KR20140103554A - Pharmaceutical composition comprising sLZIP for treating of androgen dependent prostate cancer - Google Patents

Abstract

The present invention provides a pharmaceutical composition for treating androgen dependent prostate cancer comprising sLZIP. The sLZIP may treat androgen dependent prostate cancer by being directly combined to androgen receptors and controlling increase of cancer cells.

Description

[0001] The present invention relates to a pharmaceutical composition for the treatment of androgen-dependent prostate cancer comprising sLZIP protein,

The present invention relates to a pharmaceutical composition for treating androgen-dependent prostate cancer comprising sLZIP protein.

Human leucine zipper protein (LZIP) is a bZIP transcription factor including the basic DNA binding domain, the putative transmembrane domain and the leucine zipper domain (Raggo C, et al., Mol Cell Biol 22: 5639-5649, 2002 ), Various roles such as tumor suppression and cell regulation have been suggested. The present inventors have studied a human LZIP and its use, and have identified a novel subtype of human LZIP, that is, human leucine zipper protein (sLZIP). According to the results of the study, the sLZIP protein is composed of 354 amino acids lacking the transmembrane domain (amino acids 229-245) unlike the conventional human LZIP, and is located in the nucleus. sLZIP protein has a property of inhibiting glucocorticoid-induced glucocorticoid receptor transcriptional activity, and thus can be usefully used as an active ingredient of a composition for the prevention and treatment of glucocorticoid receptor-mediated diseases such as stress disorder, inflammatory diseases and immune diseases I have said.

Prostate cancer is a cancer that has a high incidence in the western world. According to US statistics, the third highest mortality rate among men is death, resulting in about 30,000 deaths from prostate cancer. One in six American men have prostate cancer It is said to be diagnosed with cancer. In recent years, the incidence has continuously increased due to westernization of diet, increase in average life span, and development of diagnostic methods, and the World Health Organization (WHO) predicts that the number of prostate cancer patients will increase by 40% have. As a method of treating prostate cancer, surgical methods such as surgical excision, radiation therapy, and chemotherapy have been attempted in the early stage of cancer cell metastasis. Recently, new therapeutic techniques such as immunotherapy and isotope direct injection have been underway. In addition, to inhibit the progression of metastatic cancer cells, which have the greatest effect on the survival rate of prostate cancer patients, testosterone and antiandrogens are used to block the androgen hormones that are important for the proliferation of prostate cancer cells. However, since there is a problem of resistance due to increase of binding force of androgen and androgen receptor, it is urgent to develop a new therapeutic method for inhibiting the progression of prostate-derived metastatic cancer cells. For this purpose, proliferation of prostate cancer cells, The molecular or cellular target protein must be excavated. Prostate cancer has not been able to elucidate the precise pathogenesis of the prostate cancer due to the variety of causes and complexity of pathogenesis. Therefore, there is little research on the related target protein. Recently, there is a possibility that prostate cancer is related to prostate cancer using genomics and proteomics. Although many target proteins are screened, studies are still under way. However, there is no target protein that has precisely related mechanisms and functions, and only the possibility is presented.

The present inventors have studied the mechanism involved in the growth of androgen-dependent prostate cancer of sLZIP protein, which is known as a transcription factor involved in the proliferation, growth and metastasis of cancer cells, and found that the sLZIP protein inhibits the proliferation of androgen-dependent prostate cancer cells in the presence of androgen The present invention has been completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the treatment of androgen-dependent prostate cancer using sLZIP protein, which is a subtype of human leucine zipper protein.

In order to achieve the above object, the present invention provides the use of sLZIP protein for the manufacture of a medicament for the treatment of androgen-dependent prostate cancer, a method for the treatment of androgen-dependent prostate cancer comprising administering sLZIP protein to a subject, The present invention provides a pharmaceutical composition for the treatment of prostate cancer.

In one embodiment of the invention, the sLZIP protein may be one having the amino acid sequence of SEQ ID NO: 1.

In one embodiment of the invention, the composition may be a parenteral administration agent.

In one embodiment of the invention, the formulation for parenteral administration may be selected from the group consisting of non-aqueous solutions, suspensions, emulsions, and freeze-dried preparations.

The present invention also provides a pharmaceutical composition for the treatment of androgen-dependent prostate cancer, which comprises using the composition and a therapeutic agent for treating prostate cancer.

In one embodiment of the invention, the prostate cancer therapeutic agent may be selected from the group consisting of docetaxel, aviratorone, enzalutamide and bicalutamide.

The sLZIP protein of the present invention has a property of inhibiting the transcriptional activity of the androgen receptor and thus can be effectively used as an active ingredient of the composition for treating androgen-dependent prostate cancer.

