CN106924735A - Use of dopamine type 1 receptor agonist in the preparation of drugs for treating tumors - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and relates to a novel medicinal application of a dopamine 1 receptor agonist. In particular to a new application of dopamine 1 receptor agonist in preparing tumor treatment drugs, the dopamine 1 receptor agonist comprises DRD1 and DRD5 agonists, and is selected from SKF83959 or SKF38393 compounds; the invention proves that the dopamine receptor agonist SKF83959 inhibits the mTOR pathway to induce autophagy and increases ROS to promote cell death through cell culture and drug intervention tests at in vitro and in vivo levels, thereby inhibiting the growth of benign and malignant tumors. The dopamine 1 receptor agonists SKF83959 and SKF38393 can be used for preparing medicaments for treating tumors, wherein the tumors comprise benign tumors such as pituitary adenoma and malignant tumors such as malignant glioma, colorectal cancer, liver cancer, gastric cancer and the like.

Description

Use of dopamine type 1 receptor agonist in the preparation of drugs for treating tumors

technical field

The invention belongs to the technical field of biomedicine and relates to a new medicinal application of a dopamine type 1 receptor agonist, in particular to a new application of a dopamine type 1 receptor agonist in preparing tumor treatment drugs.

Background technique

According to the information released by the National Bureau of Statistics, the annual incidence of malignant tumors in my country is 3.5 million people. For this huge population, even if they receive comprehensive treatment of surgery + radiotherapy and chemotherapy, the disability rate and mortality rate are extremely high. It endangers the life and quality of life of patients; on the other hand, it also brings great economic pressure and pressure on medical resources to the country and patients' families. For example, the clinical practice shows that the malignant glioma of the brain has an average survival rate of 14.6 months after the current internationally recommended standardized treatment; another example, although the pituitary adenoma is a benign tumor, the pituitary adenoma in the normal population can be detected by random MRI examination. The detection rate of adenoma is as high as 10% to 38.5% (average 22.5%); pituitary adenomas cause endocrine disorders, such as amenorrhea-lactation and infertility, gigantism and acromegaly, Cushing's syndrome, etc.; and pituitary tumors Enlarged compression of adjacent structures leads to vision loss and hypopituitarism, which also seriously endangers the patient's survival and quality of life.

Usually, dopamine receptor agonists are the first choice for the intervention of pituitary prolactinomas, and in 80%-90% of cases, effective control of tumor volume and normalization of PRL levels can be achieved after drug treatment. The prior art discloses that dopamine receptors (dopamine receptors, DR) are divided into five types, namely DRD1, DRD2, DRD3, DRD4 and DRD5, wherein DRD1 and DRD5 are collectively referred to as D1 receptors in the industry, and DRD2, DRD3 , DRD4 is collectively referred to as D2 receptors in the industry. Studies have shown that drugs for the treatment of pituitary prolactinomas, such as bromocriptine and cabergoline, mainly stimulate DRD2, and the role of DRD5 in it is unknown. At present, DRD5 is mainly used in psychiatric research, such as schizophrenia, attention deficit hyperactivity disorder and other diseases; in recent years, some studies have found that DRD5 is related to immunity, such as DRD5 on dendritic cells can promote the regulation of CD4 ⁺ T cells autoimmunity, and upregulation of DRD5 expression can activate NK cells. In 2012, Guha et al. found that stimulating the DRD5 receptor could re-acidify cells after alkalization by chloroquine, restore lysosome degradation ability, and promote cell survival. So far, there have been no relevant reports on the role and mechanism of DRD5 in treating tumors.

