CN113413460A - Application of TRAF6 inhibitor in preparation of melanoma drugs - Google Patents
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Abstract
The invention discloses application of a TRAF6 inhibitor in preparation of a melanoma drug. The research of the invention shows that the mRNA and protein levels of PD-L1 in a melanoma cell line of TRAF6 knocked down or over expressed are changed in a consistent manner, namely TRAF6 can positively regulate PD-L1; the expression of TRAF6 can be inhibited, so that the expression of PD-L1 on the surface of a tumor cell is inhibited, the immune escape phenomenon of the combination of PD-L1 and PD-1 on the surface of a T cell is reduced, the normal immune function of the T cell is promoted, the immunity of an organism is enhanced, the tumor progress is reduced, and the immune tolerance is reduced; further, the TRAF6 inhibitor is applied to a mouse melanoma lung metastasis model as a PD-L1 regulator, and can remarkably enhance the activity of lung infiltrating T cells and effectively kill melanoma.
Description
Technical Field
The invention relates to the technical field of biological medicines, and in particular relates to application of a TRAF6 inhibitor in preparation of a melanoma drug.
Background
Melanoma, usually referred to as malignant melanoma, is a highly malignant tumor of melanocyte origin, abbreviated as malignant melanoma, which frequently occurs in the skin, also visible in mucous membranes and internal organs, accounting for about 3% of all tumors. The skin melanoma has the characteristics of strong invasiveness, easy metastasis, easy relapse and the like, and is the first cause of death of the skin tumor. In our country, the most common clinical types of cutaneous melanoma are of the limb-tip type and the mucosa type, and the incidence rate rapidly increases at 3-5% per year. The operation effect of early melanoma is better, but 60-70% of patients with later-stage skin melanoma have 5% survival rate in 5 years and extremely high malignancy degree, and the curative effects of the existing treatment means such as adjuvant interferon, conventional chemotherapy and the like are not good enough.
Currently, cancer immunotherapy is an emerging and most promising therapeutic approach following surgery, chemoradiotherapy and targeted therapy, and is also the focus and hot spot in the current field of tumor research. Tumor cell surface expression programmed death ligand 1(PD-L1), the interaction of PD-L1 with receptor programmed cell death protein 1(PD-1) on T cells prevents activation of antigen-specific T cells, in which case the tumor can escape T cell surveillance to reach tumor cell proliferation. Immune checkpoint inhibitors, represented by anti-PD-1 antibodies, are intended to release the potential of human T cells to kill tumors by eliminating PD-1 signaling pathway-mediated immunosuppression.
anti-PD-1/PD-L1 therapy is used for a variety of cancer treatments, such as melanoma, to prolong the life of a subset of patients, but this therapy is not effective in up to 60% of patients, which has been shown to be closely related to Tumor Microenvironment (TME) -mediated immune tolerance. While the density of PD-L1+ cells in metastatic melanoma is significantly related to the density of CD8+ T cells, the over-expression of PD-L1 in tumor cells is considered as a main factor for inhibiting anti-tumor immunity in Tumor Microenvironment (TME) and tumor drainage lymph nodes, and the key role of the cell expression of PD-L1 in TME is suggested. The targeted TME can relieve immunosuppression, is favorable for restoring and rebuilding the normal anti-tumor immune defense capability of a human body, and enhances the curative effect of immunotherapy.
Tumor necrosis factor receptor-associated factor 6 (TRAF 6), a member of the TNF receptor-associated factor family, was originally found in the interleukin 1(IL-1) and CD40 signaling pathways. As E3 ubiquitin ligase, TRAF6 can perform polyubiquitination modification of Lys63 ligation on its substrate, further promote the activity of its substrate kinases such as TAK1 and pkb (akt), and play an important role in innate and acquired immunity, bone metabolism, and lymph node development. Although the research progress of the relation between RAF6 and tumors is disclosed (Li Miao, Zhou Ji Jun. TRAF6 and tumors [ J ] Biotechnology report, 2017,33(6):24-31.), no report is found at present about the role of TRAF6 in expression regulation and immunosuppression of tumor cells PD-L1.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provides application of a TRAF6 inhibitor in preparing a preparation for inhibiting expression of PD-L1 on the surface of tumor cells.
The second purpose of the invention is to provide the application of the TRAF6 inhibitor in preparing a medicine for treating melanoma.
The third purpose of the invention is to provide the application of the TRAF6 inhibitor as a PD-L1 regulator in preparing tumor treatment medicines.
