CN114105870A

CN114105870A - Anti-tumor compound, application and composition containing compound

Info

Publication number: CN114105870A
Application number: CN202111423094.XA
Authority: CN
Inventors: 师健友; 王金华
Original assignee: Sichuan Peoples Hospital of Sichuan Academy of Medical Sciences
Current assignee: Sichuan Peoples Hospital of Sichuan Academy of Medical Sciences
Priority date: 2021-11-26
Filing date: 2021-11-26
Publication date: 2022-03-01

Abstract

The invention relates to an anti-tumor compound, application and a composition containing the compound. The anti-tumor compound is a compound with the structural formula I shown in the specification, can be applied to preparation of medicines, particularly medicines for treating lung cancer, and can be prepared into conventional preparations such as oral preparations and injection preparations. Can also be used as composition with other active ingredients. The antitumor compound shown in the formula I can interact with skp2, shows a strong inhibition effect, has strong antitumor activity, can inhibit proliferation, migration and invasion of human non-small cell lung cancer cells, and has great medicinal potential.

Description

Anti-tumor compound, application and composition containing compound

Technical Field

The invention relates to an anti-tumor compound, in particular to an anti-lung cancer compound and pharmaceutical application thereof.

Background

Worldwide, lung cancer is one of the major neoplastic diseases causing death, and about 180 million people die of lung cancer in 2020 by statistics, while about 85% of them are refractory non-small cell lung cancers (NSCLC). The existing clinical medical research shows that the five-year survival probability of the non-small cell lung cancer patient is only 15.9 percent, and the non-small cell lung cancer patient is the worst tumor disease after medical intervention prognosis.

Skp2 is an F-Box protein, belonging to the SCF family, and is involved in the pathogenesis of various tumor diseases. The expression of Skp2 is generally considered as a gold diagnostic marker of NSCLC, the Skp2 has close relation with the proliferation, metastasis and apoptosis of a tumor cell cycle, and if the Skp2 gene can be inhibited, the proliferation and metastasis of the tumor cell can be hopefully prevented, so that the purpose of preventing and treating the non-small cell lung cancer is achieved.

Disclosure of Invention

The invention aims to: aiming at the problem of poor treatment effect of non-small cell lung cancer in the prior art, an anti-tumor compound is provided.

In order to achieve the purpose, the invention adopts the technical scheme that:

an anti-tumor compound, which is a compound with the structural formula I.

And the application of the compound with the structural formula I in preparing medicines.

Preferably, the medicament is a medicament for the treatment of lung cancer. More preferably, the medicament is a medicament for the treatment of non-small cell lung cancer.

Further, the medicament is a conventional pharmaceutical preparation, such as an oral preparation and an injection preparation.

Further, the oral preparation is tablet, capsule, granule, fat emulsion, microcapsule, dripping pill; preferably, the injection preparation is injection or powder injection.

The invention also provides a composition containing the compound with the structural formula I as one of active ingredients.

Preferably, the composition also contains other active ingredients for treating lung cancer.

Preferably, the composition contains pharmaceutically acceptable adjuvants.

Preferably, the auxiliary agent comprises at least one of a filler, a disintegrant, a lubricant, and a binder.

Preferably, the composition is a conventional pharmaceutical formulation, such as an oral formulation, an injectable formulation. Preferably, the oral preparation can be tablets, capsules, granules, fat emulsion, microcapsules and dropping pills. Preferably, the injection preparation can be injection or powder injection.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the antitumor compound can interact with skp2, shows strong inhibition effect, has strong antitumor activity, and especially has outstanding inhibition effect on non-small cell lung cancer.

2. The anti-tumor compound can inhibit the proliferation, migration and invasion of human non-small cell lung cancer cells, and can regulate various signal pathways, so that the lung cancer cells are blocked in the G0/G1 stage. In addition, the anti-tumor compound can also induce apoptosis of human non-small cell lung cancer cells through a mitochondrial pathway.

3. The antitumor compound has high activity, can show strong apoptosis induction effect and cell aging effect at extremely low dosage, and has great medicinal potential.

Drawings

FIG. 1 shows that AAA-237 binds to Skp2 and inhibits degradation of the Skp2 substrate. A.AAA-237. SPR detection showed that AAA-237 bound to μ M with a KD of 28.77 μ M, comparable to the positive control drug SZLP1-41 with a KD (40.28 μ M). C. cetsa experiment shows that AAA-237 improved the thermal stability of Skp2 compared to the control. Western blot results show that AAA-237 can inhibit expression of Skp2 in A549 and H1299 cells, increase expression of p21cip1 and p27Kip1, and is time-dependent.

FIG. 2 shows that AAA-237 inhibits A549 and H1299 cell proliferation dose-dependently and time-dependently. A.CCK8 detection result shows that AAA-237 can inhibit the proliferation of A549 and H1299 cells. EdU-DNA synthesis experiments showed that AAA-237 inhibited DNA synthesis in A549 and H1299 cells. Scale bar 100 μm. Aaa-237 inhibited colony formation of a549 and H1299 cells. Experiments were performed in 3 replicates and data are presented as mean ± SD, × P <0.05, × P <0.01, × P <0.005 and control.

FIG. 3 is an analysis of differential expression and enrichment of A549 cells after AAA-237 treatment. The volcano diagram of the differential expression result A (the up-regulated gene is red, the down-regulated gene is green, the non-regulated gene is blue (| log2FC | is not less than 1, and the P value is not more than 0.05), the heat map of the differential expression gene B, the GO enrichment analysis of the differential expression gene C, the KEGG pathway analysis of the differential expression gene D, the protein-protein interaction E, and the network is visualized by using Cytoscape 3.6.0.

FIG. 4 shows that AAA-237 inhibited the cell cycle at g0/g1 in A549 and H1299 cells. Among them, flow cytometry analysis showed that AAA-237 blocked the cell cycle at g0/g1 in a549 and H1299 cells. Western blot results show that AAA-237 regulates the expression of cell cycle associated proteins A549 and H1299 cells. Western blot results show that AAA-237 regulates the PI3K/Akt-FOXO1 signaling pathway. qrt-PCR results showed that AAA-237 increased the expression of p21 and p27 at the mRNA level. qRT-PCR results showed that mRNA levels of p21 and p27 were reduced in AAA-237 treated cells after siRNA silencing FOXO 1. Westernblot results show that p21 and p27 protein levels are reduced in AAA-237 treated cells after siRNA silencing of FOXO 1. The experiment was repeated 3 times, data are presented as mean ± SD,. + -. P <0.05,. + -. P <0.01 and control; # P <0.05, # P <0.01, vs. AAA-237 treatment group.

