KR101274731B1 - Pharmaceutical composition for treating prostate cancer comprising resveratrol analogue HS-1793 or pharmaceutically acceptable salts thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating prostate cancer, comprising as an active ingredient HS-1793 or a pharmaceutically acceptable salt thereof, which is an analog of resveratrol. More specifically, the present invention relates to a pharmaceutical composition for treating cancer by selectively removing prostate cancer cells by inducing apoptosis by inducing proliferation and proliferation of prostate cancer cells using HS-1793, an analog of resveratrol.
The present invention enables effective cancer cell treatment by inducing specific and selective killing, particularly for Aurora B kinase deficient cancer cells.

Description

Pharmaceutical composition for treating prostate cancer comprising resveratrol analogue HS-1793 or pharmaceutically acceptable salts according to the resveratrol analogue of Hs-173, or a pharmaceutically acceptable salt thereof

The present invention relates to a pharmaceutical composition for treating prostate cancer, comprising as an active ingredient HS-1793 or a pharmaceutically acceptable salt thereof, which is an analog of resveratrol. More specifically, the present invention induces multinucleation and proliferation of prostate cancer cells using HS-1793, an analog of resveratrol, and thus induces apoptosis to selectively remove androgen-dependent prostate cancer cells to treat cancer. It relates to a composition.

Cancer (암) or carcinoma (cancer) is a translation of the English cancer, medically referring to the malignant tumor (malignant tumour) lesion (病變). English cancer comes from Latin cancer and the Greek karkinos, both of which refer to crabs. Cells are in the process of growth, differentiation, programmed apoptosis, or they have stopped growing, and these processes are tightly controlled. However, in the case of cancer cells, abnormality occurs in some of the genes of the cell, and the characteristics of the protein, which is a product of these genes, are changed, and as a result, abnormality in the regulation of cell growth occurs. Since abnormalities in cell growth regulation are accompanied by mutations in the gene, cancer is a genetic disease caused by abnormalities in the genes.

Carcinogenesis is not just a process, but occurs in multiple stages and only occurs when there are very complex reasons and requirements. The first stage of the carcinogenesis mechanism is an initiation of cancer, an irreversible reaction in which a carcinogen attacks DNA and causes mutations, and the second stage is a promotion of cancer. Stage 3 is the process of cancer progression, from benign tumors to malignant tumors, whereby the characteristics of the malignant tumors are increased. At this stage, mutations in oncogenes and cancer suppressor genes gradually increase, and chromosomal abnormalities become apparent.

The prostate is an organ that only exists in men that produces part of the semen. It is located behind the bladder and adjacent to the rectum. Prostate cancer is the most commonly diagnosed cancer in the West and the second most common disease in men who die of cancer, but in the past, the incidence was low in Asians, including Korea. However, as the dietary transition to the westernized and aging society, the frequency is increasing rapidly. According to the National Cancer Registration Project of the Ministry of Health and Welfare (1999-2001), prostate cancer is the ninth most common male cancer occurrence, corresponding to 2.42% of all male cancers, and the crude rate per 100,000 people is 5.97. The age standardized rate is 7.88. According to a report by the National Statistical Office, in 2003, prostate cancer mortality rate was 3.2 in 100,000 cases, rising to eighth place in cancer mortality, increasing the elderly population and increasing interest in prostate cancer in doctors and the general public (1999-2001). Cancer development statistics). By age, it is rare in men under 40 years old, and the incidence increases gradually with age from 50 years old, the highest in the 70s, and more than 80% is diagnosed after 65 years old.

The cause of prostate cancer is not yet known and there is no effective preventive method. Risk factors are reported to include genetic predisposition, hormonal effects, food and environmental factors.

 Resveratrol (trans-3,4 ', 5-trihydroxystilbene) is a stilbene family of polyphenols, phytoalexin, a protective substance produced by plants by abiotic and biological stresses such as UV irradiation and metal ions. phytoalexin). Research on resveratrol has been very active since it was first reported in 1970 that resveratrol was found in commercial wines [Kim, H et 2002]. It is present in various foods such as grapes, peanuts, and Audi, and it is attracting attention as a medicine and functional food material as various physiological activities such as antioxidant and anti-inflammatory effects are revealed.

