KR102325962B1 - Use for inhibiting exosome secretion or PD-L1 expression by calcium channel blockers - Google Patents

Abstract

The present invention relates to the use of inhibiting exosome secretion or PD-L1 expression by a calcium channel blocker, and at the same time inhibiting the secretion of cancer cell-derived exosomes by blocking the calcium channel, which is currently known as a target of anticancer drug development, and intracellular PD- Through the phenomenon of reducing the expression of L1, it was confirmed that it is effective in cancer treatment when used simultaneously with existing anticancer drugs or when used alone. Therefore, the present invention can be utilized for the development of a new concept anticancer drug using a drug that exhibits an immune checkpoint inhibitory effect and an exosome secretion inhibitory effect.

Description

Use for inhibiting exosome secretion or PD-L1 expression by calcium channel blockers}

The present invention relates to the use of inhibiting exosome secretion or programmed cell death-ligand 1 (PD-L1) expression by calcium channel blockers.

Exosomes are small vesicles in the membrane structure secreted by most cells. The diameter of the exosome is about 30-100 nm, and the exosome contains various types of proteins, genetic materials (DNA, RNA, miRNA), lipids, etc. derived from the cell. Exosomes do not separate directly from the plasma membrane, but rather originate in specific intracellular compartments called multivesicular bodies (MVBs) and are released and secreted out of the cell. That is, when polycystic body and plasma membrane fusion occurs, vesicles are released into the extracellular environment, which is called exosomes. It is not known exactly by what mechanism exosomes are made, but it is known that they are separated from a number of cell types and released in both normal and pathological conditions.

In addition, programmed cell death (PD-1), one of the immune checkpoint molecules, has become an attractive therapeutic target in several cancers. PD-1 is upregulated in T cells upon activation and is highly present in depleted T cells commonly seen in tumor-infiltrating lymphocytes. When the interaction between PD-1 and its ligand, PD-L1, was blocked, excellent antitumor response and clinical effects were confirmed in some patients.

Recently, research and development of various cancer treatment methods based on immunotherapy are in full swing. In addition, it is revealed that immunotherapeutic agents approved by the FDA have many advantages for the treatment of various cancers. However, due to the high cost of treatment and the disadvantage that all patients do not respond in common, it has the disadvantage that it can be used only for specific patients. Therefore, it is necessary to research and develop a new concept anticancer drug using a drug that exhibits an immune checkpoint inhibitory effect and an exosome secretion inhibitory effect.

Biochim Biophys Acta. 2016; 1863 (6 Pt B): 1385-97 (released on November 30, 2015)

An object of the present invention is an exosome secretion inhibitor or programmed cell death-ligand 1; An object of the present invention is to provide a method for screening an expression inhibitor.

Another object of the present invention is to provide a reagent composition for inhibiting exosome secretion or PD-L1 expression of cancer cells in vitro, containing a calcium channel blocker as an active ingredient.

Another object of the present invention is to provide a method for inhibiting exosome secretion or PD-L1 expression in cancer cells, comprising the step of inhibiting calcium channel activity of cancer cells in vitro.

The present invention comprises the steps of (1) contacting a test substance with cancer cells; (2) measuring the level of activity of calcium channels in cancer cells contacted with the test substance; and (3) selecting a test substance in which the degree of activity of the calcium channel is inhibited as compared with a control sample. ) expression inhibitor screening methods.

In addition, the present invention provides a reagent composition for inhibiting exosome secretion or PD-L1 expression of cancer cells in vitro, containing a calcium channel blocker as an active ingredient.

In addition, the present invention provides a method for inhibiting exosome secretion or PD-L1 expression of cancer cells, comprising the step of inhibiting the calcium channel activity of the cancer cells in vitro.

The present invention relates to the use of inhibiting exosome secretion or PD-L1 expression by a calcium channel blocker, and at the same time inhibiting the secretion of cancer cell-derived exosomes by blocking the calcium channel, which is currently known as a target of anticancer drug development, and intracellular PD- Through the phenomenon of reducing the expression of L1, it was confirmed that it is effective in cancer treatment when used simultaneously with existing anticancer drugs or when used alone. Therefore, the present invention can be utilized for the development of a new concept anticancer drug using a drug that exhibits an immune checkpoint inhibitory effect and an exosome secretion inhibitory effect.

