CN114288410B - Application of SRC inhibitor and FAK inhibitor in preparation of medicines for inhibiting lung cancer metastasis - Google Patents
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Abstract
The invention provides application of SRC inhibitor and FAK inhibitor in preparation of medicines for inhibiting lung cancer metastasis. According to research, SRC proteins and FAK proteins play an extremely important role in metastasis of lung cancer, SRC and FAK are potential lung cancer prognosis biomarkers, and are both promising treatment targets, especially in the case of lymph node metastasis or distant metastasis. The SRC inhibitor and the FAK inhibitor are combined to inhibit the signal paths (such as an epithelial-mesenchymal transition signal path and an angiogenesis signal path) related to lung cancer cell metastasis, so that migration and invasion of lung cancer cells are inhibited, and the SRC inhibitor and the FAK inhibitor are applied to preparation of medicaments for inhibiting lung cancer metastasis, so that the prepared medicaments have remarkable inhibiting effect on lung cancer cell metastasis. It has great significance for improving the clinical treatment effect of metastatic lung cancer.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to application of SRC inhibitors and FAK inhibitors in preparation of medicines for inhibiting lung cancer metastasis.
Background
Lung cancer is one of the most common malignant tumors with highest morbidity and mortality. The global morbidity and mortality of lung cancer were statistically ranked second and first in 2020. Lung cancer is largely divided into two pathological types, non-small cell lung cancer (non-small cell lung cancer, NSCLC) and small cell lung cancer (small cell lung cancer, SCLC), with NSCLC accounting for about 85% of lung cancer and SCLC accounting for about 15% of lung cancer. Where feasible, surgery remains the most prominent means of treating lung cancer. Although treatment regimens such as targeted therapy and immunotherapy have been advanced clinically in recent years, nearly 70% of NSCLC patients already have metastases at the time of diagnosis, which results in a loss of surgical opportunity and poor prognosis.
Metastasis is a difficult clinical challenge in almost all types of malignancies. Tumor metastasis is a biological process of a multi-step cascade involving tumor cell migration away from the primary focus, migration to distant organs by different means, colonization in other organs, expansion, and adaptation to the new environment of the organ being metastasized. The specific molecular mechanisms of lung cancer metastasis are currently unknown, and thus the development of targeted drugs for metastatic lung cancer is urgent.
Disclosure of Invention
The invention aims to provide application of SRC inhibitor and FAK inhibitor in preparation of medicines for inhibiting lung cancer metastasis, and application of SRC inhibitor and FAK inhibitor in preparation of medicines for inhibiting lung cancer metastasis can effectively inhibit migration and invasion of lung cancer cells, and further effectively inhibit lung cancer metastasis.
According to a first aspect of the present invention there is provided the use of an SRC inhibitor and a FAK inhibitor in the manufacture of a medicament for inhibiting lung cancer metastasis.
The non-receptor tyrosine kinase SRC (Proto-oncogene tyrosine-protein kinase Src) is a 60kD molecular weight protein encoded by the Proto-oncogene SRC and is the first oncoprotein found to have tyrosine kinase activity. From the N-terminus to the C-terminus, SRC proteins consist of SH4 domain, specific fragment, SH3 domain, SH2 domain, linker, SH1 domain (protein tyrosine kinase domain,) and carboxy-terminal regulatory tail. Previous studies have demonstrated that SRC is overexpressed and highly activated in a variety of tumor cells, and is involved in a variety of biological processes such as cell proliferation, invasion, metastasis, etc.
Focal adhesion kinase (focal adhesion kinase, FAK) is a non-receptor tyrosine kinase in cells, which is highly expressed in most tissues and has high homology in protein sequences in many species. FAK is a junction of a plurality of signal transduction pathways in cells, is involved in a plurality of biological processes such as tumor formation, proliferation, metastasis, apoptosis and cardiovascular diseases, and is one of the antitumor targets which are widely focused at present.
The SRC inhibitor and the FAK inhibitor are jointly applied to the preparation of the drug for inhibiting lung cancer metastasis, and can inhibit signal paths (such as an epithelial-mesenchymal transition signal path and an angiogenesis signal path) related to lung cancer cell metastasis, so that migration and invasion of lung cancer cells are inhibited, and further lung cancer metastasis is effectively inhibited, and the inhibition effect of the combined use of the two inhibitors is obviously better than that of a single inhibitor.
