Application of celecoxib and cetuximab in combined treatment of colorectal cancer with different KRAS genotypes

Abstract

The invention discloses application of celecoxib and cetuximab in combined treatment of colorectal cancer with different KRAS genotypes, and belongs to the field of biological medicine. Celecoxib and cetuximab are combined for use, KRAS / Cdc42 / Neutrophil elatase and RAF-MEK-ERK pathways are inhibited, the sensitivity to cetuximab targeted therapy can be remarkably improved, the cetuximab targeted therapy composition is suitable for different KRAS genotype colorectal cancer including KRAS mutant colorectal cancer and KRAS wild colorectal cancer, the combined therapy effect is obviously superior to the curative effect of cetuximab, and the cetuximab targeted therapy composition is suitable for clinical application. A new thought is provided for the treatment of the intractable and drug-resistant colorectal cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of celecoxib and cetuximab in the treatment of different KRAS genotypes of colorectal cancer. BACKGROUND

[0002] Colorectal cancer (CRC) is the third most common cancer in the world, and its incidence and mortality rate in China are increasing year by year. Metastasis and drug resistance are still the urgent challenges in the treatment of colorectal cancer. According to the latest American National Comprehensive Cancer Network (NCCN) guidelines, the first-line treatment is recommended to be FOLFOX chemotherapy based on fluorouracil, oxaliplatin and irinotecan combined with anti-vascular endothelial growth factor (VEGF) or epidermal growth factor receptor (EGFR) monoclonal antibodies (such as cetuximab or panitumumab). Recent studies have shown that a considerable proportion of colorectal cancer patients have developed resistance to cetuximab, especially KRAS mutant colorectal cancer patients, whose remission rate is lower than that of KRAS wild-type patients. Anti-epidermal growth factor receptor (EGFR) antibodies such as cetuximab are only recommended as effective treatment methods for KRAS wild-type CRC patients when combined with chemotherapy. However, their efficacy is often limited by intrinsic drug resistance, which is mainly caused by activated KRAS mutations that drive the feedback activation of downstream signaling pathways RAF-MEK-ERK, PI3K-AKT-mTOR, leading to resistance to targeted therapy and limiting its clinical application. In terms of new drugs targeting KRAS mutations, although KRASG12Cselective covalent inhibitor Sotorasib has been included in the first-line treatment of colon cancer, it faces more common KRASG12D, G12V and G13D mutations, and patients need more broad-spectrum targeted treatment regimens. Therefore, exploring a new strategy for targeted therapy combined with KRAS mutant tumors is the current research focus.

[0003] Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor, mainly used as a non-steroidal anti-inflammatory drug (NSAID) for the treatment of pain and inflammation in diseases such as osteoarthritis and rheumatoid arthritis. In addition, its potential role in cancer treatment and prevention has also been studied by an increasing number of scholars due to its anti-inflammatory and anti-proliferative effects. Some studies have explored celecoxib as a neoadjuvant therapy for a variety of cancers in the combined treatment strategy of several malignant cancers. However, the efficacy of celecoxib combined with cetuximab in the treatment of KRAS mutant CRC is not clear, and its potential mechanism needs further study.

[0004] Dormant Tumor Cells (DTCs) are cancer cells that temporarily stop proliferating but retain the ability to survive, and can remain dormant for long periods in the primary tumor or at distant metastatic sites, eventually being reactivated, leading to tumor recurrence and metastasis. Hypoxic microenvironment, chemical agents, radiotherapy, Chinese herbal medicine, etc. can induce the formation of these dormant tumor cells. In the emergency condition of exposure to therapeutic drugs, dormant tumor cells remain static and stop in G2 / M, and remain viable but not proliferative under environmental stress. When the surrounding harsh environment is improved, dormant tumor cells thus come out of dormancy, restore proliferation, leading to the recurrence or metastasis of malignant tumors. KRAS-related inflammatory signals have been shown to contribute to tumorigenesis and progression of KRAS mutant CRC, and genetic mutations / epigenetic changes such as KRAS mutations can relieve tumor dormancy. Therefore, how KRAS mutations activate tumor recurrence and metastasis and lead to drug resistance, and the molecular regulatory mechanism thereof need to be further explored. Understanding the interaction between dormant tumor cells and KRAS mutation drug resistance can provide insights for combined targeting strategies, which may potentially improve the treatment effect of KRAS mutant cancer. Based on this, the present application aims to find a new method for effectively treating KRAS mutant colorectal cancer. SUMMARY

[0005] In order to solve the above technical problems, the present application provides an application of celecoxib and cetuximab in combination for treating different KRAS genotypes of colorectal cancer.