Figure 1A shows the effect of sLZIP protein on the expression of cyclin D3 at the mRNA and protein levels of prostate cancer cells. Figure IB is a schematic representation of the possible binding sites of the sLZIP protein to the cyclin D3 promoter. Fig. 1C shows that the sLZIP protein shows the binding site of the cyclin D3 promoter, and Fig. 1D shows that the binding strength of sLZIP protein to cyclin D3 AP-1 increases in a concentration-dependent manner. FIG. 1E shows the result of sLZIP protein recruitment to the binding site in the cyclin D3 promoter, and FIG. 1F shows the result of sLZIP protein recruitment comparison of sLZIP protein and mock vector.
FIG. 2A shows the effect of inhibiting the ligand-dependent transcriptional activity of androgen receptors on the concentration of cyclin D3 in COS-7 and LNCaP cells. Figure 2B shows the effect of sLZIP protein on reporter gene activity and transcriptional activity of ligand-dependent androgen receptor depending on the presence or absence of DHT. Figure 2C shows the inhibition of sLZIP protein expression by si-sLZIP protein and the effect of DHT-dependent cyclic D3 on the androgen receptor transcription activity.
FIG. 3A shows the effect of sLZIP protein on the expression of cyclin D3, PSA and GAPDH at the mRNA level, and FIG. 3B shows the effect at the protein level, with or without DHT. FIG. 3C shows the effect of knock-down of the sLZIP protein on the expression of cyclin D3 and PSA in the presence of DHT at the mRNA and protein levels. Figure 3D shows the results of an experiment to determine whether the sLZIP protein inhibits the transcriptional activity of the androgen receptor. Figures 3E and 3F show the results of experiments using sLZIP protein to inhibit the ARE-binding ability of endogenous androgen receptor using ChIP assay.
Fig. 4 shows the result of an experiment of sLZIP protein and an androgen receptor interaction. Fig. 4A shows that, in the presence of DHT, the sLZIP protein co-immunoprecipitates with the androgen receptor and exhibits a weak signal in the absence of DHT. Figure 4B shows GST pull-down assay results in LNCaP cells. Figure 4C shows the effect of the sLZIP protein on the nuclear transactivation of the androgen receptor upon DHT stimulation upon sLZIP protein overexpression. Figure 4D shows the results of analysis of the sLZIP protein domain binding to the androgen receptor. Figures 4E and 4F show the results of analysis of the sLZIP protein mediated androgen receptor phosphorylation mechanism. Fig. 4G shows the results of analysis of the effect of the androgen receptor Ser308A mutant on the androgen receptor transcription activity. Figure 4H shows the results of an experiment of the association of sLZIP protein with androgen receptor phosphorylation induced by the cyclin D3 / CDK11 ^p58 complex. Figure 4I shows the results of immunoprecipitation performed to determine whether sLZIP protein is associated with HDAC3.
FIG. 5A shows cyclin D3 expression in response to sLZIP protein and DHT in LNCaP cells over time. Figure 5B shows the EMSA results to determine whether binding of the sLZIP protein to AP-1 (-574 / -563) is androgen-dependent. 5C shows the results of experiments on the effects of dissociation of the sLZIP protein from androgen-induced cyclin D3 promoter on the binding of androgen receptor (AR) -ARP (androgen receptor element). Figure 5D shows the results of immunoprecipitation performed to determine if the sLZIP protein modulates AR-ARE binding by interacting with the androgen receptor.
FIG. 6A shows the results showing the growth rate of LNCaP cells when the sLZIP protein was over-expressed and when the si-sLZIP protein was expressed. Figure 6B shows cell growth rates when infected with sLZIP protein, cyclin D3, sicycline D3, and sLZIP protein and sicycline D3 simultaneously. Figure 6C shows the results of a colony formation assay.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the treatment of androgen-dependent prostate cancer comprising sLZIP protein.

The sLZIP protein can be obtained as described in Patent Application No. 2009-0101278 as a subtype of LZIP lacking amino acids 229-245 in the 371 amino acid sequence of human leucine zipper protein (LZIP).

In the early stages of androgen-dependent prostate cancer among prostate cancers, cancer cells grow dependent on androgen, but tend to progress from androgen-dependent growth to androgen-independent processes as cancer progresses. The composition of the present invention has an effect of treating cancer by inhibiting the growth or proliferation of cancer cells in prostate cancer in which cancer cells grow in a stage before androgen-independent prostate cancer progresses to non-androgen-independent cancer, that is, dependence on androgen concentration .

In androgen-dependent prostate cancer, the androgen receptor is involved in the regulation of expression of various target genes as a transcription factor that plays an important role in the growth and proliferation of prostate cells and prostate cancer cells. We investigated whether the sLZIP protein, known as a transcription factor, is involved in the activity of the androgen receptor and, consequently, in the growth of the dependent prostate cancer.

Cyclin D3 in prostate cancer cells is induced by androgen stimulation (Knudsen et al, 1999; Olshavsky et al, 2008; Xu et al, 2006) and has been shown to have the ability to inhibit androgen receptor activity (Olshavsky et al, 2008; Zong et al, 2007).

Therefore, the present inventors focused on the relationship between sLZIP protein and cyclin D3 and androgen receptor. As a result, the sLZIP protein promoted the transcriptional activity of cyclin D3 (FIG. 1) and inhibited the transcriptional activity of the androgen receptor in a ligand-dependent manner (FIG. 2). Based on these results, it was determined through experiments that the sLZIP protein affects the growth and proliferation of androgen-dependent prostate cancer through the specific mechanism with the cyclin D3 and androgen receptor, and as a result, the sLZIP protein androgen Dependent prostate cancer. Specifically, sLZIP protein is androgen-dependent and binds to the second AP-1 region (-574 / -563) of the cyclin D3 promoter to induce cyclin D3 transcription and cyclin D3 to inhibit AR-ARE binding activity. The sLZIP protein also inhibits AR-ARE binding activity by reacting with DHT to dissociate from the cyclin D3 promoter and increase the phosphorylation of the androgen receptor induced by the cyclin D3 / CDK11 ^p58 complex by binding the sLZIP protein C domain to the androgen receptor I could. As a result, the sLZIP protein inhibits the proliferation and growth of androgen-dependent prostate cancer by inhibiting the binding activity of the androgen receptor and the ARE (Fig. 6). Accordingly, the present invention provides the use of sLZIP protein for the manufacture of a medicament for the treatment of androgen-dependent prostate cancer, a method for the treatment of androgen-dependent prostate cancer, which comprises administering the sLZIP protein to a subject, Lt; / RTI >

In one embodiment of the invention, the sLZIP protein may be one having the amino acid sequence of SEQ ID NO: 1.

