阅读说明：本技术 磺内酰胺-环己酮螺环衍生物1-3-51在制备治疗胃癌药物中的应用 (Application of sultam-cyclohexanone spiro derivative 1-3-51 in preparation of medicine for treating gastric cancer ) 是由 欧阳勤 古晶 王纳 高梦圆 陈应春 胡长江 杨仕明 于 2021-01-22 设计创作，主要内容包括：本发明涉及磺内酰胺-环己酮螺环衍生物1-3-51在制备治疗胃癌药物中的应用,所述药物显著抑制FOXM1的表达以及胃癌细胞的增殖、侵袭及迁移,干扰FOXM1后,其抑制增殖,侵袭及迁移能力降低。(The invention relates to an application of sultam-cyclohexanone spiro derivative 1-3-51 in preparation of a medicine for treating gastric cancer, wherein the medicine remarkably inhibits the expression of FOXM1 and the proliferation, invasion and migration of gastric cancer cells, and after FOXM1 is interfered, the proliferation inhibition, invasion and migration capabilities are reduced.)

1. Application of sultam-cyclohexanone spiro derivative 1-3-51 in preparing medicine for treating gastric cancer.

2. Use according to claim 1, characterized in that: the molecular formula of the sultam-cyclohexanone spiro derivative is C₂₄H₂₁NO₃S₂。

3. Use according to claim 1, characterized in that: the drug is an inhibitor of the protein M1 of the forkhead box.

4. Use according to claim 1, characterized in that: the drug inhibits the expression of FOXM1 in gastric cancer cells.

5. Use according to claim 1, characterized in that: the drug inhibits the expression of FOXM1 downstream target genes Cyclin D1 and MMP 9.

6. Use according to claim 1, characterized in that: the medicine inhibits the growth migration and invasion of gastric cancer cells BGC823 and MKN45 through FOXM 1.

7. Use according to claim 1, characterized in that: the medicine is administrated by an intraperitoneal injection mode.

8. Use according to claim 1, characterized in that: the dosage of the medicine is 0.5-5 mg/day/kg.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of sultam-cyclohexanone spiro derivative 1-3-51 in preparation of a medicine for treating gastric cancer.

Background

Gastric Cancer (Gastric Cancer GC) is one of the common digestive system malignancies in the world, and its high morbidity and mortality have made it a significant problem for human public health in the world, especially in developing countries, severely harming human health and wasting social resources. Currently 50% of gastric cancers are diagnosed in east asia, with most cases occurring in china. The incidence and mortality of gastric cancer are steadily increasing in the world, according to the statistics of the cancer in 2018, the incidence and mortality of gastric cancer are fifth and third in the world, and the gastric cancer is predicted to become one of the 15 major causes of death in the world by 2020 and 2030. Despite the progress of the comprehensive treatment of gastric cancer in recent years, the overall survival rate of gastric cancer patients in 5 years is still very low, which is less than 30%. The reason is that most of gastric cancer patients in China are in the advanced stage during initial diagnosis, the treatment method is limited, the chemotherapy mode is mainly combined through the operation, the recurrence rate and the metastasis rate are high, and the chemotherapy resistance of gastric cancer cells is also one of the important reasons for poor prognosis of the gastric cancer patients. Therefore, the research on novel gastric cancer targeted therapeutic drugs is very important, and the method has very important significance for improving the treatment and prognosis of gastric cancer patients.

Forkhead box protein M1(Forkhead box M1, FOXM1) is one of the members of the Forkhead superfamily, is a class of transcription factors with specific 'wing-shaped helical structure' DNA binding domains, including FOXA, FOXC, FOXM, FOXO, FOXP and other families, the Forkhead family is widely involved in the processes of life development, such as embryogenesis, proliferation, differentiation, apoptosis, transformation, tumorigenesis, life and metabolic homeostasis, has definite functions in cell proliferation and cell cycle progression, and is closely related to tumor development, wherein FOXM1 is evaluated as an annual molecule in 2010. Research shows that FOXM1 is commonly up-regulated in various tumors, such as breast cancer, lung cancer, liver cancer, osteosarcoma, nasopharyngeal carcinoma, laryngeal carcinoma, gastric cancer, ovarian cancer and the like, and tumor gene expression profiling analysis also proves that FOXM1 is one of the most frequently up-regulated genes in human malignant tumors. In gastric cancer, the activation and abnormal expression of FOXM1 pathway are closely related to malignant biological behavior and poor prognosis of gastric cancer patients, and FOXM1 is considered as a potential target for preventing and treating human cancer. The salinomycin A has been studied to induce apoptosis by inhibiting the expression of FOXM1 and further inhibiting the expression of downstream target genes Bcl-2 and Mcl-1, the cracking of caspase-3 and the like, and the thiostrepton is also reported to be used as an inhibitor of FOXM1 for the first time in 2008, and can interfere the combination of FOXM1 and DNA so as to interfere the transcription of the downstream target. Meanwhile, the research shows that the salinomycin A can also inhibit maternal embryonic leucine zipper protein kinase (MELK), and the thiostrepton can also target Peroxidoxin 3, but the specificity of the targeting FOXM1 is not high, and the clinical application is limited. Therefore, it is important to find a novel small molecule drug targeting FOXM 1.

