Application of Caspase-1 inhibitor in preparation of medicine for preventing non-small cell lung cancer brain metastasis caused by drug resistance

Abstract

The invention discloses application of a Caspase-1 inhibitor in preparation of a medicine for preventing non-small cell lung cancer brain metastasis caused by drug resistance, and belongs to the technical field of biomedicine. The invention provides an application of a Caspase-1 inhibitor in preparation of a medicine for preventing brain metastasis of cancer caused by drug resistance. The invention also provides a pharmaceutical composition for preventing brain metastasis of cancer caused by drug resistance. The pharmaceutical composition comprises the Caspase-1 inhibitor. The Caspase-1 inhibitor can prevent or reduce the occurrence of drug-resistant brain metastasis in the non-small cell lung cancer treatment process, and provides a new strategy and drug for improving the prognosis of patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of a Caspase-1 inhibitor in the preparation of a drug for preventing or reducing brain metastasis of non-small cell lung cancer caused by drug resistance. Background Art

[0002] The incidence and mortality of lung cancer are both among the top of malignant tumors. Among them, non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer. Brain metastases are the most common distant metastases of non-small cell lung cancer. The prognosis of patients with brain metastases of non-small cell lung cancer is extremely poor. However, the mechanism of brain metastases in non-small cell lung cancer remains unclear.

[0003] Currently, the clinical treatment methods for non-small cell lung cancer mainly include chemotherapy, radiotherapy, targeted therapy, immunotherapy, and combined therapy, etc. Platinum-containing chemotherapy is the main treatment method for first-line chemotherapy of non-small cell lung cancer clinically. Among them, Cisplatin is one of the most widely used chemotherapy drugs. However, the incidence of Cisplatin resistance is very high. Multiple reported studies have found that Cisplatin resistance can enhance the invasion and metastasis ability of tumor cells. And the previous in vitro and in vivo studies of the present invention have found that Cisplatin-resistant non-small cell lung cancer has stronger brain metastasis ability than its parental cells, proving that Cisplatin resistance can promote the brain metastasis of non-small cell lung cancer. The failure of chemotherapy is usually accompanied by the emergence of drug resistance and the recurrence and metastasis of tumors. Studies have shown that chemotherapy-resistant tumor cells have stronger invasion and metastasis ability than non-resistant parental cells, which may be the main reason for the rapid recurrence and metastasis of cancer patients after treatment failure. Therefore, how to prevent or reduce the occurrence of malignant brain metastases in clinical patients after conventional chemotherapy resistance has become the key to improving the prognosis of patients with non-small cell lung cancer. Currently, there are no strategies and drugs for preventing and reducing brain metastases in non-small cell lung cancer after drug resistance in clinical practice. Summary of the Invention

[0004] Aiming at the defect that there is a lack of strategies and drugs for preventing or reducing brain metastases of non-small cell lung cancer after treatment resistance clinically, the purpose of the present invention is to design and provide a new use of a Caspase-1 inhibitor that can prevent or reduce brain metastases of Cisplatin-resistant cancer cells.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0006] On the one hand, the present invention provides the use of a Caspase-1 inhibitor in the preparation of a drug for preventing brain metastases of cancer caused by drug resistance.

[0007] The use described above, wherein the Caspase-1 inhibitor is Belnacasan (VX-765).

[0008] The use described above, wherein the chemical structural formula of Belnacasan (VX-765) is as shown in the following formula (Ⅰ);

[0009]

[0010] The use described above, wherein the drug in the drug resistance is a cancer treatment drug capable of activating the caspase-1 signaling pathway.

[0011] Most cancer drugs activate the caspase-1-mediated inflammatory signal after treatment, and the caosase-1 inhibitor can inhibit the release of inflammatory factors such as IL-1β, and the release of these inflammatory factors can promote the interaction between tumors and cerebrovascular endothelial cells (for example, IL-1β can increase the expression of endothelial cell adhesion protein Vcam1 and promote the adhesion between tumors and endothelial cells), resulting in brain metastasis. Therefore, it can be expected that as long as the cancer treatment drug that can activate the caspase-1 signaling pathway develops drug resistance, it can promote brain metastasis.

