Application of ouabain to preparation of preparation for down-regulating PD-L1 level of tumor cells

Abstract

The invention discloses an application of ouabain to preparation of a preparation for down-regulating the PD-L1 level of tumor cells. The concentration of the ouabain for effectively down-regulating the PD-L1 level of the tumor cells is 100-200nmol / L. It is proved that ouabain can induce the tumor cells to generate endoplasmic reticulum stress by changing the steady state of Ca<2+> in cytoplasm, and normal glycosylation modification of the PD-L1 is interfered. After endoplasmic reticulum stress, the PD-L1 is identified by RNF19B and is subjected to ubiquitination, and finally, proteasome degradation is guided. The invention also discloses that RNF19B can influence combination of USP22 and the PD-L1 and weaken the stability of the PD-L1 in the tumor cells. According to the application, the development of tumors is inhibited by down-regulating the PD-L1 level in the tumors, a foundation is laid for research and development of related preparations for down-regulating the PD-L1 level of the tumor cells, and certain reference and enlightenment are provided for secondary development of traditional drugs in the field of tumor treatment; and a new strategy is provided for improving the technical means and the clinical curative effect of the anti-tumor epidemic therapy.

Description

technical field

[0001] The present invention relates to the application of ouabain, an inhibitor of sodium-potassium ATPase, in particular to the application of ouabain in the preparation of down-regulated tumor cell PD-L1 (Programmed Cell Death 1 Ligand 1, programmed cell death receptor ligand 1, also known as CD274 or B7-H1) level preparations, and belongs to the technical field of biomedicine and molecular biology. Background technique

[0002] PD-L1 is a glycoprotein present in the plasma membrane of tumor cells. After binding to PD-1 (Programmed Death Receptor 1, programmed death receptor-1) on the surface of T lymphocytes, it inhibits the proliferation of T cells and the release of cytokines and killing ability, eventually leading to the suppression of immune system function. Clinical trials targeting the PD-1 / PD-L1 pathway are currently being or have been carried out, and have been reported in the clinical diagnosis and treatment of certain cancers, such as melanoma,...

Examples

Embodiment 1

[0181] Example 1: Ouabain down-regulates the PD-L1 protein level in lung cancer cells in a concentration- and time-dependent manner

[0182] will be 1.0×10 5 Cells / ml of H157 cells were planted in 12-well plate (cell culture dish), 37°C, CO 2Cultivate in an incubator. After the number of cells increases to about 60%, add different concentrations of ouabain (Ouabain) solution (0-500nmol / L) to treat the cells for 24 hours, and then place the culture plate (dish) with cells on the On ice, suck away the medium; wash the cells once with pre-cooled 1×PBS, and then suck off the liquid in the plate; add an appropriate volume of cell lysate (PIC) containing 1% mixed protease inhibitors, lyse on ice for 30min, The cell lysate was collected, centrifuged at 13200r / min at 4°C for 15min, and the supernatant was taken for sample preparation. Then make a standard curve and measure the protein concentration. First, take five 1.5mL EP tubes, add 10 μL deionized water to each, and then add (0,...

Embodiment 2

[0190] Example 2: Ouabain can change the Ca in the cytoplasm 2+ The downregulation of PD-L1 by ouabain depends on the proteasome pathway but not the lysosomal pathway

[0191] H460 and A549 cells were seeded in 6-well plates (cell culture dishes) at 37°C, CO 2 After culturing in the incubator for 24 h, after adding different concentrations of ouabain (0, 100, 200 nmol / L) to treat the cells for 12 h, the cells were collected and lysed (see Example 1 for specific steps), and then passed western blot (see Example 1 for steps) 1) After detection of changes in the marker protein CHOP of ER Stress. H460 and A549 cells were seeded in 6-well plates (cell culture dishes) at 37°C, CO 2 After culturing in the incubator for 24 hours, add 1mol / L 4-PBA for pretreatment for 30 minutes, then add 200nmol / L ouabain to continue the treatment for 12 hours, collect the cells for lysis (see Example 1 for specific steps), and then pass western blot (see Example 1 for the steps) and then detect th...

