WO2017119767A1 - Composition for inhibiting anticancer drug resistance containing rare ginsenoside csh1 (rg6) as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhibiting anticancer drug resistance which contains a rare ginsenoside CSH1 (Rg6) as an active ingredient. The CSH1 (Rg6) was found to promote the binding of BRCA1 and Rad51, inhibit the expression of stress hormone-induced γ-tubulin, and inhibit the resistance to taxol in cancer cells. Therefore, the CSH1 (Rg6) is expected to be useful for drugs for inhibiting resistance to anticancer drugs in anticancer therapy, or anticancer drugs or foods.

Description

Anti-cancer drug resistance inhibiting composition containing rare ginsenoside CSH1 (Rg6) as an active ingredient

The present invention relates to a composition for inhibiting anticancer drug resistance, containing the rare ginsenoside CSH1 (Rg6) as an active ingredient.

Contrary to the commonly mentioned hypothesis, it is not clear whether mental or physical stress is actually involved in the onset or progression of cancer. In other words, the detailed molecular mechanisms of stress-related human cancers are not clear. When the human body is faced with a stress condition, stress hormones such as Cortisol and Epinephrine are released from the adrenal gland in response to pituitary gland secretive corticosteroid hormone (ACTH). An increase in cortisol causes several harmful reactions in the human body, such as depression and a decrease in the immune system. However, these reactions are not directly related to cancer initiation.

Renal cell carcinoma (RCC) is a human malignant tumor of the kidney near the adrenal gland. Although kidney cancer is known to develop for a variety of reasons, such as aging and smoking, stress is also a major cause. Serum cortisol levels are increased in kidney cancer patients. In this regard, the effect of stress hormone suppression on the immune system is generally known, but this is not enough to account for stress-induced cancer. Several epidemiological analyzes have shown that stress can increase the incidence of several types of human cancer, including colorectal and lung cancer.

Previous reports have shown that, under von Hippel-Lindau syndrome gene-deficient conditions, treatment of elevated estrogen receptor (ER-α) or estrogen may amplify the microtubule organizing center (MTOC). It has been shown to provide Taxol resistance through. VHL is directly related to ER-α and inhibits MTOC amplification. Under VHL-deficient conditions, elevated ER-α disrupts BRCA1-Rad51 binding, which inhibits MTOC amplification. Inhibition of ER-α by ER-α inhibitors such as tamoxifen or Faslodex can restore the number of centrosomes in VHL-deficient RCC cell lines. In addition, since stress hormones are derived from steroids and share a common chemical structure, the hypothesis that stress hormones promote MTOC amplification and Taxol resistance is very reasonable.

Panax Ginseng has long been used to treat many types of human diseases, including cancer and kidney dysfunction. Recently, isolated ginsenoisdes have been reported to be effective in the immune system. However, the mechanism of molecular biological action of ginsenosides on human cancers has not been identified to date. Therefore, it is very meaningful to elucidate the mechanism and effect of ginsenosides on human cancer.

The present invention provides a pharmaceutical composition for inhibiting anticancer drug resistance or a pharmaceutical composition for preventing or treating cancer, containing the rare ginsenoside CSH1 (Rg6) as an active ingredient.

In addition, the present invention is to provide a food composition for inhibiting anticancer drug resistance containing rare ginsenoside CSH1 (Rg6) as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for inhibiting cancer drug resistance containing rare ginsenoside CSH1 (Rg6) as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating cancer containing the rare ginsenoside CSH1 (Rg6) as an active ingredient.

The present invention also provides a food composition for inhibiting anticancer drug resistance, containing the rare ginsenoside CSH1 (Rg6) as an active ingredient.

The present invention relates to a food composition for inhibiting anticancer drug resistance containing rare ginsenoside CSH1 (Rg6) as an active ingredient, wherein CSH1 (Rg6) promotes the binding of BRCA1 and Rad51 and expresses stress hormone-induced γ-tubulin expression. It was confirmed that the effect of inhibiting the resistance to Taxol in cancer cells. Therefore, the CSH1 (Rg6) is expected to be usefully used in drugs, anticancer drugs or foods that inhibit anticancer drug resistance in anticancer treatment.