The first systematic study of resveratrol's cancer prevention and anticancer activity was published in 1997 by a team at the University of Chicago, USA. They found that resveratrol blocked all three stages of carcinogenesis: initiation, promotion and progression. It is also associated with the strong antioxidant activity of grapes.

Recent studies have shown that in many cancer cells, including breast, colon and lung cancers, resveratrol can inhibit cancer cell proliferation through the activity of genes that promote cell suicide. Apoptosis is a means for preventing the development of a tumor by the development of genetically damaged cells or inducing inadequate differentiation, or for the removal of cells with irreparable genetic wounds from an individual. In addition, resveratrol is known to be able to strongly block the proliferation of not only damaged cells but also rapidly dividing various human cancer cells by regulating expression of specific gene signaling systems that promote cell proliferation.

However, resveratrol itself does not have strong cytotoxicity compared to other chemotherapeutic agents, so a large amount of resveratrol is required to exhibit anticancer effects. In addition, resveratrol has photosensitivity and has a disadvantage in that its activity in the organism is limited by metabolic instability.

Thus, the present inventors confirmed the excellent anticancer effect of the resveratrol analogue 4- (6-hydroxy-2-naphthyl) -1,3-benzenediol (hereinafter referred to as HS-1793), in particular HS-1793 is an androgen-dependent cell Induced apoptosis by inducing multinucleation and ploidy of LNCaP cancer cells, which are cells deficient in Aurora B kinase, led to the present invention for treating cancer using the same.

It is an object of the present invention to treat prostate cancer with an active ingredient of HS-1793 (4- (6-hydroxy-2-naphthyl) -1,3-benzenediol) or a pharmaceutically acceptable salt thereof, which is an analog of resveratrol. To provide a suitable composition.

Another object of the present invention is to provide a pharmaceutical composition for effectively treating prostate cancer by improving cytotoxicity by administering a combination of antagonists of the resveratrol analogs, HS-1793 and Bcl-2 protein group.

The present invention is a prostate cancer using as an active ingredient HS-1793 (4- (6-hydroxy-2-naphthyl) -1,3-benzenediol) or a pharmaceutically acceptable salt thereof, which is an analog of resveratrol represented by the following formula (1): It relates to a therapeutic pharmaceutical composition. The prostate cancer is preferably an androgen dependent prostate cancer, more preferably the cancer is a composition that is an Aurora B kinase deficient cancer.

In the present invention, the pharmaceutically acceptable salt of HS-1793, which is a resveratrol analog represented by Formula 1, includes salts of acidic or basic groups which may be present in the compound of Formula 1, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, metalsulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, and methods or preparations for preparing salts known in the art It can be prepared through the process.

As used herein, the term 'androgen dependency' refers to a clinical phenomenon in which apoptosis occurs due to androgen blockade in normal and malignant tissues. Thus, drugs that block androgen in early prostate cancer are used. However, most of the prostate cancers, when these androgen-blocking drugs are administered for a long time, grow into a form that is not affected by androgen, which is called androgen-independent prostate cancer. LNCap, a cell line disclosed in the experimental example of the present invention, is an androgen-dependent prostate cancer cell, and PC-3 is an androgen-independent prostate cancer cell. As one specific example of the present invention, HS-1793 was treated for prostate cancer cells PC-3 and LNCap to observe changes in cells by live cell imaging and flow cytometric analysis. In the case of androgen-dependent cells, LNCap, it was found that the cell's ploidy and multinucleation were induced (see FIGS. 1 and 2).

As used herein, the term "aurora kinase" is a serine / trionine protein kinase, which is a protein that regulates various stages of cell division such as chromosome alignment, separation, cytoplasmic division. Mammalian cells express three types of kinases, Aurora A, B and C [Nigg EA., Et al 2001]. The three aurora kinases are very identical in structure and sequence, but are distinguished by their function [Mar Carmena., Et al 2003]. Aurora A locates spindle poles that play an important role in the separation of centrosomes, the beginning of cell division, and spindle assembly (Andrews PD., Et al 2003). Aurora C is expressed at a high rate to the extent and localizes the centrosome in migration to the late stages of the cell.

