Application of atypical retinoid A-7 in preparation of androgen receptor inhibitor and antitumor drug

Abstract

The invention discloses application of atypical retinoid A-7 in preparation of androgen receptor inhibitors and antitumor drugs, and belongs to the technical field of medicines. It is proved for the first time that AR can be degraded through a CMA pathway, and the atypical retinoid A-7 serving as an AR inhibitor can obviously reduce the AR protein level in prostate cancer cells; in addition, the Atypic retinoids 7 can be used for inhibiting the growth and proliferation capacity of the prostate cancer cells LNCaP; in addition, the invention further provides an anti-tumor medicine composition, the anti-tumor medicine composition comprises the Atypic retinoid-7 and Enzalutamide, and the anti-tumor medicine composition can be used for treating the prostatic cancer and has important clinical significance.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of atypical retinoids A-7 in the preparation of androgen receptor inhibitors and anti-tumor drugs. Background Art

[0002] Prostate cancer is the most common malignant tumor of the male reproductive system. The androgen / androgen receptor (AR) signaling pathway is the core pathway in the development of prostate cancer and is also the main target for clinical drug treatment of prostate cancer. At present, the development of castration-resistant prostate cancer after inhibiting the androgen / AR signaling pathway is a difficult problem in clinical treatment. Research on the degradation mechanism of AR may provide experimental basis and new ideas for the treatment of prostate cancer and castration-resistant prostate cancer.

[0003] Studies have reported that AR protein can be degraded by multiple pathways. Multiple E3 ubiquitin ligases and deubiquitinating enzymes in the cell jointly regulate AR degradation via the ubiquitin proteasome pathway, including MDM2, CHIP, SIAH2, USP12, and USP7. At the same time, various modifications of AR protein, including phosphorylation and SUMO (small ubiquitin-related modifier), also interact with ubiquitination modification and jointly affect AR degradation via the ubiquitin proteasome pathway. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) promotes AR protein degradation in a caspase-3-dependent manner. In addition, macroautophagy induced under hypoxic conditions also causes AR protein degradation, while macroautophagy has no significant effect on AR protein degradation under normoxic conditions.

[0004] Chaperone-mediated autophagy (CMA) is an autophagic pathway in which lysosomes selectively degrade specific intracellular proteins. Studies have shown that the CMA pathway is involved in the degradation of proteins in a variety of tumor processes, making the activation of the CMA pathway a potential tumor suppressor. For example, EPS8 was originally discovered as a substrate of epidermal growth factor receptor (EGFR), which can promote the progression of a variety of solid tumors, including colorectal cancer, thyroid cancer, cervical cancer, and squamous cell carcinoma. In a variety of tumor cells, EPS8 is enriched in lysosomes and degraded by the CMA pathway. The CMA pathway can degrade protein phosphatase 2A (CIP2A), which has tumor suppressor function, and it can prevent MYC from being degraded by the proteasome. Therefore, the CMA pathway degrades MYC indirectly. After activating the CMA pathway with chemical activators, protein substrates degraded by CMA were analyzed by protein spectrum, and it was found that substrate proteins were mostly enriched in metabolic pathways and protein translation pathways. Under nutrient deficiency, the degradation of Hexokinase II (HK2), a key factor in the glycolysis process of tumor cells, by the CMA pathway promotes metabolic disorders of tumor cells and causes cell death. In addition, as a protein quality control system, the CMA pathway is expected to be an anti-tumor treatment for the removal of abnormal proteins that promote tumor progression in cells. For example, in a variety of solid tumor cells including ovarian cancer and breast cancer, the CMA pathway is involved in the turnover of mutant TP53 (tumor protein p53) proteins; in non-small cell lung cancer, misfolded nuclear receptor corepressor (NCOR) can be degraded by the CMA pathway. Whether the CMA pathway is another pathway for AR protein degradation has not yet been reported. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to use atypical retinoids-7 in the preparation of androgen receptor inhibitors and anti-tumor drugs. The present invention proves through a large number of experiments that atypical retinoids-7 (CAS No.: 80306-38-3) can promote the degradation of androgen receptor (AR) protein, inhibit the proliferation of prostate cancer cells LNCaP, thereby curbing the development of tumors, and can be used to prepare drugs for the prevention and / or treatment of prostate cancer.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0007] The present invention provides an androgen receptor inhibitor, characterized in that the inhibitor is an atypical retinoid-7.