1 shows the result that stress hormone (Glucocoticoid hormone) induces Taxol resistance. (a) Cortisone and Cortisol blocked Taxol (TAX) -induced cell death, but did not block aldosterone. Aldosterone (5 μM), Cortisone (5 μM), Cortisol (5 μM) and Taxol (3 μM) were treated with VHL-positive C2V cells for 72 hours. Cell viability was measured by MTT assay. (b) Cortisone shows Taxol resistance similar to estrogens. ACHN cells were treated for 72 hours with indicated doses of steroid hormones. Cell viability was measured by MTT assay. (c) Cortisone and cortisol blocked Taxol-induced cell death even in non-renal cancer cell lines (lung cancer cell lines; A549, H1299, colorectal cancer cell lines; HCT116, SW480). Aldosterone (5 μM), Cortisone (5 μM), Cortisol (5 μM) and Taxol (3 μM) were treated with each cancer cell for 72 hours. Cell viability was measured by MTT assay. (d) show the specific effect of cortisone on Taxol-induced cell death. Unlike Taxol, cortisone does not show resistance to DNA damaging agents such as Adriamycin (Adr) and Etoposide (Etop). Cells were incubated for 72 hours with the indicated compounds (taxol 3 μM; cortisone 5 μM; Adr 2 μg / ml; Etop 10 μM). Cell viability was measured by MTT assay. (e) ER-α inhibition through Faslodex (FST) does not block Glucocorticoid Hormone (GH) -induced Taxol resistance. ACHN cells were incubated for 72 hours with FST (3 μM), Taxol (3 μM), Cortisone (5 μM) and Cortisol (5 μM). Cell viability was measured by MTT assay.

2 shows that stress hormones increase MTOC through the Glucocorticoid Receptor (GR). (a) Cortisol clearly induced γ-tubulin in several cell lines. Each cell was treated for 72 hours with the indicated dose of cortisol. Western blots were performed to measure γ-tubulin expression. Actin was used as a loading control. (b) GHs induce γ-tubulin independently of FST. A498 (VHL deficient cell line) was incubated with cortisone (5 μM) and cortisol (5 μM) for 24 hours with or without FST (3 μM). BRCA1 expression was increased by FST but did not affect the reduction of BRCA1 expression by GH. In addition, p53 expression was not altered by FST or GH. Actin was used as a loading control. (c and d) The number of MTOCs was increased by cortisone treatment (5 μM) in VHL-intact C2V cells. About 50 cells were counted for each condition to determine the average MTOC number and plotted. Cells were stained with anti-γ-tubulin-antibody (red) to detect MTOC and DAPI (blue) to detect DNA. (e) GR and cortisone can induce MTOC amplification. After GR transfection or cortisone (5 μM) treatment, HEK293 cells were stained with anti-γ-tubulin-antibody (red) and DAPI (blue). (f) shows the additional effect of GH and GR on MTOC amplification. About 50 cells were counted for each condition to determine the MTOC number. (g) In VHL-positive C2V cells, the overexpressed GR showed Taxol resistance. GR was transfected into C2V cells and treated with Taxol (3 μM). After 72 hours, cell viability was measured by MTT assay. (h) GR blocks Rad51-mediated Taxol-sensitivity. In VHL-deficient A498, re-sensitivity to Taxol via Rad51 overexpression was lost by GR overexpression. A498 cells were transfected with GR and / or Rad51 and incubated with Taxol for 72 hours. Cell viability was measured by MTT assay. (i) GR restored Rad51-induced γ-tubulin reduction. In addition, GR overexpression clearly reduced Rad51. GFP-labeled GR and HA-labeled Rad51 were transfected into HEK 293 cells. After 72 hours, protein levels were detected with anti-GFP and HA antibodies. (j) Clearly confirmed Rad51 reduction by cortisone treatment in GR-transfected HEK 293 cells. GFP-labeled GR was transfected with HEK 293 cells and treated with cortisone (5 μM). After 72 hours, Western blots were performed. (k) GR expression was not altered by VHL. GFP-labeled GR and HA-labeled VHL were transfected into HEK 293 cells and treated with cortisone (5 μM). After 72 hours, Western blots were performed. Actin was used as a loading control.

3 shows that GR inhibition blocks MTOC amplification. (a and b) GR antagonist Progesterone (PGT) blocked MTOC in ACHN cells. PGT (5 μΜ) treated cells blocked MTOC amplification. About 80 cells were counted for each condition to confirm the mean MTOC number (b) and photographed (a). Cells were stained with anti-γ-tubulin-antibody (red) to detect MTOC and DAPI (blue) to detect DNA. (c) PGT treatment inhibited stress hormone-induced γ-tubulin induction. Western blot analysis was performed with the indicated antibodies. Actin was used as a loading control. (d) Ketoconazole (KCZ), another chemical antagonist of GR, also blocked stress hormone effects in ACHN cells. Treatment with PGT (5 μM) and KCZ (5 μM) for 72 hours. Each protein expression was measured by Western blot. Actin was used as a loading control. (e) GR antagonists overcome cortisol induced Taxol-tolerance. Cortisol (5 μM) and Taxol (3 μM) were treated for 72 hours in ACHN cells. PGT (5 μM) and KCZ (5 μM) were treated for the same time. Cell viability was measured by MTT assay.