Aurora B is a major protein that regulates chromosomal alignment, conjugation of microtubules and kinetochore, spindle check points, and cellular division [Andrews PD., Et al 2003, Carmena M., et al 2003]. . Overexpression of Aurora B is observed in a wide range of human cancer cells [Tatsuka M., et al 1998], and sequential levels of Aurora kinase are closely linked to advanced stages of cancer [Chieffi P., et al 2006, Reiter R., et al 2006]. Accordingly, Aurora Kinase has become a good target for cancer treatment and is being evaluated as a clinical trial for cancer treatment. Several small molecule inhibitors have been developed, such as ZM447439 (Astra Zeneca), Hesperadin (Boehringer Mannheim), VX-680 (Vertex Inc.), AZD1152 (Ditchfield C, et al 2003, Wilkinson RW., Et al 2007). Cells in which Aurora B is inhibited often fail to divide cytoplasm and become diploids and multinucleated cells to become giant cells [Nair JS., Et al 2009]. Pharmacologically, disabling spindle checkpoints using inhibitors of small molecules of Aurora B causes the cells to be ploided, and the ploided cells secondaryly apoptosis. Gizatullin F, et al. 2006, Wilkinson RW, et al. 2007]. This is why Aurora B inhibitors are evaluated as clinical trials in the treatment of cancer.

In one embodiment of the present invention, the Western blot for Aurora B kinase in LNCap, an androgen-dependent prostate cancer cell, and PC-3, an androgen-independent prostate cancer cell (see FIG. 3A), shows that the expression of Aurora B kinase of LNCap It can be seen that the deficiency. In addition, in the PC-3, which expresses the expression of Aurora B, knockdown of Aurora B through siRNA (Fig. 3B) shows that the induction of multinucleation of HS-1793 is activated, and accordingly HS-1793 for Aurora B kinase deficient cancer cells. And it can be seen that the pharmaceutically acceptable salts thereof induce multinucleation, ploidy.

In another aspect, the present invention comprises a composition characterized in that the active ingredient induces multinucleation and deplomerization of cancer cells, the induction causes apoptosis.

As used herein, the term 'apoptosis' plays a very important role in the development and maintenance of homeostasis, which is an active cell death process that is controlled by genetically conserved related genes. This process is morphologically accompanied by a decrease in the specific gravity of the cell, destruction of the cell membrane, condensation of chromosomes, and the action of phagocytosis by the formation of cysts called apoptotic bodies.

As a specific example of the present invention, when 48 hours after HS-1793 was added to cells, caspase-3 was degraded to form a band in the western blot (see FIG. 1B). caspase is an important physiological mediator of apoptosis in higher animals. Caspases are synthesized as inactive enzymes in the precursor zymogen (zymogen) form with pro sites in the cell. When caspase is activated by apoptosis signal, proteolysis removes the front region located at the N-terminus of the protein from the precursor structure. The cleavage in the caspase occurs at the aspartic acid site, and another protein cleavage occurs at the C-terminus, separating it into one large unit (p20) and one small unit (p10). They form a heterozygote of p20p10 (α1β1 hetero dimer), which is then activated as a combination of α2β2 forms in which two α1β1s are gathered together. Caspase-3 is separated from the precursor 32kDa zymogen into 17 kDa and 12 kDa units, and this cleaved caspase-3 suggests apoptosis. In other words, the present invention, HS-1793 induces multinucleation and ploidy for Aurora B kinase deficient prostate cancer cells, and secondly promotes apoptosis to effectively remove cancer cells.

The present invention also includes a pharmaceutical composition for the treatment of prostate cancer comprising HS-1793 or a pharmaceutically acceptable salt thereof and Bcl-2 protein group antagonist as an active ingredient. The Bcl-2 protein family antagonist is preferably ABT-263, and ABT-263 mainly inhibits Bcl-XL.

The term 'Bcl-2 protein group' used in the present invention is a protein group that acts on the apoptosis mechanism by mitochondria and can be broadly divided into pro-suvival regulators and pro-apoptosis regulators. They act mainly on mitochondria, where pro-apoptosis regulators are involved in the outflow of cytochrome C, causing apoptosis, and pro-survival regulators, on the contrary, inhibit apoptosis by inhibiting outflow. Pro-survival modulators refer to the Bcl-2 sub-family, and pro-apoptosis modulators include the Bax and BH3 sub-family. The Bcl-2 sub-family includes Bcl-2 as well as Bcl-XL, Bcl-w, Mcl-1, and A1 / Bfl-1. Among them, Bcl-XL is a transmembrane molecule of mitochondrial membrane and plays a pivotal role in inhibiting apoptosis.

