Background
Gastric cancer is one of the most common malignant tumors of the digestive system, and has prominent morbidity and mortality in developing countries including China, and is the third leading cause of cancer-related death. In recent years, the onset of gastric cancer tends to be younger, however, the early gastric cancer discovery rate in China is low, and most patients visit middle and late stages. Infiltration of cancer cells and distant metastasis are major causes of failure in treatment of progressive gastric cancer, and as the degree of metastasis increases, the 5-year survival rate of gastric cancer patients after surgery decreases from 97% (stage IA) to 6% (stage IV), however, there is currently no effective intervention for gastric cancer metastasis.
Tumor metastasis is a multi-step process in which tumor cells invade the vasculature after shedding from primary foci, survive in the circulatory system and metastasize with the blood circulation, exude from the circulation and colonize distant organs. Since epithelial cells need to survive depending on extracellular matrix and intercellular junctions, anoikis is triggered once they shed from the extracellular matrix, so that tumor cells survive without extracellular matrix (i.e., gain resistance to anoikis), and are critical for tumor metastasis. Inducing anoikis of tumor cells may have important significance in preventing metastasis of gastric cancer.
Lopinavir (Lopinavir, LPV), also known as ABT-378, is one of the active ingredients of the compound formulation of clitoli approved by the FDA in 2000 in the united states, and is mainly used for preventing and treating Human Immunodeficiency Virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). Lopinavir, as an antiretroviral protease inhibitor, acts against HIV virus by blocking the division of Gag-Pol polyprotein, producing immature, non-infectious viral particles. In addition, lopinavir has also been tried for its clinical treatment in pneumonia caused by 2019 novel coronavirus infection. Although lopinavir is not included in standard treatment regimens, clinical data shows that administration of lopinavir intervention early in a new coronavirus infection significantly inhibits viral replication thereby reducing the risk of transmission and effectively reducing the risk of severe symptoms. However, the lopinavir is used for anti-tumor treatment, and no reports are available in the domestic and foreign documents and domestic patent inventions.
Disclosure of Invention
The invention aims to provide application of lopinavir in preparing medicines for preventing gastric cancer metastasis, and the lopinavir can inhibit gastric cancer cell metastasis, and has good application prospects under the condition that effective means for preventing and treating gastric cancer metastasis are lacking clinically at present.
The technical scheme of the invention is as follows:
The application of lopinavir in preparing medicines for preventing gastric cancer metastasis.
The medicament promotes the anoikis of AGS gastric cancer cells.
The medicine inhibits gastric cancer cell metastasis.
The medicine is administrated by intraperitoneal injection.
The administration dosage of the medicine is 10 mg/kg/time.
The lopinavir is a common clinical anti-HIV drug, and experiments prove that the lopinavir can promote the anoikis of gastric cancer cells such as AGS and the like in vitro and has a better dose response relationship. Human gastric cancer cells are injected by vein injection of the tail of the naked tail, the situation that the gastric cancer cells resist anoikis in vivo so as to finally transfer is simulated, and the in vivo experiments of the model prove that lopinavir can inhibit the transfer of gastric cancer cells. Under the condition that effective means for preventing and treating gastric cancer metastasis are lacking clinically at present, the application prospect is good.
In order to detect whether lopinavir has the effect of preventing stomach cancer metastasis, the invention firstly cultures stomach cancer cells in vitro by adopting an ultralow adsorption culture plate, simulates the survival state of the stomach cancer cells under the condition that a circulatory system does not have extracellular matrixes, further treats the cells in the culture plate by using lopinavir, and evaluates the influence of lopinavir on tumor cell anoikis in vitro by adopting a flow cytometry to detect the apoptosis condition of the stomach cancer cells, secondly simulates the condition that the stomach cancer cells resist anoikis and finally metastasize in vivo by intravenous injection of human stomach cancer cells in an animal body by using a naked tail, and further evaluates the influence of lopinavir on stomach cancer metastasis by adopting a living animal imaging technology. The lopinavir drug used in the invention can promote the in vitro cultured gastric cancer cells to lose nest and apoptosis and inhibit the transfer of gastric cancer cells in the nude mice.
