Application of SRC inhibitor and FAK inhibitor in preparation of medicine for inhibiting lung cancer metastasis
A technology of lung cancer metastasis and inhibitor, applied in the field of biomedicine, can solve the problem of unclear molecular mechanism of lung cancer metastasis, and achieve the effect of improving clinical treatment effect and inhibiting migration and invasion.
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Embodiment 1
[0036] Tumor tissues from NSCLC patients were collected, including primary lung tumors, lymph nodes, brain, and pleural metastases, and after making paraffin sections, immunohistochemical staining was performed to detect the expression of SRC protein and FAK protein. After baking, dewaxing, hydration, and blocking, add SRC or FAK primary antibody working solution dropwise and incubate overnight. The next day, add pika universal secondary antibody working solution dropwise for incubation, and add DAB (diaminobenzidine) for color development. After counterstaining with hematoxylin, hydrochloric acid alcohol differentiation, dehydration and transparency, observe under the microscope. The expression of SRC protein is as follows figure 1 As shown, the expression of FAK protein is as follows figure 2 shown. Depend on figure 1 It can be seen that compared with the primary lung lesions, the levels of SRC protein in lymph node metastases, brain metastases and pleural metastases we...
Embodiment 2
[0040]Using Flag and HA as fusion protein tags, the interaction between SRC protein and FAK protein was studied by co-immunoprecipitation (Co-IP) and western blot (Western blot, WB). Construct the truncated expression plasmids of the full length of SRC or FAK and different structural domains. After transfecting 293T cells, use RIPA buffer and protease inhibitor mixture to extract the protein, then add 20 μL protein A / G agarose beads, and incubate at 25°C 1h, to remove non-specific binders. Collect the supernatant, mix it with the mixture containing Flag antibody and HA antibody (1μg), incubate overnight at 4°C, then add 50μL protein A / G agarose beads, incubate for 2h, and then wash with fresh cold RIPA buffer 3 times to obtain protein precipitation. The protein precipitate was dissolved in the loading buffer and separated by SDS-PAGE. The experimental results of co-immunoprecipitation and western blotting were as follows: Figure 5 shown. Depend on Figure 5 It can be seen...
Embodiment 3
[0042] To investigate the influence of SRC / FAK in downstream pathways that promote 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 the American Type Culture Collection (ATCC) in 2015. The above NSCLC cells were 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 37°C, containing 5% CO 2 , and cultured under saturated humidity conditions, and the cells with a passage number less than 5 were collected for experiments.
[0043] Among them, NSCLC cells without any inhibitor treatment were used as the control group, and NSCLC cells treated with KX2-391 and PF-573228 were used as the experimental group, and the results were as follows: Figure 6 shown. Depend on Figure 6 It can be seen that the downstream signaling pathways of SRC prote...
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