CN111686111B

CN111686111B - Application of MALT1 protease inhibitor in preparation of non-small cell lung cancer therapeutic drug

Info

Publication number: CN111686111B
Application number: CN202010516594.7A
Authority: CN
Inventors: 王颖慧; 周美娟; 牛馨丽; 邓旭怡
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2023-06-13
Anticipated expiration: 2040-06-09
Also published as: CN111686111A

Abstract

The invention discloses an application of MALT1 protease inhibitor MI-2 in preparing a medicine for treating non-small cell lung cancer. MALT1 inhibitors MI-2 of the invention are effective in inhibiting the cell proliferation and migration capacity of NSCLC cell lines. In vivo experiments, the compound has excellent treatment effect on the in-situ lung cancer model of mice, effectively inhibits the growth of tumors, and can be used as an effective means for treating NSCLC for further research and development.

Description

Application of MALT1 protease inhibitor in preparation of non-small cell lung cancer therapeutic drug

Technical Field

The invention relates to the field of application of MALT1 protease inhibitors, in particular to application of MALT1 protease inhibitor MI-2 in preparation of non-small cell lung cancer therapeutic drugs.

Background

Lung cancer is the cancer of the highest incidence and mortality rate among the population, which is a growing trend. About 160 ten thousand people die annually worldwide from lung cancer, 85% of which are non-small cell lung cancers (NSCLC). In China, the incidence rate of lung cancer also shows an annual rising trend, and the new cases of 2025 are expected to be more than 100 ten thousand times, about 61 ten thousand people die of lung cancer each year, and the first place of cancer death in China is occupied. The early clinical symptoms of NSCLC patients are not obvious, the proportion of found or diagnosed is less than 15%, so that more than 80% of patients are not suitable for surgical resection because they are already at a clinically advanced stage or have poor physical function when found. Although treatment methods such as radiotherapy and chemotherapy are continuously advanced, for patients with locally advanced NSCLC, the existing combined radiotherapy and chemotherapy scheme is not obvious for improving prognosis.

Tyrosine inhibitors (TKIs) are drugs against EGFR mutated NSCLC, and are currently used in a large number of clinical applications. Clinical trial data showed that the Objective Remission Rate (ORR) was 51%, the Disease Control Rate (DCR) was 84%, and the Progression Free Survival (PFS) was 8.2 months. Cytology experiments prove that mutation of other genes in tumors can influence the effect of TKIs drugs on inhibiting proliferation of tumor cells, and researches of animal experiments show that the drugs can not effectively inhibit growth of tumors when being used alone, and at least two drugs are needed to be used simultaneously. Meanwhile, the use defects of the medicines are obvious, and after taking the medicines, the occurrence rate of adverse reactions is high, and unavoidable drug resistance can also occur. Another drug that is more commonly used is PD-1/PD-L1 antibody, which has a PFS prolongation of 4 months compared to conventional chemotherapy. However, the probability of adverse events of the immune response caused by the PD-1/PD-L1 antibody drugs is high. In addition to the two types of drugs mentioned above, drugs commonly used for treating NSCLC have problems such as drug resistance of ALK inhibitors. Therefore, the development of more effective and safer NSCLC therapeutic drugs is an important clinical problem to be solved currently.

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) was originally found in t (11; 18) (q 21; q 21) in MALT lymphomas. In lymphomas, MALT1 normally forms a CBM complex with BCL10 and CARMA1, which promotes the development and progression of lymphomas by activating NF- κb pathway. The NF- κb pathway has been shown by prior studies to be involved not only in the activation and development of B cells, but also in the development of genetic diseases, inflammatory responses and various malignant tumors in humans. Therefore, inhibitors against the NF- κb pathway are also widely used clinically as targeted drugs. Experiments show that blocking NF- κB pathway is an effective treatment in ABC-DLBCL lymphoma. In addition, NF- κB inhibition drugs are widely resistant to autoimmune and lymphoproliferative diseases. Although, it has been reported previously that MALT1 can exert a carcinomatous effect in melanoma by activating NF- κB pathway. However, the role of MALT1 in NSCLC is not yet clear, and whether it can also affect the occurrence and progression of NSCLC by affecting NF- κb pathway requires more research evidence to support.