4 shows that GR binds to Rad51 and disrupts Rad51-BRCA1 interaction. (a) Cortisone (5 μM) and cortisol (5 μM) in VHL-positive ACHN cells disrupted Rad51-BRCA1 binding interactions, but had a weak effect in VHL-negative A498 cells. For binding assays, immunoprecipitation (IP) analysis with anti-Rad51 antibodies was performed. (a) In VHL-intact ACHN cells, Rad51-BRCA1 binding was reduced by cortisone treatment. Anti-BRCA1 antibodies were used for IP analysis. (c) GR only bound Rad51, but not BRCA1. (d) Binding of GR-Rad51 was confirmed by exogenous protein. In HEK293 cells, GFP-labeled GR and HA-labeled Rad51 were overexpressed and lysates of the cells were used for IP analysis. (e) Increased binding of Rad51-GR was detected by cortisone (5 μM) treatment. IP analysis was performed with anti-GR antibodies. (f) In VHL-negative C2 cells, Taxol sensitivity induced by FST was blocked via GR overexpression. (g) For VHL-intact C2V cells with Taxol sensitivity, cortisone induced Taxol resistance was not affected by ER-α inhibition. After transfection for GR overexpression, Taxol (3 μM) and FST (3 μM) were treated. After 72 hours, MTT assay was performed to measure cell viability.

5 shows the chemical structures of the rare ginsenosides CSH1 (RG6) and RG3, estrogen and cortisone.

Figure 6 shows the effect of rare ginsenosides on Taxol-induced cell death. (a) shows the effect of several ginsenosides on Taxol-induced cell death. In C2 cells, only CSH1 (Rg6) shows Taxol sensitive effect. Ginsenosides (5 μM) and Taxol (3 μM) were treated respectively. After 72 hours, cell viability was measured by MTT assay. (b and c) PGT and CSH1 inhibited stress hormone-induced γ-tubulin expression in lung cancer cell line A549. PGT (5 μM), CSH1 (5 μM), cortisol (5 μM) and cortisone (5 μM) were treated for 72 hours. Western blots were performed to measure γ-tubulin expression. Actin was used as a loading control. Cells were stained with anti-γ-tubulin-antibody (red) and DAPI (blue). (d) GR expression was inhibited by CSH1. GR-induced Rad51 expression inhibition was restored by CSH1 treatment. (e) CSH1 promoted the interaction of BRCA1 and Rad51 and restored binding broken by GR. MYC-labeled BRCA1, HA-labeled Rad51 and GFP-labeled GR were overexpressed in HEK293 cells and cell lysates were used for IP analysis for binding assays. IP analysis was performed with anti-MYC antibodies. (f and g) CSH1 could block GR or cortisone induced MTOC amplification. About 50 cells were counted to confirm the mean MTOC number and photographed. GFP-labeled GR was transfected into HEK 293 cells. CSH1 (5 μM) and cortisone (5 μM) were treated. Cells were stained with anti-γ-tubulin-antibody (red) and DAPI (blue).

Figure 7 shows the chemical structures of CSH1-related ginsenosides CSH2 to CSH4.

FIG. 8 shows that two normal human fibroblasts were cultured for one month with or without the addition of cortisol and CSH1 at low serum concentrations to confirm that continuous treatment of stress hormones could induce transformation. to be.

