Application of GC7 in inhibition of cancer cell proliferation or metastasis

Abstract

The invention relates to application of GC7 in inhibiting proliferation or metastasis of cancer cells, in particular to inhibition of proliferation or metastasis of lung adenocarcinoma cells, and more particularly inhibition of proliferation or metastasis of the lung adenocarcinoma cells in the brain. On the basis, the GC7 can be developed into a medicine for inhibiting proliferation or migration of lung cancer cells, particularly a medicine for inhibiting brain metastasis of the lung cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to the application of GC7 in inhibiting the proliferation or metastasis of cancer cells. Background Art

[0002] Worldwide, lung cancer has been rated as the most common cancer in the past few decades. It is one of the malignant tumors with the highest incidence and mortality rates and the greatest threat to human health. Among them, most lung cancer deaths are caused by metastasis. The most common metastatic site of lung cancer is the brain, and up to 50% of lung cancers will eventually develop into brain metastases. Brain metastasis is the most common malignant tumor in the central nervous system, accompanied by poor prognosis of patients. The prognosis of patients is greatly affected after lung cancer metastasizes, with a median survival period of only 5 months, and the survival rate of untreated patients with brain metastases from lung cancer is only 4 to 7 weeks.

[0003] So far, the treatment of lung cancer patients still mainly focuses on the treatment of primary tumors. The treatment of lung cancer after metastasis, especially after brain metastasis, remains a major challenge. Therefore, it has become an urgent matter to find an efficient, mild and specific method for treating brain metastases from lung cancer. Summary of the Invention

[0004] One aspect of the present invention provides the application of GC7 in the preparation of a drug for inhibiting the proliferation or migration of lung cancer cells.

[0005] In a specific embodiment, the GC7 is used to inhibit the proliferation or migration of lung cancer cells in the brain.

[0006] In a specific embodiment, the lung cancer cells are lung adenocarcinoma cells.

[0007] Another aspect of the present invention provides the application of GC7 in the preparation of a drug for inhibiting the brain metastasis of lung cancer cells.

[0008] In a specific embodiment, the lung cancer cells are lung adenocarcinoma cells.

[0009] The chemical name of GC7 used in the present invention is N-(7-aminoheptyl)guanidine, with a CAS number of 150333-69-0, and the chemical molecular formula is as follows: .

[0010] Advantages of the present invention: The present invention discovers that GC7 can be used to inhibit lung cancer cells, especially to inhibit the proliferation or migration of lung adenocarcinoma cells, and more particularly to inhibit the proliferation or migration of lung adenocarcinoma cells in the brain. Based on this, GC7 can be developed into a drug for inhibiting the proliferation or migration of lung cancer cells, especially a drug for inhibiting the brain metastasis of lung cancer cells. Brief Description of the Drawings

[0011] Figure 1 It shows the effect of GC7 sulfate on the migration rate of A549 cells.

[0012] Figure 2 It shows the effect of GC7 sulfate on the migration rate of XW-05 cells.

[0013] Figure 3 It shows the effect of GC7 sulfate on the proliferation rate of A549 cells.

[0014] Figure 4 It shows the effect of GC7 sulfate on the proliferation rate of XW-05 cells.

[0015] Figure 5 It shows the effect of GC7 sulfate on the proliferation rate of BEAS-2B lung epithelial cells.

[0016] Figure 6 It shows the effect of GC7 sulfate on the development of XW-05 lung adenocarcinoma cells in the brain tissue of mice. Detailed implementation manners

[0017] The above content of the present invention will be further described in detail below in the form of preferred implementation cases, but it does not constitute a limitation to the present invention.

[0018] Unless otherwise specified, the strains, cell lines, plasmids and reagents in the embodiments of the present invention are all conventional products or can be purchased through commercial channels.

[0019] The GC7 used in the present invention is its sulfate form product, namely GC7 sulfate, which is purchased from MedChemExpress, abbreviated as MCE.

