WO2013113148A1 - Antitumor use of β3 adrenergic receptor blocker - Google Patents

Abstract

Disclosed is a siRNA use of the β3 adrenergic receptor blocker and the targeting β3 adrenergic receptor gene. The β3 adrenergic receptor is an important factor for regulating and controlling SIRT1, mTOR, and p53. The β3 adrenergic receptor blocker and siRNA of the β3 adrenergic receptor can be used for preparing medicine for treating diseases related to signaling pathways such as SIRT1, mTOR, and p53, for example, treating the diseases such as malignancy, hypertrophic cardiomyopathy, rheumatoid arthritis, and renal failure.

Description

 Antitumor use of β 3 adrenergic receptor blockers

 Technical field

 The invention relates to the field of signal pathways, in particular to the novel use of β 3 adrenergic receptor blockers in the preparation of therapeutic drugs.

 Background technique

 β 3 in the human body Adrenergic receptors are mainly expressed in fat cells, especially visceral fat cells, which promote fat breakdown and energy supply. A variety of agonists and blockers have been discovered that selectively act on the receptor. For β 3 The drug research of the receptor is mainly based on its lipolysis, and the β 3 receptor agonist is used as a weight loss drug. However, for β 3 There is no report on the role of receptors in the occurrence and development of malignant tumors and their potential therapeutic effects.

 Tumor suppressor gene TP53 is a key gene regulating DNA damage repair and cell cycle, TP53 It is a aging-related gene whose anti-cancer effect is closely related to aging. It controls cell senescence by regulating DNA damage, free radical generation and clearance, and p53 overactivity causes premature senescence of stem cells, p53 The modification itself is the main reason for maintaining its function. Numerous studies have shown that Sirtuin 1 (SIRT1) can be deacetylated by p53 in p53 A key role in mediated aging and anti-tumor responses. Yeast Information Regulator 2 (SIR2) Is a histone deacetylase that is dependent on nicotinamide adenine dinucleotide, SIRT1 is the most homologous family member of mammalian Sir2, SIRT1 Proteins are present in mammalian cytoplasm and nucleus and play important regulatory roles in cell survival, aging and oxidative stress. SIRT1 deacetylates p53 and inhibits its transcriptional activity. In addition SIRT1 Can be recruited to the p53 target promoter, resulting in transcriptional silencing of the hypoacetylated nucleosome.

Summary of the invention

 The invention discloses a β 3 adrenergic receptor (adrenergic beta-3-receptor, ADRB3) It is an important receptor for the regulation of SIRT1, p53, mTOR, MIF and microRNA-16 signaling pathways. The invention discloses a β 3 adrenergic receptor blocker Use to treat diseases associated with the mTOR/SIRT1/p53/MIF signaling pathway, such as cardiac hypertrophy, hypertrophic cardiomyopathy, myocardial fibrosis, pulmonary hypertension, atherosclerosis, and malignancy Wait. The present invention discloses the use of a β 3 adrenergic receptor blocker for the preparation of an antitumor drug. The present inventors have found that β 3 receptor blockers and their siRNAs can reduce tumors and their peripheral adipose tissue. SIRT1 expression leads to p53 acetylation, increases p53 transcriptional activity, increases p53 in tumor tissues, promotes tumor cell senescence, apoptosis and blocks distant metastasis. β 3 Receptor blockers also inhibit ATP production by promoting autophagy, reducing mitochondria in tumor cells. β 3 receptor blockers up-regulate miR-16-1 in leukemia cell line HL-60 miR-15a promotes apoptosis. 3 3 receptor agonists increase SIRT1 expression in heart, liver and brain tissues, reduce p53, and play an anti-aging role. The inventors also found that β 3 Receptor blockers reduce the activity of mammalian target of rapamycin (mTOR), particularly mTOR-Rictor The complex promotes mitochondrial autophagy and reduces ATP production. Therefore, β 3 receptor agonists can be used as mTOR An agonist that increases insulin secretion, improves insulin resistance, and promotes the production of new islet cells.