The D1 receptor agonists disclosed in the prior art include compounds such as SKF83959 and SKF38393, among which the molecular formula of SKF83959 is C ₁₈ H ₂₀ ClNO ₂ .HBr. Some studies have found that the compound SKF83959 has anti-Parkinson and neuroprotective properties. There are also studies that disclose that the compound SKF83959 can relieve progressive dyskinesia; its pharmacological studies first found that SKF83959 regulates striated muscle activity through PI-linked D1 dopamine (phosphatidylinositol-related D1 dopamine), and at the same time it is believed that D1-like receptor/ The PLC/IP3 pathway can become a new drug target for Parkinson's treatment; further research has supplemented the D1-like receptor/PLC/IP3 pathway, and the D5/PLC/IP3/calcium-dependent kinase IIα/brain-derived Neurotrophic factors are the pathway for SKF83959 to treat Parkinson's; and SKF83959 can not only inhibit excitatory synaptic conduction and sodium ion channels, but also down-regulate the excitability of pyramidal cells in the CA1 region of the hippocampus through membrane potential depolarization. The mechanism of SKF83959’s anti-Parkinson’s effect is partially explained; secondly, SKF83959’s neuroprotective effect can be through receptor and non-receptor pathways. In addition to the above-mentioned anti-Parkinson’s mechanism, it is also related to ERK and p38 molecular targets. Recent studies have proposed that SKF83959 is a potent allosteric regulator of sigma-1, which stimulates the activity of endogenous DHEA in a synergistic manner, inhibits BV2 microglial cells and pro-inflammatory regulators, such as TNF-α, IL-1β, etc.;

Autophagy, which means autophagy, is a general term for the degradation process of intracellular material components using lysosomes, including the degradation and reuse of long-lived proteins and some organelles, so as to maintain the internal stability of cells. Reactive oxygen species (reactive oxygen species, ROS) is a kind of toxic substances that damage cells. Recent studies have found that ROS, as a signaling molecule, participates in multiple physiological processes such as cell proliferation, differentiation and apoptosis; ROS, especially mitochondrial sources Sexual ROS, as a signaling molecule, participates in the regulation of autophagy, leading to cell survival or death in different environments; the increase of ROS can lead to the opening of mitochondrial membrane permeability transport pore (MPT), and the opening of MPT causes mitochondrial transmembrane potential (Δψm) decreases, cytochrome C is released, and then activates a series of caspase enzymes to induce apoptosis; excessive ROS in cells induces apoptosis, and a large amount of ROS will cause cell necrosis, of course, in the same tissue Necrosis and apoptosis can also occur at the same time, and the shift from apoptosis to necrosis is determined by the content of ROS in tumor cells; it has been reported in the literature that chronic stimulation of D1 receptors, such as SKF38393, can lead to an increase in neuronal NOS, Thus accelerating cell death; however, the research mechanism of tumor cell death caused by the increase of ROS caused by DRD5 activation has not been reported; whether SKF83959 has the effect of inhibiting tumor growth has not been reported yet.

Based on the current state of the art, the applicant of the present invention intends to provide a new medicinal use of dopamine type 1 receptor agonists, specifically related to the use of dopamine type 1 receptor agonists in the preparation of tumor treatment drugs.

Contents of the invention

The purpose of the present invention is to provide a new medicinal application of dopamine type 1 receptor agonist, in particular to a new application of dopamine type 1 receptor agonist in the preparation of tumor treatment drugs.

The dopamine receptor agonists described in the present invention are dopamine receptor agonists, including DRD1 and DRD5 agonists, such as SKF83959, SKF38393 and other compounds. Experiments have proved that the dopamine receptor agonists can effectively inhibit The growth of tumor cells, and promote the death of tumor cells by increasing ROS, so as to achieve the purpose of treating tumors.

In the embodiment of the present invention, the use of dopamine type 1 receptor agonists SKF83959, SKF38393 on rat MMQ and GH3 cells, N2A cells and malignant tumor cells (including glioma U87, U251, SHG66, colorectal cancer SCG7901, gastric cancer SW480) In vitro experiments were carried out, and the results showed that it had an inhibitory effect on cell growth; further transplanted tumor models confirmed that SKF83959 had an inhibitory effect on the growth of subcutaneous transplanted tumor models of GH3 cells and SCG7901 cells; the mechanism research results showed that: SKF83959 induced cell growth Autophagy occurs, and this autophagy is realized by inhibiting the mTOR pathway; at the same time, SKF83959 can promote the increase of cell ROS, thereby leading to death; the present invention also carried out the test of human-derived pituitary adenoma primary cells, using SKF83959 In primary human pituitary adenoma cells, the results show that it can significantly inhibit the growth of primary human pituitary adenoma cells.