1 is a result confirming the calcium channel inhibitor-mediated regulation of exosome secretion. a) This is the result of measuring the cell viability of cancer cells. b) Results of measurement of intracellular calcium concentration in A375 and SK-MEL-28 cells by Fluo-3AM method. Dimethyl amiloride (DMA) was used as a positive control. c) The result of measuring the number of exosomes secreted after treatment with the indicated nicardipine concentration. d) The result of measuring the number of secreted microvesicles after treatment with the indicated nicardipine concentration.
Figure 2 is a result of confirming the calcium channel inhibitor-mediated regulation of the transport-related endosomal sorting complexes (Endosomal Sorting Complexes Required for Transport; ESCRT)-dependent exosome generation and secretion. a) qRT-PCR analysis results of various genes after treatment with nicardipine for 6 hours in human melanoma A375 cells. b) Human melanoma SK-MEL-28 cells were treated with nicardipine for 6 hours, followed by qRT-PCR analysis of various genes. c) Immunoblot analysis results for the indicated proteins in human melanoma A375 cells. Beta actin was used as a loading control for whole cell lysates. ***p < 0.001, **p < 0.005 and *p < 0.05.
3 is a result confirming the calcium channel inhibitor-mediated regulation of exosome PD-L1 expression and its regulation by IFN-γ. a) Immunoblot analysis results of various proteins in whole cell lysates and isolated exosomes derived from two metastatic melanoma cell lines. b) Immunoblot analysis results of exosome proteins derived from nude mouse serum. c) After stimulation with 10ng/ml IFN-γ for 48 hours, and treatment with a calcium channel inhibitor at the indicated concentration in A375 cells, the number of secreted exosomes was measured. d) Immunoblot analysis results of PD-L1 in exosomes derived from other metastatic melanoma cells treated with nicardipine or nifedipine at the indicated concentrations after IFN-γ treatment. e) Immunoblot analysis results of PD-L1 in whole cell lysates derived from other metastatic melanoma cells treated with nicardipine or nifedipine at the indicated concentrations after IFN-γ treatment.
4 is a result confirming the calcium channel inhibitor-mediated regulation of IFN-γ-stimulated PD-L1 expression by JAK2/STAT1/IRF-1 signaling. a) After stimulation with 10ng/ml IFN-γ for 48 hours, nicardipine or nifedipine treatment in A375 human melanoma cells for 6 hours, qRT-PCR analysis results of various genes were measured. b) Stimulation with 10ng/ml IFN-γ for 48 hours, nicardipine or nifedipine treatment in SK-MEL-28 human melanoma cells for 6 hours, qRT-PCR analysis results of several genes am. c) Immunoblot analysis results for JAK2, STAT1, and IRF1 in cell lysates and nuclear fragments after IFN-γ stimulation and calcium channel inhibitor treatment.
5 is a result confirming the calcium channel inhibitor-mediated regulation of cancer cell activity. a) Results of wound healing assay in A375 cells treated with indicated concentrations of nicardipine. b) Matrigel-coated transwell invasion assay results in A375 cells treated with nicardipine.

The present inventors have identified nicardipine as a calcium channel inhibitor that inhibits the production and secretion of metastatic melanoma cell-derived exosomes without severe toxicity through library screening of FDA-approved drugs. Nicardipine, an L-type calcium channel inhibitor, is one of the most prescribed of all FDA-approved anti-hypertensive drugs. However, almost all of the mainly prescribed calcium channel inhibitors have side effects: Amlodipine for peripheral edema, dizziness, headache and fatigue, Nifedipine for headache and edema, and Nicardipine for headache and edema. There are side effects of phlebitis. In spite of these side effects, we chose nicardipine because of some unique action in hypertension. Compared to other calcium channel inhibitors, nicardipine has less negative ionotropic effects, less reflex tachycardia, and less urinary tract infections, pneumonia or bacteremia during emergency hospitalization. It is a safer and more effective drug for intravenous administration used in the treatment of severe hypertension. In addition, the present inventors confirmed that nicardipine is involved in the production and secretion of exosomes in melanoma cells, and regulation of the expression of exosomes and intracellular PD-L1, and completed the present invention.