FIG. 5 shows that AAA-237 induced apoptosis of A549 and H1299 cells in a mitochondria-dependent manner. Among them, flow cytometry analysis showed that AAA-237 increased the apoptosis rate of a549 and H1299 cells. JC-1 staining results show that AAA-237 reduces mitochondrial membrane potential in A549 and H1299 cells. Scale bar 100 μm. C. Flow cytometry analysis of caspase3/7 activity showed that AAA-237 increased caspase3/7 activity in A549 and H1299 cells. Western blot results show that AAA-237 regulates the expression of apoptosis-related proteins in A549 and H1299 cells. The experiment was performed in triplicate.

FIG. 6 is AAA-237 induced senescence in A549 and H1299 cells. Among them, the A.SA-beta-Gal test results show that the long-term aging of A549 and H1299 cells can be induced by treating the AAA-237 with low concentration. Scale bar 100 μm. B. Immunofluorescence experiments show that long-term treatment with low concentration of AAA-237 can induce the expression of the aging marker gamma H2A.X in A549 and H1299 cells. Scale bar 100 μm. rt-PCR analysis showed that low concentrations of AAA-237 increased expression of SASP factors IL1A, IL6, CXCL1, CXCL8, MMP1, and MMP10 in a549 cells. Westernblot results show that long-term treatment with low concentrations of AAA-237 increased the expression of the senescence markers γ H2a.x and p16in a549 and H1299 cells. Cck8 assay results show that quercetin reduced the viability of a549 and H1299 cells after AAA-237 treatment. Schematic model of mechanism of AAA-237 inducing NSCLC cell senescence. Experiments were performed in 3 replicates and data are presented as mean ± SD, × P <0.05, × P <0.01, × P <0.005 and control.

FIG. 7 is a graph of AAA-237 inhibiting the growth of xenograft tumors by inducing apoptosis and senescence. Wherein, a. AAA-237 reduced tumor volume. Aaa-237 reduced tumor weight. Body weight during treatment with AAA-237. Relative organ weight after aaa-237 treatment. F. Immunohistochemical analysis showed that AAA-237 reduced Ki67 expression in tumor tissues. Scale bar 50 μm. Tunel staining test shows that AAA-237 can induce tumor tissue cell apoptosis. Scale bar 100 μm. Western blot results showed that AAA-237 decreased expression of Skp2 and Bcl-2, increased expression of p27, and cleavage of caspase3, caspase9, PARP, and Bax. The expression of senescence-associated proteins γ h2a.x and p16 was also up-regulated. Data are presented as mean ± SD, × P <0.01, × P <0.005, × P <0.001 and control.

Figure 8 is a dose and time dependent manner of inhibiting cell proliferation by the inhibitor AAA-237 in a549 and H1299 cells with Skp2 inhibitor AAA-237. A.CCK8 detection result shows that AAA-237 can inhibit the proliferation of A549 and H1299 cells. Morphological changes of cells after AAA-237 treatment. Scale bar 100 μm. Hoechst staining results show that AAA-237 can induce apoptosis and senescence in A549 and H1299 cells. Scale bar 100 μm. The experiment was performed in triplicate.

FIG. 9 shows that AAA-237 inhibited migration and invasion of A549 and H1299 cells. Among them, transwell assay showed that AAA-237 inhibited the migration and invasion of A549 and H1299 cells. Scale bar 100 μm. AAA-237 regulates EMT through the Skp2/Twist axis. the expression of twist, MMP7, n-cadherin and vimentin decreased, while the expression of e-cadherin increased. Migration and intrusion mechanism schematic model for aaa-237. Experiments were performed in 3 replicates and data are presented as mean ± SD, × P <0.05, × P <0.01, × P <0.005 and control.

FIG. 10 shows that AAA-237 inhibited the cell cycle at g0/g1 in A549 and H1299 cells. Among them, flow cytometry analysis showed that AAA-237 blocked the cell cycle at g0/g1 in a549 and H1299 cells. B. Immunofluorescence experiments showed that AAA-237 increased FOXO1 expression in the nuclei of a549 and H1299 cells. Scale bar 100 μm. Experiments were performed in 3 replicates and data are presented as mean ± SD, × P <0.05, × P <0.01, × P <0.005 and control.

Figure 11 is that SASP-associated cytokines promote recruitment and activation of immune cells. Heat map of SASP-associated cytokine expression and immunoinfiltration levels in lung cancer. IL-1A is positively correlated with the level of infiltration of CD8+ T cells, monocytes and macrophages. IL-6 was positively correlated with the level of infiltration of CD8+ T cells, monocytes and macrophages. Mmp1 was positively correlated with the level of infiltration of CD8+ T cells, monocytes and macrophages. Mmp10 was positively correlated with the level of infiltration of CD8+ T cells, monocytes and macrophages.

FIG. 12 is a diagram showing the mechanism of activity of the compounds of the present invention.

Figure 13 is compound nmr spectrum.

FIG. 14 is a high resolution mass spectrum (HR-MS (ESI) m/z: calcd for C) of compound AAA-237₁₇H₂₁N₄O₂{[M+H]} +313.1665, found 313.1666), the exact molecular weight of the AAA-237 compound was verified by mass spectrometry data.

FIG. 15 is a high resolution mass spectrum of compound AAA-237.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Statistical analysis shows that: data are presented as mean ± Standard Deviation (SD) and represent at least three independent experiments. One-way anova was used to calculate the differences between the different study groups. p <0.05 is considered statistically significant.

EXAMPLE 1 Synthesis of Compounds

(1) Nicotinic acid is used as a raw material, 1 percent concentrated sulfuric acid (98 weight percent) is added dropwise in a methanol solvent under the stirring state, and the mixture is heated and refluxed for 3 hours at 85 ℃. NaHCO is added after the reaction is completed₃Adjusting the pH value to be neutral to obtain a methanol intermediate. Hydrazine hydrate is added into the intermediate, and the mixture is heated and refluxed for 2 hours at 85 ℃. After the reaction, ice water was added to the reaction solution to precipitate a precipitate. Filtering and precipitating to obtain a solid product. Wherein the molar ratio of the nicotinic acid to the hydrazine hydrate is 1: 4.