In the present invention, the dopamine receptor agonist SKF83959 has a chemical structural formula: 6-Chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3- Benzazepine-7,8-diol, molecular formula: C ₁₈ H ₂₀ ClNO ₂ .HBr, molecular weight: 398.73.

The experimental results of the present invention show that the dopamine 1 receptor agonists such as SKF83959, SKF38393 and other compounds can be used to prepare drugs for the treatment of tumors, especially drugs that can significantly inhibit tumor growth to achieve therapeutic effects; the tumor types Including benign tumors such as pituitary adenoma and neurofibroma and malignant tumor cells such as glioma, colorectal cancer, gastric cancer, etc.

For ease of understanding, the present invention will be described in detail below through specific drawings and embodiments. It should be pointed out that the specific examples and accompanying drawings are only for illustration. Obviously, those skilled in the art can make various amendments and changes within the scope of the present invention according to the description herein. These amendments and Modifications are also included within the scope of the present invention.

Description of drawings

Figure 1 shows: SFK83959 inhibits the growth of pituitary tumor cell lines MMQ and GH3, inhibits the growth of N2A cells, and also effectively inhibits the growth of malignant tumor cells, including malignant glioma U87, U251, SHG66, colorectal cancer SCG7901, and gastric cancer SW480 ; SFK83959 inhibits the growth of human primary pituitary tumor cells. SKF38393 inhibits the growth of GH3, U87, SCG7901, SW480, hepG2 cells.

Figure 2 shows: the xenograft tumor model in nude mice confirms that SKF83959 effectively inhibits the cell growth of GH3 and SCG7901.

Figure 3 shows: SKF83959 inhibits the mTOR pathway to induce autophagy, verifies the expression of LC3-II at the protein level, confirms the existence of autophagosomes under electron microscopy, and observes the formation of GFP-LC3-II under confocal microscopy. At the same time, it inhibits the expression of p-mTOR and p-4EBP1 proteins.

Figure 4 shows: SFK83959 increases cell ROS-mediated cell death, and the use of ROS inhibitor NAC can reverse cell death, and at the same time, reverse SKF83959-mediated LC3-II expression and p-4EBP1 protein inhibition.

detailed description

Example 1 SFK83959 inhibits cell line growth experiment

Experimental Materials:

Rat pituitary tumor cell lines GH3 and MMQ (purchased from ATCC), and human primary pituitary adenoma cells (removed from surgery and placed in culture medium directly for culture) were routinely cultured at 37°C and 5% CO ₂ . DMEM/F12 (Gibco) containing 2.5% fetal bovine serum (Gibco) plus 12.5% horse serum. N2A cells and other malignant tumor cells (U87, U66, U251, SCG7901, SW480, hepG2) (Chinese Academy of Sciences) were routinely cultured at 37°C and 5% CO ₂ in DMEM containing 10% fetal bovine serum (Gibco) (Gibco) medium. Dopamine type 1 receptor agonists SKF83959 and SKF38393 were purchased from Tocris (UK), the mother solution 50mM was prepared from DMSO (sigma), and the MTS assay reagent for cell viability was purchased from Promega (USA).

experimental method:

1. MTS assay to detect the activity of GH3 and MMQ cells after different concentrations of SKF83959

1) GH3 cells were suspended in conventional medium, and the cell density was adjusted to 0.7×10 ⁴ cells/well, and seeded in a 96-well culture plate to adhere to the wall overnight; MMQ cells were suspended in conventional medium, and the cell density was adjusted to 1×10 ⁴ cells Inoculate each well in a 96-well culture plate; Add medium containing different drug concentrations, GH3 cells are divided into 5 groups according to the final drug concentration (0 μM, 1 μM, 5 μM, 10 μM, 25 μM); MMQ cells are divided into 5 groups according to the final drug concentration (0 μ M, 1 μ M, 5 μ M, 25 μ M, 50 μ M) group, each group has 5 auxiliary wells, each well is 100 μ l system, and the blank control group contains DMSO 0.3%;