Antagonists of Bcl-2 include ABT-737, ABT-869, ABT-888, ABT-263, Bax inhibitor peptide P5 , Bax inhibitor peptide V5 , Bax inhibitor peptide , negative control , HA14 -1 , Obatoclax Mesylate (GX15 -070), and the like TW-37.

The term 'ABT-263' of the formula (2) used in the present invention corresponds to a BH3 mimetics mimicking the action of the PUMA protein involved in apoptosis and is a compound that inhibits the action of the Bcl-2 protein group. The PUMA protein is expressed by p53 present in the nucleus, and it is known that the expressed PUMA migrates to the cytoplasm, thereby scavenging and activating p53 present in the cytoplasm, thereby linking the apoptosis promoting function of p53 occurring in two places. .

In one embodiment of the present invention was treated with each siRNA of Bcl-2, Mcl-1, Bcl-XL belonging to the Bcl-2 protein group (see Fig. 5C), accordingly HS- in the case of knockdown of Bcl-XL It was found that the cell viability was significantly reduced during the 1793 treatment, and accordingly ABT-263 significantly inhibited Bcl-XL.

Analog HS-1793 of resveratrol of formula (1) according to the present invention and a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising said substance and Bcl-2 protein group antagonist are suitable carriers, excipients or diluents according to conventional methods. It may further include. Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erthritol, 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.

The pharmaceutical compositions according to the invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories or sterile injectable solutions according to conventional methods. have. Specifically, when formulated, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. Can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, 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 formulations and sinners. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, Uittepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention vary depending on the subject's age, sex, weight, symptoms, extent of disease, drug form, route of administration and duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.001 to 1000 mg / kg per day. The administration may be carried out once a day or divided into several times. In addition, the dosage may be increased or decreased depending on age, sex, weight, degree of disease, route of administration, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The compounds of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes such as parenteral, oral, and all modes of administration can be expected, for example, oral, rectal or intravenous. , Intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

As described above, a pharmaceutical composition for treating prostate cancer using the resveratrol analog HS-1793 or a pharmaceutically acceptable salt thereof as an active ingredient has an effect of accelerating apoptosis by inducing cell ploidy and multinucleation. The effect of cytotoxicity on androgen-dependent prostate cancer is excellent and can be used as an effective treatment for androgen-dependent prostate cancer.