SEQ ID NO: 1:

MELELDAGDQDLLAFLLEESGDLGTAPDEAVRAPLDWALPLSEVPSDWEVDDLLCSLLSPPASLNILSSSNPCLVHHDHTYSLPRETVSMDLESESCRKEGTQMTPQHMEELAEQEIARLVLTDEEKSLLEKEGLILPETLPLTKTEEQILKRVRRKIRNKRSAQESRRKKKVYVGGLESRVLKYTAQNMELQNKVQLLEEQNLSLLDQLRKLQAMVIEISNKTSSSSMYSSDTRGSLPAEHGVLSRQLRALPSEDPYQLELPALQSEVPKDSTHQWLDGSDCVLQAPGNTSCLLHYMPQAPSAEPPLEWPFPDLSSEPLCRGPILPLQANLTRKGGWLPTGSPSVILQDRYSG

In one embodiment of the invention, the composition may be a parenteral administration agent. In one embodiment of the invention, the formulation for parenteral administration may be selected from the group consisting of non-aqueous solutions, suspensions, emulsions, and freeze-dried preparations. For example, it may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, or a freeze-dried agent, but is not limited thereto. In the case of parenteral preparations, it can be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, rectal, or intradermal routes.

Formulations for parenteral administration may be prepared in an aqueous composition using techniques known to those skilled in the art. In general, such compositions include injectable formulations, for example, solutions or suspensions; Solid forms suitable for use in preparing solutions or suspensions upon addition of reconstitution medium prior to injection, such as micro- or nanoparticles; Emulsions such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions, liposomes or emulsions thereof.

The carrier may be, for example, water, ethanol, one or more polyols such as glycerol, propylene glycol, and liquid polyethylene glycol, oils such as vegetable oils such as peanut oil, corn oil, sesame oil, ≪ / RTI > or a dispersing medium. Proper fluidity can be maintained by using coatings such as lecithin or by maintaining the required particle size in the case of dispersions, or by using surfactants. It may also include isotonic agents of sugars or salts such as sodium chloride.

Solutions or dispersions of the active compounds as the free acid or free base or a pharmaceutically acceptable salt thereof may be prepared in water or other solvent or dispersion medium suitably mixed with one or more pharmaceutically acceptable excipients. Excipients include, but are not limited to, for example, surfactants, dispersants, emulsifiers, pH adjusting agents, and combinations thereof.

Suitable surfactants can be anionic, cationic, amphoteric or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylates, sulfonates and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates such as sodium dodecylbenzenesulfonate and alkyl aryl sulfonates; Dialkyl sodium sulfosuccinates such as sodium dodecylbenzenesulfonate; Dialkyl sodium sulfosuccinates such as sodium bis- (2-ethylthioxyl) -sulfosuccinate; And alkyl sulphates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, brominated seturonium, stearyldimethylbenzylammonium chloride, polyoxyethylene and coconut amines. Examples of nonionic surfactants are ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, poloxamer 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include myristoyl acetate, lauryl betaine and lauryl sulfobetaine.

In addition, the preparation may contain preservatives that inhibit the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid and thimerosal. The formulation may also contain an antioxidant capable of preventing the degradation of the active ingredient (s).

The present invention also provides a pharmaceutical composition for the treatment of androgen-dependent prostate cancer, which comprises using the composition and a therapeutic agent for treating prostate cancer. For example, the composition and the existing prostate cancer therapeutic can be administered simultaneously or sequentially.

In one embodiment of the invention, the prostate cancer therapeutic agent may be selected from the group consisting of docetaxel, aviratorone, enzalutamide and bicalutamide, but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