(2) Adding diluted hydrochloric acid into the compound A obtained in the step (1) and 4- (diethylamino) -2-hydroxybenzaldehyde in a methanol solvent at 0 ℃ and stirring for 3 minutes to generate a large amount of precipitate. Heating and refluxing for 3 hours at 85 ℃ to obtain a solid, washing with methanol, and drying to obtain a final product. Wherein the molar ratio of the compound A to the 4- (diethylamino) -2-hydroxybenzaldehyde is 1: 1. The reaction equation is as follows:

the synthesized compound AAA-237 is detected by nuclear magnetic resonance hydrogen spectrum, and the characteristic spectrum is shown in figure 13. Subjecting the synthesized product to high resolution mass spectrometry, and obtaining high resolution mass spectrogram (HR-MS (ESI) m/z: calcd for C) of compound AAA-237 by using the characteristic spectrogram as shown in FIGS. 14 and 15₁₇H₂₁N₄O₂{[M+H]} +313.1665, found 313.1666), the exact molecular weight of the AAA-237 compound was verified by mass spectrometry data. And determining the synthesized compound as a target compound AAA-237 by nuclear magnetic and mass spectrometry analysis. Thus, example 1 obtained a compound AAA-237 having a methylene hydrazide skeleton by organic synthesis, which was synthesized for the first time.

EXAMPLE 2 in order to investigate the antitumor activity and mechanism of the above-mentioned compounds, the present invention adopted the following experimental method

2.1. Cell culture

Human NSCLC cell lines a549 and H1299 were purchased from guangzhou zhenniao biotechnology limited (guangdong, china). Cells in the presence of 5 wt% CO₂Was cultured at 37 ℃ in a humidified incubator of (1), and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10 wt.% FBS and antibiotics (0.1. mu.g/mL penicillin and 0.1. mu.g/mL streptomycin).

2.2 surface plasmon resonance analysis

For Surface Plasmon Resonance (SPR) measurements, the interaction between Skp2 and AAA-237 or SZLP1-41 was detected using the Biacore T200 system (GE Healthcare Life Sciences, Marlborough, MA, USA). Briefly, Skp2 protein was immobilized on an active CM5 chip (GE Healthcare Life Sciences). Different concentrations of AAA-237 and SZLP1-41 were dissolved in running buffer, passed through the chip at specific flow rates, and KD values were measured from the generated affinity curves

2.3 Whole cell thermal Displacement analysis

For whole cell thermal displacement assay, a549 and H1299 cells were plated at 4 × 10⁵cells/mL were plated at a density of 10cm dishes. After 24 hours, cells were washed with ice-cold PBS and centrifuged. The cell pellet was resuspended in 300 μ L RIPA lysis buffer and the complete protease inhibitor mixture was added and incubated on ice for 30 min. The cell lysate was centrifuged at 12,000g for 15 minutes at 4 ℃ and the supernatant was collected. 50 μ M AAA-237 or vector control (DMSO) was incubated with A549 and H1299 cell lysate supernatants for 1 hour at room temperature. The respective lysates were divided into smaller (50 μ L) aliquots and heated separately at different temperatures (50, 55, 60, 65, 70, 75 ℃) for 5 minutes, followed by cooling at room temperature for 3 minutes. The heated lysate was centrifuged at 20,000g for 20 minutes at 4 ℃ to separate the soluble fraction from the precipitate. The supernatant was transferred to a new microcentrifuge tube and analyzed by SDS-PAGE followed by western blot analysis against Skp 2.

For whole cell thermal displacement assay (CETSA), A549 and H1299 cells were seeded at a density of 4X 105 cells/mL in 10cm dishes. After 24 hours, cells were washed with ice-cold PBS and centrifuged. The cell pellet was resuspended in 300 μ LRIPA lysis buffer and the complete protease inhibitor mixture was added and incubated on ice for 30 minutes. The cell lysate was centrifuged at 12,000g for 15 minutes at 4 ℃ and the supernatant was collected. 50 μ M AAA-237 or vector control (DMSO) was incubated with A549 and H1299 cell lysate supernatants for 1 hour at room temperature. The respective lysates were divided into smaller (50 μ L) aliquots and heated separately at different temperatures (50, 55, 60, 65, 70, 75 ℃) for 5 minutes, followed by cooling at room temperature for 3 minutes. The heated lysate was centrifuged at 20,000g for 20 minutes at 4 ℃ to separate the soluble fraction from the precipitate. The supernatant was transferred to a new microcentrifuge tube and analyzed by SDS-PAGE followed by western blot analysis against Skp 2.

2.4 cell proliferation assay

The cell proliferation rate was measured using CCK-8Kit (Beyotime, China) according to the manufacturer's instructions. Briefly, cells were plated at 3X 10 per well³Was inoculated in a 96-well plate, cultured for 24 hours, and then treated with AAA-237 for 24, 48, and 72 hours. CCK-8 was added and the plates were further incubated for 1 hour. A450 values were measured using a SpectraMaxM5 plate reader (Molecular Devices). IC50 values were calculated using GraphPadPrism7 Software (graphpad Software inc., San Diego, California, USA).

2.5Edu DNA Synthesis analysis

DNA synthesis was evaluated using the EdUApollo567 in vitro imaging kit (RiboBio, guangzhou, china) according to the manufacturer's instructions. Cells were plated at 5X 10 per well³Was inoculated in a 96-well plate, cultured for 24 hours, and then treated with AAA-237 at a concentration of 0.3, 1 or 3. mu.M for 48 hours. EdU was added at a concentration of 50. mu.M and incubated at 37 ℃ for 2 hours. Then, the cells were fixed in 4% paraformaldehyde for 30 minutes, permeabilized with 0.5% Trixon-X100 for 10 minutes, and then stained with 10. mu. MApollo567 for 30 minutes. The cells were then counterstained with Hoechst33342 for 30 minutes and photographed by fluorescence microscopy (Nikon Eclipse Ti-U).

2.6 Soft agar colony formation assay

Colony formation assays were used to determine the tumorigenicity of a549 and H1299 cells. To a 6-well plate, 2mL of 0.7% agar complete medium solution was added as the bottom layer, followed by 1mL of 0.35% agar complete medium solution containing 3,000 cells, with 0.3, 1, or 3 μ M AAA-237 added as the top layer. At 37 ℃ and 5% CO₂After two weeks of incubation, colonies were stained with MTT (3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2-H-tetrazolium bromide) solution (200. mu.L/well), photographed and counted.

2.7AnnexinV-FITC/PI staining flow cytometer analysis of apoptosis

The apoptosis rate was determined by flow cytometry using the Trans Detect Annexin V-FITC/PI apoptosis detection kit (Trans Gen, Beijing, China) according to the manufacturer's instructions. The cells were seeded in 6cm dishes and cultured for 24 hours, and then treated with AAA-237 at a concentration of 0.3, 1 or 3. mu.M for 48 hours. After harvest, cells were incubated with annexin V-FITC and PI for 15 min at room temperature. Flow cytometry was performed using BD Accuri C6(San Diego, CA, USA) to determine apoptosis and data were analyzed by FlowJov10 software.