1 is directed to the induction of multinucleation and diploidation of LNCap human prostate cancer cells of HS-1793.
1A is for cell viability 24 hours (solid) and 48 hours (hatched) after treatment of HS-1793. HS-1793 dose-dependently reduced cell viability.
Figure 1 b is a beta-actin and cleaved caspase-3 to confirm that shifted the same amount of protein shown in the Western blot.
Figure 1 c shows the cells multinucleated with HS-1793 treatment with paste staining. Below is a significant increase in the percentage of multinucleated cells on the plot (MN: multinucleated).
Figure 1d is Kim-ja staining, it can be seen that most cells treated with HS-1793 have two or more nuclei.
1E illustrates living cell imaging. Representative pictures in a time-lapse video and the time passed after the addition of HS-1793 are described. Increasing the number of nuclei indicates a continuous process.
1 f is flow cytometric analysis. A 2C DNA in the group G ₁ in the X-axis of the histogram shows the DNA of the DNA of 4C 8C in the second G ₂ (+) from G ₂ group. Drained cells with 8C are shown.
Figure 1 g shows a progressive increase in p53 protein expression following treatment of HS-1793 in western blot.
Figure 2 shows that induction of multinucleation, diploidation of HS-1793 in PC-3 cells is less effective than in LNCap.
Figure 2a is a graph of the viable cell viability after 24 hours, 48 hours after HS-1793 treatment.
FIG. 2 b is a Western blot showing that HS-1793 results in a small amount of cleaved caspase-3.
Figure 2c is the above stain test results of the multinucleated cells by HS-1793, below is the percentage of the multinucleated cells shown in the bar graph. Less multinucleated cells can be observed compared to LNCap cells. The percentage of polynucleated cells is depicted in the histogram.
2 d is a flow cytometric analysis. A 2C DNA in the group G ₁ in the X-axis of the histogram shows the DNA of the DNA of 4C 8C in the second G ₂ (+) from G ₂ group. Drained cells with 8C are shown. It can be observed that the diploidated cells corresponding to 8C are significantly less than LNCaP cells.
Figure 3 shows that the induction of multinucleation of prostate cancer cells following the treatment of HS-1793 is determined according to the expression level of Aurora B.
Figure 3a is a Western blot showing that Aurora B protein is deficient in LNCaP, while being expressed at a high rate in PC-3.
Figure 3b is a Western blot showing that siRNA corresponding to the Aurora B gene of 120 and 200 nM effectively inhibits Aurora B protein expression in PC-3 cells. PC3 cells incorporating non - target siRNA were used as experimental controls. Middle is paste dyeing. PC3 cells were cultured in the presence and absence of HS-1793, respectively. Significantly more polynucleated cells were observed in Aurora B deficient PC3 cells compared to the experimental control. The bottom is a bar graph showing the percentage of multinucleated cells. In the cells infected with siRNA, a significant polynucleation occurred compared to the control group (P <0.01).
Figure 3c is a western blot showing that Aurora is not detected in all cases in LNCaP at the top. Middle is paste staining when LNCaP is incubated with and without HS-1793 after silencing of Aurora B, respectively. The bottom is a bar graph showing the percentage of multinucleated cells. In all cases, no difference in the amount of polynucleated cells was observed (P> 0.05).
4 is a diagram showing the complementary effect on cytotoxicity of HS-1793 and Bcl-XL.
4a is a semiquantitative RT-PCR for the mRNA of Aurora B, the most representative of four independent experiments. Beta-actin was used as internal control.
Figure 4b is a Western blot showing the expression level of Aurora B between untreated cells.
4C is a percentage showing the amount of multinucleated cells in various cancer cells 48 hours after treatment with 10 μm HS-1793. In Aurora B deficient LNCap cells, the number of the multinucleated cells was significantly higher than that of the other eight cancer cells (P <0.01). In addition, T98G cancer cells in which Aurora B cells are weakly expressed can be seen to induce higher multinucleation than HS-1793 (P <0.01).
5 shows that the inhibition of the HS-1793 and Bcl-2 protein groups shows a complementary effect on cytotoxicity. When treating HS-1793 of 10㎛ alone or in combination with ABT-263
5a shows cell viability. HS-1793 When treated alone, solids and ABT263 were treated with hatching.
Figure 5b is a western blot showing that detection of caspase-3 cleaved upon treatment with HS-1793 and ABT-263 is possible within a faster time than HS-1793 alone treatment.
Figure 5c shows that the above inhibits each gene in LNPaC using siRNA corresponding to the Bcl-2, Mcl-1 and Bcl-XL genes. non-target (NT) control LNCap infected with siRNA was used as an experimental control. Solid uninfected cells. The cells infected with NT siRNA were hatched, the cells infected with siRNA of Bcl-2 were marked with dots, the cells infected with siMCl-1 were gray, and those with Bcl-XL siRNA were labeled with white dots on a black background. LNCaP cells treated with HS-1793 infected with Bcl-XL counterpart siRNA showed the most significant decrease in viability (**, P <0.01).

Hereinafter, the present invention will be described in more detail to more specifically describe the present invention. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

< Example 1  >

1-1: Reagent

The following reagents can be obtained commercially. Rabbit polyclonal anti-human Bcl-2, Mcl-1, Bcl-XL antibodies and ABT-263 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-human caspase-3 and p53 antibodies were purchased from Cell Signaling Technology (Danvers, MA). HRP-conjugated donkey anti-rabbit and sheep anti-mouse IgG antibodies were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). B-actin antibody, Hoechst 33342, dimethylsulfoxide (DMSO), RNase A, proteinase K, leupeptin, propidium iodide (PI), and Giemsa solution were purchased from Sigma (St. Louis, MO). rabbit monoclonal anti-human Aurora B was purchased from Epitomics (Burlingame, Calif.). Sulfuric acid myomycin (G418) was purchased from Calbiochem (Canada, US). RPMI-1640 and fetal bovine serum (FBS) were purchased from Gibco (Gaithersburg, MD). Western blot for reagent detection was used SuperSignal West Pico to induce chemiluminescence from Pierce (Rockford, IL).

