Itaconic acid and derivative thereof as CDK4 / 6 inhibitor and antitumor application

Abstract

The invention discloses application of itaconic acid or itaconic acid 4-octanoic acid in preparation of a medicine for treating liver cancer. The invention discloses application of itaconic acid 4-octanoic acid in preparation of a medicine for treating breast cancer. The invention discloses an application of itaconic acid or 4-octyl itaconate in preparation of a CDK4 / 6 inhibitor. Itaconic acid and itaconic acid 4-octanoic acid can mediate alkylation of CDK4 / 6, so that the activity of CDK4 / 6 kinase is further influenced. Itaconic acid and itaconic acid 4-octanoic acid further affect in-vitro proliferation of related liver cancer cells and breast cancer cells in vitro. Meanwhile, itaconic acid 4-octanoic acid can inhibit occurrence and development of breast cancer through CDK4 and CDK6, proliferation of breast cancer cells is effectively inhibited in vivo, and the itaconic acid 4-octanoic acid has the potential of improving survival of breast cancer patients.

Description

Technical Field

[0001] The present invention relates to itaconic acid and its derivatives as novel CDK4 / 6 inhibitors and anti-tumor uses, and specifically to itaconic acid and 4-octyl itaconate as novel CDK4 / 6 inhibitors and anti-tumor uses. Background Art

[0002] Itaconic acid (ITA) is an immune metabolite derived from the TCA cycle intermediate cis-aconitic acid and synthesized by aconitate decarboxylase 1 (ACOD1, also known as IRG1). In recent years, studies have shown that ACOD1 expression is upregulated in activated immune cells (such as macrophages and monocytes) in response to pathogen infection (such as bacteria and viruses), pathogen-associated molecular pattern molecules (such as LPS), cytokines (such as TNF and IFN), and damage-associated molecular patterns (such as sodium urate). ACOD1 catalyzes the decarboxylation of cis-aconitic acid to produce the anti-inflammatory metabolite itaconic acid. As a metabolite produced by the tricarboxylic acid cycle, itaconic acid can inhibit the activity of succinate dehydrogenase in mitochondria, thereby reducing the production of mitochondrial reactive oxygen species, ultimately achieving the effect of weakening the inflammatory response.

[0003]

[0004] 4-Octyl itaconate (4-OI) is a cell-permeable itaconic acid derivative that can alkylate cysteine ​​residues on Kelch-like ECH-associated protein 1 (KEAP1), activate nuclear factor erythroid 2-related factor 2 (Nrf2), and has excellent antioxidant and anti-inflammatory effects on a variety of diseases. Recently, studies have shown that alkylation of cysteine ​​residues by 4-OI can inhibit the enzymatic activity of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), leading to inhibition of aerobic glycolysis. 4-OI mainly plays an anti-inflammatory function in macrophages. Therefore, 4-OI has become a subject of great interest in biomedical research.

[0005] The essence of cancer is the abnormal activation of cell proliferation. Many oncogenes and tumor suppressor genes are directly involved in the regulation of the cell cycle. Their abnormal expression can cause the cell cycle to go out of control, leading to uncontrolled growth of cancer cells, thereby promoting the occurrence and development of cancer. For example, the increase in the amount of Cyclin D1 (CCND1) protein promotes a faster cell cycle and the occurrence of tumors. CDK4 (Cyclin-Dependent Kinase 4) is a catalytic subunit of the protein kinase complex and is important for the progression of the G1 phase of the cell cycle. The activity of CDK4 kinase exists in the G1 / S phase and is controlled by the regulatory subunit d-type cyclin and the CDK inhibitor p16 (INK4a). CDK4 kinase has been shown to be associated with the phosphorylation of the retinoblastoma gene product (Rb). Therefore, understanding the connection between the cell cycle and tumors is of great significance for the development of new strategies for treating cancer, which may include inhibiting tumor cell proliferation by regulating the cell cycle, thereby inhibiting the development of cancer.

[0006] At present, there are four CDK4 / 6 inhibitors approved worldwide, namely Pfizer's Palbociclib (Ibrance), Novartis' Ribociclib (Kisqali), Eli Lilly's Abemaciclib (Verzenio) and G1 Therapeutics' Trilaciclib (Cosela), with a global market size of US$6.992 billion in 2020. Currently, the CDK4 / 6 inhibitors approved in China are palbociclib and abemaciclib. Therefore, exploring new CDK4 / 6 inhibitors and anti-tumor applications has broad prospects and important significance.