The above object of the present invention is achieved by the following technical solutions:
according to the invention, E3 ubiquitin enzyme TRAF6 is a molecule potentially participating in regulation and control of PD-L1 by analyzing high-throughput genetic library screening data (GSE129968) and melanoma gene chip data (GSE15605) of PD-L1 in a GEO database. The research of the invention shows that TRAF6 can positively regulate PD-L1; by inhibiting the expression of TRAF6, the expression of PD-L1 on the surface of tumor cells can be inhibited, the immune escape phenomenon of the combination of PD-L1 and PD-1 on the surface of T cells is reduced, the normal immune function of the T cells is promoted, the immunity of an organism is enhanced, the tumor progress is reduced, and the immune tolerance is reduced; the TRAF6 inhibitor is used as PD-L1 regulator, can obviously improve the tumor immunotherapy effect, and has similar curative effect with PD-1 monoclonal antibody. Accordingly, the present invention provides the following uses for TRAF6 and TRAF6 inhibitors:
the use of TRAF6 in the regulation of PD-L1 expression; in particular to TRAF6 positively regulating the expression of PD-L1; more specifically, TRAF6 positively regulated the expression of PD-L1 on the surface of tumor cells.
The use of TRAF6 in the preparation of a formulation for modulating the expression of PD-L1; comprises preparing a PD-L1 expression promoter or a PD-L1 expression inhibitor.
Application of TRAF6 inhibitor in preparing preparation for inhibiting expression of PD-L1 on tumor cell surface. The TRAF6 inhibitor comprises a compound that inhibits the expression of TRAF6, an RNA interfering molecule, or a specific monoclonal or polyclonal antibody directed against TRAF 6.
The TRAF6 inhibitor can inhibit the expression of PD-L1 on the surface of melanoma cells, reduce the immune escape phenomenon of the combination of PD-L1 and PD-1 on the surface of T cells, promote the normal immune function of the T cells, enhance the immunity of an organism and reduce the tumor progress.
Application of TRAF6 inhibitor as PD-L1 regulator in preparing tumor therapeutic medicine.
Preferably, the TRAF6 inhibitor is bortezomib used in clinical medicine or shRNA for inhibiting the expression of TRAF 6.
Preferably, the optimal concentration and time for bortezomib to inhibit tumor cell surface PD-L1 expression is 30nM and 12 h; bortezomib is effective in inhibiting TRAF6 expression and PD-L1 expression.
Preferably, bortezomib is used as a PD-L1 modulator at a concentration of 0.2 mg/kg.
Preferably, the tumor is melanoma.
Compared with the prior art, the invention has the following beneficial effects:
research shows that TRAF6 can positively regulate the expression of PD-L1 on the surface of tumor cells; by inhibiting the expression of TRAF6, the expression of PD-L1 on the surface of a tumor cell can be inhibited, the endogenous PD-L1 of the tumor is down-regulated, the immune escape phenomenon of the combination of PD-L1 and PD-1 on the surface of a T cell is reduced, the normal immune function of the T cell is promoted, the body immunity is enhanced, the tumor progress is reduced, and the immune tolerance is reduced; the TRAF6 inhibitor is used as PD-L1 regulator, can obviously improve the tumor immunotherapy effect, and has similar curative effect with PD-1 monoclonal antibody. Therefore, TRAF6 is expected to become a potential new target related to the regulation and control of PD-L1 in melanoma immunotherapy, provides a new visual angle and idea for the tumor immunotherapy, and has a great application prospect.
Drawings
FIG. 1 shows screening of potential targets involved in PD-L1 regulation. A is plotted with 2474 genes with significant fold difference and their corresponding-log 10(P value) values. Genes with a high degree of significant difference greater than 2 (pink) (GSE 129968); b is a plot that calculated the correlation of 143 genes with PD-L1 expression in 12 metastatic melanomas, R greater than 0.4 representing a moderate or strong positive relationship (GSE 15605); c is an expression heat map of 26 genes with PD-L1(CD274) in 16 normal skin tissues and 12 metastatic melanoma tissues (GSE 15605). After intra-row normalization, red represents high expression and blue low expression.
FIG. 2 is a graph of PD-L1 mRNA and protein changes following TRAF6 knockdown or overexpression in two melanoma cell lines. A and D are the expression level of PD-L1 mRNA of A2058 or SK-MEL melanoma cells for knocking down or over expressing TRAF6 by utilizing a qPCR technology; b and E are the expression level of PD-L1 protein of A2058 or SK-MEL melanoma cells for knocking down or over expressing TRAF6 by using western blot detection; c and F are the detection of the expression level of PD-L1 protein of A2058 or SK-MEL melanoma cells for knocking down or over expressing TRAF6 by using flow cytometry.