[0006] In the first aspect, the present application provides a combination of cyclooxygenase-2 inhibitors and anti-vascular endothelial growth factor or epidermal growth factor receptor monoclonal antibodies, which is realized by adopting the following technical scheme.

[0007] The combination of cyclooxygenase-2 inhibitors and anti-vascular endothelial growth factor or epidermal growth factor receptor monoclonal antibodies is used for preparing a drug for treating different KRAS genotypes of colorectal cancer.

[0008] Further, the cyclooxygenase-2 inhibitor is selected from celecoxib.

[0009] Further, the anti-vascular endothelial growth factor or epidermal growth factor receptor monoclonal antibody is selected from cetuximab or panitumumab.

[0010] Further, the mass ratio of cyclooxygenase-2 inhibitors and anti-vascular endothelial growth factor or epidermal growth factor receptor monoclonal antibodies in in vivo treatment is 1:10.

[0011] Further, the different KRAS genotypes of colorectal cancer include KRAS mutant colorectal cancer and KRAS wild-type colorectal cancer.

[0012] The second aspect of the present application provides a pharmaceutical composition, which is achieved by the following technical scheme.

[0013] A pharmaceutical composition, which comprises a cyclooxygenase-2 inhibitor and an anti-vascular endothelial growth factor or epidermal growth factor receptor monoclonal antibody.

[0014] Further, the cyclooxygenase-2 inhibitor is selected from celecoxib.

[0015] Further, the anti-vascular endothelial growth factor or epidermal growth factor receptor monoclonal antibody is selected from cetuximab or panitumumab.

[0016] Further, the cyclooxygenase-2 inhibitor and the anti-vascular endothelial growth factor or epidermal growth factor receptor monoclonal antibody have a mass ratio of 1:10 in vivo treatment.

[0017] The third aspect of the present application provides a use of a pharmaceutical composition, which is achieved by the following technical scheme.

[0018] The use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating different KRAS genotypes of colorectal cancer.

[0019] The present application has the following beneficial effects.

[0020] The cyclooxygenase-2 (COX-2) inhibitor celecoxib of the present application can treat different KRAS genotypes of colorectal cancer. KRAS mutation can activate the recurrence and metastasis of dormant tumor cells, and continuously activate the KRAS downstream pathway, thereby affecting the treatment of colorectal cancer. Therefore, the present application first uses celecoxib in combination with cetuximab, which inhibits the KRAS / Cdc42 / Neutrophil elastase and MEK-ERK pathways, and can significantly increase the sensitivity to cetuximab targeted therapy, which is suitable for different KRAS genotypes of colorectal cancer (including KRAS mutant and KRAS wild type), and the effect of combination therapy is obviously better than that of celecoxib or cetuximab. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1are results of inducing KRAS mutant colorectal cancer cells with high invasion and metastasis ability of the present application; wherein, A: inducing two groups of KRAS mutant colorectal cancer cell lines (Hct116 and LoVo) to form KRAS mutant colorectal cancer cell recurrence metastasis models with high invasion and metastasis ability, the black arrow is the induced polyploid large cell, Hct116-Tre and LoVo-Tre both refer to the induced KRAS mutant colorectal cancer cell recurrence metastasis cells with high invasion and metastasis ability, Hct116-Ctr and LoVo-Ctr both refer to the untreated ordinary KRAS mutant colorectal cancer cells; B: a. scratch test to detect the migration ability of Hct116-Ctr and Hct116-Tre cells, b. scratch test to detect the migration ability of LoVo-Ctr and LoVo-Tre cells; C: a. Transwell migration experiment to detect the migration ability of Hct116-Ctr and Hct116-Tre cells, LoVo-Ctr and LoVo-Tre cells, b. Transwell invasion experiment to detect the invasion ability of Hct116-Ctr and Hct116-Tre cells, LoVo-Ctr and LoVo-Tre cells;