[0007] The most common adverse reaction of CDK4 / 6 inhibitors is neutropenia. Unlike neutropenia caused by chemotherapy, CDK4 / 6 inhibitors do not cause the death of neutrophil precursors, and rarely cause febrile neutropenia (1%-2%). In addition, the adverse reactions of CDK4 / 6 inhibitors include QT interval prolongation, venous thromboembolism (VTE), interstitial pneumonia, etc. Among them, the incidence of QT interval prolongation caused by Riboxil is the highest, with an incidence of ≥ grade 3 of 1.0%-3.1%. It can be seen that the relevant CDK4 / 6 inhibitors used clinically have certain defects.

[0008] As a derivative of metabolites in the body, 4-OI has a small molecule and has extensive potential in the research and treatment of various inflammatory and immune diseases. It has been reported in the literature that 4-OI has a strong anti-inflammatory effect and can significantly reduce the inflammatory response to a variety of pathogenic viruses (SARS-CoV-2, herpes simplex virus 1 / 2, Zika virus and vaccinia virus). At the same time, it also plays an important role in the regulation of metabolism in the body. Some molecular processes are affected by ITA, including succinate dehydrogenase (SDH) inhibition, leading to succinate accumulation and metabolic reprogramming; alkylation of protein cysteine ​​residues, inducing electrophilic stress responses mediated by Nrf2 and nuclear factor κB inhibitor ζ (IκBζ), and impaired aerobic glycolysis. In addition, the early recognition that itaconate has anti-inflammatory and immunomodulatory properties also shows that it has the potential to be used as an immunomodulatory and cytoprotective intervention for inflammation-driven diseases. Although there have been many related studies on 4-OI as a therapeutic drug for immune diseases, there have been no reports on its therapeutic regulation of tumor cell proliferation and development. Summary of the invention

[0009] The essence of cancer is the abnormal activation of cell proliferation. Many oncogenes and tumor suppressor genes are directly involved in the regulation of the cell cycle. Their abnormal expression will cause cell cycle disorders, leading to uncontrolled growth of cancer cells, thereby promoting the occurrence and development of cancer. Therefore, inhibiting the active cell cycle can inhibit the proliferation of tumor cells to a certain extent, slow down the development of cancer, and thus achieve the therapeutic effect. The inventors found that: ITA and 4-OI can mediate the alkylation modification of CKD4 / 6 and inhibit its activity; ITA and 4-OI can inhibit tumor cell proliferation in vitro, and 4-OI can inhibit tumor cell proliferation in vivo. Using Kaplan-Meier Plotter public database analysis, it was found that the expression of immune response gene 1 (IRG1, which can convert cis-citrate into itaconic acid) was significantly correlated with the survival of breast cancer patients and IRG1 was a good prognostic factor ( Figure 1 ). The purpose of the present invention is to provide a novel strategy for inhibiting CDK4 / 6 activity and propose a new use of itaconic acid and its derivative 4-octyl itaconate.

[0010] The objective of the present invention is achieved through the following technical solutions:

[0011] Use of itaconic acid or itaconic acid 4-octanoate in preparing medicine for treating liver cancer.

[0012] Preferably, the use is the use of itaconic acid or itaconic acid 4-octanoate in the preparation of a drug for treating liver cancer by inhibiting CDK4 / 6.

[0013] Use of itaconic acid 4-octanoate in preparing medicine for treating breast cancer.

[0014] Preferably, the use is the use of itaconate 4-octanoic acid in the preparation of a drug for treating breast cancer by inhibiting CDK4 / 6.

[0015] Use of itaconic acid or 4-octyl itaconate in the preparation of CDK4 / 6 inhibitors.

[0016] Preferably, the use is the use of itaconic acid or 4-octyl itaconate in the preparation of a drug for inhibiting CKD4 / 6 activity by alkylation modification of CKD4 / 6.

[0017] Further preferably, the use is that the alkylation modification site of CDK4 is cysteine ​​at position 78, 135, and 202 of the CDK4 protein, and the alkylation modification site of CDK6 is cysteine ​​at position 15 of the CDK6 protein.

