Application of lopinavir in the preparation of drugs for preventing gastric cancer metastasis

Abstract

The present invention relates to the use of lopinavir in preparing a drug for preventing gastric cancer metastasis. The lopinavir can inhibit the metastasis of gastric cancer cells and has good application prospects in the current clinical situation where there is a lack of effective means for preventing and treating gastric cancer metastasis.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of lopinavir in preparing a drug for preventing gastric cancer metastasis. Background Art

[0002] Gastric cancer is one of the most common digestive system malignancies. Its morbidity and mortality rates are particularly high in developing countries, including my country, ranking third among cancer-related deaths. In recent years, the incidence of gastric cancer has been trending toward younger patients. However, the early detection rate of gastric cancer in my country is low, with most patients presenting to the clinic in the advanced stages. Cancer cell infiltration and distant metastasis are the main causes of treatment failure for advanced gastric cancer. With increasing metastatic disease, the 5-year survival rate of gastric cancer patients after surgery decreases from 97% (stage IA) to 6% (stage IV). However, effective interventions for gastric cancer metastasis are currently lacking.

[0003] Tumor metastasis is a multistep process. After detaching from the primary tumor, tumor cells invade the vasculature, survive in the circulatory system, and metastasize through the bloodstream, eventually extravasating from the circulation and colonizing distant organs. Because epithelial cells rely on the extracellular matrix and intercellular connections for survival, anoikis is triggered once they shed from the extracellular matrix. Therefore, the ability of tumor cells to survive in the absence of the extracellular matrix (i.e., to acquire resistance to anoikis) is crucial for tumor metastasis. Inducing anoikis in tumor cells may be important for preventing gastric cancer metastasis.

[0004] Lopinavir (LPV), also known as ABT-378, is one of the active ingredients in the combination drug Kaletra, approved for marketing by the US FDA in 2000. It is primarily used to prevent and treat human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). As an antiretroviral protease inhibitor, lopinavir exerts its anti-HIV effect primarily by blocking the cleavage of the Gag-Pol polyprotein, producing immature, non-infectious viral particles. Furthermore, lopinavir has also been explored in the clinical treatment of pneumonia caused by the 2019 novel coronavirus. Although lopinavir is not included in standard treatment regimens, clinical data show that early intervention with lopinavir can significantly inhibit viral replication, thereby reducing the risk of transmission and effectively lowering the risk of severe illness. However, there are no reports of lopinavir's use in anti-tumor treatment in domestic or international literature or domestic invention patents. Summary of the Invention

[0005] The present invention aims to provide the use of lopinavir in the preparation of a drug for preventing gastric cancer metastasis. The lopinavir can inhibit the metastasis of gastric cancer cells and has good application prospects in the current lack of effective means for preventing and treating gastric cancer metastasis in clinical practice.

[0006] The technical solution of the present invention is:

[0007] Use of lopinavir in the preparation of drugs for preventing gastric cancer metastasis.

[0008] The drug promotes anoikis in AGS gastric cancer cells.

[0009] The drug inhibits the metastasis of gastric cancer cells.

[0010] The drug is administered by intraperitoneal injection.

[0011] The dosage of the drug is 10 mg / kg / time.

[0012] Lopinavir is a commonly used anti-HIV drug in clinical practice. Experiments have shown that lopinavir can promote anoikis in gastric cancer cells, including AGS, in vitro, with a good dose-response relationship. Injecting human gastric cancer cells into the tail vein of nude mice, simulating the in vivo resistance of gastric cancer cells to anoikis and the resulting metastasis, confirmed in vivo that lopinavir can inhibit gastric cancer cell metastasis. Given the current lack of effective clinical treatments for gastric cancer metastasis, this drug holds great promise for future application.

[0013] To test whether lopinavir has the effect of preventing gastric cancer metastasis, the present invention first cultured gastric cancer cells in vitro using ultra-low adsorption culture plates to simulate the survival state of gastric cancer cells in the absence of extracellular matrix in the circulatory system, and further used lopinavir to treat the cells in the culture plates. By using flow cytometry to detect gastric cancer cell apoptosis, the effect of lopinavir on tumor cell anoikis was evaluated in vitro; secondly, human gastric cancer cells were injected into the tail vein of nude mice to simulate the situation in which gastric cancer cells resist anoikis and eventually metastasize in animals, and small animal in vivo imaging technology was further used to evaluate the effect of lopinavir on gastric cancer metastasis. The drug lopinavir used in the present invention can promote anoikis of gastric cancer cells cultured in vitro and inhibit the metastasis of gastric cancer cells in nude mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The effect of lopinavir on apoptosis of AGS gastric cancer cells in suspension culture;

[0015] Figure 2 The effect of lopinavir on the metastasis of gastric cancer cells in nude mice. DETAILED DESCRIPTION

[0016] Sources of main reagents in the examples:

[0017] DMEM medium (Gibco), fetal bovine serum, PBS (Zhongshan Jinqiao), 0.25% trypsin (Gibco), Poly-HEMA (Sigma), lopinavir (MCE), Annexin V-FITC apoptosis detection kit (BD Biosciences), D-luciferin potassium salt (Solaribio), and corn oil (Biyuntian).