2) culturing the administered cells at 37° C., 5% CO ₂ for 48 hours;

3) Add 20 μl of MTS solution to each well and continue culturing in the incubator for 4 hours;

4) Measure the value of absorbance at a detection wavelength of 490 nm. Calculate the growth inhibition rate of cells. N=5, the experiment was performed in parallel three times. The results of MMQ cell activity are shown in Figure 1.A; the results of GH3 cell activity are shown in Figure 1.B;

2. MTS assay to detect the activity of SKF83959 on N2A cells and other malignant tumor cells

1) Suspend N2A cells and other malignant tumor cells in conventional culture medium, adjust the cell density to 0.7×10 ⁴ cells/well, inoculate them in 96-well culture plates and adhere to the wall overnight, add medium containing SKF83959, and divide each cell line into 2 groups , the final concentration of the drug is 25 μM, each group has 5 auxiliary wells, each well is 100 μl of the system, and the blank control group contains 0.3% DMSO;

2) culturing the administered cells at 37° C., 5% CO ₂ for 48 hours;

3) Add 20 μl of MTS solution to each well and continue culturing in the incubator for 4 hours;

4) Measure the value of absorbance at a detection wavelength of 490 nm. Calculate the growth inhibition rate of the cells. N=5, the experiment was performed in parallel three times. The results of each cell activity are shown in Figure 1.C;

3. MTS assay to detect the activity of SKF83959 on primary human pituitary tumor cells

1) Human primary pituitary tumor cells were suspended in conventional culture medium, adjusted to a cell density of 0.7×10 ⁴ cells/well, and seeded on a 96-well culture plate to adhere to the wall overnight. Add medium containing SKF83959, each cell line is divided into 2 groups, the final concentration of the drug is 25 μM, each group has 5 auxiliary wells, each well is 100 μl system, and the blank control group contains 0.3% DMSO;

2) culturing the administered cells at 37° C., 5% CO ₂ for 48 hours;

3) Add 20 μl of MTS solution to each well and continue culturing in the incubator for 4 hours;

4) Measure the value of absorbance at a detection wavelength of 490 nm. Calculate the growth inhibition rate of cells. N=5, the experiment was performed in parallel three times.

The results of cell activity are shown in Figure 1.D;

The experimental results show that SKF83959 can inhibit the growth of various tumor cells. Among them, SFK83959 inhibits the growth of pituitary tumor cell lines MMQ and GH3, and also inhibits the growth of N2A cells and other malignant tumor cells (U87, U66, U251, SCG7901, SW480) ; SFK83959 also inhibits the growth of human primary pituitary tumor cells.

The same method was used for SKF38393 experiments, and it was found that SKF38393 can also inhibit the growth of pituitary tumor cells GH3 and other malignant tumor cells (U87, SCG7901, SW480, hepG2) (as shown in Figure 1.E).

Example 2. Nude mice xenograft tumor model experiment

Experimental Materials:

Nude mice were purchased from SLAC (Shanghai), and the rest were the same as in Example 1.

experimental method:

1) Select 4-week-old nude mice;

2) Collect the logarithmically grown tumor cells, use 10ml of serum-free medium, centrifuge at 1000 rpm for 3 minutes, wash continuously for 3 times, and finally resuspend the tumor cells in serum-free medium, and adjust the density to 1×10 ⁷ Cells/ml, inoculate 100ul (i.e. 1×10 ⁶ tumor cells) in the armpit of each nude mouse, and the tumor can be palpable in the armpit of the nude mouse in about 4 days;

³ ) When the tumor volume grows to about 100mm3, divide into DMSO and SKF83959 groups, give SKF83959 (1mg/kg) intraperitoneal injection, each nude mouse weighs about 20g, and inject 1μl SKF83959 (50mM) into each nude mouse intraperitoneally + 99 μl of normal saline, administered once a day, and the size of the long axis and wide axis of the tumor (unit: mm) was measured with a vernier caliper;