≪ Example 1 >

sLZIP protein regulates prostate cancer cell cyclin D3 expression

In order to identify the target molecule of sLZIP protein in relation to prostate cancer, a differential gene expression profile in the androgen receptor-expressing prostate cancer cell line LNCaP was performed using microarray analysis. Among the genes induced by the sLZIP protein, cyclin D3 is focused on cyclin D3 because it plays an important role in the proliferation of prostate cancer cells. To investigate the effect of sLZIP protein on cyclin D3 expression, RT-PCR and western blotting of androgen receptor expressing prostate cancer cell lines LNCaP, DU145, and PC3 were performed. LNCaP, DU145 and PC3 cells were infected with mock or a predetermined amount of GFP-sLZIP protein plasmid. Total RNA was isolated and mRNA levels of cyclin D3 and sLZIP proteins were quantified using semi-quantitative RT-PCR. GAPDH was used as an internal control. Relative mRNA levels of sLZIP protein and cyclin D3 for quantitative real-time PCR arrays were analyzed by human prostate cDNA arrays (tissue scan PCaq PCR array panels I and II, Origene, Rockville, Md.). Tissue Scanning Prostate PCR assays were obtained from prostate glandular hyperplasia (BPH) and normal prostate tissue, and a normalized amount of cDNA (2 ng of cDNA normalized to β-actin) prepared from prostate cancer at different stages of progression . Panel I contains 7 normal tissues, 7 2 and 3 3 cancer tissues with 30 samples of benign prostatic hyperplasia and Panel II contains 8 normal tissues, 18 2, 19 3 and 2 It contains 4 stage cancer tissue. A detailed pathological report of the sample, including the progression of the cancer, and HE staining were from Origene? You can find it on the website. Using the SYBR Green Master Mix (Roche, Mannheim, Germany) with the approved sLZIP protein and cyclin D3 primer and control beta -actin primer from Origene (Rockville, MD), the sLZIP protein And cyclin D3 expression were measured. Total cell lysates were collected and Western blotting against anti-GFP and anti-cyclin D3 antibodies was performed. α-Tubulin was used as an internal control. As a result, it was found that sLZIP protein increases the expression of cyclin D3 in prostate cancer cells in a concentration-dependent manner at the mRNA and protein level (Fig. 1A). We then examined the regulatory mechanism of the sLZIP protein in cyclin D3 expression. The cyclin D3 promoter (-1017 bp) was investigated using Transcription Element Search System (TESS) and MOTIF software. A schematic diagram of possible binding sites of the sLZIP protein to the cyclin D3 promoter is shown in Figure IB. To evaluate the DNA binding ability of the sLZIP protein to various elements in the cyclin D3 promoter, an electrophoretic mobility shift assay (EMSA) was performed using the purified His-sLZIP protein. The 5'-end of the oligonucleotide was labeled with [γ- ³² P] ATP (Perkin Elmer, Waltham, Mass.) Using T4 polynucleotide kinase (Promega, Madison, Wis.) For EMSA. Unlabeled nucleotides were removed by passage through a Bio-Gel P-6 spin column (Bio-Rad, Inc., Hercules, Calif.) According to the manufacturer's instructions. The purified His-sLZIP protein or nuclear extract was incubated with DNA-binding reaction buffer [10 mM Tris-HCl, 500 mM KCl, 10 mM EDTA, 50% glycerol, 20 mM DTT, 1 / / ) -poly (dl-dC)] and the radiolabeled probe and the protein-DNA complex was separated from the free probe by electrophoresis on a 6% native polyacrylamide gel. Prior to loading, the gel was pre-electrophoresed in 0.5 x TBE for 30 min and electrophoresed at 180 v for 1 h. The results were recorded by autoradiography. For competition testing, the binding reaction was incubated with a 50-fold molar excess of unlabeled AP-1 or ARE binding oligonucleotide for 40 minutes before adding the radiolabeled oligonucleotide. For super shift assay, the antibody in ice was added to the reaction mixture for 30 minutes. The probes used in the EMSA are: The oligonucleotides (-464 / -457 AP-1, 5'-AAGAAACTGATTCAATGAGAA-3 '-574 / -563 AP-1, 5'-GATGTAGTGAGTCATTACATC-3' (ARE) oligonucleotide (5'-GTCTGGTACAGGGTGTTCTTTTT-3 ') was used as a probe, and the 5' -AGATCTGCAGCAAGAAGGCC-3 ' His-sLZIP protein (2 μg) was incubated with γ- ³² P-labeled oligonucleotides containing consensus binding motifs containing AP-1 and C / EBP, resulting in the sLZIP protein being a cyclin D3 1 (-464 / -457) of the cyclin D3 promoter, the third AP-1 (-464 / -457) of the cyclin D3 promoter, and the second AP- 1 (-999 / -993), or C / EBP (-660 / -652) region (Figure 1C) Implies inducing cyclin D3 transcription through the second AP-1 region of the promoter (-574 / -563). The gamma- ³² P-labeled oligonucleotide was incubated with increasing amounts of purified His-sLZIP protein. The arrow shows the shift of the His-sLZIP protein-DNA complex The DNA binding capacity to the second AP-1 (-574 / -563) region in the cyclin D3 promoter of the sLZIP protein increased in a dose dependent manner (Figure 1D). 50-fold excess of the unclassified AP-1 probe was used competitively with the complex to measure the binding specificity for AP-1 (-574 / -563) of the sLZIP protein complex. As a result of the super shift assay, Showed that the sLZIP protein specifically binds to AP-1 (-574 / -563) (FIG. 1D) after treatment with the anti-His-sLZIP protein antibody. Non-chromatin immunoprecipitation (ChIP) was then performed to assess sLZIP protein recruitment to the AP-1 (-574 / -563) binding region in the cyclin D3 promoter. For ChIP experiments, approximately 1 x ¹⁰⁷ cells per sample were assayed. The cells were washed with PBS, treated with 1% formaldehyde at 25 ° C for 10 minutes, and then added with glycine to a final concentration of 0.125 M for 10 minutes. Cells were then scraped in PBS and centrifuged at 1,000 × g for 5 minutes at 4 ° C. ChIP assays were performed by co-immunoprecipitating DNA-protein complexes with anti-HA or anti-AR antibodies and anti-rabbit IgG as internal controls. From the prepared DNA sample, the promoter region of the cyclin D3 -718 to -480 (segment size 238 bp) was amplified using the following primer pairs: forward 5'-GACAGGTCTGGGATCACAGT-3 'and reverse 5'-AGCAGCCGGTGAAGTAG-3' PSA promoter And enhancer region primers are as follows: ARE I (located at -250 to -39 in the promoter) forward 5'-TCTGCCTTTGTCCCCTAGAT-3 'and reverse 5'-AACCTTCATTCCCCAGGACT-3' ARE II (within -406 to -164 in the promoter 3 'and reverse 5'-GCTAGCACTTGCTGTTCTGC-3' ARE III (located at -4170 to -3978 in the enhancer) forward 5'-CCTCCCAGGTTCAAGTGATT-3 'and reverse 5'-GCCTGTAATCCCAGCACTT-3' HA LNCaP cells were infected with the labeled sLZIP protein expression vector. ChIP assays were performed using antibodies specific for HA-sLZIP protein by PCR. Input chromatin was used as a control for the starting material of immunoprecipitation. As a result, it was found that the sLZIP protein accumulated in the AP-1 (-574 / -563) binding region in the cyclin D3 promoter (Fig. 1E). Real-time PCR was also performed using samples from the ChIP assay. It was found that sLZIP protein aggregation into AP-1 (-574 / -563) in sLZIP protein infected cells was approximately 6-fold higher at baseline than mock vector infected cells (Fig. 1F). This result implies that the sLZIP protein directly up-regulates cyclin D3 transcription by binding directly to the cyclin D3 promoter in prostate cancer cells.