[0018] Beneficial effects of the present invention:

[0019] ITA and 4-OI can mediate the alkylation of CDK4 / 6, thereby further affecting the kinase activity of CDK4 / 6. ITA and 4-OI further affect the proliferation of related liver cancer cells and breast cancer cells in vitro, providing relevant possibilities for the treatment of cancer. At the same time, 4-OI can inhibit the occurrence and development of breast cancer through CDK4 and CDK6, and effectively inhibit the proliferation of breast cancer cells in vivo. It has the potential to improve the survival of breast cancer patients, opens up new uses for 4-OI, provides new options for the treatment of breast cancer, and has good application prospects in the development of the treatment of malignant tumors. 4-OI can BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 To analyze the correlation between the related gene IRG1 and breast cancer using the Kaplan-Meier Plotter public database.

[0021] Figure 2 Flowchart of ITA / 4-OI-mediated alkylation of CDK4 / 6.

[0022] Figure 3 The results of mass spectrometry analysis of CDK6 alkylation sites.

[0023] Figure 4 ITA / 4-OI alkylates and modifies CDK4 / 6 and inhibits their activity; A: click chemistry experimental results of CDK4 and its mutations; B: click chemistry experimental results of CDK6 and its mutations; C: experimental results of 4-OI or ITA affecting CDK4 activity and thus affecting the interaction with cyclin D1 protein (CCND1); D: CDK4 enzyme activity assay experimental results; E: CDK6 enzyme activity assay experimental results.

[0024] Figure 5The experimental results of 4-OI inhibiting tumor cell proliferation in vitro; A: IC of 4-OI on 4T1 cells 50 ; B: IC of 4-OI on MCF7 cells 50 ; C: IC of 4-OI on MCF10A cells 50 ; D: IC of Palbociclib on 4T1 cells 50 ; E: IC of Palbociclib against MCF7 cells 50 ; F: IC of Palbociclib against MCF10A cells 50 ; G: IC of 4-OI on HepG2 cells 50 .

[0025] Figure 6 These are the experimental results of ITA / 4-OI inhibiting tumor cell proliferation in vitro; A: Experimental results of different concentrations of 4-OI inhibiting HepG2 cell proliferation; B: Experimental results of different concentrations of ITA inhibiting HepG2 cell proliferation; C: Experimental results of different concentrations of 4-OI inhibiting 4T1 cell clone formation; D: Experimental results of different concentrations of 4-OI inhibiting MCF7 cell clone formation; E: Experimental results of different concentrations of 4-OI inhibiting MCF10A cell clone formation.

[0026] Figure 7 The experimental results of 4-OI inhibiting the proliferation of subcutaneous tumors; A: construction of mouse breast cancer transplant tumor model and grouping; B: mouse tumor tissue diagram; C: mouse tumor tissue volume change trend; D: mouse tumor tissue weight. DETAILED DESCRIPTION

[0027] The following experimental scheme was used:

[0028] 1. Explore 4-OI / ITA-mediated alkylation modification of CDK4 / 6 and its sites;

[0029] 2. In vitro and in vivo experiments verified that alkylation modification inhibited CDK4 / 6 activity;

[0030] 3. Alkylation modification of CDK4 / 6 disrupts its binding to the cell cycle protein CCND1;

[0031] 4. Different cancer cell lines were treated with different concentrations of 4-OI and their IC values ​​were detected 50 ;

[0032] 5. Different cancer cell lines were treated with different concentrations of 4-OI and the growth of clones;

[0033] 6. 4T1 cells were injected subcutaneously into the mammary fat pad to load the tumor in situ. After the tumor was formed, 4-OI and Palbociclib were intraperitoneally administered to observe their effects on tumor proliferation.

[0034] Example 1

[0035] ITA / 4-OI mediates alkylation of CDK4 / 6

[0036] Itaconate-alkyne (iTalk) is a specific bioorthogonal probe for quantitative and localized itaconate chemical proteomic analysis in living cells. Itaconate-alkyne is a functional analog of itaconate, has similar anti-inflammatory effects, and can mark the true target of itaconate. Itaconate-alkyne contains an alkyne group and can undergo copper-catalyzed azide-alkyne cycloaddition reaction with molecules containing azide groups. The inventors combined mass spectrometry analysis to explore proteins that can be modified by itaconate. The flow chart is shown in the figure below. Figure 2 shown.

