is known to be a central mediator that promotes TAIL-induced cell death in cancer cells resistant to TAIL treatment.

[0049] PC3 cells were pretreated with various concentrations (0, 25, 50, 100 μM) of telmisartan (Cayman Chemical) for 12 hours, then treated with recombinant TRAIL protein (100 ng / ml) and cultured for another 3 hours. After washing the PC3 cells with PBS, the PC3 cells were lysed in lysis buffer (25 mM HEPES; pH 7.4, 100 mM EDTA, 5 mM MgCl2, 0.1 mM DTT and protease inhibitor mixture). 20-30 μg of the same lysed protein was dissolved on a 10-15% SDS (sodium dodecyl sulfate (SDS)) gel and immunoblotted by a known method. The same amount of dissolved protein was dissolved on a 10-15% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was successively incubated with 5% skim milk and primary antibody. The membrane was then incubated with a specific concentration of primary antibody using dilution buffer (1% milk with PBS-Tween). Sequential secondary antibodies (1:5000) were applied and chemiluminescence reagents were used for membrane development. The primary antibodies used were:

[0050] LC3 and p62 (Sigma-Aldrich, St. Louis, MO, USA), cleaved caspase-3 and cleaved caspase-8 (Cell Signaling Technology, Danvers, MA, USA), DR5 and DR4 (Abcam, Cambridge, MA, USA), β-actin (Sigma-Aldrich)

[0051] The Western blots were visualized using a Fusion-FX7 imaging system (Vilber Lourmat, Marne-la-Vallee, France). Western blot analysis confirmed that telmisartan upregulated both death receptors, DR5, in a concentration-dependent (0, 25, 50, and 100 μM pretreatment for 12 hours) and time-dependent (100 μM pretreatment for 0, 6, 12, and 24 hours) manner (Figures 2A and 2B), confirming that telmisartan increased DR5 expression via a post-transcriptional mechanism and simultaneously stabilized DR5 protein levels by suppressing post-translational degradation.

[0052] PC3 cells on glass coverslips were treated with telmisartan at 100 μM for 12 h and then fixed in 3–4% formaldehyde. The cells were then permeabilized and blocked with 1% BSA in PBST and incubated with primary antibodies (anti-DR5, anti-p62, and anti-LC3) in a humidified chamber for 3 h at room temperature or overnight at 4°C. After washing with PBS, the cells were incubated with secondary antibodies and then stained with DAPI. Cells were observed under a fluorescent microscope, and the expression of DR5 was increased in telmisartan-treated cells (Figure 2C). The expression of cell death-related proteins, cleaved caspase-8 and cleaved caspase-3, was also increased in activation after treatment with telmisartan at 100 μM for 12 h and TRAIL at 100 ng / ml for 3 h (Figure 2D). We confirmed that telmisartan-induced upregulation of DR5 contributes to the enhancement of TRAIL-mediated cell death in TRAIL-resistant prostate cancer cells.

[0053] PC3 cells were pretreated with the specified concentrations of telmisartan (0, 25, 50, 100 μM) for 12 h, followed by TRAIL treatment for an additional 3 h. Total RNA from treated cells was analyzed by Easy-spin TM The total RNA was extracted using a total RNA extraction kit (iNtRON Biotechnology, Seoul, Korea). cDNA synthesis was performed using TaKaRa PrimeScript TM 1 st Real-time PCR was performed using a 2-strand cDNA synthesis kit (Takara Bio Inc., Tokyo, Japan) according to a known method. Real-time PCR was performed in a 20 ul reaction mixture containing 1 μl of gene-specific primers, SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), and 2 μl of cDNA. The primers used were as follows:

[0054] DR5 forward primer: 5′-ACCAGGTGTGATTCAGGTGAA-3′ (SEQ ID NO: 2) DR5 reverse primer: 5′-CCGACTTCACTTGATACTATA-3′ (SEQ ID NO: 3) GAPDH forward primer: 5′-GGAGTCAACGGATTTGGT-3′ (SEQ ID NO: 4) GAPDH reverse primer: 5′-GTGATGGGATTTCCATTGAT-3′ (SEQ ID NO:5)

[0055] All quantitative PCR reactions were performed using a CFX96 real-time PCR system (Bio-Rad). RT-PCR analysis of telmisartan-treated PC3 cells confirmed that the improved DR5 expression was at the post-translational level, as there was no significant difference in DR5 mRNA levels between telmisartan-untreated and -treated PC3 cells (Fig. 3A).