FIG. 3 is a graph of the killing effect of TRAF6 inhibitors on melanoma cells (in vitro). A is the protein level change of TRAF6 and PD-L1 after the A2058 melanoma cells are treated for 12h by using western blot to detect the bortezomib (0nM, 20nM, 30nM and 40nM) with different concentrations; b is the combination condition of A2058 cells treated by a laser confocal detection control and 30nM bortezomib for 12h and PD-1 factors; c and DThe method is characterized in that the CD8 is obtained after the A2058 melanoma cells added with 30nM bortezomib or 0.5 mu g/mL anti-PD-1 monoclonal antibody are incubated with T cells for 12h by using a flow analysis control+Proportion of T cells and IFN gamma+CD8+Levels of T cells. E is an apoptosis flow analysis chart of A2058 melanoma cells.
Figure 4 is the killing effect (in vivo) of TRAF6 inhibitor on melanoma. A is a model making and administration mode diagram of a mouse; b is a representative image of lung tissue at day 18 dissection after mouse modeling. The control group was a tumor-free group (Normal control, NC), and the experimental groups were: a tumor-producing group (PBS group), a bortezomib group (Bor group), and an anti-PD-1 monoclonal antibody group. C is hematoxylin-eosin (H) of lung tissue of each group of mice&E) Staining and histochemical staining for cell proliferation nuclear antigen (Ki67), scale: 200 mu m; d is a tumor nodule score statistical chart of each group of mice; e is the body weight record of each group of mice from molding to day 18; f and G are western blots for detecting the expression levels of TRAF6 and PD-L1 proteins in lung tissues; h and I are immunohistochemical staining of TRAF6 and PD-L1 of the lungs of each group of mice. A scale: 200 μm. J is CD8 flow-analyzed from lung tissue of mice+T cell ratio and Granzyme B, IFN gamma account for CD8+The proportion of T cells, and flow statistics thereof. K flow analysis of mouse cervical lymph, spleen and peripheral lymph CD8+The proportion of T cells and their flow statistics.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Example 1 GEO database analysis of TRAF6 as a molecule potentially modulating PD-L1
1. PD-L1 was retrieved from the GEO database (https:// www.ncbi.nlm.nih.gov/GEO /), accession number GSE129968 is the PD-L1-related high throughput genetic library screening data, this analysis was performed by introducing a human Genome-wide CRISPR/Cas9 Knock-Out (Genome-Scale CRISPR Knock-Out, GeCKO) lentiviral library into lung cancer cells, and screening both with 1. mu.g puromycinAfter week, flow sorting was performed by incubation with APC-PD-L1 flow antibody, enrichment of PD-L1highAnd PD-L1lowThe cell of (1). Then, the unsorted cell control group and PD-L1 were extracted respectivelylowCell Experimental group, PD-L1highAnd (3) performing two rounds of PCR amplification on genomic DNA of a cell experimental group, amplifying the genomic DNA containing the sgRNA, and finally performing illumina sequencing to screen out genes corresponding to the sgRNA which are obviously enriched in the experimental group so as to obtain molecules of PD-L1 with positive regulation or negative regulation. We therefore downloaded PD-L1 total norm. txt files from GSE129968, and selected the unsorted cell control and PD-L1lowData on cellular experiments. With PD-L1lowDividing the experimental group of cells by the control group to obtain the Fold difference value (Fold change), carrying out logarithmic normalization on the Fold difference value by taking 2 as a base number, and obtaining the log2The FC was set to be larger than 1 to obtain 3244 genes, 2474 genes were obtained by removing meaningless data from them, and their P values (P values) were calculated, normalized by negative logarithm with the P value being base 10, and subjected to mapping analysis using Microsoft Excel 2010. The results are shown in FIG. 1A as-log10(P value) greater than 2 is 187 highly significant divergent genes, which are also the most likely molecules that potentially regulate PD-L1.
2. Since the genes are based on data obtained from lung cancer cells, it is expected that the relationship between the 187 genes and the expression of PD-L1 in melanoma can be analyzed by using relevant clinical data of melanoma. Therefore, we downloaded the standardized file series matrix file in the GEO database melanoma gene chip data GSE 15605. If a gene corresponds to multiple gene probes, the average is taken. 144 of the 187 genes obtained in FIG. 1A were detected in this data, including PD-L1. Next, we used the expression values of 12 metastatic melanoma tissues on the gene chip to calculate the correlation between the 143 genes and the expression of PD-L1, expressed as Pearson correlation (R), and mapped the correlation number into GraphPad Prism 8. As a result, as shown in FIG. 1B, setting the correlation number to be more than 0.4 resulted in a molecule positively correlated with medium and intensity in PD-L1, for a total of 26 genes.