 The β 3 receptor blocker which can be used in the present invention is not particularly limited, and all of them can inhibit or block β 3 . Compounds of the receptor are suitable for use in the present invention. An example of a representative beta 3 receptor blocker is (but not limited to): SR59230A, and the structural formula is shown in Figure 16. With SR59230A Compounds which are parent cores are suitable for use in the present invention.

 β 3 receptor blocker of the present invention The dosage form and the preparation method are not particularly limited, and various dosage forms such as tablets, capsules, granules, sustained release agents, and injections can be prepared by a conventionally common method in the art.

 The β 3 receptor blocker of the present invention can be used for treating cardiac hypertrophy, cardiomyopathy, myocardial fibrosis, pulmonary hypertension, renal failure And drugs for malignant tumors. The β 3 receptor agonist can also be used as an agonist of SIRT1 for anti-aging.

In addition, it should be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the invention.

DRAWINGS

 Figure 1 shows that SR59230A increases the expression of p53 in MCF-7 cells;

 Figure 2 shows that SR59230A reduces SIRT1 expression in MCF-7 cells;

 Figure 3 shows BRL37344 reduces myocardial apoptosis in rats with heart failure;

 Figure 4 shows that BRL37344 reduces myocardial p53 and increases SIRT1;

 Figure 5 shows BRL37344 improving heart function in rats with heart failure;

 Figure 6 shows β-galactosidase staining in adipose stem cells;

 Figure 7 shows Rictor quantitative PCR ;

 Figure 8 shows mTOR quantitative PCR ;

 Figure 9 shows the tumor growth curve of nude mice;

 Figure 10 shows that SR59230A inhibits tumors in nude mice;

 Figure 11 shows the Annexin V/PI flow cytometry;

 Figure 12 shows that SR59230A increases miR-16-1 in leukemia cells HL-60 and K562;

 Figure 13 shows that SR59230A increases miR-15a in leukemia cells HL-60 and K562;

 Figure 14 shows that SR59230A increases the apoptosis rate of leukemia cells HL-60 and K562;

 Figure 15 shows SR59230A increases prostate cancer cell line PC-3 autophagy;

 Figure 16 shows the SR59230A structure.

detailed description

 The invention is further explained by the following examples, but the examples are not intended to limit the invention in any way.

 Example 1

 Blocking the β 3 receptor reduces SIRT1 in MCF-7 cells and increases p53 expression. Different concentrations of SR59230A MCF-7 cells were treated and cells were lysed 24 h later to extract total protein. The protein concentration was determined by BCA method, 10 ug protein was taken, and 10% SDS-PAGE was separated to transfer the protein to PVDF. On the membrane, TBST containing 4% skim milk (10 mmol/L Tris HC1, pH 7.5, 150 mmol/L NaC1, 0.1 % Tween) 20) Blocking membrane 1 h , p53 antibody ( 1 : 1000 ), SIRT1 antibody ( 1 : 1000 ), incubate at 4 °C overnight, wash the membrane and incubate with secondary antibody for 1 h , ECL color development, the experiment was repeated 3 times. The Fluorchem 8900 software analyzes the gray value of the protein band and calculates the ratio of the target band to the internal reference band (GAPDH). Results are shown in Figure 1. 2, as the concentration of SR59230A increases, p53 expression increases, while SIRT1 gradually decreases.

 Example 2

 In a rat model of myocardial infarction, excitatory beta 3 receptors increase myocardial cell SIRT1 and reduce p53 Expression, improve heart function and exert anti-apoptotic effects. The left anterior descending coronary artery was ligated in rats, resulting in a myocardial infarction model. Intraperitoneal injection of BRL37344 1mg/kg/day, heart 4 weeks later Super. Heart tissue was taken for apoptosis (tinel method) and SIRT1, p53 expression (Western blot). Take the heart and measure the ratio of left ventricular weight to total heart weight (LVW/BW ). The results are shown in Figures 3 to 5 . BRL37344 reduces myocardial p53 and increases SIRT1 , reduces cardiomyocyte apoptosis and reduces heart failure in rats LVW/BW , reverse cardiac hypertrophy and improve heart function.