Preferably, the lung cancer is non-small cell lung cancer.
Preferably, the SCR inhibitor comprises at least one of KX2-391, BMS354825, AZD-053, SKI-606, PP1, PP2, PD 166326.
Preferably, the SCR inhibitor is KX2-391.
The chemical structural formula of SRC for inhibiting KX3-391 is as follows:
preferably, the FAK inhibitor comprises at least one of PF-573228, PF-562271, GSK 2256098.
Preferably, the FAK inhibitor is PF-573228.
The FAK inhibitor PF-573228 has the following chemical structural formula:
according to a second aspect of the present invention there is provided a pharmaceutical composition for inhibiting lung cancer metastasis, the pharmaceutical composition comprising an SRC inhibitor and a FAK inhibitor.
Preferably, in the above pharmaceutical composition, the SCR inhibitor comprises at least one of KX2-391, BMS354825, AZD-053, SKI-606, PP1, PP2, PD 166326.
Preferably, in the above pharmaceutical composition, the SCR inhibitor is KX2-391.
Preferably, in the above pharmaceutical composition, the FAK inhibitor comprises at least one of PF-573228, PF-562271, GSK 2256098.
Preferably, in the above pharmaceutical composition, the FAK inhibitor is PF-573228.
Preferably, the mass ratio of SRC inhibitor to FAK inhibitor in the pharmaceutical composition is 0.5-2:0.5-2.
The beneficial effects of the invention are as follows: through researches, SRC proteins and FAK proteins play an extremely important role in metastasis of lung cancer, and SRC and FAK are potential lung cancer prognosis biomarkers and are both promising treatment targets, especially in the case of lymph node metastasis or distant metastasis. Whereas the combined use of SRC inhibitors and FAK inhibitors can inhibit signaling pathways associated with lung cancer cell metastasis (e.g., epithelial-mesenchymal transition signaling pathway and angiogenic signaling pathway), thereby inhibiting lung cancer cell migration and invasion. The two inhibitors are applied to the preparation of medicaments for inhibiting lung cancer metastasis, and the prepared medicaments have remarkable inhibiting effect on lung cancer cell metastasis. It has great significance for improving the clinical treatment effect of metastatic lung cancer.
Drawings
FIG. 1 is a diagram showing the expression of SRC protein in lung primary foci, lymph node metastases, brain metastases and pleural metastases.
FIG. 2 is a graph showing the expression of FAK protein in primary lung foci, lymph node metastases, brain metastases and pleural metastases.
FIG. 3 is a graph showing the relationship between SRC expression level and survival in NSCLC patients.
FIG. 4 is a graph showing the relationship between FAK expression level and survival in NSCLC patients.
FIG. 5 is a graph showing the results of co-immunoprecipitation of FAK and SRC proteins.
FIG. 6 is a graph showing the expression of proteins/factors associated with the down stream signaling pathway of SRC protein and FAK protein.
Fig. 7 is a graph showing the effect of SRC inhibitors and FAK inhibitors on lung cancer cell migration.
FIG. 8 is a graph showing the effect of SRC inhibitors and FAK inhibitors on lung cancer cell invasion.
Fig. 9 is a graph showing the effect of SRC inhibitor and FAK inhibitor combination on mouse lung tumor nodules.
Detailed Description
The technical features of the technical solution provided in the present invention will be further clearly and completely described in connection with the detailed description below, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Tumor tissues of NSCLC patients, including primary lung foci and lymph node, brain and pleural metastasis, were collected, paraffin sections were prepared, and immunohistochemical staining was performed to detect expression of SRC protein and FAK protein. After baking, dewaxing, hydrating and sealing, SRC or FAK primary antibody working solution is dripped for incubation overnight. The next day, the mice and rabbits were incubated with the universal secondary antibody working solution, and DAB (diaminobenzidine) was added for color development. After hematoxylin counterstain, hydrochloric acid alcohol differentiation, dehydration and transparency, observation is carried out under a microscope. The expression of SRC protein is shown in FIG. 1, and the expression of FAK protein is shown in FIG. 2. As can be seen from FIG. 1, the levels of SRC protein were significantly increased in lymph node metastasis, brain metastasis and pleural metastasis, as compared to the primary lung foci. As can be seen from FIG. 2, the results of the FAK protein are consistent with those of the SRC protein, and the levels of the FAK protein in the lymph node metastasis, brain metastasis and pleural metastasis are significantly increased as compared to the primary lung foci.