[0022] Figure 2is a mechanism research result figure of CD66b of the present application promoting migration and invasion of KRAS mutant colorectal cancer by activating KRAS-Cdc42-NE pathway; wherein, A: Western blot experiment detects protein expression of CD66b in Hct116-Ctr and Hct116-Tre and LoVo-Ctr and LoVo-Tre; B: Western blot experiment detects protein expression of KRAS, Cdc42, cathepsin D, cathepsin B, Neutrophil elastase (NE) and MMP9 in Hct116-Ctr and Hct116-Tre and LoVo-Ctr and LoVo-Tre; C: a. After Hct116-Tre is treated by small interfering RNA for Cdc42 for 24 h, cell scratch test detects migration ability of Hct116-Tre-NC group (not treated by small interference) and Hct116-Tre-Cdc42 i group cells, b. After LoVo-Tre is treated by small interfering RNA for Cdc42 for 24 h, cell scratch test detects migration ability of LoVo-Tre-NC group (not treated by small interference) and LoVo-Tre-Cdc42 i group cells; D: Immunoprecipitation experiment detects that CD66b and KRAS and Cdc42 proteins have interaction in Hct116-Tre and LoVo-Tre; E: Proximity ligation assay detects interaction of CD66b protein and KRAS protein in Hct116-Ctr and Hct116-Tre and LoVo-Ctr and LoVo-Tre, the intensity of red dot represents the strength of protein binding, the protein binding strength in Tre group in two KRAS mutant colorectal cancer cell lines is higher than that in Ctr group); F: a. After Hct116-Tre and LoVo-Tre cells are treated by small interfering RNA for CD66b for 24 h, PCR experiment detects CD66b and KRAS mRNA expression in NC group and CD66b i group cells in Hct116-Tre and LoVo-Tre cells; b.Fig. 6 shows the results of luciferase reporter assay, cell scratch assay, Transwell migration assay, Transwell invasion assay, Western blot assay, and the effects of CD66b, KRAS, and Cdc42 on the migration and invasion of Hct116-Tre and LoVo-Tre cells. Fig. 6A shows the results of luciferase reporter assay for detecting the luciferase activity in Hct116-Tre and LoVo-Tre cells treated with small interfering RNA against CD66b for 24 h. Fig. 6B shows the results of cell scratch assay for detecting the migration ability of Hct116-Tre-NC (not treated with small interfering RNA) and Hct116-Tre-CD66bi groups and LoVo-Tre-NC (not treated with small interfering RNA) and LoVo-Tre-CD66bi groups after Hct116-Tre and LoVo-Tre were treated with small interfering RNA against CD66b for 24 h. Fig. 6C shows the results of Transwell migration assay for detecting the migration ability of Hct116-Tre-NC and Hct116-Tre-CD66bi groups and LoVo-Tre-NC and LoVo-Tre-CD66bi groups after Hct116-Tre and LoVo-Tre were treated with small interfering RNA against CD66b for 24 h. Fig. 6D shows the results of Transwell invasion assay for detecting the invasion ability of Hct116-Tre-NC and Hct116-Tre-CD66bi groups and LoVo-Tre-NC and LoVo-Tre-CD66bi groups. Fig. 6E shows the results of Western blot assay for detecting the protein expression of CD66b, KRAS, Cdc42, cathepsin D, cathepsin B, Neutrophil elastase (NE), and MMP9 in Hct116-Tre and LoVo-Tre treated with small interfering RNA against CD66b for 24 h. Fig. 6F shows the results of Western blot assay for detecting the protein expression of CD66b, KRAS, Cdc42, cathepsin D, cathepsin B, Neutrophil elastase (NE), and MMP9 in Hct116-Tre and LoVo-Tre treated with small interfering RNA against KRAS for 24 h. Fig. 6G shows the results of Western blot assay for detecting the protein expression of KRAS, Cdc42, cathepsin D, cathepsin B, Neutrophil elastase (NE), and MMP9 in Hct116-Tre and LoVo-Tre treated with small interfering RNA against Cdc42 for 24 h.

[0023] Figure 3is a result figure of the present application for identifying compounds inhibiting CD66b-KRAS binding through high-throughput virtual screening; wherein, A: molecular docking shows the 3D binding model of the protein (CD66b is pink, KRAS is cyan), the key residues are represented by sticks, the hydrogen bonds are represented by yellow dotted lines, and they have good binding energy (docking score: -212.50 kcal / mol); ARG-77, ASP-154, PHE-141 and GLN-131 of KRAS form 4 hydrogen bonds with ASN-78, ILE-68, GLN-54 and ASN-53 on CD66b, respectively; B: Western blot experiment detects the expression of CD66b and KRAS in KRAS mutant colorectal cancer recurrence and metastasis cells (a, b: Hct116-Tre and LoVo-Tre) and KRAS wild-type colorectal cancer recurrence and metastasis cells (c, d: HT29-Tre and Caco2-Tre) after treatment with different concentrations of three compounds (chlorothiazide, haloperidol and celecoxib); C: molecular docking shows the 3D binding model of the protein (CD66b is pink, KRAS is cyan and celecoxib is blue), the key residues are represented by sticks, the hydrogen bonds are represented by yellow dotted lines, and celecoxib destroys the interaction between CD66b and KRAS; D: CCK8 method is used to detect the cell viability of Hct116-Tre (a) and LoVo-Tre (b) treated with 100 μM celecoxib at different time points; E: Surface plasmon resonance (SPR) is used to detect the binding affinity of KRAS and celecoxib: KRAS protein is immobilized on a CM5 chip, and Biacore T200 evaluation software (version 2.0) is used for 1:1 binding model for kinetic and affinity analysis; F: Surface plasmon resonance (SPR) is used to detect the binding affinity of CD66b and celecoxib: CD66b protein is immobilized on a CM5 chip, and Biacore T200 evaluation software (version 2.0) is used for 1:1 binding model for kinetic and affinity analysis; G: After Hct116-Tre and LoVo-Tre are treated with 100 μM celecoxib or Dmso for 24 h, the binding ability of CD66b and KRAS in Hct116-Tre and LoVo-Tre is detected by immunoprecipitation technology; H: Western blot is used to detect the expression levels of CD66b, KRAS, Cdc42, NE, cathepsin D, cathepsin B, p-ERK, ERK, p-AKT and AKT in KRAS mutant colorectal cancer recurrence and metastasis cells (a, b: Hct116-Tre and LoVo-Tre) and KRAS wild-type colorectal cancer recurrence and metastasis cells (c, d: HT29-Tre and Caco2-Tre) after treatment (PBS, cetuximab 25 μg / ml, celecoxib 100 μM, and combined treatment) for 24 h;