1-2: human prostate PC -3 lesson LNCaP  Cell culture

American type Human prostate PC-3 and LNCaP were purchased from the Culture Collection (ATCC; Rockville, MD) and 10% fetal calf serum (FCS), 20 mM HEPES buffer and 100 μg / ml gentamicin were added to RPMI-1640 medium. ) Was administered.

1-3: Resveratrol  Processing of analogs and On cell viability  A rating

10 mM of resveratrol analogs in ethanol were prepared and stored at -80 ° C until use. Cells were treated with resveratrol analogs in the range of 0-40 μm to assess cell viability after 48 hours. Cell viability was evaluated by Vi-Cell cytometer (Beckman Counter, Fullerton, CA) using an automated trypan blue exclusion assay.

1-4: karyotype analysis Apoptosis  ( apoptosis )

Cell suspensions are plated on clean fat-free glass slides and centrifuged by cytocentrifuge. Cell centrifuged samples were fixed with 4% paraformaldehyde for 10 minutes and then stained at 4 ° C. for 30 minutes using 4 μg / ml of Paste 33342 (Hoechst 33342). Cells were observed and photographed by an epifluorescence microscope. To quantify the polynucleated cells, the total cell count (300 cells per experiment) was counted by DIC microscopy (differential interference contrast).

1-5: RT - PCR

Total RNA derived from different cell lines was isolated using RNeasy Mini kit (Quiagen, Gmbh, Germany). 2 μg of total RNA was reverse transcribed using oligo-dT primers, M-MLV reverse transcriptase. In order to amplify Aurora B and B-actin, 1 to 3ul of reverse transcription PCR was cycled 30. PCR was performed using 1 uM of primer, Taq DNA polymerase of 1.25 U of dNTPs of 200 uM each. After denaturation at 95 ° C. for 2 minutes, cDNA received 30 cycles of PCR amplification. For amplification of Aurora B and B actin, PCR was performed at 95 ° C. for 40 seconds, at 60 ° C. for 40 seconds, at 72 ° C. for 100 seconds, and finally at 72 ° C. for 5 minutes. Materials amplified by PCR were electrophoresed on 1% or 1.5% agarose gel and stained with ethidium bromide. All experiments were normalized with b-actin as an internal control. The oligonucleotide primer sequence is shown below. : Aurora B forward primer 5-CGACATCTTAACGCGGCAC-3 (SEQ ID NO: 1); Reverse primer, 5-GGACGCCCAATCTCAAAGTC-3 (SEQ ID NO: 2) (50 bp fragments); B-actin forward primer, 5-TGACGGGGTCACCCACACTGTGCCCAT-3 (SEQ ID NO: 3) Reverse primer 5-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3 (SEQ ID NO: 4) (660 bp fragments). PCR was repeated at the following conditions: 2 minutes at 95 ° C., then 30 seconds at 95 ° C., 30 seconds at 60 ° C., 40 seconds at 72 ° C., 30 cycles, and finally 5 minutes at 75 ° C.

1-6: Flow Cytometry ( Flow cytometric analysis )

Flow cytometry is a technique that analyzes by particle size using a computer by passing a laser beam through the flow of particles of cells and microstructures. 0.5% Tween 20 was added to cold 95% ethanol to make 70% ethanol and added to the cell suspension. Fixed cells were pelleted and washed with 1% BSA-PBS. Cells were resuspended in 1 ml PBS with 11 Kunitz U / ml RNase and incubated at 4 ° C. for 30 minutes, washed again in BSA-PBS and resuspended in PI solution (50 μg / ml). The cells were incubated at 4 ° C. for 30 minutes in the dark and washed with PBS. DNA content was measured by Epics XL (Beckman Coulter, FL, USA), and the data was analyzed by multicycle software that can estimate cell circulation parameters and apoptosis simultaneously.