2.8 mitochondrial Membrane potential analysis (MMP,. DELTA.. psi.m)

Changes in MMPs were measured using 5,5',6,6' -tetrachloro-1, 1',3,3' -tetraethylbenzimidazolocyanine iodide (JC-1) staining. Cells were seeded in 6cm dishes, cultured for 24 hours, and treated with AAA-237 at a concentration of 0.3, 1 or 3. mu.M for 48 hours. The cells were then incubated with JC-1 at a final concentration of 10. mu.g/mL for 20 min, washed 3 times with PBS and photographed under a fluorescence microscope (Nikon eclipse Ti-U).

2.9caspase3/7 Activity assay

caspase3/7 real-time live cell fluorescence detection kit (Key Gen Bio TECH, Nanjing, China) is used for detecting caspase3/7 activity. Cells were seeded in 6cm dishes and cultured for 24 hours, then treated with AAA-237 at a concentration of 0.3 or 3. mu.M for 48 hours. After harvest, cells were incubated with caspase3/7 test solution for 30 minutes. Flow cytometry was performed on BDAccuriC6(San Diego, Calif., USA) to measure caspase3/7 activity and the data were analyzed by FlowJov10 software.

2.10RNA sequencing

A549 cells were seeded in a 6cm dish, cultured for 24 hours, and then treated with AAA-237 at a concentration of 1. mu.M for 48 hours. Cells were lysed in TRIzol reagent (Life Technologies, USA) and sent to Novogene corporation (Beijing, China) for second generation sequencing. More detailed information is provided in the supplemental material.

2.11 RNA extraction and Gene expression detection by qRT-PCR

The cells were seeded in a 6cm petri dish, cultured for 24 hours, then treated with AAA-237 at a concentration of 0.3. mu.M, 1. mu.M, 3. mu.M for 48 hours, and then lysed in TRIzol. After mixing with 1/5 volumes of chloroform, centrifugation was carried out at 12,000g for 15 minutes, the supernatants were transferred to new tubes, and an equal volume of isopropanol was added to each supernatant and gently mixed. After incubation at room temperature for 30 minutes, the mixture was centrifuged at 12,000g for 15 minutes. The precipitate was washed once with 75% ethanol and dissolved in RNase-free water at the appropriate volume. After RNA quantification, Mon Script was used according to the manufacturer's instructions^TM5XRTIII Synthesis of cDNA in a unified cocktail (Monad Biotech, Wuhan, China). Quantitative real-time RT-PCR (qRT-PCR) was performed using 2XTaq Pro Universal SYBR qPCR mastermix (Vazyme Biotech, Nanjing, China). Primers are listed in table 1, GAPDH was used as an internal control.

2.12 immunofluorescence

Cells were seeded in 96-well plates, cultured for 24 hours, and then treated with AAA-237 at a concentration of 0.3, 1, or 3 μ M for 48 hours. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS and blocked with 5% BSA (w/v) and 0.3% Triton X-100(v/v) for 1 h at room temperature. Cells were then incubated overnight at 4 ℃ with the indicated primary antibody diluted in PBS containing 1% BSA (w/v). Cells were washed 3 times with PBS and incubated with the corresponding fluorescent secondary antibody for 2h at room temperature. After washing the cells 3 times with PBS, DNA was stained with 10. mu.g/mL Hoechst33342 for 30 minutes, the cells were washed again with PBS and photographed under a fluorescence microscope (Nikon Eclipse Ti-U).

Wherein the primary antibody is: skp2, Twist, MMP7, E-cadherin, N-cadherin, Vimentin, CDK4, Phospho-CDK4, (Thr172), Cyclin D1, CDK2, Phospho-CDK2, (Thr160), Cyclin E1, p27, p21, p15, PI3K p85, p-PI3K p85, (Tyr458), Akt, p-Akt (Ser473), FOXO1, Histone 3, PARP, clear-caspase 3, clear-caspase 9, Bcl 2, Bax, gamma H2A.X, pl6, etc. are diluted by a factor of 1: 1000; beta-actin was diluted 1:2000 fold and Ki67 was diluted 1:200 fold.

2.13 Western blotting (WT)

Cells were seeded in 6cm dishes and cultured for 24 hours, followed by treatment with AAA-237 at concentrations of 0.3, 1 or 3. mu.M for 24, 48 and 72 hours. Cells were harvested and lysed with RIPA lysis buffer (Applygen, beijing, china) for 30 min at 4 ℃ to extract total protein. The BCA method (Beyotime, Guangzhou, China) was used to measure protein concentration. Equal amounts of protein were loaded and separated by 10% SDS-PAGE, then transferred to PVDF membrane (Millipore, Billerica, MA). Membranes were blocked in 5% skim milk for 2 hours at room temperature, incubated with primary antibody (as in 2.12) overnight at 4 ℃, and then with goat anti-mouse or anti-rabbit HRP conjugated secondary antibody (CWBIO, beijing, china). Immunoreactive bands were visualized using ECL western blot kit (CWBIO, beijing, china).

2.14 senescence-associated beta-gal assay

A senescence assay kit (Bio Vision inc., Milpitas, CA, USA) was used to detect senescence-associated β -galactosidase (SA- β -Gal) activity. The cells were seeded in a 12cm dish, cultured for 24 hours, and then treated with AAA-237 at a concentration of 0.3. mu.M for 1, 3, or 7 days. Cells were washed twice with PBS, fixed for 15 min, incubated overnight in freshly prepared staining solution, and then examined microscopically (Nikon Eclipse Ti-U).

2.15 in vivo antitumor Activity

Six-week-old female BALB/c-nu nude mice (Beijing Wittal river laboratory animal science and technology Co., Ltd., Beijing, China), weighing 15-18 g, were housed in a barrier facility with 12 hours light/dark cycle. All experimental procedures were performed according to the guidelines for animal care and use of the institute of medicine, and the national institutes of health, both of China and Beijing cooperative medical instituteAnimal care and institutional guidelines for use were tested (publication No. 85-23, revised 1985). A549 cells (1X 10 per mouse)⁷Individual cells) were implanted subcutaneously into the right side of the mice. After 14 days, when tumor volume increased to around 100mm3, mice were randomized into four groups (n-6) and treated with vehicle, paclitaxel (20mg/kg, ip, twice weekly for two weeks), low dose AAA-237(15mg/kg, ig, once daily for 2 weeks) or high dose AAA-237(45mg/kg, ig, once daily for 2 weeks). Tumor volume was measured using a vernier caliper and calculated according to the modified ellipsoid formula: tumor volume (mm3) ═ length × width 2 × 0.5. At the end of the experiment, all mice were euthanized, tumor tissue and other organs were taken, weighed and photographed.