[0012] The use described above, wherein the drug in the drug resistance is a platinum-containing chemotherapy drug.

[0013] The use described above, wherein the drug in the drug resistance is a cisplatin chemotherapy drug.

[0014] The use described above, wherein the cancer is a cancer with high expression of Caspase-1 before brain metastasis occurs.

[0015] The use described above, wherein the cancer is non-small cell lung cancer, breast cancer or colon cancer.

[0016] The use described above, when using mice as experimental subjects, the dosage of the Caspase-1 inhibitor is 50-100 mg / kg of the Caspase-1 inhibitor, and the drug is administered continuously for 7 days.

[0017] In a second aspect, the present invention provides a pharmaceutical composition for preventing brain metastasis of cancer caused by drug resistance, comprising a Caspase-1 inhibitor.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention discovers that Caspase-1 is highly expressed in cisplatin-resistant non-small cell lung cancer and promotes the occurrence of brain metastasis. By inhibiting the enzymatic activity of Caspase-1, the probability of its brain metastasis is reduced. Its mechanism is clear, indicating that inhibitors of Caspase-1 can prevent or reduce the occurrence of brain metastasis after drug resistance during the treatment of non-small cell lung cancer, providing new strategies and drugs for improving the prognosis of patients. Brief Description of the Drawings

[0020] Figure 1 Data showing that cisplatin resistance promotes brain metastasis in non-small cell lung cancer. Among them, A. Schematic diagram of the establishment of a nude mouse model of lung cancer brain metastasis; B. On the 7th day of modeling, the probability of brain metastasis was detected using small animal in vivo imaging technology. There were 30 mice in the A549 group and 20 mice in the A549-DDP group; C. Tumor development was shown using small animal in vivo imaging technology on the 21st day of modeling; D. Colonization was shown using small animal in vivo imaging technology on the 21st day of modeling, *P<0.05; E. IF staining of frozen serial sections was used to detect the distribution of metastatic tumor lesions. Green represents GFP, tumor cells; blue represents DAPI, cell nuclei; F. The size of brain metastasis foci was represented by the ratio of the areas of GFP and DAPI. There were 5 mice in each group, and the data were expressed as mean ± SEM, *P<0.05; G. The number of brain metastasis foci in the parental group and the A549-DDP group.

[0021] Figure 2 Data showing that knockdown of Caspase-1 inhibits the occurrence of brain metastasis in cisplatin-resistant non-small cell lung cancer. Among them, A. The expression levels of Caspase-1 and IL-1β in parental and A549-DDP cells were detected by Western blotting, with β-actin as the internal reference; B. The knockdown effect of Caspase-1 in A549-DDP cells was detected by Western blotting; C. Schematic diagram of the establishment of a brain metastasis tumor model; D. Brain metastasis in the control group (shNC) and the knockdown group (shCaspase1) was detected using small animal in vivo imaging technology on the 1st, 7th, and 14th days of modeling; E. Incidence of brain metastasis in the control group (shNC) and the knockdown group (shCaspase1) on the 1st, 7th, and 14th days of modeling.

[0022] Figure 3Data on the reduction of brain metastases in cisplatin-resistant non-small cell lung cancer by Belnacasan (VX-765). A. Western blot was used to detect the inhibitory effect of Belnacasan (VX-765) on Caspase-1 enzyme activity (left panel), and the relative protein levels were statistically analyzed by analyzing the protein gray values of Caspase-1 and its substrates IL-1β and cleaved-IL-1β (right panel). β-actin was used as an internal reference, p<0.001; B. Enzyme-linked immunosorbent assay was used to detect the levels of IL-1β-related proteins in cell culture supernatants; C. Schematic diagram of the establishment of a brain metastasis tumor model and drug administration; D. Enzyme-linked immunosorbent assay was used to detect the expression level of IL-1β in the sera of nude mice after continuous administration (Vehicle or Belnacasan (VX-765), 50 mg / kg) for 7 days. The data are expressed as mean ± SEM, p<0.01; E. After sampling, a small animal in vivo imaging system was used to detect the probability of brain metastases (GFP + ); F. The probability of brain metastases in non-small cell lung cancer was detected in the Vehicle group and Belnacasan (VX-765) of the present invention. Detailed implementation manners

[0023] The present invention will be further described below with reference to the accompanying drawings and examples.