3. Genes with a correlation coefficient of more than 0.4 and PD-L1 were input into TB tool software to plot heat maps for 16 normal skin tissues and 12 metastatic melanoma tissues (GSE15605), Row scale was set to normalized, normalized normally in the line, and gene expression trends were compared. As a result, as shown in FIG. 1C, red color means high expression, and blue color means low expression, and it can be observed that the first 8 genes are mostly red in normal tissues and mostly blue in metastatic melanoma tissues, i.e., are low expressed in metastatic melanomas. The latter 18 genes are mostly red in metastatic melanoma, i.e., highly expressed. Moreover, PD-L1 shows a trend of high expression in the metastatic melanoma, and the suggestion that the expression of PD-L1 in the metastatic melanoma has important significance. In which we observed a high expression of the specific E3 ubiquitin enzyme TRAF6 in most metastatic melanomas, and the analysis in fig. 1B concluded that it was positively correlated with PD-L1, we reasoned that TRAF6 might play a role in the regulation of PD-L1 in melanoma cells, a target potentially relevant to clinical treatment and prognosis and to PD-L1 regulation.
Example 2 TRAF6 Positive Regulation of PD-L1
1. Construction of a melanoma cell line knocking down or overexpressing TRAF6 by lentivirus packaging infection, 2X 10 cells were first introduced6HEK 293T cells were plated on 6cm cell culture dishes, supplemented with 5mL DMEM (10% FBS and 1% P/S in complete medium), and plated on 5% CO2The culture was carried out in a cell culture chamber at 37 ℃. After 18-24 h, the HEK 293T cells reach 60% -70% confluency, and the cell culture medium is changed to 3mL 5% FBS DMEM complete culture medium. Adding 475 mu L of HBS solution into a 1.5mL centrifuge tube, sequentially adding 5 mu g of packaging plasmid and 5 mu g of target plasmid (human TRAF6 no-load control plasmid, TRAF6 gene cDNA slow virus expression plasmid, human TRAF6 knockdown shRNA no-load control plasmid, human TRAF6 knockdown shRNA slow virus expression plasmid # 1 and #2), and finally dropwise adding 25 mu L of 2.5mol/L CaCl2The solution was protected from light at room temperature for 20min, and the mixture was added dropwise to the medium of HEK 293T cells, and the cells were cultured.
After 12h, the complex-mixed medium was removed to discard the old medium, and 4mL of DMEM medium containing 30% FBS was added, followed byAnd (5) continuing culturing. After 48h, 1X 106And (3) laying the melanoma cells in a 6-well plate in advance, removing the original culture medium of the cells after 18-24 hours until the cells reach 40-50% confluence, adding the harvested virus suspension for about 72 hours into the melanoma cells, adding polybrene with the final concentration of 8 mu g/mL, and continuing to culture the cells after uniform mixing. After 24h, the virus suspension was removed and the culture was continued in complete medium replaced with 10% FBS. And collecting the cell sample for subsequent analysis after 48-96 h.
2. Extracting total RNA of melanoma cell sample after lentivirus infection by Trizol method, and its application
The All-in-One First Strand cDNA Synthesis Supermix reverse transcription kit fully and uniformly mixes a reverse transcription reagent and 1 mu g of RNA according to a20 mu L system, and then places the mixture in a PCR instrument for reverse transcription to obtain cDNA. Real-time fluorescent quantitative PCR reaction was performed in a 10. mu.L system, and the level of PD-L1 mRNA was detected using β -actin as an internal control. The primer sequence of the beta-actin reference gene is as follows: a forward primer F: CCACACTGTGCCCATCTAC, respectively; reverse primer R: AGGATCTTCATGAGGTAGTCAGTC, respectively; the primer sequence of PD-L1 is: a forward primer F: GGCATTTGCTGAACGCAT, respectively; reverse primer R: CAATTAGTGCAGCCAGGT are provided. The results of fluorescent quantitative PCR are shown in FIGS. 2A and 2D, and TRAF6 knockdown or over-expression can be used to down-regulate or raise the mRNA expression level of PD-L1 to different degrees in both A2058 and SK-MEL melanoma cell lines.