1-7: Western Blot

Cell lysates were centrifuged at 14,000 rpm for 15 minutes at 4 ° C. Protein concentration in the cell lysate was measured by Bradford protein assay (Bio-Rad), the same amount of protein was separated by 7.5-15% SDS-polyacrylamide gel. Gels were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ) and reacted with each antibody. Immunostaining using antibodies was performed using SuperSignal West Pico, which promoted the substrate of chemiluminescence, which was photographed by LAS-3000PLUS (Fuji Photo Film Company, Kanagawa, Japan).

1-8: siRNA

To determine the effect of Aurora Bnockdown, the following double stranded RNA oligonucleotide was used. Sense: 5'-AAGGAGAACUCCUACCCCUGGUU-3 '(SEQ ID NO: 5), antisense: 5'-CCAGGGGUAGGAGUUCUCCUUUU-3' (SEQ ID NO: 6). For the preparation of Bcl-2, Mcl-1, and Bcl-XL, a preconfigured siRNA structure was purchased. The same number of nucleotides that did not cause genetic combination were inserted into the negative control.

All double siRNAs were synthesized by Dharmacon (Chicago, IL) and cells were transformed with siPORT Amine (Ambion, TX) in OptiMEM (Bio Sciences, Dublin, Ireland) according to the manufacturer's recommendations.

1-9: Live Cell Imaging  ( Live cell imaging )

Live cell observations after treatment of HS-1793 were performed using an integrated Delta Vision system (Applied Precision, Issaquah WA) for 24 hours in a temperature controlled chamber. Cells stained with 3-7 Hoechst 33342 were observed for at least 5 fields for multinucleation observation.

1-10: Laver  ( Giemsa  )dyeing

LNCaP cells were incubated with and without HS-1793, respectively, and fixed in 3: 1 methanol / acetic acid for 10 minutes. Slides were stained for 10 minutes in aqueous solution of Kimja for observation of polynucleation.

1-11: Statistical Analysis

Four independent in vitro experiments were performed. The statistical results were expressed as mean ± SD of the mean obtained from three independent experiments. The significance of the error was determined by the Kruskal-Wallis nonparametric test and only P values <0.05 were considered.

< Example 2 > HS -1793 is LNCap  Multinucleation Against Human Prostate Cancer Cells multinucleation ) And Drainage  ( polyploidization Cause).

HS-1793 showed dose-dependent reduction in cell viability of LNCaP, a human prostate cancer cell, by trypan blue assay (see Figure 1 A). In order to know the mechanism of cytotoxicity by HS-1793, LNCaP cells were cultured with and without HS-1793, and cell viability was decreased 48 hours after HS-1793 treatment (~ 20%). 10 μm HS-1793 was used. Western blot showed a decrease in caspase-3, indicating that HS-1793 decreased cell viability by caspase-dependent apoptosis of LNCaP cells, human prostate cancer cells (see FIG. 1B). 48 hours after the treatment of HS-1793, most LNCaP cells induce multinucleation, and therefore, it was found that HS-1793 contributed to significant cellular multinucleation (see FIGS. 1C and D). It was possible to directly observe the formation of multinucleation by using live cell imaging, and it was found that the increase in the number of nuclei of cells was a continuous process (see FIG. 1E). Using flow cytometric analysis, 48 hours after treatment, LNCaP cells were able to recognize the names of octaploid 8Cs corresponding to DNA peaks (see FIG. 1F). In addition, the expression level of P53 protein utilized as a marker of the diploidated cells was examined, and as a result, a clear increase in p53 protein expression in LNCap cells treated with HS-1793 was confirmed (see FIG. 1G).

< Example 3 > HS -1793 is PC -3 multinucleated for human prostate cancer cells multinucleation ) And Drainage  ( polyploidization Does not cause).

PC-1, a human prostate cancer cell, was also tested for HS-1793 induction of cell multinucleation. The trypanblue assay showed that HS-1793 decreased cell viability even in PC-3 in a dose dependent manner (FIG. 2A). HS-1793 at 10 μm reduced viability below 20% and a decrease in caspase-3 was seen at this concentration (FIG. 2B). However, the experiment showed that the multinucleation and ploidy effect of HS-1793 on PC-3 was significantly lower than that of LNCap. Cytomorphic observation showed that HS-1793 did not significantly induce polynucleation in PC-3. After 48 hours of treatment, the multinucleated cells corresponded to less than 10% (FIG. 2C), and HS-1793 showed a significant decrease in the multinucleating effect in PC-3 cells even in flow cytometry (FIG. 2D).