[0020] BEAS-2B cells are normal lung epithelial cells; both A549 and XW-05 are lung adenocarcinoma cells, and they are all purchased from Hefei Wanwu Biotechnology Co., Ltd., with the product numbers being Delf-10130, Delf-10116 and Delf-10145 in sequence. All cells in the embodiments of the present invention are cultured using DMEM high-glucose medium (purchased from Viva Cell, product number C3113-0500). Example 1: Effect of GC7 sulfate on the migration rate of A549 and XW-05 cells

[0021] About 5×10 were seeded in a 6-well plate culture dish 5Cells per well were seeded to reach a density of 90% after 24 h of cell culture; after 24 h of culture, the cells were scratched with a pipette tip (with moderate force to ensure a straight and clear line); the cells were gently rinsed 3 times with PBS buffer to remove the scratched cells, and the position was photographed and marked under a microscope (recorded as 0 h at this time); culture medium was added (wherein, the treatment group was added with GC7 sulfate at a final concentration of 100 μmol / L; the control group was not added with GC7 sulfate). Three replicates were set, with 3 culture wells for each replicate. Then it was placed in an incubator at 37 °C and 5% CO2 for continued culture for 24 h; photographed under a microscope (recorded as 24 h at this time); the scratch areas at 0 h and 24 h were statistically analyzed by ImageJ, and the value of (scratch area at 0 h - scratch area at 24 h) / scratch area at 0 h was regarded as the cell migration rate. The results are as Figure 1 and Figure 2 shown.

[0022] Figure 1 and Figure 2 The results of and showed that the migration rates of A549 and XW-05 cells in the GC7 sulfate treatment group were significantly lower than those in the control group, indicating that GC7 sulfate reduced the migration rates of lung adenocarcinoma cells A549 and XW-05. Example 2: Effect of GC7 sulfate on the proliferation rate of A549 and XW-05 cells

[0023] Prepare the thiazolyl blue (MTT) working solution: Add MTT solvent (purchased from Beyotime, product number C009S-2) to 25 mg of MTT (purchased from Beyotime, product number C009S-1) powder for dissolution to prepare an MTT working solution with a final concentration of 5 mg / mL.

[0024] The proliferation rates of A549 and XW-05 cells were determined by the MTT assay using an MTT cell proliferation and cytotoxicity detection kit (purchased from Beyotime, catalog number C0009S). The specific steps were as follows: 4000 cells of the same type were seeded in each well of a 96-well plate. Among them, in the treatment group, GC7 sulfate was added at a final concentration of 100 μmol / L, while in the control group, GC7 sulfate was not added. Three replicates were set up, with 4 culture wells in each replicate. The cells were incubated in an incubator at 37 °C with 5% CO2 for 24 hours; then 10 μL of MTT working solution was added to each well and incubation continued for 4 hours, during which purple formazan crystals could be seen forming in the cells; 100 μL of formazan solubilization solution (purchased from Beyotime, catalog number C009S-3) was added to each well, gently mixed, and the formazan crystals were dissolved at 37 °C. Incubation continued until all the crystals inside the cells were dissolved (usually within 3 to 4 hours); the absorbance was measured using an ELISA reader with a 570 nm filter. Then the absorbance values were normalized, that is, the control group was standardized to 1 (Relative change), and the value of the treatment group was the absorbance of the treatment group / the absorbance of the control group. This ratio was regarded as the proliferation rate of the cells in the treatment group. The results are shown in Figure 3 and Figure 4 .

[0025] Figure 3 and Figure 4 The results of Example 3: Effect of GC7 sulfate on the proliferation rate of BEAS-2B normal lung epithelial cells

[0026] Replace the A549 or XW-05 cells in Example 2 with BEAS-2B cells, and the other conditions are the same as in Example 2. The proliferation rate results of BEAS-2B cells were obtained based on the absorbance values measured by the ELISA reader and are shown in Figure 5 .

[0027] Figure 5 The results of Example 4: Effect of GC7 sulfate on the development of XW-05 lung adenocarcinoma cells in the brain tissue of nude mice

[0028] luc2-4CMV-tdTomato, abbreviated as tdT, was purchased from Shanghai Heyuan Biotechnology Co., Ltd., catalog number HY-LV-000179.