[0024] Figure 4 The figures show the results of the combined treatment of celecoxib and cetuximab of the present invention in improving the efficacy of colorectal cancer xenograft tumor models with different KRAS genotypes; wherein, A: a xenograft tumor model was constructed using KRAS mutant colorectal cancer recurrent metastatic cells (a, b: Hct116-Tre and LoVo-Tre) and KRAS wild-type colorectal cancer recurrent metastatic cells (c: HT29-Tre); B: when the tumor volume reached approximately 150 cubic millimeters, the mice were randomly divided into four groups: a control group (normal saline), a cetuximab group (10 mg / kg / day), a celecoxib group (100 mg / kg / day), and a cetuximab combined with celecoxib group (10 mg cetuximab + 100 mg celecoxib / kg / day), and the tumor volume was statistically analyzed; C: Western blot detection of CD66b, KRAS, Cdc42, NE, and cathepsin in nude mouse transplanted tumor tissues with different KRAS genotypes. D, expression levels of cathepsin B, p-ERK, and ERK;

[0025] Figure 5 The figures show the results of the combined treatment of celecoxib and cetuximab of the present invention improving the invasion and migration of colorectal cancer cells with different KRAS genotypes and the immunohistochemistry of xenograft tumor models; wherein, A: Transwell migration assay detecting the migration ability of KRAS mutant colorectal cancer recurrent and metastatic cells (Hct116-Tre and LoVo-Tre) after drug treatment (PBS, cetuximab 25 μg / ml, celecoxib 100 μM, and combined treatment) for 24 hours; B: Transwell invasion assay detecting the invasion ability of KRAS mutant colorectal cancer recurrent and metastatic cells (Hct116-Tre and LoVo-Tre) after drug treatment (PBS, cetuximab 25 μg / ml, celecoxib 100 μM, and combined treatment) for 24 hours; C: Immunohistochemistry of tumor tissue sections of the above four groups of mice; D: Measurement of body weight of the above four groups of mice. DETAILED DESCRIPTION

[0026] Through high-throughput virtual screening (HTVS), the present invention discovered for the first time that celecoxib is a new inhibitor that disrupts CD66b-KRAS binding. Further research on the treatment of KRAS wild-type and mutant colorectal cancer with celecoxib found that the combination of celecoxib and cetuximab increased the sensitivity of CRC cells with different KRAS genotypes to cetuximab by inhibiting the KRAS / Cdc42 / NE and MEK-ERK pathways, thereby inhibiting the growth and metastasis of KRAS-driven colorectal cancer.

[0027] Through mechanism research, the present invention found that CD66b and KRAS are highly expressed in KRAS-mutated colorectal cancer cells. CD66b directly targets KRAS and regulates the transcription of the KRAS promoter, thereby activating the KRAS / Cdc42 / Neutrophil elastase pathway and promoting the metastasis of KRAS-mutated CRC.

[0028] Based on the revelation of molecular mechanisms, in terms of treatment, through high-throughput virtual screening, this study discovered for the first time that celecoxib can inhibit the binding of CD66b and KRAS proteins and suppress the expression of both, effectively inhibiting the metastasis and growth of KRAS-mutant or wild-type CRC both in vitro and in vivo. Cetuximab alone has little effect on the growth of KRAS-mutant colorectal cancer tumors, while celecoxib significantly inhibits the growth of xenograft tumors. Combination therapy of celecoxib and cetuximab further minimizes tumor growth in nude mice. Combination therapy of celecoxib and cetuximab inhibits the KRAS / Cdc42 / Neutrophil elastase and MEK-ERK pathways. Therefore, this application proposes that celecoxib can be specifically used to treat colorectal cancer with different KRAS genotypes (KRAS mutant and wild-type), and that the dual-drug combination can improve colorectal cancer resistance to cetuximab, providing a new approach for the treatment of refractory and resistant colorectal cancer.