[0008] The present invention also provides a use of the above-mentioned inhibitor in preparing a medicine for preventing and / or treating androgen receptor-mediated related diseases or symptoms.

[0009] Furthermore, the androgen receptor-mediated related diseases or symptoms include prostate cancer.

[0010] The present invention also provides the use of atypical retinoid A-7 as the only active ingredient in the preparation of a drug for treating tumors, wherein the tumor is prostate cancer.

[0011] The present invention also provides the use of atypical retinoid A-7 as the sole active ingredient in the preparation of a product that inhibits the proliferation and invasion of tumor cells, wherein the tumor cells include prostate cancer cells.

[0012] The present invention also provides the use of atypical retinoid A-7 in the preparation of a drug for increasing the sensitivity of anti-tumor drugs to treat tumors, wherein the anti-tumor drug is enzalutamide and the tumor is prostate cancer.

[0013] The present invention also provides an anti-tumor pharmaceutical composition, characterized in that the pharmaceutical composition comprises atypical retinoid A-7 and enzalutamide, and the tumor is prostate cancer.

[0014] The present invention also provides the above-mentioned medicine and medicine composition for inhibiting tumor cell growth or promoting tumor cell death.

[0015] Furthermore, any of the above-mentioned drugs and pharmaceutical compositions are characterized in that the drugs and pharmaceutical compositions also include a pharmaceutical carrier suitable for preparing anti-tumor drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] The present invention proves for the first time that AR can be degraded through the CMA pathway; Atypical retinoids-7 can significantly reduce the level of AR protein in prostate cancer cells; and Atypical retinoids-7 can inhibit the growth and proliferation ability of prostate cancer cells LNCaP; in addition, Atypical retinoids-7 combined treatment can promote the sensitivity of prostate cancer cells to Enzalutamide. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The autophagy inhibitor increases the protein expression level of AR in prostate cancer cells. Among them, (A) After treating two prostate cancer cell lines with different concentrations of chloroquine for 2 hours, the cells were collected for protein extraction and the changes in AR protein levels were detected, with β-Actin as the internal reference; B The changes in AR protein levels when the two prostate cancer cell lines were treated with leupeptin (Leu, 10 μM) and chloroquine for 1 hour and 2 hours, with β-Actin as the internal reference; (C) In the presence of DHT (10 -7 M) Changes in AR protein levels after treatment with chloroquine in two prostate cancer cell lines without stimulation or after stimulation, with β-Actin as the internal reference; (D) Real-time quantitative fluorescence PCR experiment was performed after treatment with 50mM chloroquine in two prostate cancer cell lines, and the results showed that chloroquine treatment did not affect the mRNA expression level of AR. Data are expressed as mean ± standard deviation (n=3), and one-way analysis of variance and Tukey test were used for comparison between groups; ns express P >0.05.

[0019] Figure 2 AR interacts with HSC70 and LAMP2A, key factors in the CMA pathway, and AR protein is affected by them. (A) In DHT (10 -7 M) Total proteins of LNCaP and CWR22RV1 cells were collected under unstimulated or stimulated conditions, and precipitated using AR antibody or control IgG. The test results showed that AR could bind to HSC70 and LAMP2A under DHT stimulation or not. GAPDH was used as an internal reference. (B) FLAG-HSC70 was transfected into LNCaP and CWR22Rv1 prostate cancer cell lines to detect AR expression levels. The medium was changed 4 hours after transfection, and cells were collected for protein extraction 24 hours later. β-Actin was used as an internal reference. (C) siRNA was transfected into LNCaP and CWR22Rv1 prostate cancer cell lines to silence LAMP2A. Cells were collected 12 hours later to extract proteins to detect AR expression levels. β-Actin was used as an internal reference.