Disclosure of Invention

The invention aims to provide application of sultam-cyclohexanone spiro derivative 1-3-51 (hereinafter referred to as compound 1-3-51) in preparation of a medicine for treating gastric cancer, wherein the medicine remarkably inhibits the expression of FOXM1 and the proliferation, invasion and migration of gastric cancer cells, and after FOXM1 is interfered, the proliferation, invasion and migration inhibition capacity of the medicine is reduced.

The technical scheme of the invention is as follows:

application of sultam-cyclohexanone spiro derivative 1-3-51 in preparing medicine for treating gastric cancer.

The molecular formula of the sultam-cyclohexanone spiro derivative is C₂₄H₂₁NO₃S₂The structural formula is as follows:

the drug is an inhibitor of the protein M1 of the forkhead box.

The drug inhibits the expression of FOXM1 in gastric cancer cells.

The drug inhibits the expression of FOXM1 downstream target genes Cyclin D1 and MMP 9.

The medicine inhibits the growth migration and invasion of gastric cancer cells BGC823 and MKN45 through FOXM 1.

The medicine is administrated by an intraperitoneal injection mode.

The dosage of the medicine is 0.5-5 mg/day/kg.

The applicant has constructed a number of molecular scaffolds with novel structures through studies of chemical synthesis methodology. Wherein, the complex molecular structure of part of compounds has the characteristics of natural products, and shows potential drug-like properties. In the synthesis methodology research carried out in the previous period, a high-selectivity asymmetric [3+3] cyclization reaction is developed, and a series of sultam-cyclohexanone spiro derivatives are constructed. In the process of evaluating the general biological activity of the products, the applicant finds that the representative compounds 1-3-51 can effectively inhibit the proliferation and invasion migration of gastric cancer cells and promote apoptosis, and further mechanism research finds that the representative compounds obviously inhibit the expression of FOXM1, which suggests that 1-3-51 may be a novel FOXM1 small-molecule inhibitor, and provides a certain theoretical basis and thought for the design of a targeted FOXM1 small-molecule inhibitor.

The applicant adopts a CCK-8 kit to detect BGC823 and MKN45 cells IC50 after 1-3-51 treatment; detecting the expression of gastric cancer cells FOXM1 by Western blot; RT-qPCR detects the mRNA expression of FOXM1 of gastric cancer cells and downstream molecules thereof, namely Cyclin D1 and MMP 9; flow cytometry to determine cycle status; the Transwell experiment detects the migration and invasion capacity of gastric cancer cells; FOXM1 shRNA plasmid and its idle plasmid are transfected in gastric cancer cells BGC823 and MKN45, and changes of gastric cancer cell activity, migration, invasion and the like are detected in CCK8 experiment and Transwell experiment respectively. Experiments prove that the compounds 1-3-51 can inhibit the expression of FOXM1 and the expression of molecules such as downstream target genes Cyclin D1 and MMP9 in gastric cancer cells, so that the proliferation, migration and invasion capacities of the gastric cancer cells are reduced, and a new thought is provided for a novel micromolecule anticancer drug targeting FOXM 1.

Drawings

FIG. 1 shows that 1-3-51 decreased levels of FOXM1 protein and mRNA in gastric cancer cells; wherein, fig. 1A: 1-3-51; FIG. 1B: 1-3-51BGC823 and MKN45 cells are respectively treated for 48h, and then an OD450 value is measured by CCK 8; FIG. 1C: the expression of FOXM1 protein and mRNA after BGC823 and MKN45 cells are treated by different concentrations of 1-3-51; FIG. 1D: time gradient 1-3-51 expression of FOXM1 protein and mRNA after BGC823 and MKN45 cells were treated.