The present invention comprises the steps of (1) contacting a test substance with cancer cells; (2) measuring the level of activity of calcium channels in cancer cells contacted with the test substance; and (3) selecting a test substance in which the degree of activity of the calcium channel is inhibited as compared with a control sample. ) expression inhibitor screening methods.

Specifically, the PD-L1 expression inhibitor may inhibit the expression of PD-L1 in cancer cells or the expression of exosome surface PD-L1 secreted from cancer cells, but is not limited thereto.

Preferably, the cancer cell may be a melanoma cell, but is not limited thereto.

The term "test substance" used while referring to the screening method of the present invention affects the expression level of a gene, affects the expression or activity of a protein, or affects the binding between proteins. It refers to an unknown candidate substance used in screening. The sample includes, but is not limited to, chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural product extracts.

In addition, the present invention provides a reagent composition for inhibiting exosome secretion or PD-L1 expression of cancer cells in vitro, containing a calcium channel blocker as an active ingredient.

Specifically, the calcium channel blocker may be at least one selected from the group consisting of nicardipine, nifedipine, and amlodipine, but is not limited thereto.

Specifically, the PD-L1 expression inhibitor may inhibit the expression of PD-L1 in cancer cells or the expression of exosome surface PD-L1 secreted from cancer cells, but is not limited thereto.

Preferably, the cancer cell may be a melanoma cell, but is not limited thereto.

In addition, the present invention provides a method for inhibiting exosome secretion or PD-L1 expression in cancer cells, comprising the step of inhibiting the calcium channel activity of the cancer cells in vitro.

Specifically, the calcium channel blocker may be at least one selected from the group consisting of nicardipine, nifedipine, and amlodipine, but is not limited thereto.

Specifically, the PD-L1 expression inhibitor may inhibit the expression of PD-L1 in cancer cells or the expression of exosome surface PD-L1 secreted from cancer cells, but is not limited thereto.

Preferably, the cancer cell may be a melanoma cell, but is not limited thereto.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Cell Lines and Cell Culture

All melanoma cancer cells were obtained from the American Type Culture Collection (ATCC) and cultured at 37° C. in a humid atmosphere _{with 5% CO 2 and 95% air using the suggested culture medium.} A375 and B16F10 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic/antifungal solution. SK-MEL28 cells were cultured in Minimum Essential Medium with Earle's Balanced Salt (MEM/EBSS, Hyclone).

2. MTT proliferation assay

A375, SK-MEL28 and B16F10 cell lines (50,000 cells/well) were inoculated into 24-well cell culture plates and cultured for 24 hours. The medium was replaced by adding various concentrations of calcium channel blockers to the serum-free medium, and incubated for an additional 24 hours. Then, 0.5 mg/mL MTT solution was added, and incubated at 37° C. for 3 hours. 500 μl of 100% isopropanol was added, and the mixture was incubated with slow stirring at room temperature. 200 μl of the mixed solution was transferred to a 96-well plate, and absorbance was measured at 495 nm.

3. exosomes separation

To isolate exosomes, each cell culture supernatant obtained from A375, SK-MEL-28 and B16F10 cells was centrifuged sequentially at 300 × g/3 min, 2500 × g/20 min and 10000 × g/30 min. did. Then, the supernatant was filtered through a 200 nm syringe filter, and centrifuged at 120000×g/90 min. The exosome pellet was resuspended in PBS and centrifuged again at 120000×g/90 min. Finally, for the next experiment, the isolated exosome pellet was resuspended in PBS or 1× RIPA buffer.

4. Nanoparticle tracking analysis ( NTA )

The exosome suspension obtained from the cell culture supernatant was analyzed using a Nano-Sight LM10 instrument (NanoSight, Malvern). For this analysis, monochromatic laser light at 405 nm was applied to the diluted exosome suspension. Video was taken for 30 seconds at a rate of 30 frames/s, and exosome movement was analyzed using NTA software version 2.3 (NanoSight). The settings after capture of NTA were optimized, kept constant between samples, and then each video was analyzed to calculate the concentration.