[0037] The click chemistry experimental process is as follows: In a 6 cm dish, HepG2 cells were cultured with 250 μM 4-OI or an equal amount of DMSO (as a control group) at 37°C for 24 hours, and then co-cultured with 100 μM iTalk (dissolved in DMSO) at 37°C for 12 hours. 1 mL of ice lysis buffer (lysis buffer: 1×PBS containing 1% TritonX-100 and 0.1% sodium dodecyl sulfate SDS) was added to each dish, and the cells were lysed on ice to obtain cell lysate; 100 μL of cell lysate was taken from each treatment group, and ultrasonicated on ice (ultrasonic power 4 kHz, 3 times in total, each time for 3 seconds). After the ultrasonication, the cells were centrifuged at 4°C and 16200g for 10 minutes, and the supernatant was taken. Click chemistry system (0.1mM biotin azide-biotin, 1mM CuSO4, 1mM TCEP, 0.1mM TBTA) was added to the supernatant, incubated for 1h at room temperature and protected from light, centrifuged at 4600g for 5min at room temperature, discarded the supernatant, and the precipitate was washed twice with cold methanol by rotation, each washing for 10min, centrifuged at 4600g for 5min at room temperature after each washing, and the precipitate was retained. The precipitate was dissolved in 10μL of 1×PBS containing 2% SDS and 10mM dithiothreitol (DTT), heated at 95℃ for 5min, and then diluted with 90μL 1×PBS (containing 1.2% SDS), 30μL streptavidin beads were added to each sample, incubated for 2h at room temperature by rotation, and then the streptavidin beads were washed five times by rotation with 1mL 1×PBS containing 0.2% SDS at room temperature. Sample preparation: Centrifuge at room temperature and 4600g for 5 minutes, retain the streptavidin beads at the bottom, add 50μL 1.5×SDS loading buffer to each sample, cook at 95℃ for 5 minutes, take 30μL for SDS PAGE gel electrophoresis detection and identification, cut the protein strip, and hand it over to Wuhan BGI Gene Technology Testing Service Company for mass spectrometry analysis (LC-MS). According to the mass spectrometry results, iTalk was found to be able to identify cyclin-dependent kinases 4 and 6 (CDK4 / 6).

[0038] Studies have shown that CDK4 / 6 proteins play an important role in the cell cycle, especially their regulatory mechanism in the transition from G1 to S phase. Cyclin D and CDK4 / 6 promote the phosphorylation of RB1 protein, release E2F transcription factor, activate Cyclin E and CDK2 complex, and finally push cells from G1 phase to S phase through interaction with cell cycle inhibitors such as P27 and P21. Among them, proteins such as P15, P16, P18, and P19 regulate cell cycle progression by inhibiting the activity of Cyclin D / CDK4 / 6 complex.

[0039] Example 2

[0040] HepG2 cells were plated in a 6 cm dish and cultured to a density of about 70%, and then transfected with Flag-CDK6 plasmid (provided by Sangon Biotechnology (Shanghai) Co., Ltd.). After 12 h, the medium was changed and cultured for another 36 h. Then 250 μM 4-OI (dissolved in DMSO) was added. After culturing at 37 °C for 24 h, the cells were washed with PBS and scraped. The cells were centrifuged at 4 °C and 1000 rpm for 3 min, the supernatant was discarded, and 1 mL of cold IP lysis buffer (20 mM Tris HCl (pH 8.0), 250 mM NaCl, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 0.2% NP-40, 1 mM NaF, 10% glycerol, 2 mM EDTA, protease inhibitor phenylmethylsulfonyl fluoride) resuspended, and ultrasonically disrupted cells (ultrasonication on ice 3 times, ultrasonic power 4kHz, ultrasonication 3s each time, standing for 3s between two ultrasonications), centrifuged at 4℃, 12000rpm for 10min, collected the supernatant, which was the untreated cell lysate input sample; added 10μL of beads with Flag tags to the supernatant, incubated overnight at 4℃ shaker, washed the beads (using 1mL cold IP lysis buffer each time, rotating and washing at 4℃ shaker for 10min, a total of 3 times), retained the bottom Flag beads, added 30μL 1.5×SDS loading buffer, boiled the sample at 95℃ for 5min, took 25μL for SDS PAGE gel electrophoresis detection and identification, and cut the protein strip and handed it over to Wuhan BGI Gene Technology Testing Service Company for mass spectrometry analysis (LC-MS). The results are as follows Figure 3 As shown, according to the mass spectrometry results, the site where CDK6 can be modified by 4-OI is the 15th cysteine.