FIG. 2 shows that 1-3-51 reduces the expression of mRNA levels of the downstream target genes Cyclin D1 and MMP9 of FOXM1 in gastric cancer cells; wherein, fig. 2A: after BGC823 and MKN45 cells are treated by a concentration gradient of 1-3-51, the expression conditions of FOXM1 downstream target genes Cyclin D1 and MMP9 mRNA are treated; FIG. 2B: after BGC823 and MKN45 cells are treated by a time gradient of 1-3-51, the expression conditions of FOXM1 downstream target genes Cyclin D1 and MMP9 mRNA are treated;

FIG. 3 is a graph showing the effect of 1-3-51 on growth cycle, migration and invasion of gastric cancer cells; wherein, fig. 3A: flow cytometry measured cell cycle compared to DMSO group; FIG. 3B: compared with DMSO group, cell migration and invasion are detected by Transwell experiment;

FIG. 4 is a graph showing the detection of 1-3-51 changes in proliferation, migration and invasion of FOXM1 partial knockout cells in BGC823 cells, where FIG. 4A: detecting the expression change of FOXM1 protein after the FOXM1 is knocked down in the BGC823 cells by Western blot; FIG. 4B: the CCK-8 experiment detects the change of cell proliferation capacity before and after 1-3-51 knockdown of BGC823 FOXM 1; FIG. 4C: the Transwell experiment detects the cell migration and invasion changes before and after 1-3-51BGC823 FOXM1 knockdown.

Detailed Description

Reagents used in this example:

human gastric cancer cell lines BGC823 and MKN45 were purchased from Shanghai Rich and balanced organisms;

high-sugar DMEM medium and RPMI 1640 medium were purchased from HyClone, USA;

fetal bovine serum was purchased from Gibco, usa;

anti-human FOXM1 antibody was purchased from Cell signaling technology, usa;

anti-human TUBULIN primary antibody, goat anti-rabbit and goat anti-mouse secondary antibody marked by HRP, CCK8 kit, BCA protein concentration determination kit, RIPA lysate and crystal violet are purchased from Shanghai Bin Yuntian company in China;

paraformaldehyde was purchased from Dr. USA;

TRIzol, a reverse transcription kit and SYBR Green reagent were purchased from TAKARA, Japan;

FOXM1 shRNA and its empty plasmid were purchased from Santa corporation, USA;

transfection reagent Lipofectamine^TM2000 and plasmid mini-cartridges were purchased from Invitrogen, usa;

APC Annexin V Apoptosis Detection Kit with PI from BioLegend, USA;

PVDF membrane, ECL chemiluminescence kit from Millipore, Germany.

Example 1 preparation, detection and extraction of cells

1.1 cell culture

Human gastric cancer cells BGC823 and MKN45 are cultured in DMEM high-sugar medium and RPMI 1640 medium (containing 10% fetal calf serum, 100U/mL penicillin and 10mg/L streptomycin) respectively under the following culture conditions: saturated humidity, 37 ℃ and 5% CO₂Culturing in a constant temperature incubator. When cells grew to 80-90%, 1: 3, digestion passage.

1.2 CCK8 detection of cellular Activity

After seeding cells at 4000/well in 96-well plates, 10 μ L CCK8 was added to each well at harvest according to CCK8 instructions. After addition of CCK81h, the cell activity was measured by optical density at a wavelength of 450nm in a microplate reader.

1.3 extraction of Total cellular protein

The treated cells are discarded with culture medium, washed twice with precooled PBS, PBS is discarded as much as possible, a proper amount of RIPA (strong) lysate containing protease inhibitor is added into each hole, the cells are scraped by using cell scrapers, transferred into a new EP (Eppendorf) tube, shaken on an oscillator for 30s every 1min for 3 times, cracked on ice for 30min, then centrifuged for 15min at 4 ℃, 14000 rpm, and finally the supernatant is extracted to obtain total protein and the concentration of the total protein is measured by using a BCA (burst amplification assay).