Meanwhile, clinical data and protein expression information of NSCLC patients in TCGA and GEO databases are utilized to analyze prognosis conditions of the NSCLC patients, a KM-plot survival curve is drawn, the relation between SRC expression quantity and survival time in the NSCLC patients is shown in figure 3, and the relation between FAK expression quantity and survival time in the NSCLC patients is shown in figure 4. As can be seen from fig. 3, the survival of the patient with high SRC expression in the primary lung cancer is relatively short, which suggests that the high SRC expression in the primary lung cancer suggests that the patient is ill-prognosticated. As can be seen from fig. 4, the survival of patients with high expression of FAK in the primary foci of lung cancer is also relatively short, indicating that high expression of FAK in the primary foci of lung cancer suggests poor prognosis for patients.
The above results demonstrate that SRC and FAK play a key role in the progression and metastasis of NSCLC.
Example 2
The interaction between SRC protein and FAK protein was studied using Co-immunoprecipitation (Co-IP) and Western Blotting (WB) with Flag and HA as fusion protein tags. Truncated expression plasmids of the full length and different domains of SRC or FAK were constructed, and after transfection of 293T cells, proteins were extracted using RIPA buffer and protease inhibitor cocktail, and then 20. Mu.L of protein A/G agarose beads were added and incubated for 1h at 25℃to remove non-specific binders. The supernatant was collected, mixed with a mixture (1. Mu.g) containing Flag antibody and HA antibody, incubated overnight at 4℃and then 50. Mu.L of protein A/G agarose beads were added, incubated for 2h, and then washed 3 times with fresh cold RIPA buffer to give a protein precipitate. Protein pellet was dissolved in loading buffer and separated using SDS-PAGE, and experimental results of co-immunoprecipitation and immunoblotting are shown in FIG. 5. From fig. 5, it can be seen that the tyrosine kinase domain of FAK protein is capable of interacting with the SH2 domain of SRC protein. The above results demonstrate that there is an interaction between SRC protein and FAK protein, both of which play an important role in metastasis and progression of lung cancer.
Example 3
To investigate the effect of SRC/FAK in downstream pathways promoting tumor metastasis, western blot analysis was performed using NSCLC cells. The human NSCLC cell lines H1975 and H460 used in this example were purchased from American type culture Collection (American Type Culture Collection, ATCC) in 2015. The NSCLC cells are stored in DMEM medium or RPMI-1640 medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), penicillin (100 μg/mL) and streptomycin (100 μg/mL), and placed in an incubator at 37deg.C with 5% CO 2 Culturing under saturated humidity, and collecting cells with passage number less than 5 for experiment.
The results of the experiment in which NSCLC cells not treated with any inhibitor were used as a control group and NSCLC cells treated with KX2-391 and PF-573228, respectively, were used as an experimental group, are shown in FIG. 6. As can be seen from fig. 6, the SRC protein is similar to the downstream signaling pathway of FAK protein, where one pathway is the epithelial-mesenchymal transition signaling pathway, which is inhibited by treatment with both inhibitors, and appears to be up-regulated by ECD (epithelial cadherin), and down-regulated by NCD (N-cadherin), vimentin (Vimentin); the other common signal pathway is an angiogenesis pathway, and after treatment with two inhibitors, the angiogenesis pathway is inhibited, which is shown to be down-regulated by all of the pro-angiogenic factors VEGF (vascular endothelial growth factor), MMP9 (matrix metalloproteinase 9) and MMP2 (matrix metalloproteinase 2). The above results demonstrate that both SRC inhibition and FAK inhibitors are capable of inhibiting signaling pathways associated with tumor metastasis (epithelial-mesenchymal transition signaling pathway and angiogenic pathway), thereby inhibiting metastasis of lung cancer.