[0041] Example 3

[0042] ITA / 4-OI alkylates CDK4 / 6 and inhibits its activity

[0043] according to Figure 4 A. Figure 4 B was divided into groups, and HepG2 cells were transfected with wild-type Flag-CDK4 plasmid and mutant plasmid Flag-CDK4 C78A 、Flag-CDK4 C135A 、Flag-CDK4 C202A 、Flag-CDK4 C215A (respectively, the cysteine ​​at positions 78, 135, 202, and 215 of the CDK4 protein were mutated to alanine) and the wild-type Flag-CDK6 plasmid and the mutant plasmid Flag-CDK6C15A (The cysteine ​​at position 15 of CDK6 protein was mutated to alanine). The above plasmids were provided by Sangon Biotechnology (Shanghai) Co., Ltd. 48 hours after plasmid transfection, click chemistry experiments were performed. The experimental process is shown in Example 1. After sample preparation, 15 μL was taken for Western Blot detection, where the Post-Click Input sample group was an untreated cell lysate, and the SA-Beads sample group was a total protein that could be captured by streptavidin magnetic beads. It was found that the itaconic acid derivative 4-OI can modify CDK4 by alkylation. After mutating the cysteine ​​at positions 78, 135, and 202 of the CDK4 alkylation site to alanine (C78A, C135A, C202A), 4-OI was unable to alkylate CDK4 ( Figure 4 A). After the cysteine ​​at position 15 of the CDK6 alkylation site was mutated to alanine (C15A), CDK6 would not be alkylated ( Figure 4 B) The above experimental results provide new clues to explain the mechanism by which itaconate exerts its regulatory function of alkylating CDK4 / 6.

[0044] HepG2 cells were plated in three 6 cm dishes and cultured to a density of about 70% and then transfected with Flag-CDK4 plasmid. After 12 h, the medium was changed and cultured for 36 h. The cells were divided into control group (Ctrl), 4-OI treatment group, and ITA treatment group. The 4-OI treatment group was treated with 100 μM 4-OI for 8 h, the ITA treatment group was treated with 4 mM ITA for 12 h, and the control group was treated with the same volume of DMSO as the 4-OI treatment group for 12 h. After the treatment, the cells were washed with PBS and scraped, centrifuged at 4°C and 1000 rpm for 3 min, the supernatant was discarded, and the cell pellet was washed with 600 μL IP The cells were resuspended in buffer (with the protease inhibitor phenylmethylsulfonyl fluoride) and ultrasonically disrupted (ultrasonication on ice 3 times, ultrasonic power 4kHz, each ultrasonication 3s, and standing for 3s between two ultrasonications), centrifuged at 4℃ and 12000rpm for 10min, and the supernatant was collected, which was the untreated cell lysate input sample; an equal amount of beads with Flag labels were added to the supernatant, incubated overnight at 4℃ on a shaker, and the beads were washed to obtain IP samples; the input samples and IP samples were detected by WB, and the results are shown in Figure 4 C, where the Input sample group is untreated cell lysate, and the IP sample group is total protein captured by Flag-tagged beads. The results show that 4-OI or ITA affects the activity of CDK4 and further affects the interaction between CDK4 and its target protein cyclin D1 (CCND1).