Disclosure of Invention

The invention aims to provide an application of MALT1 protease inhibitor in preparing medicines for treating non-small cell lung cancer.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided:

the application of MALT1 protease inhibitor in preparing medicine for treating lung cancer is provided.

Further, the lung cancer is non-small cell lung cancer.

Further, the MALT1 protease inhibitor is MI-2.

In a second aspect of the invention, there is provided:

use of MALT1 protease inhibitors for the preparation of a medicament for cell proliferation and migration.

Further, the cells are non-small cell lung cancer cells.

Further, the non-small cell lung cancer cells include lung adenocarcinoma cells and large cell lung cancer cells. Of course, other non-small cell lung cancer (NSCLC) line cells can be reasonably replaced according to actual requirements.

Further, the lung adenocarcinoma cell line is A549, and the large cell lung cancer cell line is H460. Of course, other lung adenocarcinoma cell types and large cell lung cancer cell types can be reasonably replaced according to actual requirements.

Further, the MALT1 protease inhibitor is MI-2.

In a third aspect of the invention, there is provided:

use of MALT1 protease inhibitor in the preparation of NF- κb pathway inhibitor.

Further, the MALT1 protease inhibitor is MI-2.

The beneficial effects of the invention are as follows: in an in vitro experiment, a lung adenocarcinoma cell line A549 and a large cell lung cancer cell line H460 are taken as research objects, and the MALT1 inhibitor can inhibit the cell proliferation and migration capacity of an NSCLC cell line; in an in vivo experiment, a mouse in-situ lung cancer model is taken as an experimental object, and the MALT1 inhibitor can inhibit the growth of tumors and finally is used as an effective means for treating NSCLC.

Drawings

FIG. 1 is the effect of MI-2 on proliferation of A549 (A) and H460 (B) cells;

FIG. 2 is the effect of MI-2 on the clonogenic capacity of A549 (A) and H460 (B) cells (statistical plot on the right);

FIG. 3 is the effect of MI-2 on A549 (A) and H460 (B) cell migration (right panel is a statistical plot);

FIG. 4 is the effect of MI-2 on lung cancer in situ in mice: (a) the intensity of the fluorescent signal in the lungs of the mice at the end of the administration; (B) Is a statistical plot of the weekly mouse pulmonary fluorescence signal intensity; (C) a subscopic view of tissue sections after mice were sacrificed;

FIG. 5 is MI-2 inhibits activation of NF- κB pathway: (a) a549 cells; (B) H460 cells.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to the following specific embodiments. It should be understood that the detailed description is presented herein for purposes of illustration only and is not intended to limit the invention.

The experimental materials and reagents used, unless otherwise specified, are those conventionally available commercially.

Description of the Experimental procedures

1. Cell culture

A549 and H460 cells were selected as subjects at 37 ℃ with 5% co ₂ Subculturing was performed under culture conditions using DMEM containing 10% new born calf serum and 100U/ml penicillin-streptomycin. When the cell confluency is as long as 70% -80%, the cells are passaged and used for subsequent experiments.

2. Cell proliferation assay

Cells were seeded in 96-well plates and after 24h replaced with fresh medium containing different concentrations of MI-2. After 24h 10 μl CCK-8 was added to each well and the wells were incubated at 37deg.C with 5% CO ₂ After incubation for 2h in the cell incubator, the OD was read at 450nm using an enzyme-labeled instrument.

3. Cloning formation experiments

Inoculating 500 cells in 6-well plate, changing into fresh culture medium containing MI-2 at different concentration after 24 hr, placing at 37deg.C and 5% CO ₂ Incubate in cell incubator for 10-14 days. After clone formation, 3.7% formaldehyde was used for fixation and stained with 0.1% crystal violet. The number of colonies containing more than 50 cells was counted and statistically analyzed.

4. Cell migration experiments

When the cells grow to logarithmic phase, the cells are digested and counted to obtain 4x10 ⁴ Individual cells were resuspended in 200 μl DMEM. The transwell chamber was placed in a 24-well plate, 750. Mu.l fresh medium was added to the lower chamber, 200. Mu.l cell suspension was added to the upper chamber, and the plate was placedAt 37℃with 5% CO ₂ After 16h incubation in the cell incubator, 3.7% formaldehyde was used for fixation and stained with 0.1% crystal violet. 5 different fields of view were selected under a high power microscope to take pictures and counted using Image J software. The counting results were statistically analyzed.