[0020] Figure 3 Atypical retinoids-7 reduces the level of AR protein in prostate cancer cells and inhibits the growth and proliferation of prostate cancer cells LNCaP. (A) After adding different concentrations of Atypicalretinoids-7 to LNCaP and CWR22RV1 cell lines for 12 hours, the cells were collected for protein extraction and the changes in AR protein levels were detected. β-Actin was used as an internal reference. (B) 2×10 4 LNCaP cells were cultured and atypical retinoids-7 and Enzalutamide were added. After 5 days in culture, the cells were fixed and stained. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0022] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0023] Example Experiment of atypical retinoid A-7 as androgen receptor inhibitor and anti-tumor drug.

[0024] Main reagents and materials.

[0025] RPMI1640 cell culture medium (Gibco, USA), fetal bovine serum (Clark, USA), DEPC water (Novozyme), Trizol (Novozyme), reverse transcription kit (Novozyme), real-time fluorescence quantitative PCR kit (Novozyme), protease inhibitor (Roche), protein concentration determination kit (Keygen Biotechnology), PVDF membrane (Merck Millipore), protein marker (Thermo), HSC70 antibody (Proteintech), LAMP2A antibody (abclonal), AR antibody (Proteintech), β-Actin antibody (Proteintech), GAPDH antibody (abclonal), FLAG antibody (Bethyl), chloroquine (ClQ) (Sigma), Leupeptin (Sigma), jetPRIMETM plasmid transfection kit (Polyplus-transfection), Atypical retinoids-7 (MCE Company, dissolved in DMSO to make a 10 mM stock solution), Enzalutamide (MCE Company, dissolved in DMSO to make a 20 mM stock solution).

[0026] siRNA was designed and ordered by Sigma, and the sequence is as follows: siLAMP2A#1 sense strand: 5ʹ-GCAGUGCAGAUGACGACAATT-3ʹ; siLAMP2A#1 antisense strand: 5ʹ-UUGUCGUCAUCUGCACUGCTT-3ʹ; siLAMP2A#2 sense strand: 5ʹ-GCCUUGGCAGGAGUACUUATT-3ʹ; siLAMP2A#2 antisense strand: 5ʹ-UAAGUACUCCUGCCAAGGCTT-3.

[0027] The primers were synthesized by Ivitrogen and the sequences are as follows: AR-F:5'-TCTTTGTCGTCTTCGGAAATGT-3'; AR-R:5'-AAGCTCTCCTTCCTCCTGTA-3'; 18S-F:5'-TTGACGGAAGGGCACCACCAG-3'; 18S-R:5'-GCACCACCACCCACGGAATCG-3'.

[0028] The pCDNA3-FLAG-HSC70 expression plasmid was constructed by our laboratory.

[0029] Experimental methods.

[0030] 2.1 Cell transfection.

[0031] Human prostate cancer cell lines LNCaP and CWR22Rv1 were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee and were identified as correct by STR. The cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 100 u / mL penicillin-streptomycin in a 5% CO2 incubator at 37°C.

[0032] After the cells were treated, they were collected on ice using a cell scraper and centrifuged to discard the supernatant. After adding TNE lysis buffer containing protease inhibitors to the cell pellet, the cell pellet suspension was sonicated using an ultrasonic instrument and centrifuged at 12000 r for 20 min at 4°C. The supernatant was aspirated into a new EP tube, the protein concentration was measured using a BCA kit, and Western Blot was performed with 20 μg of total protein.

[0033] 2.1.1 Western Blot.

[0034] Prepare the Running Buffer required for electrophoresis, take out the prepared protein sample, let it stand at 4℃ and wait for it to melt. After melting, boil it in boiling water for 5 min, and briefly centrifuge the denatured protein sample. Add protein samples and markers to the electrophoresis gel channel, and perform constant voltage electrophoresis at 80 V. The voltage can be adjusted to 110 V when the sample is electrophoresed to the separation gel. After the electrophoresis, the membrane can be transferred. Prepare the Transfer buffer according to the formula, take out the gel after electrophoresis, and clamp the PVDF membrane excited by methanol into the transfer clamp in the order of sponge-filter paper-gel PVDF membrane-filter paper-sponge, and place the gel on the negative electrode side. Each transfer tank corresponds to 200mA constant current transfer for 2 h; put the transferred PVDF membrane into 5% skim milk prepared with PBST and block it for 1 h. The blocked PVDF membrane is first washed with PBST, and then cut according to the experimental requirements, diluted with PBST according to the corresponding antibody instructions, and incubated with the primary antibody overnight with the antibody incubation box. The PVDF membrane incubated overnight was washed with PBST and washed three times on a horizontal shaker, each time for 10 minutes. The required secondary antibody was diluted with PBST according to the primary antibody, and incubated at room temperature for 1 hour. After the incubation, it was washed with PBST and washed three times on a horizontal shaker, each time for 10 minutes; then the luminometer was used to emit light and collect photos.