9 is the result for CSH1 showing a similar effect to FST. (a) shows inhibitory effect of CSH1 on estrogen-induced cell proliferation. Proliferation of ER-α positive MCF-7 cells was dose-dependently increased by estrogen-treatment. Similar to FST, CSH1 inhibited estrogen induced proliferation in MCF-7. Compounds were treated as indicated. After 72 hours, MTT assay was performed to measure cell viability. (b) Inhibition of γ-tubulin expression by FST was seen only in MCF-7 cells, but CSH1 reduced γ-tubulin in both MCF-7 and MDA-MB-468 cells. (c) In ER-α transfected HEK293 cells, ER-α mediated transcriptional activity was reduced by CSH1 and CSH3. To measure ERE-luciferase activity, the ERE-luciferase vector and ER-α were cotransfected into HEK 293 cells. (d) Similar results were obtained with MCF-7 cells. Like FST, CSH1 effectively inhibited ERE luciferase activity. ERE-luciferase vectors were transfected into ERα-positive MCF7 cells and ERα-negative MDA-MB-468 cells. ERE-luciferase analysis was performed 4 hours after each compound treatment. (e) In VHL-negative C2 cells, Taxol-induced cell death by CSH1 showed a concentration dependent sensitivity similar to FST. (f) As a result of Western blot analysis, expression of γ-tubulin and ER-α was decreased by the treatment of CSH1, but Rad51 expression was increased. (g) Expression of ER-α was inhibited by CSH1. ER-α was transfected into VHL-positive ACHN cells. After transfection, CSH1 (5 μM) was treated. After 72 hours, Western blots were performed. (h and i) In VHL-negative C2 cells, MTOC amplification was inhibited by rare ginsenosides similar to FST. About 50 cells were counted for each indicated condition to confirm the mean MTOC number and photographed. FST (5 μM), CSH1 (5 μM) and CSH3 (5 μM) were treated to VHL-negative C2 cells. After 72 hours, IF staining was performed. Cells were stained with anti-γ-tubulin-antibody (red) and DAPI (blue).

10 is a schematic diagram showing that the GR pathway is activated in response to stress hormones, and MTOC amplification and chromosomal instability are induced under stress conditions. It is one of the estimated tumorigenic mechanisms for stress-induced cancer. However, unlike the ER-α pathway, GR is not regulated by VHL. CSH1, a rare ginsenoside, has a steroid-like backbone, which blocks GR as well as ER-α pathway-induced MTOC amplification and Taxol sensitivity. Therefore, it can be used as a cancer prevention strategy and anticancer drug.

The present inventors therefore focused on the tumor forming effects of stress hormones, in particular on MTOC amplification and anticancer drug resistance. Since several ginsenosides have stress hormone-related chemical structures, the effects of ginsenosides on MTOC amplification and Taxol resistance have been investigated and the present invention has been completed.

The present invention provides a pharmaceutical composition for inhibiting anticancer drug resistance, containing the rare ginsenoside CSH1 (Rg6) as an active ingredient.

In detail, the anticancer drug resistance may be induced by a stress hormone, but is not limited thereto.

Specifically, the rare ginsenoside CSH1 (Rg6) inhibits γ-tubulin expression induced by stress hormones, promotes binding of BRCA1 and Rad51, and amplifies the microtubule organizing center (MTOC). Can be suppressed.

Preferably, the stress hormone may be Cortisone or Cortisol, but is not limited thereto.

Preferably, the anticancer agent may be Taxol, but is not limited thereto.

The present invention also provides a pharmaceutical composition for preventing or treating cancer containing the rare ginsenoside CSH1 (Rg6) as an active ingredient.

Preferably, the cancer may be cancer having anticancer drug resistance, but is not limited thereto.

Preferably, the anticancer agent may be Taxol, but is not limited thereto.

Preferably, the cancer may be kidney cancer, lung cancer, colon cancer or breast cancer, but is not limited thereto.

In one embodiment of the invention, the pharmaceutical composition is any one selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or solutions according to conventional methods Formulations of

In another embodiment of the present invention, the pharmaceutical composition is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents commonly used in the manufacture of pharmaceutical compositions It may further comprise one or more additives selected from the group consisting of diluents, dispersants, surfactants, binders and lubricants.

Specifically, the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.

According to one embodiment of the invention the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal Via the route can be administered to the subject in a conventional manner.

The preferred dosage of the pharmaceutical composition may vary depending on the condition and weight of the subject, the type and severity of the disease, the drug form, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

The present invention provides a food composition for inhibiting anticancer drug resistance containing rare ginsenoside CSH1 (Rg6) as an active ingredient.

In detail, the anticancer drug resistance may be induced by a stress hormone, but is not limited thereto.

Specifically, the rare ginsenoside CSH1 (Rg6) inhibits γ-tubulin expression induced by stress hormones, promotes binding of BRCA1 and Rad51, and amplifies the microtubule organizing center (MTOC). Can be suppressed.

Preferably, the stress hormone may be Cortisone or Cortisol, but is not limited thereto.

Preferably, the anticancer agent may be Taxol, but is not limited thereto.

The present invention also provides a food composition for preventing or improving cancer containing rare ginsenoside CSH1 (Rg6) as an active ingredient.

Preferably, the cancer may be cancer having anticancer drug resistance, but is not limited thereto.

Preferably, the anticancer agent may be Taxol, but is not limited thereto.

Preferably, the cancer may be kidney cancer, lung cancer, colon cancer or breast cancer, but is not limited thereto.