[0024] Example 1:

[0025] To evaluate whether cisplatin resistance promotes the occurrence of brain metastases in non-small cell lung cancer, the present invention established a mouse model of lung cancer brain metastases using parental and cisplatin-resistant A549 cell lines.

[0026] (1) Culture of parental and cisplatin-resistant cell lines: The non-small cell lung cancer cell line A549 used in the present invention was purchased from ATCC (cat.#CRM-CCL-185), and the A549-DDP cells were obtained by screening in the laboratory (drug resistance index: ~18). DMEM medium containing 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin double antibody was used and cultured at 37°C and 5% CO2. Among them, 1 μg / mL of cisplatin was added additionally to the medium of A549-DDP cells to maintain growth. A549-GFP-Luci and A549-DDP-GFP-Luci cells were constructed using lentivirus for subsequent in vivo imaging and immunofluorescence analysis.

[0027] (2) Establishment of a mouse model of lung cancer brain metastasis: In this invention, the above-mentioned parental and drug-resistant cells are used to construct a nude mouse model of lung cancer brain metastasis by injecting tumor cells into the left ventricle (the method is the same as that of Arguello F, et al., 1992): Anesthetize nude mice aged 5 - 6 weeks appropriately, place them on their backs, disinfect the chest, and use a syringe of 0.5 mL (27 - 28G needle) to insert the needle at a 45° angle 6 mm into the left ventricle 3 mm to the right of the sternum at the second intercostal space. When obvious blood return is seen and the blood pulsates with the heartbeat, slowly and intermittently push 3×10 6 tumor cells suspended in 100 μL of PBS into the heart, and then slowly withdraw the needle after injection ( Figure 1 A). According to the above method, we established nude mouse models of brain metastasis using control A549-GFP-Luci and A549-DDP respectively. At 7 days and 21 days after modeling, the probability and size of brain metastasis were detected by small animal in vivo imaging. After 50 days, the samples were taken and the size of brain metastasis foci was further measured by immunofluorescence of serial brain sections. At 7 days after modeling (the time point when lung cancer cell extravasation can be detected in the brain parenchyma), the results of in vivo imaging showed that cisplatin resistance promoted the probability of brain metastasis in non-small cell lung cancer ( Figure 1 B). After 21 days of modeling, the development and colonization of tumors were observed by small animal in vivo imaging technology, and the results showed that cisplatin resistance promoted the growth of lung cancer brain metastasis ( Figure 1 C - D). At about 50 days after modeling, the number and size of brain metastasis foci were detected by analysis of serial brain tissue sections, and the results showed that cisplatin resistance promoted the colonization and proliferation of non-small cell lung cancer brain metastasis cells in the brain ( Figure 1 E - G).

[0028] (3) Immunofluorescence staining: In this invention, the mice with lung cancer brain metastasis are deeply anesthetized, then the mice are quickly decapitated, the brain tissues are dissected and immersed in ice-cold PBS, and then washed three times. The mouse brain tissues are fixed in 4% PFA at 4°C for 24 hours, and then sequentially transferred to 15% and 30% sucrose buffer overnight for dehydration at 4°C. After 24 hours, they are taken out and embedded in OCT. Using the method of frozen serial sectioning, cut into thin slices 30 μm thick, take 1 slice every 10 slices to make a brain section set and store it in the antifreeze solution. At room temperature, place it on a SuperFrost Plus slide (Fisher), then stick the slice in 1×PBS, directly seal it with a mounting medium containing DAPI, air dry and examine under the microscope to analyze the probability of brain metastasis, the number and size of brain metastasis foci (since A549 used in this experiment is a cell line stably expressing GFP, there is no need to stain the tumor cells). The experimental results showed that compared with the parental cells, cisplatin resistance of A549-DDP promoted the occurrence of brain metastasis in non-small cell lung cancer.