[0029] The present patent application is further described below with reference to the accompanying drawings.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials, reagents, etc. used in the following preparation examples and examples are all commercially available unless otherwise specified.

[0031] 1. Inducing KRAS mutant colorectal cancer cells with high invasive and metastatic abilities

[0032] Hypoxic microenvironment, chemical reagents, radiotherapy, Chinese medicine monomers, etc. can all induce the formation of these dormant tumor cells. In this study, the Chinese medicine monomer arsenic trioxide was used to induce a model of recurrence and metastasis of KRAS mutant colorectal cancer cells (Hct116 and LoVo). The specific method is as follows: Hct116 and LoVo cells were cultured in a T25 culture flask with complete RPMI-1640 medium. When the tumor cells reached 80-90% confluence, arsenic trioxide at a concentration of 80 μm was added to the T25 culture flask and incubated at 37 ° C in a 5% CO2 incubator for 24-48 hours. Most normal-sized cancer cells were killed, while a few cells survived. The size of these cells was three times that of normal cancer cells. After about 10 to 15 days, the surviving large cells produced smaller, highly invasive daughter cells through asymmetric division. These cells are tumor cell models that undergo recurrence and metastasis. This application will refer to them as Hct116-Tre and LoVo-Tre ( Figure 1 A).

[0033] The scratch test and Transwell migration and invasion assay showed that the KRAS mutant colorectal cancer cells after induction had higher metastasis and invasion ability than the control cells ( Figure 1 B, 1C). Thus, a model of recurrence and metastasis of KRAS mutant colorectal cancer cells has been successfully established, which will be used to further explore the mechanism of recurrence and metastasis.

[0034] 2. Mechanisms by which CD66b promotes migration and invasion of KRAS-mutant colorectal cancer by activating the KRAS-Cdc42-NE and MEK-ERK pathways

[0035] KRAS-related inflammatory signaling has been shown to contribute to the tumorigenesis and progression of KRAS-mutant CRC, and KRAS mutations can release tumor dormancy. Therefore, how KRAS mutations activate tumor recurrence and metastasis and lead to drug resistance, and its molecular regulatory mechanisms need further exploration.

[0036] In KRAS mutant colorectal cancer, the expression levels of KRAS and Cdc42 were significantly increased compared with the control group ( Figure 2 B), scratch test and Transwell migration and invasion assay showed that knockdown of Cdc42 could significantly reduce the migration and invasion ability ( Figure 2C). In summary, the KRAS-Cdc42 axis is activated in KRAS mutant colon cancer cell lines, promoting tumor metastasis and invasion. However, the specific molecules that activate the KRAS signaling pathway need further exploration. Therefore, the present application further performed co-immunoprecipitation and proximity ligation experiments, which were performed using in situ proximity ligation detection probes (from Thermo Fisher Scientific, product numbers DUO92002 and DUO92004, USA, according to the manufacturer's protocol). The specific operation steps are as follows: PDCs were placed on glass coverslips, fixed with 4% formaldehyde, and then incubated in a preheated humidity chamber at 37°C for 60 minutes to treat the blocking solution and incubated with the primary antibody overnight at 4°C. After washing, the cells were incubated with PLA probes MINUS (anti-mouse) and PLUS (anti-rabbit) at 37°C for 1 hour. Ligation and amplification were performed using an in situ detection kit. Cell nuclei were stained with DAPI. Images were captured by fluorescence scanning microscopy, and red spots indicate interactions between endogenous proteins. The experiment showed that the expression level of CD66b was significantly increased compared with the control group ( Figure 2 A), CD66b has endogenous interactions with KRAS or Cdc42 ( Figure 2 DE). Knockdown of CD66b reduces the expression of KRAS mRNA, and the dual luciferase reporter gene assay was used. Specifically, PROMO and JASPAR were used to predict the CD66b binding motif in the KRAS promoter region. The full-length and truncated sequences of the KRAS promoter were synthesized and then inserted into the pGL3-Basic vector (Genecfps, China). The cells were co-transfected with siRNA targeting the CD66b luciferase reporter plasmid (KRAS) for 48 hours. Luciferase activity was assessed using a dual luciferase assay kit (Yeasen, 11402ES60, China) according to the manufacturer's instructions. The experimental results showed that CD66b, as a transcription factor, directly binds to the KRAS promoter, activates the KRAS / Cdc42 pathway, and thus promotes the metastasis of KRAS mutant colorectal cancer in vitro ( Figure 2 F). Scratch wound healing and Transwell migration and invasion assays demonstrated that knockdown of CD66b reduced the metastatic and invasive ability of KRAS-mutated CRC cells ( Figure 2 GI). Based on the above, CD66b can lead to the continuous activation of KRAS. Next, it is necessary to explore whether activated KRAS will affect other downstream proteins that promote tumor metastasis. Previous studies have shown that the downstream signaling pathways of oncogenic KRAS are mainly composed of MAPK (RAF-MEK-ERK) and PI3K-AKT pathways. In this study, neutrophil elastase was overexpressed in the KRAS mutant colorectal recurrence and metastasis model ( Figure 2B), the expression of proteins KRAS, Cdc42 and Neutrophil elastase all decreased by knocking down the expression of CD66b, the expression of Cdc42 and Neutrophil elastase all decreased by knocking down the expression of KRAS, the expression of Cdc42 and Neutrophil elastase decreased by knocking down the expression of Cdc42, Neutrophil elastase, in general, CD66b promotes the migration and invasion of KRAS mutant colorectal cancer by activating the KRAS-Cdc42-NE pathway Figure 2 J-L). In summary, CD66b can be a viable therapeutic target for KRAS mutant colorectal cancer.