5. exosomes Protein concentration measurement

Exosome protein concentration was measured using micoBCA assay (#23235, ThermoFisher). To quantify protein concentration, exosomes were isolated through fractional ultracentrifugation. Exosome proteins were extracted using 1× RIPA buffer (#50-188, Merch Millipore). The microBCA assay was performed according to the manufacturer's instructions.

6. RNA Extraction and qRT - PCR

Total RNA from several melanoma cells was extracted using TRIzol reagent (#15596026; Invitrogen) according to the manufacturer's recommendations. Then, using RT-premix (K2041, Bioneer), a total of 2ug RNA was reverse transcribed. qRT-PCR gene expression analysis was completed through 3 biological replicates using gene-specific qRT-PCR oligonucleotides. RT-PCR reaction was performed using SYBR premix (#4368577; ThermoFishers) as ABI stepOne plus instruments (Applied Bioscience). Using the same amount of cDNA template, each sample was PCR amplified. After the initial step in a thermal cycler at 95°C for 15 minutes, PCR amplification was performed 40 times at 95°C for 15 seconds and at 60°C for 1 minute, followed by melting curve analysis to confirm the specificity of the PCT product. did. Baseline and threshold values were adjusted according to the manufacturer's recommendations. The primer sequences used for the reverse-transcription polymerase chain reaction (RT-PCR) are shown in Table 1 below.

Gene forward primer Reverse primer RAB27A 5'-AGT TGA TGG AGC GAA CTG CT-3' 5'-CCC TAC ACC AGA GTC TCC CA-3' RAB7 5'-GAT GGT GGA TGA CAGGCT AGT-3' 5'-CGA GAGACT GGA ACC GTTCCT-3' RAB5 5'-ACG GGC CAA ATA CGG GAA AT-3' 5'-TCA AAC TTT ACC CCA ATG GTA CT-3' PD-L1 5'-AGAGCTACGAGCTGCCTGAC-3' 5'-TGCTTGTCCAGATGACTTCG-3' JAK2 5'-TGC CGG TAT GAC CCT CTA CA-3' 5'-ACC AGC ACT GTA GCA CAC TC-3' STAT1 5'-GTT ATG GGA CCG CAC CTT CA-3' 5'-CAG TGA ACT GGA CCC CTG TC-3' IRF1 5'-AAA GTC GAA GTC CAG CCG AG-3' 5'-CAG AGT GGA GCT GCT GAG TC-3' Beta-actin 5'-TTC CTG GGC ATG GAG TCC TGT GG-3' 5'-CGC CTA GAA GCA TTT GCG GTG G-3'

7. western blotting

Proteins collected from whole cells were digested by SDS-PAGE, transferred onto nitrocellulose membranes, bound with primary antibodies, and reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies. . Blots were detected with enhanced chemiluminescence (ECL) detection reagents (Thermo scientific, Waltham, MA, USA) and quantified using ECL hyperfilm (AGFA, Morstel, Belgium). The primary antibodies used were as follows. anti-CD63 (ab68418, 1:1000; Abcam), Alix (ab56932, 1:1000; Abcam), Filotillin-1 (#3253, 1:1000; CST), beta-actin (4670, 1:3000; Cell Signaling) Technology (CST)), Rab27a (ab55667, 1:1000; Abcam), Rab5 (ab18211, 1:1000; Abcam), Rab7 (ab50533, 1:1000; Abcam), PD-L1 (#13684, 1:1000; Cell Signaling Technology (CST)), JAK2 (#3230, 1:1000; Cell Signaling Technology (CST)), STAT1 (#14994, 1:1000; Cell Signaling Technology (CST)), IRF-1 (#8478, 1 :1000; Cell Signaling Technology (CST)).

8. Wound Healing and Invasion Assays

To perform wound healing assays, A375 cells were seeded into 24-well cell culture plates and cultured overnight. The next day, the monolayer was wounded through repeated scratches with a 200 μl pipette tip, and the medium was changed to remove cell debris. Each wound site was imaged at 0 h and re-imaged after 24 h. The average wound healing value was determined as the average of the values measured three times for the wound site.

Cell invasion assays were performed using a cell culture insertion chamber (CLS3364; Corning). The chamber was coated using basement membrane Matrigel (50 μl of 20% matrigel/filter, #354248; Corning). Melanoma cells were inoculated into the upper chamber at 10,000 cells/well, and the invasive cells were attached and stained with crystal violet. After the membrane was mounted on a glass slide, images of the cells were captured using a microscope (×4 magnification).