3. Add 100. mu.L of RIPA lysate to approximately 2X 106The melanoma cell sample is fully blown and placed on ice for lysis for 30min, and protein supernatant is collected after centrifugation at 12000rpm for 10 min. Protein concentration was quantified using Braford staining solution after 10-fold dilution of the protein solution. According to the protein concentration, the amount was 1. mu.g/. mu.L using ultrapure water and 5 × loading Buffer, followed by boiling at 100 ℃ for 20 min. 10% SDS-PAGE gel, electrophoresis, 60V, 1.5h, then membrane transfer, 330mA, 90min, blocking with 5% skimmed milk powder for 1h, adding TRAF6, PD-L1 and beta-actin antibody (diluted according to the specification), and incubating overnight at 4 ℃. TBST was washed 3 times and incubated with the corresponding secondary antibody for 1 h. TBST cleaning for 3 times, adding developer to the film, and placing the film into a chemiluminescence apparatusAnd (5) line development. Results as shown in fig. 2B and 2E, significant down-regulation or elevation of the protein levels of PD-L1 can occur when TRAF6 is knocked down or over expressed in a2058 and SK-MEL melanoma cell lines.
4. The melanoma cells of different groups are digested and prepared into cell suspensions, after PBS is washed, 100 mu of LAPC-PD-L1 flow type antibody (the ratio of the antibody to staining solution is 1:100) is added into each tube and mixed evenly, the mixture is incubated on ice for 15min, the staining solution is removed by centrifugation at 1000rpm and 4 ℃ for 5min, and 300 mu L-500 mu L of PBS is re-suspended in each tube. The membrane protein level of PD-L1 was analyzed by flow cytometry, and fig. 2C shows that the peak of PD-L1 clearly shifts to the left, i.e., exhibits a reduced level, when TRAF6 is reduced compared to the positive control group. FIG. 2F shows that the peak of PD-L1 shifts significantly to the right, i.e., has an increasing trend, when TRAF6 is overexpressed. The above results all indicate that TRAF6 can positively regulate PD-L1.
Example 3 TRAF6 inhibitors may enhance CD8 by downregulating PD-L1 expression+T cell Activity (in vitro)
1. The above results have demonstrated that TRAF6 can positively modulate PD-L1, and thus inhibition of TRAF6 is expected to reduce endogenous PD-L1 and thereby impair tumor immune escape. Since TRAF6 plays an important role in innate immunity and adaptive immunity, it is necessary to select proper TRAF6 inhibitors that exert tumor killing effect without significantly affecting the normal immunity of the body. According to the reports in the literature, parthenolide or quinine and the like can effectively inhibit TRAF6 activity so as to inhibit cancer cell proliferation, and the proteasome inhibitor MG132 can slow the tumor cell proliferation by inhibiting the expression of TRAF 6. In addition, the reversible proteasome inhibitor bortezomib can inhibit the activity of proteasome 26S subunits, reduce the degradation of inhibitor IkB of NF-kB, promote the combination of the IkB and the NF-kB to inhibit the activity of the NF-kB, reduce the secretion of myeloma cell growth factors such as IL-6 and the like, so the bortezomib is approved to be used for treating multiple myeloma, and other researches prove that the bortezomib can promote TRAF6 autophagy-mediated lysosome degradation so as to reduce TRAF 6. Bortezomib is used as a clinical drug, and the safety is better guaranteed than other reported drugs, so that the bortezomib is selected as a TRAF6 inhibitor, and whether the bortezomib can down-regulate endogenous PD-L1 or not is observed. We treated A2058 cells with DMSO and 20nM, 30nM and 40nM bortezomib, respectively, collected protein samples after 12h, and western blots to detect the corresponding protein levels, as shown in FIG. 3A, when cells were treated with 30nM bortezomib for 12h, the protein levels of TRAF6 and PD-L1 in A2058 cells were effectively reduced, demonstrating that 30nM was applied for 12h at the appropriate concentration and time.
2. To further verify whether binding to PD-1 is reduced after bortezomib reduces PD-L1, a control and 30nM bortezomib are used for washing melanoma cells for 12h, the melanoma cells are incubated with 5 mug/mL recombinant human PD-1-mFC chimeric protein (1:50) at 37 ℃ for 15min, the melanoma cells are incubated with a goat anti-mouse Cy3 secondary antibody at room temperature for 30min after being fixed and sealed, and the cells are sealed after DAPI staining and photographed and detected by a confocal laser microscope. FIG. 3B demonstrates that A2058 cells treated with 30nM bortezomib for 12h were also effective in reducing binding to PD-1, consistent with the results above.