[0037] III. Identification of compounds inhibiting the binding of CD66b-KRAS through high-throughput virtual screening

[0038] Based on the direct action of CD66b on KRAS to activate the KRAS signaling pathway, developing drugs that can block the interaction between CD66b and KRAS may provide a new treatment strategy for KRAS mutant colorectal cancer. In addition, cetuximab as an epidermal growth factor receptor inhibitor has limited clinical efficacy in patients with KRAS mutant colorectal cancer. Whether used alone or in combination with other KRAS pathway inhibitors, it may provide a new strategy for treating KRAS mutant cancer.

[0039] The present application further carries out molecular docking experiment, the active ingredient compound structure obtained from FDA launch Drug Library and Hit Locator Library is introduced into ChemBio3D 14.0 software, the spatial conformation of active ingredient is adjusted, the optimized energy is calculated, and is saved as mol2 format. After processing by AutoDockTools 1.5.6, the file is saved as pdbqt format. The three-dimensional crystal structure of target protein CD66b and KRAS is downloaded from Uniprot. Water molecules and organic matter in the target protein are removed by Notepad2, and then the target protein is introduced into AutoDockTools 1.5.6 for hydrogenation, charge distribution and atom type addition. Save the pdbqt format file. Use AutoDockVina for molecular docking, and draw the docking results with Pymol 2.6. The experimental results show that CD66b and KRAS have good binding energy (docking score: -212.50 kcal / mol). ARG-77, ASP-154, PHE-141 and GLN-131 of KRAS can form 4 hydrogen bonds with ASN-78, ILE-68, GLN-54 and ASN-53 on CD66b respectively Figure 3 A).