Statistical analysis: data are presented as mean ± Standard Deviation (SD) and represent at least three independent experiments. One-way anova was used to calculate the differences between the different study groups. p <0.05, statistically significant.

Results of mechanism experiment of antitumor active agent of example 3 Compound

1 AAA-237 binds to Skp2 and inhibits degradation of Skp2 substrates

The test results confirmed Skp2 to be the target of AAA-237 in vitro by performing Surface Plasmon Resonance (SPR) assays. By surface plasmon resonance analysis. The specific method is as follows, for Surface Plasmon Resonance (SPR) measurements, the interaction between Skp2 and AAA-237 or SZLP1-41 was detected using the Biacore T200 system (GE Healthcare Life Sciences, Marlborough, MA, USA). The Skp2 protein was immobilized on an active CM5 chip (GE Healthcare Life Sciences). Then, AAA-237 and SZLP1-41 were dissolved in running buffer at different concentrations, passed through the chip at specific flow rates, and KD values were measured from the generated affinity curves.

The test results showed that AAA-237 bound Skp2 with a KD of 28.77. mu.M and the positive control drug SZLP1-41 with a KD of 40.28. mu.M. It can be seen that AAA-237 binds to Skp2 and inhibits degradation of the Skp2 substrate. The result is shown as B in FIG. 1.

Whether Skp2 is the endogenous target of AAA-237 in the cellular environment was investigated by a CETSA assay using the whole cell thermal shift assay (CETSA), which is based on the observation that the melting curve of a particular protein can be generated in cell lysates, resulting in a significant change in the melting temperature (Tm) of the drug AAA-237 when bound to the protein. The experiment was performed with reference to the 2.3 whole cell thermal displacement analysis in example 2.

The results of the experiment show that AAA-237 increased the thermal stability of Skp2 relative to the control, indicating that AAA-237 binds directly to intracellular Skp2, as shown in FIG. 1C.

The potential effect of AAA-237 on Skp2 protein expression was investigated by Western immunoblotting (WB) method, with specific reference to the 2.13 Western blotting method in example 2. Experimental results show that AAA-237 can significantly inhibit expression of Skp2 in NSCLC cells in a time-dependent manner, as shown by D in fig. 1. The most well-known substrates in Skp2 are the cell cycle inhibitors p21Cip1 and p27Kip1, Skp2 is able to induce ubiquitination of p21Cip1 and p27Kip1, leading to degradation of these proteins. The experimental results show that AAA-237 incubation increased the expression levels of p21Cip1 and p27Kip1 in a549 and H1299 cells, as shown in D in fig. 1. This indicates that AAA-237 binds to Skp2, thus inhibiting expression of Skp2 and degradation of p21Cip1 and p27Kip 1.

2 AAA-237 inhibits NSCLC proliferation

To determine the effect of AAA-237 on NSCLC cell viability and proliferation, CCK8 experiments were performed and changes in cell morphology were imaged, with reference to the 2.1CCK8 proliferation assay in example 2.

The experimental results showed that AAA-237 inhibited proliferation of a549 and H1299 cells, and was dose and time dependent, as shown in a in fig. 2. The AAA-237 half inhibitory concentration of A549 was 3. mu.M at 24h, 2.5. mu.M at 48h, and 0.7. mu.M at 72 h. AAA-237 of H1299 was 3.9. mu.M at 24H, 1.8. mu.M at 48H, and 1.1. mu.M at 72H, as shown in A in FIG. 8. The morphology of a549 and H1299 cells after AAA-237 treatment was dose-dependent, as shown in fig. 8B.

The effect of inhibiting cell migration was tested by reference to the 2.13 western blot assay of example 2. Western blot results show that AAA-237 treated 24h inhibited the endothelial-mesenchymal transition process by modulating the expression of Twist, thereby inhibiting the metastasis of NSCLC cells (as shown in C-D in FIG. 9).

The test for whether AAA-237 has an effect on cellular DNA synthesis was evaluated with reference to the 2.5Edu-DNA synthesis assay in example 2. The results show that after AAA-237 treatment, DNA synthesis was significantly inhibited in both a549 and H1299 cells, and was dose-dependent, with the results shown in fig. 2, B.

The effect of AAA-237 on the colony formation of NSCLC cells was evaluated with reference to the 2.6 soft agar colony formation assay in example 2. The results are shown in figure 2, C, where AAA-237 inhibited both a549 and HA5299 cell colony formation in a dose-dependent manner. AAA-237 was shown to inhibit growth of NSCLC cells in vitro. Meanwhile, AAA-237 treatment for 24H was also found to significantly inhibit migration and invasion of A549 and H1299 cells, and was dose-dependent, with the results shown in A-B in FIG. 9.

AAA-237 cell inhibition was studied by reference to the Western blot 2.13 in example 2. The results show that AAA-237 treated for 24h inhibited the endothelial-mesenchymal transition process by modulating the expression of Twist, thereby inhibiting the metastasis of NSCLC cells (shown in fig. 9C-D).

Differential gene expression and enrichment analysis of A549 cells treated by 3 AAA-237

To investigate the mechanism of effect of AAA-237 on NSCLC, the differentially expressed genes of A549 cells treated with 1. mu. MAAA-237 for 48h and control cells were examined by RNA-seq method with reference to 2.10RNA sequencing in example 2.

RNA-seq results showed that PI3K/Akt and FOXO signaling pathways were involved in NSCLC cell regulation following AAA-237 treatment (shown in FIG. 3, E). Compared with the gene expression of the cells of the control group, 757 up-regulated genes and 1242 down-regulated genes were found in the AAA-237 treated cells (| log2FC | ≧ 1 and p ≦ 0.05, shown in FIG. 3A-B). The results show that AAA-237 decreased the phosphorylation levels of PI3K and Akt in a549 and H1299 cells. Western blot and immunofluorescence analysis were performed with reference to 2.12 immunofluorescence and 2.13 Western blot in example 2. The results indicated that the expression of nuclear FOXO1, representing transcriptional activity, was up-regulated (shown as C in fig. 4, and B in fig. 10). This increased the expression of the downstream genes p21Cip1 and p27Kip1 at the transcriptional level and induced cell cycle arrest (shown in FIG. 4D).

Gene Ontology (GO) enrichment analysis of these Differentially Expressed Genes (DEGs) showed that the genes enriched in the Biological Process (BP) class were involved in regulation of the ERK1 and ERK2 cascades, regulation of signaling receptor activity, and response to lipopolysaccharide (shown in C in fig. 3). In the Cellular Component (CC) class, DEGs are involved in the protein extracellular matrix, MCM complex and chromosome center (shown in C in fig. 3). In the Molecular Function (MF) class, deg is involved in hijacked molecular function, viral receptor activity and carboxylic acid binding (shown in fig. 3, C). The results of the KEGG pathway enrichment analysis showed that this gene was enriched in pathways associated with cancer, cellular senescence, DNA replication, cell cycle and FOXO signaling pathways (shown in D-E in fig. 3).