[0045] In order to further explore whether 4-OI affects the activity of CDK4 in vitro, we first performed GST (glutathione protein), GST-Rb (792-928aa), GST-CDK4, GST-CDK6, fusion protein, and four CDK4 mutant fusion proteins GST-CDK4 C78A , GST-CDK4 C135A , GST-CDK4 C202A , GST-CDK4 C215A and GST-CDK6 C15A Protein purification. The recombinant plasmids containing GST tags (pGEX, pGEX-CDK4, pGEX-CDK6, pGEX-RB (792-928aa), pGEX-CDK4 C78A , pGEX-CDK4 C135A , pGEX-CDK4 C202A , pGEX-CDK4 C215A , pGEX-CDK6 C15A , purchased from Sangon Biotech (Shanghai) Co., Ltd.) were transformed into Escherichia coli BL21, and cultured in 100 mL LB medium, with the addition of 1 mM isopropyl-β-D-thiogalactoside (IPTG) as inducer, and cultured at 16°C for 16 h to promote the expression of GST fusion protein. Centrifuge at room temperature and 8000 rpm for 5 min, and collect the cell pellet. Each protein was resuspended in 10 ml 4°C lysis buffer (50 mM Tris HCl (pH 7.5), 0.05% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM DTT), and the cells were lysed by ultrasound on ice (ultrasound power 4 kHz, 3 times in total, 3 seconds each time, 8 seconds between two ultrasounds, 10 minutes in total) to release GST fusion protein; centrifuge at 4°C, take the supernatant, and add the supernatant to the column containing 100 μL glutathione (GSH) affinity resin. The GST tag will bind to GSH, allowing the fusion protein to be specifically captured. Use cold washing buffer (100mM Tris HCl (pH8.0), 150mM NaCl, 1mM EDTA) to remove non-specifically bound impurities. Wash each protein three times with 1mL of washing buffer each time. Elute the GST fusion protein with elution buffer containing 10mg glutathione per ml, collect the eluted protein sample, and obtain the purified GST protein and CDK4, CDK6, Rb (792-928aa), CDK4 with GST tags. C78A 、CDK4 C135A 、CDK4 C202A 、CDK4 C215A 、CDK6 C15AProtein (due to the large molecular weight of retinoblastoma protein Rb, purification is difficult, and the present invention truncates and purifies the main active site 792-928 amino acids of the Rb protein).

[0046] Equal amounts of purified GST (control protein), CDK4 protein, and four CDK4 mutant proteins were used. C78A 、CDK4 C135A 、CDK4 C202A 、CDK4 C215A The cells were incubated with an equal amount of GST beads that can specifically bind to the purified protein at 4°C for 4 h, and reacted with 250 μM 4-OI at 37°C for 1 h. The cells were reacted with an equal volume of DMSO with 4-OI for 1 h as a control. After the reaction, the cells were washed three times with 1 mL PBS, and incubated with 20 mM ATP, an equal amount of GST-RB (792-928aa) protein and Reaction Buffer. The ATP utilization rate was measured. The results are shown in the table. Figure 4 D, indicating that 4-OI affects the activity of CDK4, but does not affect the activity of CDK4 with cysteine ​​mutations at positions 135 or 202.

[0047] Equal amounts of purified GST (control protein), CDK4 protein, and CDK6 C15A After incubation with an equal amount of GST beads that can specifically bind to the purified protein at 4℃ for 4h, the cells were reacted with 250μM 4-OI at 37℃ for 1h, and an equal volume of DMSO with 4-OI was used as a control for 1h. After the reaction, the cells were washed three times with 1ml PBS, and 20mM ATP, an equal amount of GST-RB (792-928aa) protein and Reaction Buffer were added for incubation. The ATP utilization rate was measured. The results are shown in Figure 4 E, shows that 4-OI affects the activity of CDK6, but does not affect the activity of CDK6 with cysteine ​​15 mutation.

[0048] In summary, it is shown that 4-OI can inhibit CDK4 / 6-mediated cell cycle regulation and provide a new strategy for the treatment of tumors.

[0049] Example 4

[0050] ITA / 4-OI inhibits the proliferation of tumor cells in vitro

[0051] Four cell lines, namely, human liver cancer cell HepG2 and breast cancer cell 4T1, MCF7, and MCF10A, were selected as experimental subjects. Each cell line was cultured at a rate of 2.5 × 10 3The cells were inoculated into 96-well plates (three replicates per group). After the cells adhered to the wall, they were treated with a dose gradient (0-800 μM) of 4-OI or Palbociclib for 48 h. 10 μL of 5 mg / mL MTT reagent was added to each well. 100 μL of DMSO was added 2 h later. The absorbance of the solution was detected by an ELISA reader, and the IC 50 .