The food composition of the present invention is variously used as a food composition for inhibiting anticancer drug resistance, the food composition comprising the composition of the present invention as an active ingredient, various foods, for example, beverages, gum, tea, vitamin complex, It may be prepared in the form of powder, granules, tablets, capsules, sweets, rice cakes, bread and the like. Since the food composition of the present invention has little toxicity and side effects, it can be used with confidence even for long-term use for the purpose of prevention. When the composition of the present invention is included in a food composition, the amount may be added at a ratio of 0.1 to 100% of the total weight. Herein, when the food composition is prepared in the form of a beverage, there is no particular limitation other than the food composition in the proportion indicated, and may include various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and common sugars such as polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol. can do. Examples of the flavourant include natural flavourant (tautin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators Stabilizing agents, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, etc. These components may be used independently or in combination The ratio of such additives is not so critical, but 100 weight of the composition of the present invention. It is generally selected in the range of 0.1 to 100 parts by weight per part.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Experimental Example

The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

1. Cell Lines and Reagents

ACHN (VHL +) and A498 (VHL−) cells were purchased from the Korean Cell Line Bank. A549, HEK293, MCF-7 and MDA-MB-468 were purchased from the American Cell Line Bank (ATCC, Manassas, VA). Other renal cancer cell lines (UMRC2; C2, UMRC2 / VHL; C2V) are described by Dr. Jung, YJ (Pusan University). Human fibroblasts were purchased from Coriell Cell Repositories (New Jersey, USA). ACHN, A498, HEK293, MCF-7 and MDA-MB-468 were maintained in liquid DMEM medium containing 10% FBS and 1% antibiotic in a 37 ° C. growth chamber. HCT116 cell line Obtained from Vogelstein B (Johns Hopkins University). A549, HCT116 was maintained in RPMI-1640 with 10% FBS and antibiotics. Estrogen (250155), Fulvestrant (I4409), Taxol (T7402), Ketoconazole (UC280), Progesterone (P0130), Cortisone (C2755) and Cortisol; H4001 ) Was purchased from Sigma (Missouri, USA). Antibodies against Actin (sc-1616), ER-α (sc-8002), GFP (sc-7392) and HA (sc-9996) were purchased from Santa Cruz (California, USA). Anti-γ-tubulin (T6557) was purchased from Sigma (Missouri, USA) and anti-glucocorticoid receptor (GR) Ab (12041) was purchased from Cell signaling (Massachusetts, USA). Rad51 (05-530), BRCA1 (07-434) were purchased from Milliopore (Darmstadt, Germany).

2. Vector and Transfection

GFP-tagged GR (GR-GFP) and HA-tagged Rad51 (Rad51-HA) vectors were purchased from Addgene. Myc-tagged wild type BRCA1 (BRCA1-Myc) Obtained from Livingston, DM (Harvard Medical School). pVHL animal cell expression vector is Obtained from Jung, YJ (Pusan University). Transfection for animal cell expression of the vectors was performed using a Jetpei transfection agent (Polyplus New York, USA). Briefly, the vector (1.5 μg) was mixed with 1.5 μl Jetpei reagent dissolved in 150 mM NaCl solution. The mixture was reacted at room temperature for 15 minutes. After the reaction, the mixture was added to the cells. After 3 hours, serum-free medium was replaced with medium containing 10% FBS.

3. Weston Blot  Assays and Protein Interaction Studies

Proteins were extracted from cells using RIPA buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS and 10% sodium deoxycholate). Samples were applied to SDS-PAGE and transferred to PVDF membrane. The blotted membrane was reacted with the primary antibody for 1 hour at 4 ° C., and HRP-conjugate species-matched secondary antibodies were reacted for 1 hour at room temperature. Peroxidase activity was detected through chemi-luminescence with an ECL kit (Intron, Seoul, Korea). For immunoprecipitation (IP) analysis, first whole cell lysates were reacted with appropriate antibody for 4 hours at 4 ° C, followed by protein A / G agarose beads (Invitrogen, California, USA) for 2 hours at 4 ° C. Reacted. After centrifugation and washing with RIPA, the precipitated immuno-complex was subjected to SDS-PAGE and Western blot analysis.

4. Immunofluorescence  dyeing

Cells were placed in a cover glass and transfected with the indicated vectors or treated with the indicated compounds. After fixing for 30 minutes with Me-OH, cells were reacted with blocking buffer [PBS + anti-human-Ab (1: 500)] for 1 hour. After washing with PBS, cells were reacted for 4 hours with Anti-γ-tubulin antibody (1: 100 to 200) dissolved in blocking buffer, and FITC-conjugated or Rhodamine-conjugated secondary antibody (1) dissolved in blocking buffer. : 500) for 2 hours. Nuclei were stained with DAPI. After rinsing with PBS, the cover glass was mounted in mounting solution (Vector Laboratories, Cambridgeshire, UK). Immunofluorescence signals were detected by fluorescence microscopy (Zeiss, Jena, Germany).