[0035] 2.2 RNA extraction.

[0036] All experimental consumables must be siliconized or treated with RNAase inhibitors before use.

[0037] Use a 6 cm culture dish to culture cells. When the cell density reaches 80% to 90%, blow 1 mL trizol evenly, collect the cells and transfer them to an EP tube and let it stand for 5 min. Add 1 / 5 volume of chloroform, shake vigorously to mix, and let it stand at room temperature for 5 min. Then centrifuge at 4℃ and 12000 r for 15 min. After centrifugation, liquid stratification can be seen. Take the supernatant (about 400 μL) and transfer it to a new siliconized EP tube. Add an equal volume of isopropanol to the transferred supernatant, let it stand at room temperature for 10 min, and centrifuge at 12000 r at 4℃ for 10 min. After centrifugation, a white precipitate will appear. Add 1 mL 75% ethanol to wash (prepared with DEPC water), flick the bottom of the tube to float the precipitate, centrifuge at 12000 r at 4℃ for 5 min, discard the supernatant and keep the precipitate, open the tube cap, and dry for 5 min. Add an appropriate amount of DEPC water (about 30-50μL) according to the amount of precipitate, and dissolve it in a water bath at 55-60℃. Measure the concentration after dissolution.

[0038] Reverse transcription system: Table 1 Reverse transcription system .

[0039] Calculation method for RNA solution volume: V RNA = mass required for reaction system (μg) / C RNA .

[0040] 2.2.1 Quantitative real-time PCR (qPCR): Table 2 qPCR system .

[0041] The product after reverse transcription was diluted ten-fold with sterile ultrapure water and used as a qPCR template. The system prepared according to the above table was vortexed and centrifuged, and amplified and analyzed using the LightCycler 96 instrument.

[0042] 2.3 Co-immunoprecipitation (Co-Ip).

[0043] 2.3.1 Pretreatment of beads.

[0044] Take a certain amount of mixed beads, add 1 mL of Lysis buffer, mix with a rotary mixer for 10 min, then centrifuge at 1500 r at 4°C for 5 min, repeat three times, and store at 4°C.

[0045] Table 3 Buffer formula required for Co-IP .

[0046] 2.3.2 Preparation of input: The method is the same as that of Western Blot protein sample preparation.

[0047] 2.3.3 Experimental process

[0048] Prepare the determined protein concentration with lysis buffer to 600 μL, transfer to a siliconized EP tube, add the corresponding antibody (determine the concentration according to the antibody manual), mix with rotation for 4 hours, add 30 μL of pretreated and mixed beads, and rotate and mix overnight at 4℃. Wash the beads the next day, centrifuge the mixed beads overnight at 1500 r4℃ for 5 min, discard the supernatant, add an equal volume of wash buffer pre-added with PI, and rotate and mix for 10 minutes. After repeating three times, add an equal volume of 2× loading buffer to the beads precipitate from the last centrifugation, let it stand on ice for 5 minutes, and then cook the sample and perform Western Blot experiment at the same time as the previously prepared input sample. The experimental results are detected in the same way as Western Blot.

[0049] 2.4 Clone formation.

[0050] LNCaP cells were seeded in 12-well plates, with 2×10 4 cells. Atypicalretinoids-7 and Enzalutamide were added to the plate. After 5 days of culture, the cells were fixed with 4% paraformaldehyde, washed with PBS, and then stained with Coomassie Brilliant Blue R250 at 4°C overnight. After that, the cells were washed with PBS and photographed.

[0051] Experimental results.

[0052] 3.1 Autophagy inhibitors increase the protein expression level of AR in prostate cancer cells.