[0040] To inhibit the binding of CD66b and KRAS, the present application utilizes the binding site between the two to screen high-efficiency inhibitors of CD66b-KRAS protein interaction. High-throughput virtual screening was performed using the FDA drug library and Hit Locating Library containing about 200,000 small molecule compounds. The protein and compound library were first pre-processed, then virtual screening was performed, and the screening results were sorted out. According to the docking score, chemical chemical principles and Lipinski's five rules, three compounds (chlorothiazide CAS No.: 77-36-1; haloperidol CAS No.: 548-73-2; celecoxib CAS No.: 169590-42-5) were selected for further verification. Among the three compounds, only celecoxib significantly inhibited the expression of CD66b and KRAS in a dose-dependent manner by Western blotting Figure 3 B). Therefore, celecoxib was selected for subsequent experiments. In addition, the present application conducted a molecular docking study on the binding of celecoxib to CD66b and KRAS. The sulfonamide group in celecoxib can form three hydrogen bonds with GLY-138, GLU-162 and LYS-165 on KRAS, with bond lengths of 2.2, 2.1 and 2.8 angstroms, respectively, which indicates that celecoxib disrupts the interaction of CD66b-KRAS Figure 3 C). Next, the present application treated Hct116 and LoVo high-invasive and metastatic KRAS mutant colorectal cancer cell recurrence and metastasis cells with 100 μM celecoxib for 24 h, and then used CCK8 to detect cell viability at different time points, and the results showed that celecoxib exhibited a significant inhibitory effect on the proliferation of these cell lines in a dose-dependent manner Figure 3D). In addition, to further validate the interaction of celecoxib with KRAS and CD66b, surface plasmon resonance (SPR) assay was performed by immobilizing KRAS and CD66b protein domains on the chip, respectively. Specifically, Biacore T200 system (GE Healthcare Life Sciences, Uppsala, Sweden) was used to quantitatively measure the interaction between the compound and the target protein. The purified target proteins KRAS and CD66b were directly immobilized on a carboxymethylated 5 (CM5) sensor chip, respectively, and then different concentrations of celecoxib were used as analytes for multi-cycle kinetic detection. The carboxyl groups on the CM5 chip (Cytiva) were first activated at 25°C with a mixture of EDC and NHS solution (Cytiva) at a flow rate of 10 μL / min for 7 min; then the target protein was injected into sodium acetate buffer (10 mM; pH 4.5) to immobilize the protein on the chip to a content of 3000-RU; finally the chip was blocked with ethanolamine. Multi-cycle kinetics, the target protein was directly immobilized on the CM5 chip, and then the gradient-diluted analyte was detected in turn, 1 concentration / cycle, with DMSO solvent correction, to detect the affinity and kinetics. Temperature: 25°C, injection: 30 uL / min, binding time 90 s, dissociation time 90 s, running buffer: 5% DMSO PBS-P. All results were analyzed by kinetics / affinity fitting analysis according to the 1:1 model, and the data were analyzed using Biacore evaluation software Biacore T200 Evaluation Software (T200 version 2.0). The results showed that the binding constant of celecoxib with KRAS was 0.93 μM, and the binding constant with CD66b was 0.53 μM Figure 3 E、3F). In addition, it was next explored whether these compounds could inhibit the protein-protein interaction between CD66b and KRAS. After Hct116-Tre and LoVo-Tre were treated with 100 μM celecoxib or Dmso for 24 h, the ability of CD66b and KRAS to bind in Hct116-Tre and LoVo-Tre was detected by co-immunoprecipitation technology, and the results showed that celecoxib significantly inhibited the interaction of endogenous CD66b with KRAS Figure 3 G).

[0041] Four, the therapeutic effect of celecoxib and cetuximab combination therapy on different KRAS genotypes of colorectal cancer cells

[0042] Most of the KRAS mutant CRC patients show resistance to EGFR inhibitors such as cetuximab. The present application further explores whether the combination of celecoxib and cetuximab can increase the sensitivity of different KRAS genotypes of colorectal cancer. The cells were divided into four groups for drug intervention for 24 hours, including the control group treated with the same dose of Dmso, the cetuximab group (25 μg / ml), the celecoxib group (100 μM), and the combination of cetuximab and celecoxib group (25 μg / ml cetuximab + 100 μM celecoxib). The Transwell experiment verified that cetuximab had no obvious inhibitory effect on the growth of two KRAS mutant colorectal cancer cells. As expected, both KRAS mutant cell lines were resistant to cetuximab treatment Figure 5 A). In contrast, the novel inhibitor of CD66b-KRAS interaction, celecoxib, produced significant growth inhibition in KRAS mutant Hct116 and LoVo Tre, and the combination of cetuximab and cetuximab maximally blocked the migration and invasion of cells in vitro Figure 5 A-B). In addition, KRAS downstream signaling pathways include MAPK (RAF-MEK-ERK) and PI3K-AKT pathways. Western blotting experiments showed that the use of celecoxib alone and the combination of cetuximab and celecoxib treatment both inhibited the CD66b / KRAS / Cdc42 / NE pathway and the CD66b / KRAS / MEK / ERK pathway, and the inhibition of these two pathways was significantly inhibited in two colorectal cancer cell lines with or without KRAS mutation (KRAS mutant colorectal cancer recurrent metastatic cells Hct116-Tre, LoVo-Tre and KRAS wild-type colorectal cancer recurrent metastatic cells HT29-Tre and Caco2-Tre) Figure 3 H). In contrast, the AKT / P-AKT pathway was up-regulated in the cetuximab group and the combination treatment group, indicating that the combination of cetuximab and celecoxib did not inhibit tumor metastasis through the AKT / P-AKT pathway but through the RAF-MEK-ERK pathway. In summary, these data suggest that celecoxib not only inhibits CD66b-KRAS interaction but also inhibits the CD66b / KRAS / Cdc42 / NE or RAS / MEK / ERK pathway, which may be an important mechanism for inhibiting the recurrence and metastasis of different KRAS genotype colorectal cancer cell lines.