The results show that AAA-237 decreased the phosphorylation levels of PI3K and Akt in a549 and H1299 cells. Western blot and immunofluorescence analysis were performed with reference to 2.12 immunofluorescence and 2.13 Western blot in example 2. The results showed that up-regulation of nuclear FOXO1 expression, representing transcriptional activity (shown in fig. 4C, fig. 10B), increased the expression of downstream genes p21Cip1 and p27Kip1 at the transcriptional level and induced cell cycle arrest (shown in fig. 4D). Furthermore, the levels of p21 and p27 in NSCLC cells treated with AAA-237 after miRNA silencing FOXO1 were significantly reduced at both mRNA and protein levels as expected (shown in fig. 4 as E-F).

Furthermore, the levels of p21 and p27 in NSCLC cells treated with AAA-237 after miRNA silencing FOXO1 were significantly reduced at both mRNA and protein levels as expected (as shown in E-F in fig. 4). In conclusion, AAA-237 can inhibit PI3K/Akt signal pathway in A549 and H1299 cells, enhance the activity of transcription factor FOXO1, regulate the expression of p21Cip1 and p27Kip1, and promote the block of cell cycle at G0/G1 in NSCLC cells.

4 AAA-237 inhibits cell cycle at G0/G1 checkpoint by modulating Skp2-Cip/Kip and PI3K/Akt-FOXO1 signaling pathways

4.1 cell cycle analysis was performed in A549 and H1299 cells.

Reference example 2 Annexin V-FITC/PI staining flow cytometer analysis of apoptosis assay Annexin V-FITC/PI staining apoptosis assay: the apoptosis rate was determined by flow cytometry using the Trans Detect Annexin V-FITC/PI apoptosis detection kit (Trans Gen, Beijing, China) according to the manufacturer's instructions. The cells were seeded in 6cm dishes and cultured for 24 hours, and then treated with AAA-237 at a concentration of 0.3, 1 or 3. mu.M for 48 hours. After harvest, cells were incubated with annexin V-FITC and PI for 15 min at room temperature. Flow cytometry was performed using BD Accuri C6(San Diego, CA, USA) to determine apoptosis and data were analyzed by FlowJov10 software.

The results show that AAA-237 blocked the cell cycle in a dose and time dependent manner (as shown in A in FIG. 4, and as shown in A in FIG. 10).

4.2 to investigate the potential mechanism of AAA-237, the expression levels of G0/G1 cell cycle checkpoint-related proteins in AAA-237 treated A549 and H1299 cells were examined using the Westernblot assay, referred to the 2.13 Western blot assay in example 2.

Expression levels of CDK2, p-CDK2, cyclinE e1, CDK4, p-CDK4 and cyclinD were reduced in AAA-237 incubated a549 and H1299 cells (as shown in fig. 4B). The role of the Cip/Kip family of cyclin-dependent kinase inhibitors (CKI), such as p21Cip1 and p27Kip1, in the negative regulation of the cell cycle has been well documented. Skp2 plays a key role in mediating CKIs ubiquitin dependence in degradation. The results show that the expression of p21Cip1 and p27Kip1 in a549 and H1299 cells increases after treatment with Skp2 inhibitor AAA-237, and is dose and time dependent (as shown in D in fig. 1). This indicates that AAA-237 can modulate the Skp2-Cip/Kip pathway, thereby inhibiting the CDK2/cyclin E and CDK4/cyclin D complex in NSCLC cells.

To investigate whether AAA-237 could induce apoptosis in NSCLC cells, A549 and H1299 were stained with Hoechst33342, according to 2.12 immunofluorescence assay in example 2. Following treatment with AAA-237, nuclei were concentrated and vacuoles appeared, indicating that these cells were still in the early stages of apoptosis (shown as C in FIG. 8). Referring to the test method of example 2, annexin V-EGFP/PI double staining was used to analyze apoptosis by flow cytometry. As a result, AAA-237 induced apoptosis of A549 and H1299 cells in a dose-and time-dependent manner. 5A) In that respect

To further investigate the mechanism of AAA-237 inducing apoptosis, changes in mitochondrial membrane potential (MMP,. DELTA.. psi.m) and caspase-3/7 activity were determined by reference to the 2.8 mitochondrial membrane potential analysis (MMP,. DELTA.. psi.m) method in example 2.

The results showed that MMP was detected by JC-1 staining 48H after treatment of A549 and H1299 cells with AAA-237. In A549 and H1299 cells, the ratio of JC-1 monomer/JC-1 polymer increases with increasing concentration of AAA-237 (as shown by B in FIG. 5).

Reference example 2.9caspase3/7 activity assay. Caspase-3/7 activity was detected by flow cytometry and the results showed that AAA-237 treatment increased caspase-3/7 activity. As shown at C in fig. 5).

The expression of apoptosis-related proteins was examined by reference to the 2.13 western blot assay in example 2. Wherein the primary antibody is: skp2, Twist, MMP7, E-cadherin, N-cadherin, Vimentin, CDK4, Phospho-CDK4, (Thr172), Cyclin D1, CDK2, Phospho-CDK2, (Thr160), Cyclin E1, p27, p21, p15, PI3K p85, p-PI3K p85, (Tyr458), Akt, p-Akt (Ser473), FOXO1, Histone 3, PARP, clear-caspase 3, clear-caspase 9, Bcl 2, Bax, gamma H2A.X, pl6, etc. diluted 1:1000 times; beta-actin was diluted 1:2000 fold and Ki67 was diluted 1:200 fold.

Western blotting for detecting the expression of apoptosis-related protein, and the test result shows that after AAA-237 treats A549 and H1299 cells, the levels of PARP, semi-caspase 3, semi-caspase 9 and Bax are increased, and the expression of PARP and Bcl-2 is reduced. Shown as D in fig. 5). This evidence supports the hypothesis that AAA-237 induces apoptosis by up-regulating the pro-apoptotic protein Bax and down-regulating the anti-apoptotic protein Bcl-2.