[0029] The XW-05 cells were first infected with lentivirus luc2-4CMV-tdTomato to label the XW-05 cells with red fluorescence and luciferase tags, aiming to facilitate subsequent in vivo fluorescence imaging and photography.

[0030] Approximately 5×10 5 XW-05 cells per well were seeded in a 6-well plate culture dish. Among them, in treatment group 1, GC7 sulfate with a final concentration of 100 μmol / L was added to the cell culture medium and incubated at 37 °C for 24 h; the difference between treatment group 2 and treatment group 1 was that the incubation time was 48 h, and the control group was incubated for 48 h without adding GC7 sulfate. After incubation, the cells were collected and counted.

[0031] The cells from the above treatment group 1, treatment group 2, and control group were used for in vivo brain injection of BALB / c Nude female nude mice. Three replicates were set, with 1 nude mouse as 1 replicate. The specific operation was as follows: Using the three-dimensional coordinate system defined by the external auditory canal outside the mouse skull as a reference point to determine the brain injection position. Localization of the Bregma point: Select a position in the middle of the brain, disinfect it with iodine or alcohol, longitudinally cut the scalp, separate the skin left and right with hemostatic forceps, scrape off the cranial periosteum with a scalpel, or corrode the cranial periosteum with hydrogen peroxide. After completely removing the periosteum, the Bregma point was revealed. The Bregma point value was read with a brain locator as the origin O of the three-dimensional coordinates. For the localization of the lateral ventricle position, starting from the origin, move 2 mm to the right and 2 mm upward, which is the injection point. Gently drill through the skull with a needle, and use a 10-μL microinjector for injection. The injection depth was approximately between 2.5 mm and 3 mm. The injection dose was 1×10 6 cells / 10 μL. Slowly inject the cell suspension into the mouse brain. The injection time was about 30 min. After injection, stop the needle for 10 min, and then slowly pull out the syringe needle. Suture the mouse wound. After the mouse wakes up, place it in the culture room for feeding. On the 2nd, 4th, and 6th days after injection, use a multi-dimensional real-time awake animal in vivo optical imaging system (PHOTON IMAGERTM OPTIMA) to take in vivo fluorescence images of the test animals. The PhotoAcquisition software was used for optical signal acquisition, and the M3 Vision analysis software was used for processing, quantification, and output of the acquisition results. The fluorescence signal values of lung cancer cells in the brains of the test animals could be obtained through the two software, and thus the proliferation and migration of lung cancer cells in the brains of the test animals could be evaluated. Draw a line graph based on the obtained fluorescence values, and compare the logarithms of the ratios of the fluorescence values of the three groups of mice on the 6th day and the 2nd day pairwise. Use the unpaired t-test for difference analysis to reflect the growth of lung cancer cells in the brains of the three groups of mice. The results are as Figure 6 shown.

[0032] Figure 6 The results showed that, compared with the control group (Ctrl), after incubating XW-05 with GC7 sulfate for 24 h and then injecting it into the brains of nude mice by stereotactic brain injection, there was no significant difference in the proliferation and migration of XW-05 in the mouse brains; while after incubating XW-05 with GC7 sulfate for 48 h and then injecting it into the brains of nude mice by stereotactic brain injection, both the proliferation and migration of XW-05 in the mouse brains were significantly reduced.

Claims

1. Use of GC7 in the preparation of a drug for inhibiting the proliferation or migration of lung cancer cells.

2. The application according to claim 1, characterized in that, The GC7 is used for inhibiting the proliferation or migration of lung cancer cells in the brain.

3. The application according to claim 1, wherein The lung cancer cells are lung adenocarcinoma cells.

4. Use of GC7 in the preparation of a drug for inhibiting the brain metastasis of lung cancer cells.

5. The application according to claim 4, wherein The lung cancer cells are lung adenocarcinoma cells.

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