[0052] The results of the cell proliferation assay are shown in Figure 5 , Figure 6 The IC values ​​of 4-OI on breast cancer cells 4T1, MCF7, and MCF10A were 50 They are 0.2386mM, 0.1952mM, 0.4mM ( Figure 5 A. Figure 5 B. Figure 5 C); IC of 4-OI against HepG2 cancer cells compared with breast cancer cells 4T1 and MCF7 50 Higher ( Figure 5 G), IC 50 The IC values ​​of the positive control drug Palbociclib for 4T1, MCF7, and MCF10A are 0.6761 mM. 50 They are 0.2386mM, 0.1952mM, 0.4mM ( Figure 5 D- Figure 5 F). Under high concentrations of 4-OI or ITA treatment, the proliferation ability of HepG2 cells was significantly inhibited ( Figure 6 AB). Compared with the blank control group, 4-OI significantly inhibited the clone formation rate of 4T1 cells. As the drug concentration gradient increased, the clone formation rate decreased ( Figure 6 C); When MCF7 cells were treated with different concentrations of 4-OI, the number of clones formed decreased significantly, and the clone formation rate decreased with the increase of 4-OI concentration ( Figure 6 D). The clone formation rate of MCF10A cells decreased with the increase of 4-OI concentration ( Figure 6 E). This indicates that 4-OI may inhibit the proliferation of breast cancer and liver cancer cells by affecting the tumor cell cycle.

[0053] Example 4

[0054] High-dose 4-OI inhibits tumor cell proliferation in mice

[0055] Establishment of breast cancer transplant model: 4T1 cells in logarithmic phase were digested with 0.25% protease for 2-3 min, culture medium was added to terminate digestion, centrifuged at 1000 rpm for 3 min, supernatant was removed, and cell concentration was adjusted to 1×10 with PBS. 6 / mL to obtain a tumor cell suspension. Under sterile conditions, tumor cells were inoculated into the fat pad of the fourth nipple of the right side of BALB / c mice using a 1mL syringe, with an inoculation volume of 0.1mL / mouse. Observe the general living conditions of the mice daily. If a tumor mass as large as a soybean is seen at the inoculated site after 5 days, it means that a breast cancer transplant tumor model has been successfully established. At this time, the mice were randomly divided into 4 groups, namely the control group (injected with 60% β-cyclodextrin prepared in normal saline according to the weight of the mice), the Palbociclib group (dissolved in DMSO, 25 mg / kg, intraperitoneal administration), the 4-OI high-dose group (dissolved in 60% β-cyclodextrin prepared in normal saline, 80 mg / kg, intraperitoneal administration) and the 4-OI low-dose group (40 mg / kg, intraperitoneal administration), with 6 mice in each group ( Figure 7 A). The drug was administered once a day. On the 22nd day after administration, the nude mice were anesthetized with 0.5% sodium pentobarbital, and the tumor tissues were removed by dissection after tissue perfusion ( Figure 7 B). The tumor volumes of mice in the Palbociclib group, 4-OI high-dose group, and 4-OI low-dose group were significantly smaller than those in the control group ( Figure 7 C). The tumor tissues of the dissected mice were weighed, and it was found that compared with the control group, the tumor tissue weights of the mice in the Palbociclib group and the 4-OI high-dose group (80 mg / kg) were significantly reduced, but there was no significant difference in the tumor tissue weights of the mice in the 4-OI low-dose group (40 mg / kg) ( Figure 7 D) indicates that the administration of 4T1 to 4-OI in breast cancer transplanted tumor model mice can inhibit the development of tumor volume in the model mice.

[0056] In summary, 4-OI can effectively inhibit tumor cell proliferation both in vivo and in vitro, and can significantly reduce tumor proliferation in mice; this therapeutic effect may be achieved by inhibiting the cell cycle through high concentrations of 4-OI.

Claims

1. Use of itaconic acid or itaconic acid 4-octanoate in the preparation of drugs for treating liver cancer.

2. The method according to claim 1, characterized in that: Use of itaconic acid or itaconic acid 4-octanoate in preparing a drug for treating liver cancer by inhibiting CDK4 / 6.

3. Use of itaconic acid 4-octanoate in the preparation of drugs for treating breast cancer.

4. The method according to claim 3, characterized in that: Use of itaconate 4-octanoic acid in preparing a drug for treating breast cancer by inhibiting CDK4 / 6.

5. Use of itaconic acid or 4-octyl itaconate in the preparation of CDK4 / 6 inhibitors.

6. The use according to claim 5, characterized in that: The use is the use of itaconic acid or 4-octyl itaconate in the preparation of a drug for inhibiting the activity of CKD4 / 6 by alkylation modification of CKD4 / 6.

7. The use according to claim 6, characterized in that: The alkylation modification sites of CDK4 are cysteine ​​at positions 78, 135, and 202 of the CDK4 protein, and the alkylation modification site of CDK6 is cysteine ​​at position 15 of the CDK6 protein.

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