Embodiment 3

[0194] Example 3: RNF19B interacts with PD-L1

[0195] HEK293FT cells were seeded in 6 cm cell culture dishes at 37 °C, CO 2 After culturing in the incubator for 24 hours, HEK293FT cells were transfected with pcDNA3.1-FLAG-PD-L1 / pcDNA3.1-FLAG-PD-L1(3NQ), pcDNA3.1-V5-RNF19B plasmids, and 2 Add 200 μL of medium to each 1.5mL EP tube, calculate according to the experimental requirements, add the plasmid and transfection reagent twice the mass of the plasmid, pipette five times respectively, and let stand for 5min; put two 1.5 Mix the liquid in the mL EP tube, blow and beat five times to make it fully mixed, and let it stand for 20 minutes; absorb a part of the original culture medium, add the above-mentioned mixed and let the liquid dropwise, shake it to make it evenly distributed; After 6 hours of transfection, change to fresh medium with 5% serum, and continue to cultivate for 24 hours. Add 20 μmol / L MG132 to treat the cells 6 hours before collecting them, then place the cult...

technical field

[0001] The present invention relates to the application of ouabain, an inhibitor of sodium-potassium ATPase, in particular to the application of ouabain in the preparation of down-regulated tumor cell PD-L1 (Programmed Cell Death 1 Ligand 1, programmed cell death receptor ligand 1, also known as CD274 or B7-H1) level preparations, and belongs to the technical field of biomedicine and molecular biology. Background technique

[0002] PD-L1 is a glycoprotein present in the plasma membrane of tumor cells. After binding to PD-1 (Programmed Death Receptor 1, programmed death receptor-1) on the surface of T lymphocytes, it inhibits the proliferation of T cells and the release of cytokines and killing ability, eventually leading to the suppression of immune system function. Clinical trials targeting the PD-1 / PD-L1 pathway are currently being or have been carried out, and have been reported in the clinical diagnosis and treatment of certain cancers, such as melanoma,...

Examples

Embodiment 1

[0181] Example 1: Ouabain down-regulates the PD-L1 protein level in lung cancer cells in a concentration- and time-dependent manner

[0182] will be 1.0×10 5 Cells / ml of H157 cells were planted in 12-well plate (cell culture dish), 37°C, CO 2Cultivate in an incubator. After the number of cells increases to about 60%, add different concentrations of ouabain (Ouabain) solution (0-500nmol / L) to treat the cells for 24 hours, and then place the culture plate (dish) with cells on the On ice, suck away the medium; wash the cells once with pre-cooled 1×PBS, and then suck off the liquid in the plate; add an appropriate volume of cell lysate (PIC) containing 1% mixed protease inhibitors, lyse on ice for 30min, The cell lysate was collected, centrifuged at 13200r / min at 4°C for 15min, and the supernatant was taken for sample preparation. Then make a standard curve and measure the protein concentration. First, take five 1.5mL EP tubes, add 10 μL deionized water to each, and then add (0,...

Embodiment 2

[0190] Example 2: Ouabain can change the Ca in the cytoplasm 2+ The downregulation of PD-L1 by ouabain depends on the proteasome pathway but not the lysosomal pathway

[0191] H460 and A549 cells were seeded in 6-well plates (cell culture dishes) at 37°C, CO 2 After culturing in the incubator for 24 h, after adding different concentrations of ouabain (0, 100, 200 nmol / L) to treat the cells for 12 h, the cells were collected and lysed (see Example 1 for specific steps), and then passed western blot (see Example 1 for steps) 1) After detection of changes in the marker protein CHOP of ER Stress. H460 and A549 cells were seeded in 6-well plates (cell culture dishes) at 37°C, CO 2 After culturing in the incubator for 24 hours, add 1mol / L 4-PBA for pretreatment for 30 minutes, then add 200nmol / L ouabain to continue the treatment for 12 hours, collect the cells for lysis (see Example 1 for specific steps), and then pass western blot (see Example 1 for the steps) and then detect th...

Embodiment 3

[0194] Example 3: RNF19B interacts with PD-L1

[0195] HEK293FT cells were seeded in 6 cm cell culture dishes at 37 °C, CO 2 After culturing in the incubator for 24 hours, HEK293FT cells were transfected with pcDNA3.1-FLAG-PD-L1 / pcDNA3.1-FLAG-PD-L1(3NQ), pcDNA3.1-V5-RNF19B plasmids, and 2 Add 200 μL of medium to each 1.5mL EP tube, calculate according to the experimental requirements, add the plasmid and transfection reagent twice the mass of the plasmid, pipette five times respectively, and let stand for 5min; put two 1.5 Mix the liquid in the mL EP tube, blow and beat five times to make it fully mixed, and let it stand for 20 minutes; absorb a part of the original culture medium, add the above-mentioned mixed and let the liquid dropwise, shake it to make it evenly distributed; After 6 hours of transfection, change to fresh medium with 5% serum, and continue to cultivate for 24 hours. Add 20 μmol / L MG132 to treat the cells 6 hours before collecting them, then place the cult...

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