Example 4
The effect of SRC inhibitors (KX 2-391), FAK inhibitors (PF-573228), and combinations thereof (KX 2-391+PF-573228) on the migratory and invasive abilities of NSCLC cells was studied using transwell experiments. Will be about 2 x 10 3 The NSCLC cells were resuspended in 200. Mu.L of DMEM serum-free medium, inoculated into the upper chamber of a transwell well, and KX2-391, PF-573228 or KX2-391+PF-573228 (at a drug concentration of 10. Mu. Mol/L) were added thereto, and the lower chamber was filled with DMEM medium containing 15% fetal bovine serum using NSCLC cells not treated with any inhibitor as a control.
Wherein, when a migration experiment is carried out, NSCLC cells are directly planted in an upper chamber and are added with inhibitor for treatment; in the invasive test, 40. Mu.L of Matrigel gel was coated on the bottom of the chamber, and after standing at 37℃for 1h, NSCLC cells were inoculated and inhibitor was added for treatment after gelling.
The results of the cell migration experiment are shown in FIG. 7. As can be seen from FIG. 7, after SRC inhibitor KX2-391 or FAK inhibitor PF-573228 treatment, migration of both NSLCL cells was inhibited, and the combined use of SRC inhibitor KX2-391 and FAK inhibitor PF-573228 was more remarkable in the migration inhibition effect on NSCLC cells.
The results of the cell invasion assay are shown in figure 8. As can be seen from FIG. 8, after SRC inhibitor KX2-391 or FAK inhibitor PF-573228 treatment, invasion of both NSCLC cell lines was inhibited, and the combined use of SRC inhibitor KX2-391 and FAK inhibitor PF-573228 showed more remarkable invasion inhibiting effect on NSCLC cells.
The results show that the SRC inhibitor and the FAK inhibitor can inhibit the migration and invasion of lung cancer cells when used independently, and the migration and invasion inhibition effect of the SRC inhibitor and the FAK inhibitor on the lung cancer cells is more obvious when used in combination, which indicates that the SRC inhibitor and the FAK inhibitor can obviously inhibit the migration and invasion of lung cancer when used in combination, thereby achieving the effect of inhibiting the metastasis of lung cancer.
Example 5
Female nude mice of 6 weeks of age were randomly divided into 2 groups of 6 animals each. H1975 cells were resuspended in PBS buffer to adjust the cell concentration to 10 7 And injected into mice via tail vein. Each mouse was injected with 200. Mu.L of liquid, 2X 10 6 Cells/cells. 4 weeks after implantation, intraperitoneal administration (i.e., simultaneous injections of SRC inhibitor KX2-391 and FAK inhibitor PF-573228 in the abdominal cavity) was initiated. Mice injected with PBS buffer were used as control groups, and mice injected with 5mg/kg KX2-391+PF-573228 were used as experimental groups, administered once every 3 days for a total of 2 weeks. After reaching the experimental end point, the mice were sacrificed, the lungs of the mice were dissected, the lung tumor nodules were visually observed, and the number and size of tumor nodules were measured. Finally, the tumors were pelleted and HE stained (i.e. hematoxylin-eosin staining method), and the tumor tissue was observed under a microscope, and the results are shown in FIG. 9.
As can be seen from the staining results of FIG. 9, H1975 lung cancer cells were injected into mice via tail vein, and after 4 weeks, the mice had several scattered metastatic tumor nodules formed in the lungs, and the number and size of the mice' lung tumor nodules could be significantly reduced by the combined treatment of KX2-391 and PF-573228 inhibitors.
The results of the above examples show that SRC and FAK are both potential lung cancer prognosis biomarkers, and are both promising therapeutic targets, especially in the case of lymphatic or distant metastasis, whereas SRC inhibitors and FAK inhibitors can inhibit signaling pathways (such as epithelial-mesenchymal transition signaling pathways and angiogenesis signaling pathways) related to lung cancer cell metastasis, thereby inhibiting migration and invasion of lung cancer cells, and the two inhibitors are applied in preparation of drugs for inhibiting lung cancer metastasis, have significant inhibitory effects on lung cancer cell metastasis, and are significant in improving clinical therapeutic effects of metastatic lung cancer.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention, but these modifications or substitutions are all within the scope of the present invention.
Claims (1)
1. Use of KX2-391 in combination with PF-573228 for the manufacture of a medicament for inhibiting metastasis of non-small cell lung cancer.
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