 Example 3

Blocking the β 3 receptor increases β-galactosidase in adipose stem cells and promotes stem cell aging. Rat adipose-derived stem cells were treated with BRL37344 and SR59230A for 10 ^-7 M each. After 24 h, paraformaldehyde was fixed and stained and photographed according to the β-galactosidase staining kit. The results are shown in Figure 6. Compared to SR59230A, BRL37344 reduced β-galactosidase in adipose-derived stem cells, suggesting that the β 3 adrenergic receptors that agonize adipose-derived stem cells can play an anti-aging role.

 Example 4

Exciting beta 3 receptors reduces myocardial cell mTOR-Rictor complexes. Rat cardiomyocytes were treated with BRL37344 and SR59230A for 10 ^-7 M each. After 8 h, total RNA was extracted and reverse transcribed. Quantitative PCR was performed to detect mTOR and Rictor gene expression. The results are shown in Figures 7 and 8: BRL37344 reduced the mTOR-Rictor complex compared to the control group.

 Example 5

SR59230A reduces the volume of transplanted tumors in MCF-7 nude mice. Five-week-old, female nude mice were injected subcutaneously with 10 ⁷ MCF-7 cells to establish a transplanted tumor model. After 10 days, the tumor volume was about 250 mm ³ , randomized into groups of 8 rats. The treatment group was given SR59230A 100nmol/n, intraperitoneal injection once every 2 days for 5 weeks; the control group was given the same volume of solvent. Tumor volume was calculated by measuring the length, width and height of the tumor with a vernier caliper before and after each administration. The tumor growth curve is shown in Fig. 9. Compared with the control group, the transplanted tumor of the SR59230A group grew slowly. At the end of treatment, the tumor volume was 42.03% of the control group, and the tumor inhibition rate was 57.97%. The relative tumor growth rate T/C. (%) is 39.8%. Paraffin-embedded sections and protein samples of tumor tissues were taken, and the expressions of phospho-p70S6K (Thr389), phospho-4E-BP1 (Thr37/46) and phospho-mTOR (Ser2448) were detected by immunohistochemistry and immunoblotting. The results showed that SR59230A inhibited mTOR activity and decreased the expression of phospho-p70S6K, phospho-4E-BP1 and phospho-mTOR.

 Example 6

 SR59230A and siRNA targeting β 3 receptors promote MCF-7 breast cancer cells, H1975 and H1299 Apoptosis of lung cancer cells. The most reasonable target sequence was selected from the DNA sequence of the β 3AR gene by a computer program, and then the DNA fragment of the target sequence was artificially synthesized and cloned into a vector. Human source The U6 promoter was cloned into the pcDNA3/neo vector and used to clone the shRNA DNA fragment. Transfection of plasmid vector into cells with lipofectin2000 48h Afterwards, siRNA was expressed in cells, Annexin V/PI double staining, and apoptosis rate was detected by flow cytometry. As shown in Figure 11, silencing of β 3AR increased the apoptosis rate of MCF-7 cells ( 51.1±9.3% vs 19.5±4.4%, P <0.01), G1 phase increased cells (72.6±4.2% vs 38.5±3.5%, P <0.01). The apoptotic rate of SR 59230A group (32.0±4.9%) was also significantly increased compared with the blank group (P <0.01). Similar results were obtained for H1975 and H1299 lung cancer cells, both of which showed SR59230A and siRNA targeting β 3 receptors promoting H1975 and H1299 Apoptosis

 Example 7

SR59230A promotes apoptosis by increasing miR-16-1 and miR-15a in leukemia cells HL-60 and K562. After treatment of leukemia cells HL-60 and K562 with 10 ^-7 M SR59230A for 8 h, total microRNA was extracted, and after reverse transcription, quantitative PCR was performed to detect the expression of miR-16-1 and miR-15a. Flow cytometry was used to detect the early apoptosis marker Annexin V / PI and apoptosis rate. MitoTracker Red CMXRos mitochondrial probe detects mitochondria number, JC1 mitochondrial membrane potential, monodansylcadaverine (MDC) staining for autophagosomes, transmission electron microscopy for autophagosomes and counting, ATP kit for detection of intracellular ATP Level. The results are shown in Figures 12, 13 and 14. Compared with the control group, SR59230A significantly increased the expression of miR-16-1 and miR-15a in HL-60 and K562, and increased the apoptotic rate (*P<0.01). SR59230A can decrease the mitochondrial membrane potential of HL-60 and K562 cells, induce mitochondrial autophagy, reduce mitochondrial content, and decrease intracellular ATP levels.