6 AAA-237 induces senescence in NSCLC cells

The AAA-237 induced NSCLC cell senescence assay was performed with reference to the 2.14 senescence-associated β -gal assay in example 2. Some NSCLC cells showed increased nuclear morphology following AAA-237 treatment (shown as C in FIG. 8). To investigate whether AAA-237 was able to induce cellular senescence, SA- β -Gal activity (senescence cell marker) was measured. The number of SA- β -gal positive cells increased with long-term treatment of a549 and H1299 cells with low concentration of AAA-237 for 3 days and 7 days compared to the control group (shown as a in fig. 6). The experimental results show that Skp2 is also involved in DNA damage repair mechanisms.

6.2 the study found that AAA-237 inhibited expression of Skp2, resulting in DNA damage.

Detection was performed by reference to the immunofluorescence assay method 2.12 of example 2. Wherein the primary antibody is: skp2, Twist, MMP7, E-cadherin, N-cadherin, Vimentin, CDK4, Phospho-CDK4, (Thr172), Cyclin D1, CDK2, Phospho-CDK2, (Thr160), Cyclin E1, p27, p21, p15, PI3K p85, p-PI3K p85, (Tyr458), Akt, p-Akt (Ser473), FOXO1, Histone 3, PARP, clear-caspase 3, clear-caspase 9, Bcl 2, Bax, gamma H2A.X, pl6, etc. diluted 1:1000 times; beta-actin was diluted 1:2000 fold and Ki67 was diluted 1:200 fold.

Immunofluorescence results showed that phosphorylation levels of H2AX (γ H2AX) were increased in a549 and H1299 cells in long-term AAA-237 treated H1299 cells, which is an early marker of DNA damage (shown as B in fig. 6). Expression of the senescence markers γ H2AX and p16 was also increased in a549 and H1299 cells after AAA-237 treatment (shown in C in fig. 6).

6.3 RNA extraction and gene expression detection by qRT-PCR.

RNA extraction and gene expression detection analysis were performed by the method described for RNA extraction and gene expression detection by qRT-PCR in reference example 2.11. The primer sequences used during the qRT-PCR analysis are shown in the following table.

TABLE 1 primer sequences

PCR analysis showed a significant increase in the levels of SASP-associated cytokines (IL-1A, IL-6, CXCL1, CXCL8, MMP1 and MMP10) in a549 and H1299 cells following long-term exposure to AAA-237 (shown in D in figure 6). As described above, AAA-237 induces senescence of A549 and H1299 cells by DNA damage and displays the SASP phenotype.

Senescent cells in tumors have immune-activating properties through secretion of SASP-associated cytokines that promote recruitment and activation of immune cells into the tumor microenvironment. Therefore, we evaluated the correlation of SASP-associated cytokine expression with the level of immunoinfiltration in lung cancer by using the TIMER 2.0 database.

The heat map shows that the SASP-associated cytokines IL-1A, IL-6, CXCL1, MMP1 and MMP10 are associated with immunoinfiltration (shown in a in figure 11). Expression of IL-1A, IL-6, MMP1, and MMP10 was positively correlated with the level of infiltration of CD8+ T cells, monocytes, and macrophages (shown as B-E in FIG. 11).

However, accumulation of senescent cells in the tumor microenvironment will primarily lead to tumor recurrence and metastasis. Quercetin is a selective aging drug, and can eliminate aging cells; thus, the effect of quercetin on the viability of AAA-237-induced senescent a549 and H1299 cells was examined. As shown in fig. 6, E, quercetin significantly reduced the viability of AAA-237 treated a549 and H1299 cells, particularly at high concentrations (60 μ M).

Taken together, the results of the study indicate that low concentrations of AAA-237 treatment can induce senescence by DNA damage. Senescent cells secrete SASP-associated cytokines IL-1A, IL-6, CXCL1, CXCL8, MMP1 and MMP10, which recruit and activate CD8+ T cells, monocytes and macrophages. The aging drug quercetin can selectively eliminate aged a549 and H1299 cells. As shown at F in fig. 6.

7. In vivo antitumor Activity test

To confirm that AAA-237 is active in anti-NSCLC, xenograft models were established in vaccinated nude mice, which were then treated with 15 or 45mg/kg AAA-237 daily for 14 days or 20mg/kg paclitaxel twice a week for 14 consecutive days. Specific animal test methods were performed as described with reference to 2.15 in vivo antitumor activity in example 2.

The results of in vivo antitumor experiments show that: intraperitoneal injection of AAA-237, shown in FIGS. 7A-C, significantly reduced tumor volume and weight. Paclitaxel group showed Tumor Growth Inhibition (TGI), low AAA-237 and high AAA-237, 48%, 55% and 64%, respectively (Table 2). The results indicate that AAA-237 may inhibit dose-dependent growth in a tumor-like manner. No significant weight loss or abnormal behavior was observed in nude mice during AAA-237 treatment (shown as D in FIG. 7). In addition, in the AAA-237 treated group, the color and texture of important organs such as heart, liver, spleen, lung and kidney were not significantly changed, nor was there a significant difference in relative organ weight (as shown in E in FIG. 7) compared to the control group. No significant systemic toxicity was observed in the physiological blood test, and the results of the physiological blood test in AAA-237 treated mice are shown in table 3, with data expressed as mean ± standard deviation, n ═ 6. P <0.05, P <0.01vs. control.

TABLE 2 antitumor Activity of AAA-237 against Lung cancer A549 cells in xenograft mouse model

Grouping	Dosage/mg. kg^-1	Quantity (end/start)	Tumor weight/g	TGI(％)
					Blank control group	-	6/6	0.39±0.09	-
Tax	20	6/6	0.20±0.06**	48
					AAA-237 Low dose	15	6/6	0.18±0.05***	55
AAA-237 high dose	45	6/6	0.14±0.03****	64

n 6, mean ± SD, P <0.01, P <0.001, P <0.0001 vs. blank. TGI: tumor (volume) inhibition rate.

TABLE 3 Biochemical examination of mouse blood

Abbreviations in the tables are as follows. WBC: white blood cell count, 10⁹L; RBC: erythrocyte count, 10¹²L; PLT: platelets, 10⁹L; HGB: hemoglobin, g/L; HCT: hematocrit,%; MCV: mean red blood cell volume, fL; MCH: mean corpuscular hemoglobin amount, pg; MCHC: mean hemoglobin concentration of red blood cells, g/L; RDW: width of red blood cell volume distribution,%; PCT: platelet volume percent,%; MPV: platelet mean volume, fL; PDW: platelet distribution width,%; LYM, lymphocyte count, 10⁹L; MON: monocyte count, 10⁹L; NEUT: neutrophil count, 10⁹L; LYM%: lymphocyte fraction,%; MON%: monocyte fraction,%; NEUT%: neutrophil ratio,%; EOS%: eosinophil ratio,%. n is 6, mean ± sd, # p<0.05，**p<0.01。

To investigate the anti-tumor mechanism of AAA-237 in vivo, Ki-67 immunohistochemistry and TUNEL (tdt-mediated dUTP nick end labeling) fluorescent staining was performed in tumor tissues. The fluorescent staining test and the 2.13 western blot test were performed by referring to the immunofluorescence assay method of 2.12 in example 2.