3. Next, we extracted T cells from blood collected from normal healthy people to co-culture with melanoma cells. First we prepared a dilution of CD3 antibody by adding 10. mu.g of CD3 antibody to 1mL of PBS one day in advance, added the antibody dilution to a 6cm cell culture dish, and placed in a biosafety cabinet to ventilate overnight. The next day the Ficoll fraction was added to a 50mL centrifuge tube tilted at 45 ℃ and the collected blood was added slowly along the centrifuge tube wall (blood: Ficoll fraction 2:1) and centrifuged at 1000rpm for 40min at 24 ℃ to separate the blood into four layers. Carefully sucking the intermediate cloud layer into a new 50mL centrifuge tube to obtain PBMC (peripheral mononuclear blood cells), adding RMPI 1640 culture medium (culture medium: PBMC cells 2:1), centrifuging at 1000rpm and 4 ℃ for 10min, removing supernatant, washing with PBS, and counting. Extracting T cells according to a MojoStort T cell extraction kit, suspending the extracted T cells in 5mL of RMPI 1640 medium containing 10% FBS, placing the RMPI 1640 medium in a 6cm cell culture dish coated with a CD3 antibody, adding IL-2 cytokine with a final concentration of 5 ng/mu L, continuously culturing and activating the RMPI medium in a carbon dioxide incubator for 72h, then co-culturing the RMB and A2058 cells according to a ratio of 12:1, simultaneously adding 30nM bortezomib or 0.5 mu g/mL PD-1 antibody for co-incubation, carefully collecting the supernatant of the T cells of each hole in a 1.5mL EP tube during sample collection, centrifuging at 1000rpm and 4 ℃ for 5min, removing the supernatant, washing the PBS twice, and incubating the supernatant with anti-human FITC-CD3, BV anti-human 421-CD4 and anti-human PerCP/Cyanine5.5-CD8 antibodies for 15min on ice to complete flow-type surface staining.
After washing with PBS, 150. mu.L of the fixative was added to each tube and mixed well, and incubated at room temperature for 20 min. Washing with PBS, adding 150 μ L of membrane-breaking solution into each tube, mixing, centrifuging at 1000rpm, 4 deg.C for 5min, and removing membrane-breaking solution. mu.L of anti-human APC/Cyanine7-IFN γ flow antibody (antibody: membrane-disrupting solution: 1:100) was added to each tube, incubated at room temperature for 20min, centrifuged at 1000rpm, 4 ℃ for 5min, and the supernatant discarded. After PBS washing, the samples were resuspended in 300. mu.L to 500. mu.L PBS and analyzed by flow analysis for CD8+Proportion of T cells and IFN γ on CD8+Proportion of T cells. The experimental results in FIG. 3C demonstrate that CD4 is higher in the bortezomib-treated group than in the control group+T cells and CD8+The T cell proportion is not changed significantly, and the anti-PD-1 monoclonal antibody treatment group is slightly increased. However, FIG. 3D shows that the bortezomib-treated group and the anti-PD-1 antibody-treated group significantly potentiate IFN γ+CD8+T cell ratio, suggesting that 30nM bortezomib can effectively activate the killer CD8+T cells.
4. In addition to measuring the level of activation of T cells, we also examined killing of a2058 cells. Staining the collected melanoma cell sample according to the apoptosis flow kit specification, as shown in fig. 3E, the late apoptosis ratios of 30nM bortezomib and anti PD-1 monoclonal antibody are higher than those of the control group, which indicates that bortezomib can effectively kill melanoma cells and has similar killing effect to anti-PD-1. In conclusion, bortezomib can down-regulate tumor endogenous PD-L1 by inhibiting TRAF6, thereby strengthening T cell activity and better eliminating melanoma cells.
In conclusion, bortezomib, although a broad-spectrum drug, was tested at 30nM to simultaneously lower the protein levels of TRAF6 and PD-L1, demonstrating that bortezomib could pass this pathway and thereby down-regulate PD-L1. 30nM was also effective in activating CD8 in melanoma cell co-culture with T cells+T cells and more effective tumor killing, suggesting that bortezomib can passInhibiting TRAF6 down-regulating PD-L1, promoting killing CD8+T cells are activated to inhibit tumor immune escape. Therefore, TRAF6 inhibition and thus down-regulation of tumor endogenous PD-L1 can be used as a potential new strategy for tumor immunotherapy.