9. In vivo in vivo ) mouse study

All animal experiments were performed according to the protocol approved by the Institutional Animal Care and Use Committes (IACUC) of Kyungpook National University (KNU). To establish a human melanoma xenograft model in nude mice, A375 cells (5 × 10 ⁶ cells in 50ul matrigel with 50ul PBS) were injected into the flanks of 7-week-old female BALB/C nude mice. Tumors were measured every 2 days using a digital caliper, and tumor volume was calculated as follows: (width ² × length)/2. Mice were euthanized when the longest tumor size reached 2.0 cm on the 21st day or even before 21 days after cell inoculation. Immediately after euthanasia, blood samples were collected using cardiac puncture, and exosomes were isolated.

10. Statistical Analysis

For statistical analysis, an unpaired two-tailed students t-test was used in an experiment comparing two sets of data. Error bars in the graph represent mean ± standard deviation. All in vitro experiments were performed in triplicate. A p value less than 0.05 was considered statistically significant. *, ** and *** indicate p values of <0.05, 0.005 and 0.001, respectively. NS indicates no significance. Data analysis was performed using PRISM 6 software (GraphPad Software, Inc.).

< Example 1> exosomes discovery of secretion inhibitory drugs

The intracellular calcium concentration increases rapidly upon multivesicular body (MVB) maturation as well as CD63 ⁺ , CD9 ⁺ and Alix ^{+ exosome release from cancer cells.} Therefore, in order to discover a target drug that reduces cell-derived exosome secretion by regulating calcium levels, a cell-based high-throughput analysis system for analyzing 24 calcium channel inhibitors from 1000 FDA-approved drugs was developed. developed. Meanwhile, the present inventors selected highly metastatic melanoma A375 cells capable of secreting a large amount of exosomes. In an initial screening, we tested drugs at concentrations of 10 uM and 20 uM that inhibit cell-derived exosome secretion, but decreased calcium levels increased cell death, indicating that calcium is not only an essential element for cell survival, but also rarely This is because all cellular mechanisms are regulated by intracellular calcium. Finally, we selected the L-type calcium channel inhibitor nicardipine at 0.5 uM and 1 uM concentrations, comparable to those used to treat hypertension, reducing intracellular calcium levels by 40%-60% and reducing the cellular Toxicity is about 5%. In conclusion, nicardipine is an effective FDA-approved drug administered orally and intravenously for the treatment of hypertension and angina without systolic action.

< Example 2> exosomes inhibiting secretion and production nicardipine

To determine the dose at which nicardipine and nifedipine do not show cytotoxicity, and to evaluate the effect on exosome production and secretion, three melanoma cell lines, A375, SK-MEL-28 (human melanoma) and B16F10 A dose response cell viability assay was performed in (mouse melanoma). While no effect was confirmed in nifedipine, 3 types of melanoma cell lines exposed to nicardipine for 24 hours resulted in apoptosis of 5% compared to the control group (FIG. 1a). Since intracellular calcium regulates signaling in normal cellular homeostasis, a decrease in calcium levels leads to cell death. As a result of treating cells with calcium channel inhibitors at the indicated concentrations, the present inventors confirmed that nicardipine and nifedipine reduced calcium levels by 25-30% in two types of hypermetastatic melanoma cells (FIG. 1b). Clinically used calcium channel blockers have high affinity for drug binding domains and interact with activation gates of L-type calcium channels. The binding drug disrupts the normal voltage-dependent cycle of the channel through dormant and inactive states. Accordingly, the present inventors confirmed the effect of nicardipine in the regulation of CaV 1.2 expression expressed from melanoma cancer cells. Nicardipine had no significant effect on CaV 1.2 protein expression, indicating that intracellular calcium level regulation via nicardipine occurs by modulating calcium channel activity. The present inventors analyzed the regulation of exosome secretion by nicardipine in primary embryonic fibroblast NIH-3T3, which is reported as a cell line that secretes a lot of exosomes, and nicardipine down-regulates exosome secretion by 15-20%. appeared to be It has been reported that NIH-3T3 cells are highly sensitive to T-type and L-type calcium channels, and nicardipine appears to affect the regulation of exosome secretion. Therefore, the present inventors confirmed the effect of nicardipine on the regulation of exosome secretion inhibition in A375, a cell line that secretes a lot of exosomes. In addition, the present inventors also confirmed the effect on the inhibition of microvesicles secretion in three different melanoma cells. Surprisingly, nicardipine treatment significantly inhibited exosome and microvesicle secretion in a dose-dependent manner without cytotoxic effects in melanoma cells ( FIGS. 1C and 1D ).