1.4 extraction of RNA

Removing the culture medium of the treated cells, washing the treated cells twice by using precooled PBS, removing the PBS as much as possible, adding 1mL of TRIzol into each hole, standing for 1min, blowing to crack the cells, collecting the cells, shaking the cells on a shaker every 1min for 30s for 3 times, and cracking the cells on ice for 5 min; adding 200 μ L of chloroform into the lysate, slightly mixing, and standing at room temperature for 15 min; centrifuging: 12000 g at 4 ℃ for 15 min; taking the supernatant, putting the supernatant into an EP tube without DNA and RNA enzyme, adding 0.5mL of isopropanol, uniformly mixing, and standing at room temperature for 5-10 min; centrifuging: 12000 g at 4 ℃ for 10 min; the supernatant was discarded, 1mL of 75% ethanol was added, and centrifuged: 4 ℃, 8000g and 5 min; centrifuging again, carefully sucking up residual alcohol, and air drying; adding 50 μ L DEPC water, dissolving the precipitate, and mixing; and (5) measuring the concentration.

1.5 reverse transcription

The reaction system is prepared from 5 XgDNA Eraser buffer2.0 μ L, gDNA Eraser1.0 μ L, RNA1.0 μ g, and RNaseFreedH₂Make up to 10 μ L of O (reaction conditions: 42 ℃, 10 min); the following system was added to the above reaction product: 5 XPrimeScript Buffer2(for Real Time) 4.0. mu.L, PrimeScriptRT Enzyme Mix I1.0. mu.L, RT Primer Mix 1.0. mu.L, RNase Free dH₂O4.0. mu.L (reaction conditions: 37 ℃, 15min, 85 ℃, 5 s); to the reaction product cDNA, 20. mu.L of DEPC water was added for use.

1.6 RT-q PCR

The following system is prepared: TB Green Premix Ex Taq II 10.0. mu.L, cDNA 2.0. mu.L, upstream and downstream primers 0.8. mu.L each, Rox 0.4. mu.L, DEPC water 6. mu.L, a total volume of 20. mu.L (reaction conditions: 95 ℃, 15s, 58 ℃, 30s), 40 cycles, 3 duplicate wells set, and the final values were plotted and statistically analyzed using Prism 8.

1.7 WesternBlot

The loading amount of the protein is 30 mug, and the protein is separated by 10 percent SDS-PAGE gel electrophoresis; transferring protein to PVDF membrane by wet transfer method, sealing with 5% skimmed milk powder for 30min-1h, adding primary antibody at 4 deg.C overnight (FOXM 11: 1000, TUBULIN 1: 10000), incubating secondary antibody at room temperature for 1h (goat anti-mouse 1: 5000, goat anti-rabbit 1: 10000), and displaying the result by ECL chemiluminescence method.

1.8 Transwell Chamber assay for detecting cell invasion and migration

Invasion experiment steps: 2.5mg/mL of Matergel was pipetted into the upper chamber of the Transwell chamber and placed in an incubator at 37 ℃ for 2 h. Resuspending gastric cancer cells in serum-free cell culture medium at a cell concentration of 2 × 10⁵/mL, 200. mu.L of the cell culture medium was aspirated and added to the upper chamber, and 550. mu.L of the cell culture medium containing serum was added to the lower chamber, and the resulting mixture was placed at 37 ℃ in 5% CO₂Culturing in an incubator for 24 h. The chamber was washed with PBS, fixed with formaldehyde, stained with 0.1% crystal violet, and the number of cell attacks, i.e. the number of cell membrane penetrations, was recorded under the mirror. The cell migration experiment is the same as the invasion experiment, and the Matergel coating step is not needed.

1.9 flow cytometry measurement of cell cycle

The treated cells are discarded with culture medium, washed twice with precooled PBS, discarded with PBS as much as possible, collected and washed for 2 times, fixed overnight with 70% alcohol at 4 ℃, washed twice with precooled PBS, added with 300 mu LPI and dyed for 30min in dark place, and analyzed for cell cycle rate by an up-flow cytometer.

1.10 statistical analysis

Statistical analysis was performed using GraphPad Prism 8 software, data are expressed as x ± s, comparisons of differences between groups were performed using analysis of variance, and differences of P <0.05 were statistically significant.

