KR20110004985A - Inhibitor for refractory prostate cancer comprising resveratrol - Google Patents

Abstract

PURPOSE: An inhibitor for refractory prostate cancer containing resveratrol is provided to suppress progression of prostate cancer and to enhance drug efficiency. CONSTITUTION: An inhibitor for refractory prostate cancer contains resveratrol. The inhibitor is combined with flutamide. The resveratrol is isolated from grape, mulberry, peanut, raspberry, cranberry or cuspidati Radix. The resveratrol suppresses or prevents growth of prostate cancer. The inhibitor for refractory prostate cancer is manufactured in the form of oral, parenteral or local administration.

Description

Inhibitor of regrowth of prostate cancer containing resveratrol {INHIBITOR FOR REFRACTORY PROSTATE CANCER COMPRISING RESVERATROL}

The present invention relates to inhibitors of reverse growth promotion of prostate cancer comprising resveratrol, and more particularly, to the treatment of prostate cancer, flutamide-resistant or prostate cancer which appears when the patient is administered flutamide, a hormone therapy. It relates to a reverse growth promoting inhibitor of prostate cancer comprising resveratrol that inhibits reverse growth promotion.

Prostate cancer is the most common cancer among men in the West and has a high incidence. In Korea, the incidence of prostate cancer is increasing rapidly. The most important causes of prostate cancer are age, race and family history. In addition to genetic predisposition, hormones, dietary habits, and chemicals such as herbicides are also known to play an important role in the onset.

The most helpful methods for diagnosing prostate cancer are prostate specific antigens (PSAs) and rectal balance tests. In general, if the prostate specific antigen level is 4.0 ng / ml or more, it is determined that it is not normal. If a hard nodule is touched on a rectal exam, prostate cancer is suspected first. If serum prostate-specific antigens are increased or abnormal signs are observed on rectal balance examination, prostate biopsy using transrectal ultrasound confirms prostate cancer.

Treatment of prostate cancer depends on the stage of the disease. Local cancer is treated for the purpose of radical treatment, but for metastatic cancer spread to other organs, local treatment is meaningless and systemic treatment is performed.

Surgical treatment and radiation therapy are used as curative treatments for prostate cancer, and hormonal therapy is an adjuvant therapy. In recent years, the combination of radiation therapy and hormonal therapy is widely used, and prostate specific antigen (PSA) is a glycoprotein serine protease that is specific to prostate tissue. Become a useful cancer marker.

In hormonal therapy of prostate cancer, surgical or medical castration suppresses the production of male hormones and at the same time adds flutamide, a drug that inhibits receptors that act on target tissues. However, flutamide treatment, which is an existing treatment for prostate cancer, causes side effects of prostate cancer when resistance or high concentration occurs.

In patients taking flutamide as a primary hormonal therapy for prostate cancer, there is a side effect of initially showing cancer treatment effect and resistance to flutamide or paradoxical stimulation. They have found that resveratrol can inhibit and prevent paradoxical prostate cancer growth-promoting or resistant prostate cancer due to flutamide as described above in the hormone therapy treatment of prostate cancer.

SUMMARY OF THE INVENTION An object of the present invention is to provide an inhibitor for promoting reverse growth of prostate cancer comprising resveratrol which inhibits the promotion of reverse growth of prostate cancer due to flutamide.

Another object of the present invention is to provide an inhibitor for promoting reverse growth of prostate cancer, including resveratrol, which inhibits the progression of hormone refractory prostate cancer.

It is another object of the present invention to provide an inhibitor for promoting reverse growth of prostate cancer comprising resveratrol, which can be co-administered with flutamide.

Hereinafter, the present invention will be described in detail.

The present invention is a reverse growth promoting inhibitor of prostate cancer including resveratrol that inhibits the reverse growth promotion of prostate cancer due to flutamide.

Hereinafter, the present invention will be described in more detail.

Casodex or flutamide, which are existing treatments for prostate cancer, may be a trigger for prostate cancer when resistant or elevated in concentration for long-term treatment.

According to the present invention, it is possible to provide an inhibitor comprising resveratrol that is able to resist or prevent paradoxical stimulation, which may occur when treating prostate cancer with flutamide, or paradoxical stimulation. have.

In the present invention, the inhibitor may inhibit or prevent the progression of hormone refractory prostate cancer.

In addition, in the present invention, the inhibitor may be concurrently administered with flutamide to inhibit or prevent the promotion of reverse growth of prostate cancer due to flutamide.

Resveratrol is well known as the main constituent of grapes, and in addition to grapes, it is mainly abundant in audi, peanuts, raspberries, cranberries, and esoteric roots. Resveratrol is known to be effective for antioxidant activity, anti-aging and heart health, and has been reported to inhibit or prevent the growth of prostate cancer.

In the present invention, resveratrol can be used both in natural form, in commercialized form, or in the form of a synthetic product.