5. In-situ lung cancer model of mice

BALB/c female nude mice were purchased for 4-6 weeks and experiments were performed one week after the nude mice were acclimatized. Anesthesia with isoflurane, mice were placed in right lateral position on a laboratory bench, 4x10 ⁶ Individual a549-Luc cells were injected directly into the left lung of the mouse at 45 ° along the posterior axillary line with a 29G insulin needle at a depth of about 3-5mm. After one week of cell inoculation, the mice were randomly divided into two groups, a control group and an MI-2 group, wherein the control group was 3 and the MI-2 group was 4, according to body weight. From one week after cell inoculation, MI-2 groups were intraperitoneally injected with MI-2 (dissolved using 5% DMSO in PBS) at a dose of 25mg/kg per day, and control groups were given equal volumes of 5% DMSO in PBS for a total of 3 weeks. Tumor growth In mice was observed using a multi-mode In vivo small animal imaging system (In-vivo Fx Pro, bruker), and D-sodium fluorescein salt was injected at a dose of 150mg/kg once a week prior to imaging, and tumor growth was analyzed based on fluorescence intensity and area calculations.

6. Statistical analysis

Statistical analysis was performed using SPSS 20.0 software. Using independent sample one-way analysis of variance, all data were statistically significant using a two-sided test and analysis of variance alignment with p <0.05, p <0.01, p < 0.001.

Experimental results

MI-2 inhibits NSCLC cell proliferation and migration

In A549 and H460 cells, treatment was performed with different concentrations of MI-2 for 24H, and proliferation and migration capacity of NSCLC cells was examined by CCK-8, clonogenic assay, and transwell migration assay, respectively. As shown in FIG. 1, the CCK-8 experimental results show that the cell proliferation capacity is continuously reduced with the increase of MI-2 dose, and the difference is statistically significant. As shown in FIG. 2, the results of the clone formation experiment for 14 days show that the larger the MI-2 dose, the smaller the clone number, and the cells cannot form clones under the condition of large dose, and the difference has statistical significance. As shown in FIG. 3, the results of the Transwell migration experiments revealed that the ability of cells to pass through the chamber decreased significantly with increasing MI-2 dose, and the differences were statistically significant. The above results demonstrate that MI-2 can effectively inhibit proliferation and migration of NSCLC cells, thereby achieving the purpose of inhibiting the development of NSCLC.

MI-2 inhibits tumor growth in vivo

And detecting the influence of MI-2 on tumor growth in vivo by establishing an in-situ model of the lung cancer of the mice. The results showed that after 3 weeks of continuous dosing, the pulmonary fluorescence signal intensity of mice in the dosing group began to decline and remained stable after one week of self-administration (fig. 4A-4B), while the pulmonary fluorescence signal intensity of mice in the control group continued to increase, and that the pulmonary tissue sections of mice in the MI-2 group after sacrifice showed less actual lung changes than in the control group, and that the alveoli were in a normal morphology (fig. 4C), indicating that MI-2 could inhibit the development of lung tumors in mice.

MI-2 inhibits activation of NF- κB pathway

In A549 and H460 cells, MI-2 with different concentrations is used for processing for 24 hours, and the expression condition of the NF- κB pathway key protein p-p65 is detected through a western blot experiment. As shown in FIG. 5, the expression of p-p65 decreased with increasing MI-2 dose. The above results indicate that MI-2 may regulate proliferation and migration capacity of NSCLC by inhibiting activation of NF- κB pathway.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

Use of MI-2 as sole active substance for the preparation of a medicament for the treatment of non-small cell lung cancer, said MI-2 being capable of inhibiting proliferation and migration of non-small cell lung cancer cells and of inhibiting NF- κb pathway.
2. The use according to claim 1, wherein the non-small cell lung cancer cells comprise lung adenocarcinoma cells, large cell lung cancer cells.
3. The use according to claim 2, wherein the lung adenocarcinoma cell line is a549 and the large cell lung carcinoma cell line is H460.