5. MTT  analysis

To measure cell viability, cells were treated with the indicated compounds for 4 days. For MTT assay, cells were reacted with 0.5 mg / ml MTT solution (Calbiochem, Darmstadt, Germany) at 37 ° C. for 4 hours. After removal of the remaining solution, the precipitate was dissolved in 200 μl DMSO and quantified by measuring at 540 nm absorbance.

6. Luciferase ( Luciferase ) analysis

To measure ER-α promoter activity, ERE-Luc vectors were transfected into cells for 24 hours and cells were treated with the indicated compounds. After washing with wash buffer (Promega, Wisconsin, USA), cells were lysed with lysis buffer (Promega, Wisconsin, USA). Luciferase activity was measured by a luminometer (MicroDigital, Gyeonggi-do, South Korea).

7. Statistical Analysis

All results were expressed as mean + s.e.m., with at least n = 4 per group. To obtain statistical significance, Student's t-test was performed.

< Example  1> Taxol  Stress hormones that cause resistance

Cortisol, a stress hormone and related hormones (cortisone and aldosterone), originated from cholesterol and exhibited a chemical structure similar to estrogen, so their biological effects on Taxol-induced cell death were tested. Although not aldosterone, cortisone and cortisol showed Taxol resistance in both RCC cell lines similar to estrogens (FIG. 1A). Their inhibitory effect was dose-dependent (FIG. 1B). Stress hormones can affect many types of tissues and cells. We have identified the effects of glucocorticoid hormones in lung and colorectal cancer cell lines, and cortisol and cortisone are Taxol-induced cells in a dose-dependent manner. Results similar to inhibiting death were obtained (FIG. 1C). In this cell line, aldosterone did not change Taxol-sensitivity even at high doses. Next, the inventors confirmed the effects of cortisone and cortisol on other types of anticancer agents. Similar to estrogens, cortisol and cortisone did not change their sensitivity to Adriamycin or etoposide (FIG. 1D). Since cortisol / cortisone-induced taxol resistance is known to be obtained by the ER-α / Est signaling mechanism, we treated the ER-α inhibitor Fulvestrant (FST) and measured Taxol-sensitivity. . However, FST did not block cortisone / cortisol-induced Taxol resistance, indicating that the effects of these hormones on Taxol-induced cell death are caused by ER-α independent mechanisms (FIG. 1E).

< Example  2> MTOC  Stress hormones that promote amplification

Since synergistic expression of γ-tubulin can overcome Taxol-induced cell death, we first measured γ-tubulin expression. As expected, cortisol / cortisone certainly induced γ-tubulin in all test cell lines (FIG. 2A) and FST did not block γ-tubulin induction (FIG. 2B). In addition, in the above experiments, the inventors found that BRCA1 was decreased in response to cortisol and cortisone (FIG. 2B). Indeed, a decrease in BRCA1 has been identified in Est-mediated γ-tubulin induced conditions. However, cortisone could induce γ-tubulin in ER-α negative cell lines. In addition, the hormone could promote MTOC amplification. Indeed, cortisone-treatment could increase the average number of mitotic MTOCs from 2 to 3 (FIGS. 2C and 2D).

< Example  3> MTOC  Glucocorticoid Receptors May Promote Amplification

Cortisol and cortisone are glucocorticoid hormones, and their signaling is mediated by glucocorticoid receptors (GR), so we have identified the association of GR to MTOC amplification. Transfection with GR alone was able to increase MTOC numbers as well as cortisone treatment (FIGS. 2E and 2F). GR can also block Taxol-induced cell death (FIG. 2G). Thus, we identified the effect of GR on Rad51-mediated Taxol sensitivity. Previous studies have found that Rad51 overexpression can regain sensitivity in Taxol-resistant ER-α elevated cells and VHL deficient cell lines. Interestingly, GR overexpression could reduce Rad51 to block Rad51-mediated Taxol re-sensitivity (FIGS. 2H and 2I). Indeed, GR overexpression and cortisone treatment reduced endogenous Rad51 expression (FIG. 2J). However, unlike ER-α, GR expression did not change with VHL status. VHL overexpression or knock down did not affect GR expression, and FST did not affect GR expression (FIG. 2K). The results indicate that stress hormones induce MTOC amplification, and γ-tubulin elevation is caused by GR through VHL independent mechanism.