[0056] Example 3. Confirmation of upregulation of DR5 by autophagy inhibition by telmisartan To confirm that the upregulation of apoptotic receptors is at the post-translational level, PC3 cells were treated with cycloheximide (CHX), a potent protein stabilizer that inhibits new protein synthesis. After pretreatment with cycloheximide (CHX) for various times (0, 1, 3 hours), the cells were pretreated with telmisartan (100uM) for 12 hours, then treated with recombinant TRAIL protein (100ng / ml) and cultured for another 3 hours. Western blotting, as described in Example 2, confirmed that CHX treatment reduced DR5 levels, whereas telmisartan treatment stabilized DR5 levels, stabilizing the post-translational level of DR5 (Figure 3B).

[0057] Cells were pretreated with various concentrations of telmisartan (0, 25, 50, 100 μM) for 12 hours, then treated with recombinant TRAIL protein (100 ng / ml) and cultured for another 3 hours, or pretreated with 50 μM telmisartan for various times (0, 6, 12, 24 hours), then treated with recombinant TRAIL protein (100 ng / ml) and cultured for another 3 hours. Western blotting was performed on the cells treated as above to confirm the conversion of LC3-I to LC3-II and the level of p62, which reflects the formation of autophagosomes, which is the beginning of autophagy. As a result, it was confirmed that the conversion of LC3-I to LC3-II was induced, the level of p62 was increased, and lysosomal degradation was inhibited (Figure 4A and Figure 4B).

[0058] Cells were treated with telmisartan or chloroquine (CQ), which inhibits autophagy at a later stage, as a positive control group, and immunocytochemistry and transmission electron microscopy were performed to observe the autophagic endoplasmic reticulum. Immunocytochemistry was performed in the same manner as in Example 2 on cells that had been pretreated with telmisartan (50 μM) or chloroquine (20 μM), then treated with recombinant TRAIL protein (100 ng / ml), and further cultured for 3 hours. As a result, it was confirmed that the fluorescence levels of DR5 and LC3 increased in both telmisartan- and chloroquine-treated cells, indicating the accumulation of autophagic bodies (Figure 4C).

[0059] Transmission electron microscopy (TEM) analysis confirmed autophagy in telmisartan-treated cells. After pretreatment with 50 μM telmisartan (Cayman Chemical) for 12 h, transmission electron microscopy was performed as follows. Cells treated as above were fixed in a solution of 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and 2% paraformaldehyde (EMS, USA) in 0.05 μM sodium chalcodylate (pH 7.2, Electron Microscopy Sciences) for 2 h at 4 °C. Samples were then further fixed in 1% osmium tetroxide (Electron Microscopy Sciences) for 1 h at 4 °C. Dehydration was performed by sequential treatment with alcohol solutions of increasing concentrations (25%, 50%, 70%, 90%, and 100%) for 5 min each. Samples were embedded in epoxy resin (Embed 812, Electron Microscopy Sciences) for 48 h at 60 °C according to the manufacturer's instructions. Ultrathin sections (60 nm) were obtained using an LKB-III ultratome (Leica Microsystems GmbH, Wetzlar, Germany) and then stained with 0.5% uranyl acetate (Electron Microscopy Sciences) for 20 min at room temperature and with 0.1% lead citrate (Electron Microscopy Sciences) for 7 min. Images were taken at 10,000x magnification with a Hitachi H7650 electron microscope (Hitachi, Ltd., Tokyo, Japan). Transmission electron microscopy (TEM) analysis confirmed that autophagy was substantially suppressed in telmisartan-treated cells (Figure 4D). Overall, telmisartan suppressed the inhibition of lysosomal degradation, increased DR5 expression, and improved TRAIL sensitivity.