< Example 3> ESCRT -Dependence exosomes regulating production and secretion nicardipine

In order to confirm the effect of calcium channel inhibitors on the production of transport-related endosomal sorting complexes (Endosomal Sorting Complexes Required for Transport; ESCRT) dependent exosomes, the present inventors analyzed nicardipine-mediated regulation of genes involved in exosome generation. did. The ESCRT mechanism is very important in regulating not only multivesicular body (MVB) maturation but also intraluminal vesicle (ILV) formation. After nicardipine treatment, factors important for MVB maturation and ILV formation such as Alix, Hrs, and Flotilin-1 were inhibited in a dose-dependent manner. The amounts of RAB27A and RAB5 proteins expressed in cancer cells were increased compared to normal cells. As a result of qRT-PCR analysis, expression of RAB family (RAB5, RAB7, RAB27A) genes in A375 and SK-MEL-28 cells was inhibited by calcium channel inhibitor treatment ( FIGS. 2A and 2B ). In addition, after nicardipine treatment, it was confirmed that the amounts of RAB27A, RAB7, RAB5A and Alix proteins in A375 cells were down-regulated while maintaining dose dependence ( FIG. 2c ). Taken together, these results support that nicardipine regulates exosome production and secretion through an ESCRT-dependent mechanism.

< Example 4> for PD-L1 expression and PD-L1 stimulation IFN regulating the response of -γ. nicardipine

Exosomes contain various molecules such as microRNA, mRNA, and protein that are generated and secreted through a defined intracellular migration pathway, and are transmitted to recipient cells, and ultimately induce tumor development, metastasis, and therapeutic drug resistance. . PD-L1, a membrane-bound protein, is present in cancer cell-derived exosomes and plays a role in cancer formation. Meanwhile, in order to confirm the regulation of intracellular PD-L1 expression by the calcium channel blocker, the present inventors treated HEK293, MCF-10A and NIH-3T3 normal cells, and collected proteins for Western blotting analysis. As a result, nicardipine did not show an inhibitory effect on PD-L1 on normal cell expression levels. Then, the present inventors collected exosomes and intracellular proteins from several melanoma cells after nicardipine treatment, and analyzed by Western blotting. As a result, nicardipine showed elevated expression regulation in exosome PD-L1 compared to the intracellular level (FIG. 3a).

To verify the in vitro assay, the present inventors confirmed the effect of nicardipine on PD-L1 expression in vivo. As reported in a previous study, 1 mg/kg nicardipine administered every 6 hours showed an anti-hypertensive effect, and 5 mg/kg/day nicardipine intraperitoneal administration for 3 days had an anti-neuroinflammatory effect. showed Based on this, the present inventors selected human melanoma xenograft Balb/c nude immunodeficient mice, and confirmed the effect of nicardipine on the regulation of circulating exosome PD-L1 expression. After daily intraperitoneal administration of nicardipine (5 and 10 mg/kg/day) for 14 consecutive days, pathological signs including weight change, abnormal behavior and unexpected death were identified. Blood was collected from the mice for exosome isolation, and then human PD-L1 protein was detected through Western blotting. The present inventors measured the expression of circulating exosome PD-L1 regulated by nicardipine treatment from cancer cell-derived exosomes in the serum of mice ( FIG. 3b ).

Melanoma cell-derived exosome PD-L1 was greatly stimulated following IFN-γ treatment, and correspondingly, the exosomes were shown to increase binding to PD-1 to inactivate T-cell mediated cytotoxicity. . Therefore, in order to confirm the effect of IFN-γ treatment on exosome secretion, the present inventors treated A375 cells with IFN-γ (10ng/ml), nicardipine and nifedipine in combination. As a result, IFN-γ treatment could not quantitatively enhance exosome secretion, but both calcium channel inhibitors significantly down-regulated exosome secretion ( FIG. 3c ).