Detailed Description
The main reagent sources in the examples are:
DMEM medium (Gibco), fetal bovine serum, PBS (huperzia serrata), 0.25% pancreatin (Gibco), poly-HEMA (Sigma), lopinavir (MCE), annexin v-FITC apoptosis assay kit (BD Biosciences), D-luciferin potassium salt (Solaribio), corn oil (bi cloud).
Human gastric cancer AGS cells were purchased from ATCC, nude mice from the collection of the division of the biotechnology of the medicine and health, and fed to the experimental animal center of the army university of army (SPF grade).
Example 1 in vitro experiments to examine the effect of lopinavir on gastric cancer cell anoikis
1.1 The experimental method comprises the following steps:
(1) Lopinavir drug preparation:
50mg of lopinavir from MCE company was added to 1.6ml of DMSO and after complete dissolution at a concentration of 50mM, split into 100 ul/branch and stored at-80 ℃.
(2) Cell culture plate coating:
Weighing 1g of Poly-HEMA, adding 100ml of 95% ethanol, shaking and dissolving until the solution is clear, adding 1ml of prepared solution into each hole of a 6-hole plate, placing the solution in a 65 ℃ incubator for air drying, placing the incubator in an ultra-clean workbench for ultraviolet irradiation sterilization for standby, and rinsing the hole plate for one time by adopting PBS before use.
(3) Cell inoculation and treatment:
Cells in logarithmic growth phase were taken and trypsinized to prepare single cell suspensions, 5x104 cells per well were inoculated after cell counting, the liquid in the culture wells was supplemented to 2ml with complete medium, and cells in the culture wells were treated with 25 μm or 50 μm lopinavir for 48 hours (control group added with equal volume of DMSO).
(4) Flow cytometry detects apoptosis:
Cell suspensions in the well plate were collected and centrifuged to obtain a cell pellet, after rinsing the pellet with PBS, 100ul of 1xBinding buffer heavy suspension pellet containing 2ulAnnexinV-FITC and 2ul of PI was added, incubated for 15min in the absence of light, and then detected on a machine.
1.2 Experimental results:
The Annexin V-FITC single positive and Annexin V-FITC/PI double positive cell ratio are calculated to reflect the apoptosis rate, and the result shows that the suspension culture apoptosis rate of the DMSO group is 13.2%, the apoptosis rate of the lopinavir treatment group with 25 mu M is 29.55%, and the apoptosis rate of the lopinavir treatment group with 50 mu M is 60.09%, which indicates that lopinavir can significantly promote the anoikis apoptosis of gastric cancer cells.
Example 2 in vivo experiments to examine the effects of lopinavir on gastric cancer metastasis
2.1 The experimental method comprises the following steps:
(1) Lopinavir drug preparation:
10mg lopinavir from MCE company was added to 5ml corn oil and the concentration was 2mg/ml after complete dissolution and was prepared immediately prior to each animal administration.
(2) Constructing a vein gastric cancer model of the tail of the naked mouse and performing drug intervention:
Taking gastric cancer cells in logarithmic growth phase of stably expressing firefly luciferase gene, preparing single cell suspension by pancreatin digestion, centrifuging to obtain cell precipitate, rinsing with PBS once, re-suspending the precipitate with PBS and counting cells, finally diluting to obtain single cell suspension with concentration of 5x107/ml, injecting 100ul of the cell suspension into each nude mouse through tail vein in SPF environment, randomly dividing the injection into two groups, injecting 150ul corn oil into abdominal cavity of control group, and injecting 150ul (1) of prepared medicine (about 10mg/kg body weight) into abdominal cavity of lopinavir treatment group.
Every other day, 6 weeks after continuous administration, each nude mouse was intraperitoneally injected with D-luciferin potassium salt at 150mg/kg body weight, and 10 minutes later, the mice were examined by a living animal imager.
2.2 Experimental results:
nude mice of lopinavir treated group showed significantly reduced fluorescence intensity and area under the small animal in vivo imager compared to control group.