≪ Example 2 >

Inhibitory effect of sLZIP protein on the ligand-dependent transcriptional activity of androgen receptor

To examine the ability to inhibit androgen receptor activity, the androgen receptor negative cell line COS-7 and androgen receptor expressing cell line LNCaP cells were seeded in a 12-well dish of 10% charcoal-stripped medium for 24 hours. COS-7 and LNCaP cells were co-infected with the androgen receptor (0.25 [mu] g), pMMTV-Luc (0.5 [mu] g), and [beta] -galactosidase (0.1 [mu] g) in the presence of cyclin D3 (Fig. 2A). COS-7 and LNCaP cells were transiently transfected with androgen receptor (0.25 [mu] g) and pMMTV-Luc (0.5 [mu] g) increasing the amount of sLZIP protein expression plasmid (Fig. 2B). LNCaP cells were infected with sLZIP protein, cyclin D3, si-sLZIP protein and / or sicycline D3 (Fig. 2C). The primers for sLZIP protein, cyclin D3, and AR were used to generate siRNAs for scrambled control, human sLZIP protein and cyclin D3. The sequence of the siRNA for sLZIP protein is 5'-CGUCGUUGAACAUUCUCAG-3 '(sense) and 5'-CUGAGAAUGUUCAACGACG-3' (antisense). The sequence of the siRNA targeting cyclin D3 is 5'-CUGGAUCGCUACCUGUCUU-3 '(sense) and 5'-AAGACAGGUAGCGAUCCAG-3' (antisense). The sequence of the siRNA for AR is 5'-CACAAGUCCCGGAUGUACAdTdT-3 '(sense) and 5'-UGUACAUCCGGGACUUGUGdTdT-3' (antisense). For RNA interference experiment, cells were plated in a 6-well plates at a cell density of 5 × 10 ⁵ cells per well each. After 24 hours, cells were infected with siRNA according to the manufacturer's instructions using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif.). After 24 hours of infection, cells of 10% charcoal-stripped medium were treated with 10 nM DHT (dihydrotestosterone) or ETOH for 24 hours and recovered for luciferase assay. The luciferase activity is normalized to the? -Galactosidase activity. Each experiment was performed three times and the data were expressed as mean ± standard deviation. Statistical evaluation was performed using one-way ANOVA. Data were considered statistically significant at p <0.05. All statistical analyzes were performed with a computer program Prism (Graph Pad Software, La Jolla, Calif.). The same applies to all statistical analyzes below.

Experimental results showed that, in COS-7 and prostate cancer cell line LNCaP cells, cyclin D3 inhibited the ligand-dependent transcriptional activity of the androgen receptor in a concentration-dependent manner (FIG. 2A). The sLZIP protein affected reporter gene activity and the transcriptional activity of ligand-dependent androgen receptors depending on the presence or absence of DHT (dihydrootestosterone), a ligand of the androgen receptor (Fig. 2B). In the absence of DHT, overexpression of sLZIP protein did not affect reporter gene activity, whereas ligand - dependent androgen receptor transcriptional activity was inhibited by 80% in proportion to the sLZIP protein dose. To determine whether endogenous sLZIP protein is associated with androgen receptor transcriptional activity, we used siRNA (si-sLZIP) for sLZIP protein and siRNA (si-cyclin D3) for cyclin D3. Inhibition of sLZIP protein expression by si-sLZIP protein in LNCaP cells enhanced the transcriptional activity of androgen receptor. Similarly, DHT-dependent cyclic D3 enhanced the transcriptional activity of androgen receptors (Figure 2C). This result indicates that the sLZIP protein negatively regulates the transcriptional activity of the androgen receptor.

&Lt; Example 3 >

Regulation of cyclin D3 and prostate-specific antigen expression induced by androgen in the sLZIP protein