< Example  4> stress hormone-mediated MTOC  In control suppression GR  Preferred Effects of Inhibitors

To confirm the association of GR in MTOC amplification, we tested the effect of progesterone (PGT) on stress hormone-induced MTOC amplification. PGT is well known as an antagonist of GR. Treatment of PGT inhibited γ-tubulin induction as well as stress hormone-induced MTOC amplification (FIGS. 3A, 3B and 3C). Since PGT is also a hormone, we used ketoconazole (KCZ), another chemical antagonist of GR. Consistent with PGT, KCZ also inhibited γ-tubulin induction and a decrease in Rad51 and BRCA1 (FIG. 3D). In addition, the inhibitors overcome cortisol induced Taxol-resistant (FIG. 3E).

< Example  5> On Rad51  In combination BRCA1 - Rad51  Destroying bond GR

To determine how the glucocorticoid hormone promotes MTOC amplification, the effect of the hormone on Rad51-BRCA1 binding was confirmed. Indeed, the binding between BRCA1-Rad51 in ACHN cells intact with VHL was clearly reduced by the glucocorticoid hormone (FIG. 4A). Instead, the binding in VHL-deficient A498 was weak and appeared to be slightly reduced by glucocorticoid hormones (FIG. 4A). Isolation of BRCA1-Rad51 by cortisone was confirmed by IP analysis using BRCA1 Ab (FIG. 4B). One of BRCA1 and Rad51 is the binding target of GR. To confirm which of BRCA1 and Rad51 was the target of GR, IP analysis was performed using GR. Unlike ER-α, which is equally involved in the two proteins, GR showed binding affinity only in Rad51 (FIG. 4C). Binding between Rad51 and GR was confirmed by exogenous protein (FIG. 4D). Although binding of Rad51 and GR was detected without cortisone, GH promoted binding (FIG. 4E), presumably due to GR transition to the nucleus. To eliminate the association of ER-α in the reaction, GR-induced taxol resistance was measured under FST-treatment conditions. While FST shows Taxol sensitivity in VHL-deficient C2, GR can be found to overcome FST-induced sensitivity (FIG. 4F). In addition, in C2V where VHL is intact, FST does not block GR-induced Taxol resistance (FIG. 4G). The results indicate that GR / GH signaling can disrupt Rad51-BRCA1 binding through an ER-α independent mechanism.

< Example  6> Taxol Rare ginsenosides susceptible to induced cell death

We have found that adequate inhibition of stress hormone signaling may indicate sensitivity to Taxol as well as inhibition of MTOC regulation (FIG. 3). Thus, the present inventors searched for candidate compounds capable of inhibiting the stress hormone-GR network. In Chinese medicine, Panax Ginseng is used for cancer and kidney disease. Indeed, some types of ginsenosides, especially rare ginsenosides CSH1 (RG6), have a chemical structure very similar to estrogen or cortisone (FIG. 5). Therefore, the effect of Rg6 on Taxol-induced cell death was tested in comparison with other common ginsenosides. Interestingly, CSH1 clearly showed sensitivity to Taxol in C2 cells. However, other common ginsenosides did not show a clear effect (FIG. 6A). In addition, CSH1 showed a similar effect in A498 cells. To get more detailed information on this, the effects on CSH1-related ginsenosides (CSH2 to CSH4; FIG. 7) were identified. In VHL-deficient C2 cells that are resistant to Taxol by ER-α elevation, CSH1 and CSH3 have increased Taxol sensitivity similar to FST. However, C2V (taxol sensitive cell line) did not show additional sensitivity to Taxol. Thus, the present inventors confirmed the effect of CSH1 on γ-tubulin induction by cortisol and cortisone. Similar to PGT, CSH1 inhibited stress hormone-induced γ-tubulin expression as well as MTOC amplification in human lung cancer cell line A549 (FIGS. 6B and 6C). The present inventors confirmed the beneficial effect of CSH1 in human colon cancer cell line HCT116. The results indicate that CSH1 can inhibit MTOC amplification regardless of cancer cell type.