[0029] Example 2:

[0030] To explore the role of Caspase-1 in cisplatin-resistant brain metastases, the present invention uses specific shRNA of Caspase-1 to knockdown the expression of Caspase-1 in A549-DDP.

[0031] (1) Western blot was used to verify the expression of Caspase-1 and IL-1β in parental cells and drug-resistant cells: Proteins were lysed and extracted with 1×SDS, denatured in a metal bath at 100 °C, and then subjected to SDS-PAGE gel electrophoresis (constant voltage: 80 V for 30 minutes in the stacking gel and about 1 hour at 120 V in the separating gel). Then, a nitrocellulose (NC) membrane was used for membrane transfer (constant current: 220 mA for 90 minutes). The membrane was blocked with 5% skim milk at room temperature for 1 hour, followed by overnight incubation with the primary antibody diluted in TBST at 4 °C. The membrane was washed three times with TBST for 10 minutes each time, then incubated with the secondary antibody diluted in TBST at room temperature for 1 hour, and washed three times with TBST for 10 minutes each time. Finally, the protein levels of Caspase-1 and IL-1β were detected by developing with the GV6000 instrument, with β-actin as the internal reference. The results are as Figure 2 shown in A, indicating that the protein levels of Caspase-1 and IL-1β in A549-DDP cells were significantly higher than those in parental cells.

[0032] (2) Construction of control and A549-DDP cell lines with stable knockdown of Caspase-1: The present invention uses lentivirus to construct control (shNC) and A549-DDP cell lines with stable knockdown of Caspase-1 (shCaspase-1). The knockdown effect of Caspase-1 in cisplatin-resistant cells A549-DDP was verified by Western blot: The method was the same as (1) above. The results are as Figure 2 shown in B.

[0033] (3) Construction of cisplatin-resistant brain metastasis mice with knockout of Capsase-1: A brain metastasis model was constructed using the stable knockdown cells constructed in (2) above, with the method the same as step (2) in Example 1. Each nude mouse was injected with 0.5*10 6 tumor cells, and the probability of brain metastasis was detected by small animal in vivo imaging technology on days 1, 7, and 14 after modeling ( Figure 2 C). The results of in vivo imaging 24 hours later showed that the brain metastasis models were all successfully established ( Figure 2 D). The results of in vivo imaging on days 7 and 14 showed that knockdown of Capsase-1 significantly inhibited the occurrence of brain metastasis in cisplatin-resistant cells ( Figure 2 D-E). The experimental results of in vivo imaging showed that compared with the control, knockdown of Caspase-1 significantly reduced the probability of brain metastasis in non-small cell lung cancer. There were 7 mice in the shNC group and 5 mice in the shCaspase1 group.

[0034] Example 3:

[0035] Demonstrate that inhibitors of Caspase-1 reduce the probability of brain metastasis in cisplatin-resistant non-small cell lung cancer.

[0036] (1) Western blot was used to verify that Belnacasan (VX-765) (Cat. No.: HY-13205, MCE) reduced the accumulation of cleaved-IL-1β: A549-DDP cells were seeded in 6-well plates. When the cell density reached about 80%, 20 μM of Benacasan (VX-765) was added and the cells were treated for 24 hours. In the control group, an equal volume of DMSO (solvent control) was added. Using the method of Western blot, the protein levels of Caspase-1, IL-1β, cleaved-IL-1β, and β-actin as an internal reference were detected as in step (1) of Example 2. The results of Western blot are shown in Figure 3 Figure A, indicating that Belnacasan (VX-765) could inhibit the protease activity of Caspase-1 in vitro cells, reduce the protein level of cleaved-IL-1β, and did not change the protein levels of Caspase-1 and IL-1β.