[0053] The lysosomal inhibitor chloroquine can increase the pH value in lysosomes and inhibit the degradation function of lysosomes. After treating two AR-positive prostate cancer cell lines, LNCaP and CWR22Rv1, with different concentrations of chloroquine, the protein expression of AR in cells was observed. The results showed that after chloroquine treatment, the level of full-length AR protein increased, and its effect was concentration-dependent ( Figure 1 A). Similar results were observed when leupeptin, an inhibitor of acidic proteases in lysosomes, was combined with chloroquine to inhibit lysosomal function ( Figure 1 B). In addition, chloroquine can cause an increase in intracellular AR protein levels regardless of DHT stimulation ( Figure 1 C). At the same time, chloroquine treatment did not significantly affect the mRNA expression level of AR ( Figure 1 D). The above experimental results indicate that chloroquine causes protein accumulation after the degradation of AR protein in the lysosomal pathway is inhibited. Since macroautophagy does not affect the degradation of AR protein under normoxic conditions, it suggests that AR can be degraded by other autophagic pathways.

[0054] 3.2 Key factors in the CMA pathway, HSC70 and LAMP2A, interact with AR and affect AR protein expression levels.

[0055] There are two key proteins in the process of CMA degradation of target substrate proteins. HSC70 is responsible for recognizing the KFERQ-like motif of the degraded protein, binding to the motif and promoting the binding of the degraded protein to LAMP2A on the lysosomal membrane, so that the degraded protein enters the lysosome to achieve protein degradation. The interaction between these two proteins and the degraded protein enables CMA to specifically degrade the degraded protein. Co-IP experiments of endogenous proteins were performed using two cell lines, LNCaP and CWR22Rv1, respectively. The results showed that in both cell lines, AR can bind to HSC70 and LAMP2A ( Figure 2 A). These results indicate that AR has the prerequisites for degradation by the CMA pathway. Overexpression of HSC70 in LNCaP and CWR22Rv1 cell lines caused a decrease in AR protein levels ( Figure 2 B). In addition, in LNCaP and CWR22Rv1 cell lines, after silencing LAMP2A protein expression using two different siRNA targets, AR protein levels increased, further proving that AR can be degraded through the CMA pathway ( Figure 2 C).

[0056] 3.3 Atypical retinoids-7 reduces the AR protein level in prostate cancer cells and inhibits the growth and proliferation of prostate cancer cells LNCaP.

[0057] In both LNCaP and CWR22Rv1 cell lines treated with different concentrations of Atypical retinoids-7, it was shown that as the concentration of the activator increased, the protein level of intracellular AR decreased ( Figure 3 A). The growth and proliferation of prostate cancer cells after treatment with Atypicalretinoids-7 showed that Atypicalretinoids-7 could inhibit the cloning ability of prostate cancer cells LNCaP. In addition, combined treatment with Atypical retinoids-7 could further enhance the inhibitory effect of the AR inhibitor Enzalutamide on the growth and proliferation of prostate cancer cells ( Figure 3 B).

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An androgen receptor inhibitor, characterized in that The inhibitor is an atypical retinoid-7.

2. Use of the inhibitor according to claim 1 in the preparation of a medicament for preventing and / or treating androgen receptor-mediated diseases or symptoms.

3. The use according to claim 2, characterized in that: The androgen receptor-mediated related diseases or symptoms include prostate cancer.

4. Use of atypical retinoid A-7 as the sole active ingredient in the preparation of a drug for treating tumors, wherein the tumor is prostate cancer.

5. Use of atypical retinoid A-7 as the sole active ingredient in the preparation of a product that inhibits the proliferation and invasion of tumor cells, wherein the tumor cells include prostate cancer cells.

6. Use of atypical retinoid A-7 in the preparation of a drug for increasing the sensitivity of an anti-tumor drug to treat a tumor, wherein the anti-tumor drug is enzalutamide and the tumor is prostate cancer.

7. An anti-tumor drug composition, characterized in that: The pharmaceutical composition comprises atypical retinoid A-7 and enzalutamide, and the tumor is prostate cancer.

8. The drug and pharmaceutical composition according to claims 6 and 7 inhibit tumor cell growth or promote tumor cell death.

9. The drug and pharmaceutical composition according to any one of claims 6 to 7, characterized in that: The medicine and composition also include a pharmaceutical carrier suitable for preparing anti-tumor medicine.

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