D-type cyclin is important for androgen-dependent growth of prostate cancer cells and androgen stimulation increases gene expression of the cyclin D population (Knudsen et al, 1999; Xu et al, 2006). For the luciferase reporter assay, the effect of sLZIP protein on the expression of prostate specific antigen (PSA) in response to endogenous cyclin D3 and androgen, in a ligand dependent manner, was examined. LNCaP cells were infected with the desired amount of sLZIP protein and treated with ETOH or 10 nM DHT for 24 hours in 10% charcoal-strip medium. Total RNA was extracted from the cells and analyzed for mRNA (FIG. 3A) and protein levels (FIG. 3B) of cyclin D3, AR and PSA by RT-PCR and Western blotting. GAPDH and a-tubulin were used as internal controls. The sLZIP protein increased the expression of cyclin D3 in a ligand dependent manner. However, as a result of observing the DHT-dependent PSA induction, the sLZIP protein inhibited the expression of PSA at the mRNA level in the presence of DHT (Fig. 3A). Expression at the mRNA level of cyclin D3 and PSA in the presence of varying amounts of sLZIP protein was also measured. As a result, the effect of sLZIP protein on the expression of cyclin D3 and PSA was dose-dependent (FIG. 3A). The results at the protein level were also confirmed. The inhibitory effect of sLZIP protein on PSA expression was observed at the protein level in the presence of DHT (Fig. 3B). As a result, it was confirmed that sLZIP protein also dose-dependently inhibits PSA expression at the protein level (Fig. 3B). The effect of knockdown of the sLZIP protein on the expression of cyclin D3 and PSA in the presence of DHT was analyzed at the mRNA and protein levels (Figure 3C). LNCaP cells were infected with si-sLZIP protein and treated with 10 nM DHT or ETOH in 10% charcoal-strip medium for 24 hours. MRNA and protein levels were analyzed by RT-PCR and Western blotting. (Sc) siRNA scrambled with internal control was used. As a result, it was found that the sLZIP protein inhibited the expression of endogenous PSA ligand-dependent through the negative regulation of the androgen receptor transcription activity.

sLZIP protein inhibited the transcriptional activity of the androgen receptor by directly interfering with the DNA binding ability of the androgen receptor (Fig. 3D). LNCaP cells were infected with sLZIP protein, cyclin D3, and / or sicycline D3 and stimulated with 10 nM DHT for 24 hours in 10% charcoal-strip medium. Nuclear extraction was prepared and incubated with a gamma- ³² P-labeled 15-bp palindromic ARE sequence. Competitive assays were performed using a 50-fold molar excess of unlabeled ARE. For super shift assay, the anti-androgen receptor antibody was added to the reaction mixture for an additional 40 minutes in ice. Arrows indicate the movement of the AR-ARE complex. The DNA binding activity of the androgen receptor was induced by DHT, and the binding of the androgen receptor was destroyed by a 50-fold excess of unlabeled ARE oligonucleotide. AR-ARE complex formation was inhibited in the presence of sLZIP protein and in the presence of sicycline D3 in response to DHT, indicating that the overexpression of sLZIP protein in the presence of DHT inhibited the binding of the androgen receptor to DNA. The effects of cyclin D3 and sLZIP proteins on complex formation by reaction with DHT were confirmed using a super shift assay. As a result, both cyclin D3 and sLZIP protein inhibited complex formation.

ChIP assay was used to test whether the sLZIP protein inhibits the ARE-binding ability of the endogenous androgen receptor (Fig. 3E and 3F). LNCaP cells were infected with the HA-mock or HA-sLZIP protein plasmid and treated with 10 nM DHT for 6 hours. Soluble chromatin was prepared and immunoprecipitated with anti-androgen receptor antibody or IgG as negative control. The precipitated DNA was quantitated by RT-PCR (Fig. 3E). The relative recruitment of the androgen receptor to the PSA promoter was analyzed by a densitometer (Fig. 3F). DHT promoted the binding of the endogenous ARE to the androgen receptor. However, the sLZIP protein reduced the recruitment of the androgen receptor to the PSA promoters (ARE I and ARE II) and the enhancer (ARE III) (FIGS. 3E and 3F). This corresponds to a previously unknown mechanism by which the sLZIP protein inhibits androgen receptor-mediated transcriptional activity.

<Example 4>

Interaction of sLZIP protein with androgen receptor and role as androgen receptor co-inhibitor

To investigate the potential role of the sLZIP protein in the regulation of androgen receptors, the interaction between the sLZIP protein and the androgen receptor was examined. HEK 293T cells were infected with the androgen receptor (AR), mGST-mock or mGST-sLZIP protein plasmids. Immunoprecipitation was performed with a cell lysate using an anti-androgen receptor antibody and the precipitated beads were Western blotted. In the presence of DHT, the sLZIP protein co-immunoprecipitated with the androgen receptor and showed a weak signal in the absence of DHT (Fig. 4A). A GST pull-down assay was performed on LNCaP cells (Figure 4B). LNCaP cells were infected with the mGST-sLZIP protein. Cells were infected with 10 nM DHT for 24 hours. The cell lysate was pulled down using glutathione beads. The protein bound to the beads was analyzed by 10% SDS-PAGE and immunoblotted (FIG. 4B). In the absence of DHT, a weak signal was detected, whereas in the presence of DHT the endogenous androgen receptor interacted with the sLZIP protein (Fig. 4B).

The effect of sLZIP protein on the nuclear translocation of androgen receptor upon DHT stimulation upon sLZIP protein overexpression was tested (Fig. 4C). For immunohistochemistry, tissue array kits from Super Bio Chips Laboratories (Seoul, Korea) were used. A human prostate tissue array containing 40 tumor tissues and 10 normal tissue samples was used for immunostaining for anti-sLZIP protein and anti-cyclin D3 antibody. Samples were either fixed in formalin or embedded in paraffin or sectioned for histological examination. Hematoxylin eosin stained sections were tested to assess histopathologic characteristics of lesions.