Reporter gene assays produce reporter vectors by cloning the promoter or regulatory element of a specific gene into the 5'-region of the reporter gene, which can be used to generate appropriate reporter vectors from E. coli to mammalian cells. After introduction into cells, various treatments such as simultaneous introduction of a vector expressing transcription factors, treatment of active compounds, and the like followed by activity of reporter enzymes transcribed and translated by changes in promoter activity By quantifying the degree of gene expression.

Androgen-sensitive diseases, namely those in which the onset or development is aided by androgen activity, are known, including prostate cancer. Androgen receptor (androgen receptor) dependent prostate cancer cell line by measuring the transcriptional activity of the androgen receptor by the reporter gene analysis method described above can confirm the inhibition of resveratrol resistant prostate cancer or reverse growth promotion.

In the prostate cancer cell line treated with flutamide alone, the transcriptional activity of the prostate cancer cell line was inhibited at low concentrations, but the transcriptional activity was increased when the concentration of the flutamide was increased. According to the present invention, it can be seen that the transcriptional activity is inhibited when the resveratrol is treated in a concentration-dependent manner with respect to the transcriptional activity of the androgen receptor at a certain flutamide concentration.

Treatment of androgen receptor-dependent substances such as dihydrotestosterone and interleukin-6 in androgen receptor-dependent prostate cancer cell lines increases the transcriptional activity of androgen receptors. At this time, when the low concentration of flutamide is treated, the transcriptional activity is inhibited. As the concentration of the flutamide increases, the transcriptional activity of the androgen receptor is rather increased (refractory phase). This phenomenon proves that the inducers of prostate cancer are increased at high concentrations of flutamide, and the resveratrol concentration-dependent treatment resulted in a significant inhibition of the androgen receptor transcriptional activity.

The anti-proliferation inhibitor of prostate cancer due to flutamide containing resveratrol according to the present invention is not particularly limited in the formulation, and according to the purpose of the conventional oral, parenteral, topical administration, etc. Can be formulated.

In the preparation for oral administration of powders, granules, tablets, capsules and the like, binders, suspending agents, disintegrating agents, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavoring agents and the like can be used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. In the case of injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers and the like can be used in combination.

Flutamid containing resveratrol according to the present invention, the inhibitor for promoting reverse growth of prostate cancer inhibits the progression of prostate cancer in patients with hormone-resistant prostate cancer and also doubles the current efficacy even in combination with conventional therapeutics. As it can be shown above, it is expected to be able to prolong the cure and lifespan of patients in terms of preventive effect.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Reporter Gene Analysis

1. Reagent

RPMI-1640 and fetal bovine serum (FBS) were purchased from invitrogen. The jetPEI DNA transfection reagent was purchased from Polyplus transfection, and the PSA-luc and pDsRed genes were purchased from Clonetech. Luciferase Assay Detection system was purchased from promega.

2. Cell

LNCaP-FGC cells were purchased from ATCC, grown in RPMI-1640 medium containing 10% FBS, and incubated in a 37 ° C. CO ₂ incubator.

3. LNCaP-FGC Prostate Cancer Cell Construction and Experimental Method

LNCaP-FGC cells were aliquoted into RPMI-1640 medium containing 10 ml of 10% FBS at 1 × 10 ⁶ cells per 100 mm dish and incubated in a CO ₂ incubator at 37 ° C. for about 24 hours. The DNA / transfection reagent complex is then mixed for 15 minutes by mixing the two genes with the reagents for co-transfection of the pDsRed and PSA-luc genes with the jetPEI transfection reagent in 4ug increments. After dropping, droplets of DNA / transfection reagent complexes were added dropwise to LNCaP-FGC cells which were replaced with fresh medium (10 ml). After incubation for 24 hours, cells containing the genes were selected by treatment with the pDsRed selective antibiotic G418.

LNCaP-FGC cells permanently delivered with the PSA-luc gene obtained by the above method were dispensed in a 96 well plate at 20,000 per well and incubated in a CO ₂ incubator at 37 ° C. for about 24 hours. Samples were processed by combining concentrations according to the experimental purpose.

Example 1

Treatment with flutamide concentrations of 3.1, 6.25, 12.5, and 25 uM alone, mixed with 25 uM of flutamide and 6.2 and 12.5 uM of resveratrol, were treated in LNCaP-FGC prostate cancer cells with permanent delivery of PSA-luc gene and 37 for 12 hours. Incubated in a CO ₂ incubator.

After removing the cell culture solution and lysing it with 20 ul of cell lysis buffer of Luciferase assay kit, 50 ul of the substrate of the luciferase assay kit was measured by luminometer (Luminometer, Berthold). Gene expression was quantified and the results are shown in FIG. 1.

As shown in Figure 1, the androgen receptor-dependent prostate cancer cell line androgen receptor transcriptional activity was found to be increased by treatment alone, such as flutamide 6.25, 12.5, 25uM. The transcriptional activity of the androgen receptor at 25 uM flutamide concentration was inhibited by the concentration-dependent treatment of 6.25 and 12.5 uM resveratrol.