4. After 11 days of administration, the nude mice were routinely treated, the tumors were weighed and photographed, and the tumor weight and volume were counted. Tumor volume = long axis x wide axis ² x 1/2; the results of GH3 cell in vivo experiments are shown in Figure 2.A , 2.B, and 2.C, and the results of in vivo experiments on SCG7901 cells are shown in Figures 2.D, 2.E, and 2.F. The experimental results showed that in vivo animal experiments, SKF83959 inhibited the cell growth of tumor cells GH3 and SCG7901.

Example 3, SKF83959 induces autophagy by inhibiting the mTOR pathway

Experimental Materials:

RIPA Lysis Solution, PMSF, Loading Buffer (5×), BCA Protein Quantification Kit, and DAPI were purchased from Beyontien Biotechnology Research Institute (Jiangsu); Protease Inhibitor Cocktail was purchased from Merck (Germany). BSA was purchased from sigma (9048-46-8). PBS, 0.5% Trypsin-EDTA were purchased from Gibco. Antibody: Antibody LC3(sigma L7543), Antibody GAPDH(Huaan ARH4156), Antibody PARP(CST#9542), Antibody p-4EBP1(CST#2855), Antibody p-mTOR(CST#5536), Anti-rabbit IgG(CST 14708), Anti-rabbit IgG-Peroxidase antibody (sigma A0545); strong chemiluminescence kit was purchased from Bio-rad company.

experimental method:

1) GH3 cells were suspended in conventional medium, and the cell density was adjusted to 4×10 ⁵ cells/well, inoculated in a 6-well culture plate to adhere to the wall overnight, each well contained 2ml of medium, the final concentration of the drug was 25 μM, and they were treated for 0 h respectively. , 12h, 24h, 48h. 0h is the control group, containing DMSO 0.3%, cultured at 37°C, 5% CO ₂ ;

2) Western Blot detection of the expression of LC3-II, p-mTOR, p-4EBP1

(1) Protein extraction:

Add PMSF (100mM, 1:100) and Protease Inhibitor Cocktail Set III (1:200) to RIPA lysate before use;

Collect the cells according to the drug action time point. When collecting the cells, remove the culture medium in the 6-well plate, wash the cells gently with PBS, digest the GH3 cells with 0.05% trypsin for 10 seconds, remove the trypsin, and add 1ml of complete medium to neutralize. Collected in a 1.5ml EP tube, centrifuged at 2000rpm for 5min, discarded the supernatant, added 100μl of lysate in an ice bath for 30min; centrifuged at 12000rpm for 20min after lysing; absorbed the supernatant for quantification, and at the same time took part of the supernatant and added 1/4 volume of the sample Buffer (5×), cook at 100°C for 10 minutes; denatured protein is stored in a -80°C refrigerator;

(2) Loading amount: BCA protein quantification box quantifies the protein samples extracted from each group, and the protein sample loading amount after correction is 40 μg;

(3) Electrophoresis: 80V constant voltage electrophoresis for 30 minutes to concentrate the sample; 120V electrophoresis for 1.5 hours to separate the sample, and judge the degree of electrophoresis according to the marker;

(4) Transfer film: use wet transfer, 170mA, constant current transfer film for 2 hours;

(5) Antibody hybridization: first block with 5% BSA on a shaker at room temperature for 1 hour; dilute the primary antibody (LC3 1:5000; GAPDH1:2000; p-mTOR 1:1000; p-4EBP1 1:1000; PARP 1:1000), hybridize the transferred membrane overnight at 4°C in the hybridization tank; wash the membrane 3 times with TBST (shaking at room temperature, 2 mL each time, 5 min); dilute the secondary antibody with blocking solution, and shake in the hybridization tank at room temperature Bed hybridization for 1 hour; chemiluminescent detection after washing the membrane three times with TBST;