HEK 293T cells were infected with GFP-sLZIP protein and HA-AR. After 24 hours, cells were fixed with 4% paraformaldehyde and incubated with anti-androgen receptor antibody for 1 hour. And then incubated for one hour with Texas Red conjugated anti-rabbit antibody. The preparation was then washed and contrasted with DAPI. Without ligand treatment, the sLZIP protein was present only in the nucleus whereas the androgen receptor was present in both cytoplasm and nucleus (Fig. 4C). Following ligand stimulation, the androgen receptor was enriched in the nucleus and the over-expressed sLZIP protein co-localized with the androgen receptor in the nucleus (Fig. 4C). As a result, the sLZIP protein interacted with the androgen receptor in a ligand-dependent manner.

The sLZIP protein domain binding to the androgen receptor was analyzed (Figure 4D). Various deletion variants of the wild type and sLZIP proteins were made and the androgen receptor binding domain was analyzed by GST pull down experiments. A variety of deletion mutants including wild type and sLZIP protein N (1-228), sLZIP protein C (229-354), and sLZIP protein CC (297-354) were expressed in 293T cells as HA-labeled androgen receptors. The sLZIP proteins N, C, and CC represent the subdomains of the sLZIP protein. Deletion variants of wild-type and mGST-sLZIP proteins were transiently transfected with 293T cells. The expression of the mGST-sLZIP protein was measured by Western blotting (Fig. 4D). sLZIP protein N and sLZIP protein CC were not, while the sLZIP protein C domain retained the ability to interact with androgen receptors similar to the wild-type sLZIP protein. This indicated that the sLZIP protein C domain was associated with androgen receptor binding.

sLZIP protein to phosphorylate the androgen receptor. HA-labeled androgen receptor and two mutants HA-AR Ser308D (cyclin D3 / CDK11 ^p58 Complex-induced active phosphorylation) and HA-AR Ser308A (negative phosphorylated form) were expressed in 293T cells with the GST-sLZIP protein wild-type plasmid. HEK 293T cells were infected with mGST-sLZIP protein, HA-labeled AR, HA-AR S308D or HA-AR S308A. Cells were treated with 10 nM DHT for 24 hours. Immunoprecipitation was performed with total protein (Fig. 4E). The AR Ser308A variants did not bind the sLZIP protein whereas wild type androgen receptor and AR Ser308D variants interacted with the sLZIP protein (Fig. 4E). Cells were treated with 10 nM DHT for 24 h for fluorescence microscopy analysis. HEK 293T cells were transiently transfected with sLZIP protein and androgen receptors were grown in cover slips. After 5 hours of infection, cells were grown in RPMI 1640 containing 10% charcoal-strip FBS. After 24 hours of medium exchange, the cells were either treated with 10 nMDHT or not treated. After 24 hours, the cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.2% Triton-X 100 for 5 minutes. Cells were incubated with 1% BSA for 1 hour and incubated with polyclonal anti-GFP, anti-AR and anti-HA antibodies for 1 hour. After washing with PBS, cells were incubated with anti-rabbit or anti-mouse antibody (Santa Cruz, Calif.) Conjugated with Texas Red for 1 hour. The coverslips were washed, counted with PBS and assayed using Axiovert 100 (Carl Zeiss, Jena, Germany). The preparation was washed and contrasted with DAPI. The subcellular localization of the androgen receptor variants and sLZIP protein was observed using fluorescence microscopy. Wild type AR, AR Ser308D and AR Ser308A variants were located in the nucleus with sLZIP protein (Fig. 4F).

The effect of AR Ser308A variant on the transcriptional activity of androgen receptor was analyzed (Fig. 4G). COS-7 cells were infected with sLZIP protein, HA-AR or HA-AR S308A. After 24 hours of infection, cells were treated with 10 nM DHT or ETOH in 10% charcoal-strip medium for 24 hours and recovered for luciferase assays. In the AR Ser308A mutant, the inhibitory effect of the sLZIP protein on the androgen receptor transcriptional activity was not observed (FIG. 4G). This result implies that the sLZIP protein is co-localized in the nucleus with wild-type AR and AR variants. However, in the presence of androgen, the sLZIP protein specifically interacted with wild type androgen receptor and AR Ser308D variants.

The association of the sLZIP protein with the androgen receptor phosphorylation induced by the cyclin D3 / CDK11 ^p58 complex was tested (Fig. 4H). LNCaP cells were infected with mGST-sLZIP protein and ^{immunoprecipitated} with cell lysate using anti-CDK11 ^p58 antibody. An in vitro kinase assay was performed as described above (Fig. 4H). As a result of the CDK11 ^p58 kinase assay, the sLZIP protein increased the phosphorylation of the androgen receptor in the presence or absence of DHT (Fig. 4H).

Cyclin D3 is known to bind directly to AR and HDAC3 (Olshavsky et al, 2008). Immunoprecipitation was performed to determine if the sLZIP protein was associated with HDAC3 (Fig. 4I). LNCaP cells were infected with the mGST-sLZIP protein. After 24 hours of infection, the cells were treated with 10 nM DHT for 24 hours. Immunoprecipitation was performed with cell lysate. As a result, it was found that the sLZIP protein binds to HDAC3 in the absence of DHT and reacts with DHT in the presence of DHT (FIG. 4I). This result indicates that the sLZIP protein functions as a co-inhibitor by interacting with HDAC3 and the cyclin D3 / CDK11 ^p58 -phosphorylated androgen receptor.