Example 2

0.2, 0.4, 0.8, 1.6, 3.1, 6.25, 12.5, 25 uM flutamide treatment in the presence of 1 nM of dihydrotestosterone (DHT) or 25 uM flutamide and 6.2, 12.5 uM in the presence of DHT 1 nM PSA-luc genes were treated in permanently delivered LNCaP-FGC prostate cancer cells and cultured in a CO ₂ incubator at 37 ° C. for 12 hours.

After removing the cell culture solution and lysing 20 ul of the cell elution buffer of the luciferase assay kit, 50 ul of the substrate of the luciferase assay kit was measured by luminometer (Berthold) to quantify the gene expression, and as a result Is shown in FIG. 2.

As shown in Figure 2, treatment with dihydrotestosterone in androgen receptor dependent prostate cancer cell line increased the transcriptional activity of the androgen receptor. At this time, treatment with low concentrations (0.2, 0.4, 0.8, 1.6 uM) of flutamide inhibited the transcriptional activity, but higher concentrations (6.2, 12.5, 25 uM) resulted in increased transcription activity, with resveratrol 6.2 and 12.5 uM. As a result, it was confirmed that androgen receptor transcriptional activity was inhibited.

Example 3

Interleukin-6 (IL-6) 10 ng / ml with or without flutamide concentrations 3.1, 6.25, 12.5, 25 uM flutamide in the presence or absence of 10 ng / ml and 10 ug / of interleukin-6 (IL-6) In the presence of ml, 25uM flutamide, resveratrol 6.25, 12.5uM was mixed, treated with LNCaP-FGC prostate cancer cells permanently delivered with PSA-luc gene, and incubated in a CO ₂ incubator at 37 ° C for 12 hours.

Cell cultures were removed and lysed by treatment with 20 ul of the cell elution buffer of the Luciferase Assay Kit, and then 50 ul of the substrate of the Luciferase Assay Kit was measured by luminometer (Berthold) to quantify gene expression. Is shown in FIG. 3.

Treatment of prostate cancer cell line with another factor involved in androgen receptor transcriptional activity, interleukin-6 (IL-6), increased the transcriptional activity of androgen receptors and increased concentrations of 3.1, 6.25, 12.5, and 25uM of flutamide. Depending on the cooperative androgen receptor transcriptional activity is increased. As a result of treatment of resveratrol at 6.25 and 12.5 uM concentration in the presence of 10ug / ml of interleukin-6 and 25uM flutamide, it was confirmed that androgen receptor transcriptional activity was inhibited.

Example 4

Treatment of 0.8, 1.6, 3.1, 6.25, 12.5, 25 uM of flutamide in the presence of 1 nM and IL-6 10 ng / ml or 25 uM flutamide and resveratrol 6.25, 12.5 uM in the presence of DHT 1 nM and IL-6 10 ug / ml Were mixed and treated in cells and incubated in a CO ₂ incubator at 37 ° C. for 12 hours.

Cell cultures were removed and lysed by treatment with 20 ul of the cell elution buffer of the luciferase assay kit, and 50 ul of the substrate of the luciferase assay kit was measured by luminometer (Berthold) to quantify gene expression. Is shown in FIG. 4.

Treatment of androgen receptor-dependent prostate cancer cell lines with androgen and interleukin-6 (IL-6) resulted in a cooperative increase in the transcriptional activity of androgen receptors and treatment with low concentrations (0.8, 1.6, 3.1 uM) of flutamide. The transcriptional activity was inhibited, and at higher concentrations (6.25, 12.5, 25uM), the transcriptional activity increased more cooperatively. At this time, resveratrol was treated with 6.25 and 12.5 uM, and as a result, it was confirmed that androgen receptor transcriptional activity was inhibited.

The present invention is not limited to the technical spirit of the specific examples, test examples, drawings or tables described above, and the general knowledge in the technical field to which the invention belongs without departing from the spirit of the invention claimed in the claims. Of course, anyone with a variety of modifications can be made, as well as such changes are within the scope of the claims.

FIG. 1 is a graph showing gene expression levels (Androgen Receptor (AR)) luciferase activity when treated with flutamide alone and when treated with resveratrol simultaneously in prostate cancer cell lines.

FIG. 2 is a graph showing the amount of gene expression expressed by luciferase activity when flutamid alone and resveratrol were simultaneously treated in prostate cancer cell lines in the presence of dihydrotestosterone.

3 is a graph showing the amount of gene expression as luciferase activity when flutamid alone and resveratrol were simultaneously treated in prostate cancer cell line in the presence of interleukin-6.

FIG. 4 is a graph showing the amount of gene expression expressed by luciferase activity when flutamid alone and resveratrol were simultaneously treated in prostate cancer cell lines in the presence of dihydrotestosterone and interleukin-6.

Claims (3)

A reverse growth promoting inhibitor of prostate cancer comprising resveratrol, which inhibits the reverse growth promotion of prostate cancer due to flutamide. The inhibitor of claim 1, wherein the inhibitor inhibits the progression of hormone refractory prostate cancer. The inhibitor of claim 1 or 2, wherein the inhibitor can be co-administered with flutamide at the same time.

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