< Example  4> HS Induction of multinucleation of prostate cancer cells following treatment of -1793 is determined by the expression level of Aurora B.

Whether the expression level of aurora b determines the degree of multinucleation induction of prostate cancer cells of HS-1793 was examined experimentally. Aurora Bdml expression of PC-3 was very high while Western Blot expression of Aurora B of LNCap was basically deficient (FIG. 3A), and knocking-down of Aurora B resulted in PC-3. We tested whether prostate cancer cells could be susceptible to the multinucleation action of HS-1793. SiRNA corresponding to the Aurora B gene significantly reduces the expression of Aurora B in PC-3 cells (see FIG. 3B), which siRNA is PC-3 cells treated with HS-1793 compared to the control group and the experimental control group. Increased polynucleation significantly (see FIG. 3C). In addition, when the Aurora B siRNA was applied to LNCaP, it was tested whether the treatment of HS-1793 affects the multinucleation of LNCaP, and the amount of LNCap polynucleation was not changed (see FIG. 3D).

< Example  5> HS Multinuclearized by -1793 Cell mass  Inversely proportional to the extent of Aurora B expression.

Reverse transcriptase PCR was performed to examine the relationship between the expression of Aurora B and the incidence of multinucleation by HS-1793, using a specific primer of Aurora B. Reverse transcriptase PCR showed a weak expression of Aurora B mRNA in LNCaP and T98G cancer cells, unlike each of the seven cancer cells capable of clearly expressing Aurora B mRNA (see FIG. 4A). Western blot also showed that the expression level of Aurora B protein in T98G was much weaker than in the other seven cancer cells (FIG. 4B), and the amount of polynucleated by HS-1793 treatment in T98G was highly expressed. It was found to be significantly higher than other cancer cells (FIG. 4C). However, the amount of polynucleated at T98G was significantly lower than LNCap lacking Aurora B, indicating that the amount of polynucleated cells by HS-1793 treatment was inversely related to the expression level of Aurora B.

< Example  6> HS -1793 and Bcl - XL Inhibition of has a complementary effect on cytotoxicity.

We tested whether the combination of ABT-263 and HS-1793, which negatively regulates the Bcl-2 protein group, including Bcl-XL, can produce significant cytotoxicity. The cell viability of LNCaP did not decrease when only 1 μm of ABT-263 was treated, but the cell viability of 1 μm of ABT-263 and 10 μm of HS-1793 was significantly higher than that of HS-1793 alone. It can be seen that the decrease (see Fig. 5A). Moreover, the cleaved caspase-3 could be obtained at the time of treatment of the combination earlier than that of HS-1793 alone (FIG. 5B). In addition, siRNAs corresponding to the Bcl-2, Mcl-1, and Bcl-XL genes were used to identify which proteins in the Bcl-2 protein group, in particular, HS-1793 had a complementary effect. The siRNAs of the Bcl-2, Mcl-1, and Bcl-XL genes all reduced cell viability and further reduced cell viability upon treatment of HS-1793 (see FIG. 5C). In particular, the treatment of siRNA of Bcl-XL maximized the cell viability of HS-1793, and thus the combination of ABT-263 and HS-1793 significantly inhibited Bcl-XL.

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Claims (11)

delete delete delete delete delete 4- (6-hydroxy-2-naphthyl) -1,3-benzenediol (4- (6-hydroxy-2-naphthyl) -1,3-benzenediol) represented by Formula 1 below and Formula 2 below A pharmaceutical composition for the prevention or treatment of androgen-dependent prostate cancer, which lacks Aurora B kinase, comprising a combination of ABT-263, an antagonist of Bcl-2 protein group, as an active ingredient.
(Formula 1)

(2)

delete delete delete The composition of claim 6, wherein the ABT-263 inhibits Bcl-XL in the Bcl-2 protein group.
The composition according to claim 6, wherein the active ingredient is 0.001 to 1000 mg / kg.

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