[0037] (2) ELISA was used to detect the secretion of IL-1β: First, cell culture supernatant samples were prepared. A549-DDP was seeded in 6-well plates. When the cell density reached about 80%, serum-free DMEM containing 20 μM of Benacasan (VX-765) or an equal volume of DMSO (solvent control) was added. After 24 hours, the cell culture supernatant was collected, filtered through a 0.22 μm cell filter, and reserved. The level of IL-1β in the medium was detected using an ELISA kit for IL-1β (cat.#ab217608, abcam) according to the instructions. The results of ELISA are shown in Figure 3 Figure B, indicating that Belnacasan (VX-765) could inhibit the protease activity of Caspase-1 in vitro cells and reduce the secretion of cleaved-IL-1β protein. It was demonstrated that Belnacasan (VX-765) could inhibit the enzyme activity of Caspase-1 in vitro cells and reduce the secretion of IL-1β.

[0038] (3) Treatment of cisplatin-resistant brain metastasis mice with Belnacasan (VX-765): A brain metastasis model was constructed using A549-DDP-GFP cells, and the method was the same as in step (2) of Example 1 above. Each nude mouse was injected with 1×10 6Tumor cells. One day after modeling, the mice were randomly divided into two groups: a control group and an inhibitor group. Through intraperitoneal injection, 50 mg / kg Benacasan (VX-765) and Vehicle (PBS) for the control group were administered continuously for 7 days ( Figure 3 C). On the 9th day of modeling, tail vein blood was taken to obtain serum, and the content of IL-1β in the serum was detected by enzyme-linked immunosorbent assay. The results of the enzyme-linked immunosorbent assay showed that Benacasan (VX-765) could inhibit the enzyme activity of Caspase-1 in vivo and reduce the content of IL-1β in the serum ( Figure 3 D). The mice were sacrificed 50 days after modeling to obtain the mouse brains, and the brain metastasis probability was detected by small animal in vivo imaging technology. The results showed that compared with the Vehicle group, Benacasan (VX-765) could reduce the brain metastasis probability of cisplatin-resistant cells in vivo ( Figure 3 E-F), n = 8 in the Vehicle group, and n = 8 in the Belnacasan group.

[0039] Conclusion: The feasibility of the present invention has been verified through in vitro and in vivo experiments. The in vitro experiments verified the inhibitory effect of the Caspase-1 inhibitor on the enzyme activity of Caspase-1 by Western blot and enzyme-linked immunosorbent assay; the in vivo experiments confirmed that the specific shRNA and inhibitor of Caspase-1 could significantly reduce the incidence of brain metastasis by constructing a mouse model of lung cancer brain metastasis. The experimental results showed that the Caspase-1 inhibitor could prevent or reduce the probability of brain metastasis of cisplatin-resistant non-small cell lung cancer.

Claims

1. Use of a Caspase-1 inhibitor in the preparation of a drug for preventing brain metastasis caused by cancer due to drug resistance.

2. The use according to claim 1, characterized in that, The Caspase-1 inhibitor is Belnacasan (VX-765).

3. The use according to claim 2, characterized in that, The chemical structural formula of the Belnacasan (VX-765) is shown as the following formula (Ⅰ); 4. The use according to claim 1, characterized in that, The drug in the drug resistance is a cancer treatment drug capable of activating the caspase-1 signaling pathway.

5. The use according to claim 1, wherein, The drug in the drug resistance is a platinum-containing chemotherapeutic drug.

6. The use according to claim 1, characterized in that, The drug in the drug resistance is a cisplatin chemotherapeutic drug.

7. The use according to claim 1, wherein The cancer is a cancer with high expression of Caspase-1 before brain metastasis occurs.

8. The use according to claim 1, characterized in that, The cancer is non-small cell lung cancer, breast cancer or colon cancer.

9. The use according to claim 1, characterized in that, When using mice as experimental subjects, the dosage of the Caspase-1 inhibitor is 50 - 100 mg / kg of the Caspase-1 inhibitor, and it is administered continuously for 7 days.

10. A pharmaceutical composition for preventing brain metastasis caused by drug resistance in cancer, characterized in that, Contains a Caspase-1 inhibitor.

Images