Example 2 Compounds 1-3-51 inhibit the expression of FOXM1 in gastric cancer cells

Chemical Synthesis method (He X L, Xiao Y C, Du W, et al. endogenous selective formulations of Ketone and Cyclic 1-Azadienes by Cascade amine-amine Catalysis [ J ]. Chemistry-A European Journal,2015. DOI: 10.1002/chem.201404550) Compound 1-3-51 was constructed as shown in FIG. 1A, and half inhibitory concentration (IC50) of 1-3-51 promoting gastric cancer apoptosis at 48 hours was detected by CCK-8 kit, and the results showed that IC50 of Compound 1-3-51 was 5.429. + -. 0.225. mu.M and 5.169. + -. 0.239. mu.M, respectively, in BGC and MKN45 cells (FIG. 1B). Then, the two gastric cancer cells were treated with DMSO, 2.5. mu.M and 5. mu.M compounds 1-3-15 respectively, and the expression of FOXM1 protein and mRNA in the gastric cancer cells was detected by using Western Blot and RT-qPCR using compounds 1-3-51, and the results showed that compounds 1-3-51 inhibit the expression of FOXM1 protein and mRNA in a concentration-dependent manner (FIG. 1C). Subsequently, the gastric cancer cells were treated with 1-3-515 μ M for 0h, 24h, and 48h, and thus showed the same time-dependent inhibition of FOXM1 protein and mRNA expression (fig. 1D).

Example 3 Compounds 1-3-51 inhibit expression of the downstream target genes Cyclin D1, MMP9 of FOXM1 in gastric cancer cells

The results of treating two kinds of gastric cancer cells including Cyclin D1 and MMP9 with DMSO, 2.5. mu.M and 5. mu.M respectively and detecting mRNA expression of FOXM1 downstream target genes Cyclin D1 and MMP9 by RT-qPCR revealed that compounds 1-3-51 inhibit the expression of Cyclin D1 and MMP9 mRNA in a concentration-dependent manner (FIG. 2A). Subsequently, the gastric cancer cells were treated with 1-3-515. mu.M compounds for 0h, 24h, and 48h, and the time-dependent inhibition of Cyclin D1 and MMP9 mRNA expression was also shown (FIG. 2B).

EXAMPLE 4 Compounds 1-3-51 affect gastric carcinoma cell cycle, inhibit cell migration and invasion

The above two gastric cancer cells were treated with DMSO, 2.5 μ M, and 5 μ M, respectively, and the effect of compound 1-3-51 on the gastric cancer cell cycle was examined by flow cytometry, and it was shown that compound 1-3-51 blocked the gastric cancer cells at the G1 stage in a concentration-dependent manner (fig. 3A). Then, after treating the above two kinds of gastric cancer cells with DMSO and 5. mu.M of the compound 1-3-51, the migration and invasion abilities of the gastric cancer cells were verified by Transwell experiments, and the results showed that the number of the gastric cancer cells treated with the compound 1-3-51 penetrated the cell chamber membrane was significantly reduced compared to the control group (FIG. 3B). The above results indicate that the compounds 1-3-51 can arrest gastric cancer cells at the G1 stage and inhibit gastric cancer cell migration and invasion.

Example 5 knock-down of FOXM1 significantly reduced the effect of compound 1-3-51 on gastric cancer cell proliferation, migration and invasion

To further verify whether compounds 1-3-51 exert anticancer effects by inhibiting FOXM1 expression, applicants further used an interference plasmid to knock down the expression of FOXM1 in cells. FOXM1 interference plasmid shFOXM1 and control plasmid shNC were transfected into BGC823 cells respectively, and Western Blot experiments confirmed that FOXM1 expression was successfully interfered (FIG. 4A). Subsequently, BGC823 cells transfected with shFOXM1 or shNC were treated with 5 μ M of compound 1-3-51, and after 48 hours, cell activity was examined by CCK8, which showed a significant decrease in cell activity after interfering with FOXM1, and a significant decrease in the apoptosis-promoting effect of compound 1-3-51 (fig. 4B). Transwell experimental results also showed that compound 1-3-51 had a significantly reduced ability to inhibit cell migration and invasion after interfering with FOXM1 (fig. 4C). Taken together, the results indicate that compounds 1-3-51 can exert anticancer effects by inhibiting FOXM 1.

As a result: IC50 of the compounds 1-3-51 after the compounds act on BGC823 cells and MKN45 cells for 48 hours are 5.429 +/-0.225 mu M and 5.169 +/-0.239 mu M respectively, so that mRNA expression of FOXM1 and downstream genes Cyclin D1 and MMP9 of the FOXM1 can be remarkably inhibited, and proliferation, invasion and migration of gastric cancer cells can be remarkably inhibited. Interfering with FOXM1 results in a decrease in the ability to inhibit proliferation, invasion and migration.