(6) Chemiluminescence detection: use enhanced chemiluminescence (ECL method) to detect the target protein;

The experimental results are shown in Figures 3.A and 3.B, and ImajeJ counts the relative expression levels of p-4EBP1, p-mTOR and GAPDH (as shown in Figure 3.C);

Electron microscope inspection

According to the above experimental method, 48 hours after the administration of GH3 cells, the cells were collected in a 15ml centrifuge tube, centrifuged at 2000rpm for 10min, then added to 2.5% glutaraldehyde, and detected by the electron microscope center, the results are shown in Figure 3.D;

Confocal detection of LC3 expression

1) Drug-treated cells: GH3 cells were suspended in conventional culture medium, and the cell density was adjusted to 3×10 ⁵ cells/well, seeded in a 6-well culture plate, and adhered overnight. Discard the culture medium in the 6-well plate, and replace it with fresh medium containing SKF83959 with a final concentration of 25 μM; replace the control group with fresh medium containing 0.3% DMSO, and culture at 37°C and 5% CO ₂ for 48 hours;

2) Cell flakes: Discard the culture medium in a 6-well plate, digest with 0.05% trypsin for 20 seconds, discard the trypsin, neutralize with 1ml of complete medium, collect the cells, and fling the cells onto a glass slide with a spinner, 1500 rpm, 15min ;

3) Fix and permeabilize: circle the cell area with an oil-based pen, add 100 μl acetone to fix and permeabilize it for 5 minutes, shake the cells at room temperature, wash the cells 3 times with TBST, 5 minutes each time;

4) Sealing: add 200 μl of 0.1% BSA to the cell area, put the slide in a wet box, and seal at room temperature for 1 hour;

5) Antibody hybridization: Dilute LC3 (1:100) with 0.1% BSA, add 100 μl primary antibody dilution to the cell area, put it in a wet box, and incubate overnight; shake the cells at room temperature, wash the cells 4 times with TBST, 5 min each time; 0.1% Dilute the secondary antibody with BSA (1:100), add 100 μl of the secondary antibody diluent to the cell area, put it in a wet box, and incubate at room temperature for 1 h; shake the cells at room temperature, wash the cells 4 times with TBST, 5 min each time. Anti-fluorescent quenching agent for sealing;

6) Observe under a laser confocal fluorescence microscope, take pictures and count LC3 green fluorescent spots (as shown in Figure 3.E).

The results showed that after SKF83959 acted on GH3 cells, it inhibited the expression of p-mTOR and p-4EBP1, inhibited the mTOR signaling pathway, promoted the expression of autophagy-related protein LC3, and induced the formation of autophagosomes.

Example 4, ROS-mediated SFK83959-induced cell death test

Experimental Materials:

ROS inhibitor NAC and ROS detection kits were purchased from Beyontien Biotechnology Research Institute (Jiangsu), and the rest of the experimental consumables were the same as above.

experimental method:

1. Flow cytometric detection of ROS levels in GH3 cells after SKF83959 treatment

1) Drug treatment: GH3 cells were suspended in conventional culture medium, and the cell density was adjusted to 3×10 ⁵ cells/well, seeded in a 6-well culture plate, and adhered overnight. Add fresh drug-containing medium to divide into 4 groups, respectively DMSO, SKF83959, NAC, SKF83959+NAC group, so that the final concentration of SKF83959 is 25 μM, the final concentration of NAC is 10 μM, and the blank control group contains 0.3% DMSO. Cultivate for 48 hours at 37°C and 5% CO ₂ ;

2) Cell collection: When collecting cells, remove the culture medium in the 6-well plate, gently wash the cells with PBS, digest the GH3 cells with 0.05% trypsin for 10 seconds, remove the trypsin, add 1ml of complete medium to neutralize, blow gently Scattered, collected in 1.5ml EP tube, centrifuged at 800rpm for 5min, and discarded the supernatant;

3) Loading probes: Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000, so that the final concentration is 10 μmol/L. Suspend the collected cells in diluted DCFH-DA at a cell concentration of 1 million to 20 million/ml, and incubate in a cell culture incubator at 37°C for 20 minutes. Mix by inverting every 3-5 minutes to make the probe fully contact with the cells. Wash the cells three times with serum-free cell culture medium to fully remove DCFH-DA that has not entered the cells;

4) Flow cytometric detection: The cells loaded with the probes were subjected to flow cytometric detection with an excitation wavelength of 488nm and an emission wavelength of 525 (as shown in Figure 4.A); SKF83959 can increase the level of intracellular ROS.