[0018] Human gastric cancer AGS cells were purchased from ATCC, and nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd. and raised in the Experimental Animal Center of Army Medical University (SPF grade).

[0019] Example 1 In vitro assay to detect the effect of lopinavir on anoikis in gastric cancer cells

[0020] 1.1 Experimental Methods

[0021] (1) Preparation of Lopinavir:

[0022] 50 mg of lopinavir purchased from MCE was added to 1.6 ml of DMSO and fully dissolved to a concentration of 50 mM. The solution was then dispensed into 100 μl tubes and stored at -80°C.

[0023] (2) Cell culture plate coating:

[0024] Weigh 1g Poly-HEMA, add 100ml 95% ethanol, and shake to dissolve until the solution is clear. Add 1ml of the prepared solution to each well of a 6-well plate and place it in a 65℃ incubator to air-dry. After air-drying, place it in a clean bench and sterilize it with UV irradiation. Rinse the plate once with PBS before use.

[0025] (3) Cell inoculation and treatment:

[0026] Cells in logarithmic growth phase were obtained and digested with trypsin to prepare single cell suspension. After cell counting, 5x10 4 The liquid in the culture wells was supplemented to 2 ml with complete culture medium, and the cells in the culture wells were treated with 25 μM or 50 μM lopinavir for 48 hours (the control group was added with an equal volume of DMSO).

[0027] (4) Flow cytometry detection of cell apoptosis:

[0028] Collect the cell suspension in the well plate and centrifuge to obtain the cell pellet. After rinsing the pellet with PBS, add 100ul of 1xBinding buffer containing 2ul Annexin V-FITC and 2ul PI to resuspend the pellet. Incubate in the dark for 15min and then detect on the instrument.

[0029] 1.2 Experimental Results

[0030] The proportions of AnnexinV-FITC single-positive and AnnexinV-FITC / PI double-positive cells were calculated to reflect the cell apoptosis rate. The results showed that the apoptosis rate of suspended cultured cells in the DMSO group was 13.2%, the apoptosis rate of cells in the 25μM lopinavir treatment group was 29.55%, and the apoptosis rate of cells in the 50μM lopinavir treatment group was 60.09%, indicating that lopinavir can significantly promote anoikis in gastric cancer cells.

[0031] Example 2: In vivo experiments to detect the effect of lopinavir on gastric cancer metastasis

[0032] 2.1 Experimental methods:

[0033] (1) Preparation of Lopinavir:

[0034] 10 mg of lopinavir purchased from MCE was added to 5 ml of corn oil and fully dissolved to a concentration of 2 mg / ml. The solution was prepared immediately before administration to the animals.

[0035] (2) Construction of a nude mouse tail vein gastric cancer model and drug intervention:

[0036] Gastric cancer cells in logarithmic growth phase that stably express firefly luciferase gene were obtained, digested with trypsin to prepare single cell suspension, and cell pellet was obtained by centrifugation. After rinsing once with PBS, the pellet was resuspended in PBS and the cells were counted. The final concentration was 5x10 7 Each nude mouse was injected with 100ul of the cell suspension via the tail vein in an SPF environment. After the injection, the mouse was randomly divided into two groups. The control group was injected with 150ul of corn oil intraperitoneally, and the lopinavir-treated group was injected with 150ul of the drug prepared in (1) (about 10 mg / kg body weight) intraperitoneally.

[0037] The drug was administered once every other day for 6 consecutive weeks, and then each nude mouse was intraperitoneally injected with 150 mg / kg body weight of D-luciferin potassium salt. Ten minutes later, the drug was detected using a small animal in vivo imaging device.

[0038] 2.2 Experimental Results

[0039] Compared with the control group, the nude mice in the lopinavir-treated group showed significantly reduced fluorescence intensity and area under the small animal in vivo imaging instrument.

Claims

1. Use of lopinavir in the preparation of a drug for preventing gastric cancer metastasis, wherein the drug promotes anoikis in gastric cancer cells.

2. The use according to claim 1, characterized in that: The drug inhibits the metastasis of gastric cancer cells.

3. The use according to claim 1, characterized in that: The drug is administered by intraperitoneal injection.

4. The use according to claim 1, characterized in that: The dosage of the drug is 10 mg / kg / time.

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