Immunohistochemistry results show that Ki-67 expression is reduced in tumor tissues after AAA-237 treatment. Ki-67 is a proliferation-associated antigen involved in mitosis and essential for cell proliferation. This suggests that AAA-237 inhibits the proliferation of cancer cells in vivo (shown as F in FIG. 7). TUNEL staining can be used to detect nuclear DNA fragmentation in tissues late in apoptosis. We found that treatment with AAA-237 promotes nuclear DNA fragmentation, thereby inducing apoptosis in tumor tissues (shown as G in FIG. 7).

Western blot results also show that AAA-237 inhibited expression of Skp2, thereby increasing expression of p27Kip1 in tumor tissues, and thereby inhibiting tumor growth. AAA-237 can also induce apoptosis in tumor cells by cleaving PARP, caspase9 and caspase3, increasing Bax expression, and decreasing Bcl-2 expression. The expression of senescence-associated protein p16 and the DNA damage marker γ h2a.x was also significantly up-regulated, indicating that AAA-237 was also able to induce tumor cell senescence (as shown in H in fig. 7).

Taken together, the in vivo data indicate that AAA-237 inhibits tumor growth by modulating the Skp2-Kip pathway, inducing tumor cell apoptosis and senescence.

Discussion of Experimental results

In the present study, the biophysical binding of AAA-237 to Skp2 was confirmed and the mechanism of action of AAA-237 in vitro was further studied, indicating that it inhibits the proliferation, migration and invasion of human NSCLC cells. In mechanism, AAA-237 inhibits the cell cycle at G0/G1 by regulating the Skp2-Cip/Kip and PI3K/Akt-FOXO1 signaling pathways. CDK4/cyclinD and CDK2/cyclinE were reported to promote cell passage through G1 and S phases. The activity of CDK-cyclin complexes depends on a balance between cyclins and cyclin-dependent kinase inhibitors (CKIs), such as p27Kip1 and p21Cip1(Skp2 substrates). Overexpression of Skp2 induced ubiquitination and sustained degradation of p21Cip1 and p27Kip1, while low levels of Skp2 reduced their degradation. The research of the invention finds that the expression of CDK4, p-CDK4, cyclinD1, CDK2, p-CDK2 and cyclin E is reduced and the expression of p21Cip1 and p27Kip1 is increased in A549 and H1299 cells after AAA-237 treatment.

In addition, differentially expressed genes were identified by RNA-seq, and the FOXO1 signaling pathway was found to be involved in cell cycle regulation after AAA-237 treatment. It is known that FOXO1, a transcription factor, depends on the expression of p21Cip1 and p27Kip1 as target points, plays an important role in preventing cell cycle, and thus significantly inhibits cell proliferation. Experimental study verifies that AAA-237 is treated

The regulatory role of signaling pathways in cell cycle arrest and demonstrates that AAA-237 increases nuclear FOXO1 expression by inhibiting the activation of the PI3K/Akt signaling pathway. This increased its transcriptional activity, increasing the expression of p21Cip1 and p27Kip 1. FOXO1 silenced with siRNA significantly reversed the expression of p21Cip1 and p27Kip1 in AAA-237 treated NSCLC cells. In conclusion, our studies show that AAA-237 inhibits the cell cycle at G0/G1 by modulating the Skp2-Cip/KipPI3K/Akt-FOXO1 signaling pathway.

In addition, it was found in the experiment that continued incubation of NSCLC cells with low concentrations of AAA-237 induced senescence. AAA-237 inhibits expression of Skp2, which leads to accumulation of nuclear DNA damage, stimulates the activity of γ H2AX, and in turn triggers initiation of cellular senescence. The tumor suppressor protein p16Ink4a is frequently transcriptionally activated in senescent cells and is one of its major regulators. In senescent cells, cell cycle arrest is associated with increased levels of p 16. sup. INK4 a. The expression level of p16Ink4a was increased after AAA-237 treatment, indicating that DNA damage caused by AAA-237 might activate p16Ink4 a. p16Ink4a induces cell cycle arrest at the G0/G1 checkpoint by activating p21CIP1 mediated senescence.

AAA-237 induced senescence of NSCLC cells and increased SA- β -Gal activity and γ h2a.x and p16 expression. In AAA-237 treated lung cancer cells, increased secretion of SASP cytokines such as IL-1A, IL-6, CXCL1, CXCL8, MMP1 and MMP10 further promoted infiltration of CD8+ T cells, monocytes and macrophages. AAA-237 induces senescence in some cases, while the senescence drug quercetin can selectively eliminate senescent a549 and H1299 cells. Thus, AAA-237 induced aging treatment in combination with the aging drug quercetin may be beneficial to NSCLC patients.

And (4) conclusion: the Skp2 inhibitor AAA-237 shows a remarkable growth inhibition effect on NSCLC cells in vivo and in vitro, and is related to cell cycle arrest in the G0/G1 stage by regulating Skp2-Cip/Kip and PI3K/Akt-FOXO1 signal pathways. AAA-237 also exerts an anti-tumor effect on NSCLC by triggering apoptosis and DNA damage-induced senescence. Therefore, the novel Skp2 inhibitor AAA-237 may be a promising therapeutic agent for NSCLC.

Claims

1. An anti-neoplastic compound having the formula I:

formula I.

2. The use of a compound of formula I according to claim 1 for the preparation of a medicament.

3. The use of a compound of formula I according to claim 2 for the preparation of a medicament for the treatment of lung cancer.

4. The use of a compound of formula I according to claim 3 for the preparation of a medicament for the treatment of non-small cell lung cancer.

5. The use of a compound of formula I according to claim 2 for the preparation of a medicament, wherein the medicament is a conventional pharmaceutical formulation, such as an oral formulation, an injectable formulation.

6. The use of a compound of formula I according to claim 5 for the preparation of a medicament, wherein the oral formulation is a tablet, capsule, granule, fat emulsion, microcapsule, drop pill; preferably, the injection preparation is injection or powder injection.

7. A composition comprising as one of the active ingredients a compound of formula I.

8. The composition of claim 7, wherein the composition further comprises an additional active ingredient for treating lung cancer.

9. The composition of claim 7, wherein the composition comprises a pharmaceutically acceptable adjuvant.

10. The composition of claim 9, wherein the auxiliary agent comprises at least one of a filler, a disintegrant, a lubricant, and a binder.