 Example 8

The β 3 receptor regulates mitochondrial autophagy. Human prostate cancer cells PC-3, SR59230A and BRL37344 were treated with 10 ^-7 M for 18 h, MDC and mitochondrial probe MitoTracker Red CMXRos, labeled at 37 °C for 20 min, washed 3 times with PBS, and fluorescence was detected by confocal microscopy and flow cytometry. . Western blot was used to measure LC3 II and Beclin-1. RESULTS: SR59230A significantly reduced mitochondria, increased autophagosomes, and increased LC3 II and Beclin-1 expression.

 to sum up:

 The present invention discloses a signaling pathway of β 3 adrenergic receptors, namely β 3 receptor regulation mTOR , SIRT1 , p53 The expression and activity of other proteins are important for the occurrence and development of tumors, metabolic diseases, immune system diseases, and aging-related diseases. β 3 receptor can be used as a therapeutic target by regulating β 3 Receptors, treating the above diseases.

 The siRNA forward sequence targeting the β 3 adrenergic receptor gene is shown in SEQ ID NOS: 1-8.

 5'-ctggctaggttatgccaat-3' SEQ ID NO: 1;

 5'-cagctctcttgccccatgg-3' SEQ ID NO: 2;

 5'-cagctctcttgccccatgg-3' SEQ ID NO: 3;

 5'-taccg ccaacaccagtggg-3' SEQ ID NO: 4;

 5'-cgtgttcgtgacttcgctg-3' SEQ ID NO: 5;

 5'-ccgcgctgctgtgccttc-3' SEQ ID NO: 6;

 5'-ggggtgcccgcctgcggcc-3' SEQ ID NO: 7;

 5'-caccgggccctg tgcacct-3' SEQ ID NO: 8.

Claims (10)

1. The use of β 3 adrenergic receptors in the regulation of SIRT1, mTOR and p53 signaling pathways.
2. The use of β 3 adrenergic blockers in the preparation of SIRT1 , SIRT3 and SIRT4 inhibitors.
3. The use of β 3 adrenergic receptor blockers in the preparation of p53 agonists or mTOR inhibitors.
4. 3 3 adrenergic receptor blockers in the preparation of therapeutic SIRT1 , mTOR , p53 , MIF and microRNA-16 Applications in drugs for signaling pathway-related diseases.
5. The use of β 3 adrenergic blockers in the preparation of drugs for the treatment of cardiac hypertrophy, hypertrophic cardiomyopathy, myocardial fibrosis, pulmonary hypertension, Alzheimer's disease and malignancies.
6. As a therapeutic target, β 3 adrenergic receptors block β 3 by detecting the amount and activity of β 3 adrenergic receptors in tumors and surrounding tissues. Treatment of adrenergic receptors to treat tumors.
7. The application of β 3 adrenergic receptor blockers in the preparation of tumor multidrug resistance reversal agents.
8. The application of β 3 adrenergic receptor blockers in the preparation of malignant tumors such as breast cancer, leukemia, lung cancer, liver cancer, colon cancer, pancreatic cancer and prostate cancer.
9. siRNA targeting the β 3 adrenergic receptor gene The application in the preparation of drugs for breast cancer, leukemia, lung cancer, liver cancer, colon cancer, pancreatic cancer, prostate cancer malignant tumor; the positive sequence of siRNA is shown in SEQ ID NOs: 1-8.
10. β 3 The use of adrenergic receptor blockers in the preparation of anti-atherosclerosis, anti-coronary stent restenosis or coronary drug stent coating drugs.

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