< Example  7> Blocking stress hormone-induced dysfunction CSH1

To identify more detailed biological effects on CSH1, we observed the effect of CSH1 on Rad51 and GR expression. CSH1 inhibited GR expression and inhibited the reduction of Rad51 by GR-overexpression (FIG. 6D). In addition, CSH1 promoted the interaction of BRCA1 and Rad51 and was able to overcome their binding broken by GR (FIG. 6E). Based on these results, the inventors confirmed the number of MTOCs. As expected, the increase in MTOC by cortisone or GR overexpression was completely inhibited by CSH1 treatment (FIGS. 6F and 6G). Since CSH1 blocks MTOC amplification and inhibition of γ-tubulin regulation, it is expected that the compound may be usefully used for cancer prevention. In addition, two normal human fibroblasts were cultured for one month with or without the addition of cortisol and CSH1 at low serum concentrations to determine if continuous treatment of stress hormones could induce transformation. After one month, cells were seeded in 96 well plates and incubated for an additional two weeks without serum. Under severe conditions, normal fibroblasts survived only 2 wells. In contrast, most of them survived cortisol-treated cells (61/96 wells, 85/96 wells). Treatment with CSH1 under these conditions significantly reduced survival (FIG. 8). The results indicate that under adverse conditions cortisol can promote cell survival and that inhibition of the GR pathway through CSH1 can overcome inadequate cell survival due to stress-hormone induction.

< Example  8> shows a similar effect to FST CSH1

Similar to FST, CSH1 exhibits sensitivity to Taxol in C2 cells, thus confirming the possibility that CSH1 could replace FST. First, estrogen-induced cell proliferation was observed. Similar to FST in ER-α positive MCF-7 cell line, CSH1 was able to block estrogen-induced cell proliferation (FIG. 9A). In addition, CSH1 was able to inhibit estrogen-induced-γ-tubulin and ER-α (FIG. 9B). However, they did not alter basic Rad51 expression (FIG. 9B). In addition, ER-α-mediated transcriptional activity was reduced by CSH1 in exogenous ER-α transfected 293 cells (FIG. 9C). In addition, it was confirmed that ERE-Luc activity was strongly reduced by FST by CSH1 in MCF-7 (FIG. 9D). The results indicate that CSH1 may act as an ER-α inhibitor. Taxol-sensitivity was also confirmed. Similar to the dose-dependent sensitivity of FST, CSH1 also increased Taxol-induced cell death with dose in C2 cells (FIG. 9E). In addition, CSH1 also increased Rad51 expression like FST in VHL-deficient C2 cells (FIG. 9F). FST can also inhibit ER-α expression, so this was also tested. Exogenous ER-α was clearly reduced by CSH1 (FIG. 9G), indicating that CSH1 can function very similar to FST. Finally, MTOC amplification was observed compared to FST capable of restoring MTOC. CSH1 as well as CSH3 recovered MTOC (FIGS. 9H and 9I). These results indicate that CSH1 as well as CSH3 can be used as one of the FST replacement compounds.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

1. A pharmaceutical composition for inhibiting anticancer drug resistance, containing the rare ginsenoside CSH1 (Rg6) as an active ingredient.
2. The pharmaceutical composition for inhibiting anticancer drug resistance according to claim 1, wherein the anticancer drug resistance is induced by a stress hormone.
3. The method of claim 1, wherein the rare ginsenoside CSH1 (Rg6) inhibits γ-tubulin expression induced by stress hormones, promotes the binding of BRCA1 and Rad51, and microtubule organizing center (MTOC) A pharmaceutical composition for inhibiting anticancer drug resistance, characterized by inhibiting amplification.
4. The pharmaceutical composition for inhibiting anticancer drug resistance according to claim 2 or 3, wherein the stress hormone is cortisone or cortisol.
5. The pharmaceutical composition of claim 1, wherein the anticancer agent is Taxol.
6. A pharmaceutical composition for preventing or treating cancer, containing the rare ginsenoside CSH1 (Rg6) as an active ingredient.
7. 7. The pharmaceutical composition for preventing or treating cancer according to claim 6, wherein the cancer is cancer having anticancer drug resistance.
8. 8. The pharmaceutical composition for preventing or treating cancer according to claim 7, wherein the anticancer agent is Taxol.
9. The pharmaceutical composition for preventing or treating cancer according to claim 6, wherein the cancer is kidney cancer, lung cancer, colon cancer, or breast cancer.
10. A food composition for inhibiting anticancer drug resistance, containing the rare ginsenoside CSH1 (Rg6) as an active ingredient.
11. The food composition of claim 10, wherein the anticancer drug resistance is induced by a stress hormone.
12. The method of claim 10, wherein the rare ginsenoside CSH1 (Rg6) inhibits γ-tubulin expression induced by stress hormones, promotes binding of BRCA1 and Rad51, and microtubule organizing center (MTOC). A food composition for inhibiting anticancer drug resistance, characterized by inhibiting amplification.
13. The food composition of claim 11 or 12, wherein the stress hormone is cortisone or cortisol.
14. 11. The anticancer drug resistance food composition of claim 10, wherein the anticancer agent is Taxol.

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