[0060] Example 4. Confirmation of the effects of blocking autophagy at a later stage TRAIL is a cytokine that specifically binds to cancer cells through receptors containing specific apoptotic domains (DR4 and DER5) on the cell surface, inducing a series of caspase-cascade events, ultimately causing cell death. Thus, increased levels of surface DR5 increase TRAIL sensitivity. In Figure 2, we confirmed that telmisartan increases the expression of DR5. Cells were treated with telmisartan or chloroquine (CQ), which inhibits autophagy at a later stage, as a positive control.

[0061] 1.0 × 10 PC3 cells 4 After seeding in 12-well with 100 μM telmisartan, culturing at 37 ° C for 24 hours, the following groups were examined for cell death: no treatment group, 50 μM telmisartan pre-treated group for 12 hours, 20 μM chloroquine (Sigma-Aldrich) pre-treated group for 12 hours, telmisartan and chloroquine pre-treated simultaneously for 12 hours, 20 μM chloroquine (Sigma-Aldrich) pre-treated group for 12 hours, recombinant TRAIL protein (100 ng / ml) treated group for 3 hours, recombinant TRAIL protein (100 ng / ml) treated group for 3 hours, and recombinant TRAIL protein (100 ng / ml) treated group for 3 hours. As a result, both telmisartan and chlorchitin induced TRAIL sensitivity (Figure 5A to Figure 5C).

[0062] The effect of telmisartan treatment on surface DR5 levels was further investigated. Untreated cells, cells pretreated with telmisartan 50 μM or chloroquine 20 μM, and then treated with recombinant TRAIL protein (100 ng / ml) and cultured for an additional 3 h were analyzed by flow cytometry. Surface DR4 and DR5 were analyzed by FACS analysis using PE-conjugated antibodies specific for the individual receptors. Cells were blocked with 1% goat serum, cultured with 10 μg / mL anti-DR4-PE or anti-DR5-PE (control mouse IgG1-PE and IgG2b-PE, respectively), and analyzed by flow cytometry. Flow cytometry analysis confirmed a dose-dependent increase in surface DR5 induced by telmisartan in PC3 cells (Figure 6A). Furthermore, the inhibition of autophagy at the late stage correlated with an increase in DR5 levels on the surface of PC3 cells (Figure 6B). Western blotting of isolated plasma membranes from telmisartan and chlorexine-treated prostate cancer cells revealed increased levels of DR5 at the plasma membrane (Figure 6C). Notably, blocking autophagy at the late stage amplified TRAIL sensitivity, confirming the role of autophagy in reducing surface DR5 levels while simultaneously inhibiting lysosomal autophagic degradation and increasing sensitivity to TRAIL.

Claims

1. A composition for enhancing the activity of TRAIL against prostate cancer, comprising telmisartan.

2. The composition of claim 1, wherein the telmisartan enhances TRAIL sensitivity to prostate cancer.

3. The composition of claim 1, wherein the prostate cancer is TRAIL-resistant prostate cancer.

4. The composition of claim 1, wherein the telmisartan increases the expression level of DR5 in TRAIL-resistant prostate cancer.

5. The composition of claim 1, wherein the telmisartan inhibits lysosomal degradation in TRAIL-resistant prostate cancer.

6. A pharmaceutical composition for preventing or treating prostate cancer, comprising telmisartan and TRAIL.

7. The pharmaceutical composition according to claim 6, wherein the Telmisartan is 0.1 μM to 200 μM and the TRAIL is 1 mg / ml to 200 mg / ml.

8. The composition of claim 7, wherein the prostate cancer is TRAIL-resistant prostate cancer.

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