Example 4 TRAF6 inhibitor is effective in killing melanoma (in vivo)
1. We hope to further investigate whether TRAF6 inhibition in vivo in a physiological environment could increase tumor infiltration killing CD8+The proportion of T cells and the effective killing of melanoma cells, and the anti-PD-1 monoclonal antibody treatment is prepared as a positive control. Therefore, the SPF grade C57BL/6 mice of 6-8 weeks old are purchased from Beijing Wintonlifa experiment technology Limited and are bred in the sterile environment of the experimental animal center of east school district of Zhongshan university. 2X 10 through the tail vein6A mouse melanoma lung metastasis model was constructed by injecting B16F10 mouse melanoma cells into C57BL/6 mice. The day after the tumorigenesis, the mice were randomly divided into 3 groups of 5 mice each, i.e., a tumorigenic group (PBS group), a bortezomib group (Bor group), and an anti-PD-1 monoclonal antibody group, while 5 mice without tumorigenesis were prepared as blank controls (Normal control, NC group). Body weights were recorded starting on the day of neoplasia, and every other day thereafter. The administration is started the next day after the tumor formation, the dosage of the Bor group is 0.2mg/kg, the dosage of the anti-PD-1 monoclonal antibody group is 150 mu g/mouse, and the administration is performed once every three days later. The sample was prepared on day 18 after molding. The dosing profile is shown in figure 4A.
From the day of modeling to the period of dissection we recorded the body weight curves of the mice, and figure 4E shows that the body weights of the four groups of mice all have a small upward trend, indicating that the body weights of the mice are not significantly affected by the dose of 0.2mg/kg bortezomib and 150 μ g/mouse anti-PD-1 single drug.
At day 18 after the model creation, the mice were anesthetized and dissected, and the lungs of the mice were photographed to directly observe the growth of melanoma, and fig. 4B shows that the Bor group and the anti-PD-1 monoclonal antibody group can effectively inhibit the growth of melanoma compared with the PBS group.
Then, the melanoma nodules of the lung of the mouse are calculated under a dissecting microscope, and classification is well performed, wherein the melanoma nodules are classified into 4 grades: the grade I is less than 0.5mm, the grade II is less than or equal to 0.5mm and less than 1mm, the grade III is less than or equal to 1mm and less than or equal to 2mm, the melanoma nodule fraction of the mice is I multiplied by 1+ II multiplied by 2+ III multiplied by 3+ IV multiplied by 4, the melanoma nodule fractions of the mice in all groups are calculated through the formula, as shown in figure 4D, the melanoma nodule fractions of the PBS group are far greater than those of the Bor group and the anti-PD-1 monoclonal antibody group, and the Bor group and the anti-PD-1 monoclonal antibody group have no significant difference.
Then we performed hematoxylin-eosin staining and Ki67 staining on the dissected part of lung tissue of the mice to preliminarily observe the pathological condition of the lung tissue, and as a result, as shown in FIG. 4C, the nuclei of the lung tissue of NC group, i.e., normal mice, were smaller and the tissue was looser, while in PBS group, the nuclei of the tumor part were larger and very dense, which clearly contrasts with the normal tissue beside the tumor. The tumor parts of the Bor group and the anti-PD-1 monoclonal antibody group are obviously smaller than those of the tumor-producing group, which shows that the bortezomib and the anti-PD-1 monoclonal antibody can effectively inhibit melanoma. Ki67 staining indicates that the immunohistochemical staining of the tumor part Ki67 in the PBS group is dark brown yellow, the area is far larger than that of the Bor group and the anti-PD-1 monoclonal antibody group, and the staining condition is consistent with that of hematoxylin-eosin. Experimental results prove that 0.2mg/kg of bortezomib can effectively prevent the progress of mouse melanoma and can achieve the curative effect similar to that of anti-PD-1 monoclonal antibody treatment.