2. MTS detects the activity of GH3 cells after the action of ROS inhibitors and SKF83959

1) GH3 cells were suspended in conventional culture medium, adjusted to a cell density of 0.7×10 ⁴ cells/well, and seeded on a 96-well culture plate to adhere to the wall overnight. Add fresh medium containing medicine and divide into 4 groups, namely DMSO, SKF83959, NAC, and SKF83959+NAC groups, so that the final concentration of SKF83959 is 25 μM, and the final concentration of NAC is 10 μM. There are 5 secondary wells in each group, and the blank control group contains DMSO. 0.3%. The administered cells were cultured for 48 hours at 37°C and 5% CO ₂ ;

2) Add 20 μl of MTS solution to each well and continue culturing in the incubator for 4 hours;

3) Measure the value of absorbance at a detection wavelength of 490 nm. Calculate the growth inhibition rate of cells. N=5, the experiment was repeated three times; the results of cell viability are shown in Figure 4.B, the results showed that the inhibition of ROS level could partially reverse the cell death induced by SKF83959.

3. Western Blot detection of the expression of LC3-II and p-4EBP1 after the ROS inhibitor and SKF83959 acted on GH3 cells. GH3 cells were suspended in conventional medium, and the cell density was adjusted to 3×10 ⁵ cells/well and seeded on a 6-well culture plate. wall overnight; adding fresh drug-containing medium into 4 groups, respectively DMSO, SKF83959, NAC, SKF83959+NAC group, so that the final concentration of SKF83959 was 25 μM, the final concentration of NAC was 10 μM, and the blank control group contained 0.3% DMSO. The cells after administration were cultured for 48 hours at 37°C and 5% CO ₂ ;

Protein extraction, western blot detection of LC3 and p-4EBP1 protein expression, the method is the same as in Example 3;

The experimental results showed that inhibiting the generation of ROS could partially reverse the increase of LC3 expression and the decrease of 4EBP1 phosphorylation activity induced by SKF83959. (As shown in Figure 4.C).

The above experimental results show that ROS plays an important role in the process of cell death induced by SKF83959-induced autophagy.

Claims (7)

1. purposes of the receptoroid activator of dopamine 1 in anti-tumor medicine is prepared.

2. the purposes as described in claim 1, it is characterised in that described Dopamine receptor_1 excitomotor includes DRD1 and DRD5 activators, selected from SKF83959 or SKF38393 compounds.

3. the purposes as described in claim 1, it is characterised in that described dopamine-receptor stimulant SKF83959, its chemical structural formula is：6-Chloro-2,3,4,5-tetrahydro-3-methyl-1- (3-methylphenyl) -1H-3-benzazepine-7,8-diol, molecular formula：C18H20ClNO2.HBr, molecular weight：398.73.

4. the purposes as described in claim 1, it is characterised in that the described receptoroid activator of dopamine 1 suppresses growth of tumour cell.

5. the purposes as described in claim 1 or 4, it is characterised in that described tumour cell is MMQ and GH3 cells, N2A cells and malignant cell include glioma U87, U251, SHG66, colorectal cancer SCG7901, liver cancer hepG2 cells, stomach cancer SW480 cells.

6. the purposes as described in claim 1, it is characterised in that described anti-tumor medicine is treatment pituitary adenoma or the carcinoid medicine of neurofibroma.

7. the purposes as described in claim 1, it is characterised in that described anti-tumor medicine is the medicine for treating glioma, colorectal cancer, liver cancer or stomach cancer malignant tumour.