[0043] Five, the combination of celecoxib and cetuximab improves the efficacy of different KRAS genotype colorectal cancer xenograft tumor models

[0044] Given that CD66b plays a key role in promoting oncogenic KRAS signaling, this application explores its potential as a therapeutic target for KRAS-induced malignancies. Most colorectal cancer patients develop resistance to cetuximab due to KRAS mutations. In order to further explore the therapeutic potential of combined therapy with celecoxib and cetuximab in vivo, two KRAS mutant colorectal cancer recurrent metastatic cells Hct116-Tre and LoVo-Tre and one KRAS wild-type colorectal cancer recurrent metastatic cell HT29-Tre were used to construct a colon cancer xenograft model with different KRAS genotypes in nude mice. Five-week-old female BALB / c nude mice were purchased from Beijing Weitong Lihua Company. All animal experiments were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee of Tianjin United Medical Center. The induced recurrent metastatic cells (5×10 6 cells / 100 μL) were subcutaneously injected into the right groin of 5-week-old female BALB / c nude mice. When the tumor volume reached approximately 150 cubic millimeters, the mice were randomly divided into four groups: a control group (normal saline), a cetuximab group (10 mg / kg), a celecoxib group (100 mg / kg), and a cetuximab combined with celecoxib group (10 mg cetuximab + 100 mg celecoxib / kg / day). There were 5 mice in each group. All groups received an intraperitoneal injection of 200 μL once a day. Tumor volume and weight were measured three times a week. After approximately 17 days of treatment, the mice were sacrificed and the tumors were removed, followed by immunohistochemistry and protein analysis.

[0045] After cell implantation, all mice received either saline, cetuximab, celecoxib, or a combination of celecoxib and cetuximab. Cetuximab alone had little effect on the growth of Hct116 and LoVo tumors with KRAS mutations, while celecoxib significantly inhibited the growth of xenograft tumors with and without KRAS mutations. The combination of celecoxib and cetuximab produced the greatest suppression of tumor growth in nude mice ( Figure 4 A, B). There was no significant difference in body weight between the control group and the treatment group ( Figure 5 D). Western blot analysis confirmed that celecoxib inhibited the growth of colorectal cancer cells with different KRAS genotypes (regardless of whether they had KRAS mutations) by blocking the CD66b / KRAS / Cdc42 / NE pathway or the RAS / MEK / ERK pathway ( Figure 4C). Cetuximab sensitizes KRAS mutant cancers to celecoxib by impairing activation of both pathways. Next, the expression of CD66b was detected by immunohistochemical detection method. Similar to the results of Western blot detection, the expression of CD66b in colorectal cancer tissues was low after the combined treatment of celecoxib and cetuximab, regardless of the presence of KRAS mutation Figure 5 C). In summary, celecoxib has therapeutic potential for KRAS mutant tumors.

[0046] The embodiments of the present specific implementation are the preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Therefore, any equivalent changes made in the structure, shape, and principle of the present application should be covered within the protection scope of the present application.

Claims

1. The use of a cyclooxygenase-2 inhibitor and a monoclonal antibody against vascular endothelial growth factor or epidermal growth factor receptor in combination for the preparation of drugs for the treatment of colorectal cancer with different KRAS genotypes.

2. The use according to claim 1, characterized in that: Celecoxib was used as the cyclooxygenase-2 inhibitor.

3. The use according to claim 1, characterized in that: Monoclonal antibodies against vascular endothelial growth factor or epidermal growth factor receptor are cetuximab or panitumumab.

4. The use according to claim 1, characterized in that: The mass ratio of cyclooxygenase-2 inhibitor and monoclonal antibody against vascular endothelial growth factor or epidermal growth factor receptor for in vivo treatment was 1:

10.

5. The use according to claim 1, characterized in that: Colorectal cancer with different KRAS genotypes includes KRAS mutant colorectal cancer and KRAS wild-type colorectal cancer.

6. A pharmaceutical composition, characterized in that: The composition includes a cyclooxygenase-2 inhibitor and a monoclonal antibody against vascular endothelial growth factor or epidermal growth factor receptor.

7. The pharmaceutical composition according to claim 6, characterized in that: Celecoxib was used as the cyclooxygenase-2 inhibitor.

8. The pharmaceutical composition according to claim 6, wherein: Monoclonal antibodies against vascular endothelial growth factor or epidermal growth factor receptor are cetuximab or panitumumab.

9. The pharmaceutical composition according to claim 6, characterized in that: The mass ratio of cyclooxygenase-2 inhibitor and monoclonal antibody against vascular endothelial growth factor or epidermal growth factor receptor for in vivo treatment was 1:

10.

10. Use of the pharmaceutical composition according to any one of claims 6 to 9 in the preparation of drugs for treating colorectal cancer of different KRAS genotypes.

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