&Lt; Example 5 >

binding of the sLZIP protein to the androgen-dependent cyclin D3 promoter

Cyclin D3 expression is increased by DHT stimulation through mTOR activity (Xu et al, 2006). To investigate the mechanism of the effect of sLZIP protein on cyclin D3 expression, we examined the expression of cyclin D3 over time by sLZIP protein in LNCaP cells in response to DHT. LNCaP cells were infected with mGST-mock or mGST-sLZIP protein (2 [mu] g). After 24 hours, cells in charcoal-strip medium were treated with 10 nM DHT for a predetermined period. Total cells were harvested and Western blotting was performed. Cyclin D3 expression in response to DHT increased with time and in the absence of DHT, sLZIP protein induced the expression of cyclin D3 (Fig. 5A). However, the degree of cyclin D3 expression induced by sLZIP protein was not affected by DHT treatment time (FIG. 5A). PSA was used as a positive control for testing the effects of DHT. EMSA was performed to determine whether the binding of sLZIP protein to AP-1 (-574 / -563) was androgen-dependent. Prepare for nuclear extraction And incubated with radiolabeled oligonucleotides containing the AP-I motif in the cyclin D3 promoter. Competitive reactions were performed using 50-fold molar excess of unlabeled AP-1. As a result, it was found that the sLZIP protein was androgen-dependent and regulated cyclin D3 expression (Fig. 5B). The effect of dissociation of the sLZIP protein from androgen-induced cyclin D3 promoter on AR-ARE binding capacity was examined. Nuclear extracts were harvested and EMSA was performed using a gamma- ³² P-labeled ARE probe. A competitive reaction was performed using a 50 fold molar excess of unlabeled ARE (Figure 5C). Immunoprecipitation was performed to determine whether the sLZIP protein modulates AR-ARE binding by interacting with AR. Immunoprecipitation was performed with a cell lysate using an anti-androgen receptor antibody. Proteins bound to the beads were analyzed by 10% SDS-PAGE and immunoblotted using anti-GST antibodies. (Fig. 5D). As a result, it was found that the sLZIP protein reacts with DHT to dissociate from the cyclin D3 promoter and binds to the cyclin D3 / CDK11 ^P58 complex as a co-inhibitor, thereby inhibiting AR-ARE binding activity (FIGS. 5C and 5D).

&Lt; Example 6 >

Effect of sLZIP protein on the proliferation of androgen-dependent prostate cancer cells

Cyclin D3 expression and its interaction with androgen receptors have examined whether the sLZIP protein affects the growth of androgen-dependent prostate cancer cells. The cell proliferation assays were based on the MTT method. After infection of the plasmid, cells were seeded into 96-well plates at a density of 1 x 10 ³ cells / well. Cells were treated or not with 10 nM DHT for 72 hours. MTT was added, incubated for 1 hour, and dimethylsulfoxide was added to dissolve the formazan crystals. Absorption was measured at 595 nM using a microplate reader reader (Bio-Rad). Each experiment was repeated three times and the statistical analysis was as described above.

LNCaP cells were infected with sLZIP protein, si-sLZIP protein, cyclin D3 or sicycline D3. Cells in 10% charcoal-strip medium were assessed for cell viability via MTT assay without or with 10 nM DHT for a given period (Figs. 6A and B). When the sLZIP protein was over-expressed and si-sLZIP protein was expressed, the growth rate of LNCaP cells was examined (Fig. 6A). LNCaP cells overexpressing sLZIP protein in the presence of DHT had a decreased growth rate compared to the control (Fig. 6A). However, the cells expressing the si-sLZIP protein increased the rate of cell growth. sLZIP protein, cyclin D3, sicycline D3, and sLZIP protein and sicycline D3 (Fig. 6B). In the presence of DHT, cyclin D3 inhibited the growth of LNCaP cells and sicycline D3 restored cell growth (Fig. 6B). When co-infected with sLZIP protein and sicycline D3, the growth of androgen-dependent prostate cancer cells was partially restored (Fig. 6B). These results were confirmed by performing a colony forming assay and a cell growth assay (FIG. 6C). LNCaP cells were infected with sLZIP protein, si-sLZIP protein, cyclin D3 or sicycline D3. For colony formation assay, cells were seeded in a 6-well plate at a density of 1 × 10 ⁴ cells / well and kept for 24 hours for adhesion. The cells were then either treated with 10 nM DHT or not treated. After 10 days, the colonies were fixed with fixative (methanol: acetic acid = 3: 1) at room temperature for 10 minutes and stained with 0.01% crystal violet (Fig. 6C). This suggests that sLZIP protein plays an important role in the growth of androgen-dependent prostate cancer cells.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Pharmaceutical composition comprising sLZIP protein for treating          of androgen dependent prostate cancer <130> P120955 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 0 <212> PRT <213> human small leucine zipper protein

Claims (6)

A pharmaceutical composition for treating androgen-dependent prostate cancer, comprising human small leucine zipper protein (sLZIP).
The pharmaceutical composition according to claim 1, wherein the human minor leucine zipper protein has the amino acid sequence of SEQ ID NO: 1.
The pharmaceutical composition according to claim 1, wherein the composition is a preparation for parenteral administration.
The pharmaceutical composition according to claim 4, wherein the preparation is selected from the group consisting of non-aqueous and non-aqueous solutions, suspensions, emulsions and freeze-dried preparations.
A pharmaceutical composition for the treatment of androgen-dependent prostate cancer, which comprises using the composition of any one of claims 1 to 4 and a therapeutic agent for prostate cancer. 6. The pharmaceutical composition according to claim 5, wherein the therapeutic agent for prostate cancer is selected from the group consisting of docetaxel, aviratorone, enalutamide and bicalutamide.

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