However, the present inventors collected exosomes and intracellular proteins from several metastatic melanoma cells treated with calcium channel inhibitors as well as IFN-γ, and confirmed IFN-γ-mediated PD-L1 expression enhancement through western blotting. As a result, IFN-γ promoted the elevated expression of PD-L1 compared to the control group, and despite IFN-γ-mediated stimulation, both calcium channel inhibitors showed PD- of melanoma cell-derived exosomes and intracellular proteins. L1 expression levels were reduced ( FIGS. 3D and 3E ). In addition, both nicardipine and nifedipine negatively regulated the expression of the exosome marker CD63, which was found at a level similar to the result of regulating exosome secretion from melanoma cells ( FIG. 3e ).

< Example 5> JAK2 / STAT1 / IRF -1 through signaling IFN Nicardipine mediating -γ-induced PD-L1 expression regulation

The biological consequences of IFN-γ mainly occur through the activation of intracellular molecular signals by the JAK/STAT pathway, which regulates the transcription of numerous genes and ultimately regulates various biological responses. By binding to the receptor IFN-γ, it induces phosphorylation of STAT1 through JAK2 and promotes the induction of the transcription factor IRF1, thereby regulating the expression of PD-L1 protein. To highlight the regulation of intracellular PD-L1 expression by calcium channel inhibitors after IFN-γ-mediated stimulation, the present inventors identified expression regulation of key factors of JAK/STAT signaling. We collected intracellular protein and nuclear fractions after IFN-γ (10 ng/ml) mediated stimulation, treated A375 cells with nicardipine and nifedipine at indicated concentrations, and performed Western blotting and qRT-PCR analysis. . We analyzed the regulation of PD-L1, JAK2, STAT1, IRF1 gene expression by two calcium channel inhibitors from other metastatic melanoma cells after IFN-γ stimulation. As a result, IFN-γ showed higher stimulation in A375 cells than SK-MEL-28, and nifedipine more inhibited PD-L1, JAK2, STAT1, IRF1 gene expression than nicardipine (Fig. 4a and Fig. 4a and Fig. 4b). The present inventors confirmed stimulation by IFN-γ compared to the control group, and the two calcium channel inhibitors significantly regulated the expression of JAK2 and IRF1, and as a result of Western blotting analysis, nifedipine could reduce STAT1 expression by 10%. There was (Fig. 4c). These results indicate that the PD-L1 expression level is decreased through JAK/STAT signaling, depending on the intracellular calcium level.

< Example 6> nicardipine mediated cancer cell activity regulation

In a previous study, nicardipine at a concentration of about 10 uM was used in the analysis of microglial cell metastasis to confirm the anti-neuroinflammatory effect. There have been no reports to confirm the anticancer effect of nicardipine. Accordingly, the present inventors confirmed nicardipine-mediated regulation of cancer cell activity through wound healing and metastatic activity ( FIGS. 5A and 5B ). Through in vivo experiments, nicardipine exhibited anti-metastatic activity in melanoma cancer cells in a dose-dependent manner.

Claims (11)

delete (1) contacting the test substance to the melanoma cells;
(2) measuring the intracellular calcium level in melanoma cells contacted with the test substance; and
(3) compared with a control sample, comprising the step of selecting a test substance in which the intracellular calcium level is suppressed, programmed cell death-ligand 1 (PD-L1) expression in melanoma cells Or a PD-L1 expression inhibitor screening method, characterized in that the suppression of the exosome surface PD-L1 expression secreted from melanoma cells. delete delete delete Contains nicardipine or nifedipine as an active ingredient, inhibits PD-L1 expression in melanoma cells in vitro, or expresses exosome surface PD-L1 secreted from melanoma cells A reagent composition for inhibiting delete delete delete treating melanoma cells in vitro with nicardipine or nifedipine to suppress intracellular calcium levels, wherein PD-L1 expression in melanoma cells or melanoma cells A method of inhibiting the expression of exosome surface PD-L1 secreted by delete

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