2. We need to further know whether bortezomib can effectively kill melanoma by inhibiting TRAF6 endogenous to down-regulate the expression of melanoma cells PD-L1, enhancing in vivo anti-tumor immunity. Therefore, we tested the protein expression levels of TRAF6 and PD-L1 in mouse lung tissues by western blot. Fig. 4F and 4G show that TRAF6 and PD-L1 protein levels in the PBS group were significantly elevated compared to the NC group, while Bor group significantly reduced both protein levels, TRAF6 in the anti-PD-1 mab group was slightly reduced, but not as significant as Bor group, while PD-L1 levels were significantly down-regulated. It has been reported in literature that anti-PD-1 monoclonal antibody can eliminate tumor cells which are over-expressed by PD-L1 and have hypomethylated PD-L1 level or inhibit the expression of PD-L1 through epigenetic regulation, which may explain the down-regulation effect of PD-L1 caused by anti-PD-1 monoclonal antibody treatment. However, the results show that bortezomib can effectively inhibit TRAF6 expression and PD-L1 expression. In addition, the immunohistochemical staining of TRAF6 and PD-L1 is carried out on partial lung tissues of the mice at the same time, the brown yellow represents protein expression, the shade of the color represents the strength of the protein expression, and the results in FIG. 4H and FIG. 4I are consistent with the results of western blot detection. All show that the bortezomib can inhibit TRAF6 in melanoma to reduce the expression of endogenous PD-L1, so that melanoma cells are effectively killed.
3. Whether endogenous PD-L1 reduces the ability to activate anti-tumor immunity in the body to kill melanoma cells requires further investigation. Preparing a cell suspension from a part of dissected lung tissue, centrifuging at 1500rpm at 4 ℃ for 5 min; and (3) removing the supernatant, adding 2mL of erythrocyte lysate into each part, mixing uniformly, incubating on ice for 5min, centrifuging to remove the supernatant, dividing the lung tissue cell suspension of each sample into three parts after the PBS is resuspended, centrifuging to remove the supernatant, incubating each tube of each part with an anti-mouse FITC-CD3 antibody, an anti-mouse APC-CD4 antibody and an anti-mouse BV421-CD8 flow antibody, and performing flow detection. FIG. 4J the results of the study show that the Bor group and anti-PD-1 group significantly increased the tumor infiltration of CD8 in lung tissue compared to the PBS group+T cell proportion, and has significant difference, and the Bor group and the anti-PD-1 group have no significant difference.
4. Centrifuging the other two lung tissue cell suspensions to remove supernatant, co-incubating the supernatant with FITC-CD3 antibody and anti-mouse BV421-CD8 flow antibody, fixing and breaking membranes, co-incubating the supernatant with anti-mouse APC-IFN gamma or anti-mouse AF647-Granzyme B flow antibody at room temperature for 20min, washing with PBS, and then suspending the sample in 300-500. mu.L PBS for flow analysis. The results are shown in FIG. 4J, where Granzyme B and IFN γ account for CD8 in the Bor group+The proportion of T cells is obviously increased compared with that of the PBS group, and the T cells are obviously different; while the Granzyme B level in the anti-PD-1 monoclonal antibody group is increased very significantly, the IFN gamma level is up-regulated but has no significant difference, but the activation of CD8 can be also shown+A T cell; and the Bor group and the anti-PD-1 monoclonal antibody group have no significant difference. The results show that bortezomib can increase lung tumor infiltration CD8+The proportion and the activity of the T cells strengthen the anti-tumor immunity in an organism, thereby better killing melanoma cells and having the curative effect similar to that of anti-PD-1 monoclonal antibody.
5. Furthermore, we needed to test whether bortezomib treatment negatively affected other sites in mice, so we dissected the cervical, spleen and peripheral lymph nodes of mice, incubated with anti-murine FITC-CD3 antibody, anti-murine APC-CD4 antibody, anti-murine BV421-CD8 flow antibody and then detected their CD4 by flow analysis+And CD8+T cell proportion. The results showed that the PBS group had CD8 in the cervical, spleen and peripheral lymph nodes compared to the NC group+The proportion of T cells is reduced, but only the cervical lymph nodes have significant difference; CD8 of these three sites in the Bor group+The T cell ratio was not significantly changed compared to the PBS group, suggesting that low doses of bortezomib did not affect other immunity in vivo; CD8 of cervical and peripheral lymph nodes in anti-PD-1 monoclonal antibody group+The increase in T cell proportion was significant and significantly different, but spleen changes were not significant. The results are combined, and the results show that the low dose of bortezomib does not influence the normal immune function in the body, and can increase the T cells at the tumor infiltration part so as to kill melanoma more effectively.
Claims (9)
- Use of TRAF6 in the regulation of PD-L1 expression.
- Use of TRAF6 in the preparation of a formulation for modulating PD-L1 expression.
- Use of a TRAF6 inhibitor in the preparation of a formulation for inhibiting the expression of PD-L1 on the surface of tumor cells.
- The application of the TRAF6 inhibitor in preparing melanoma treatment medicines is characterized in that the TRAF6 inhibitor can inhibit the expression of PD-L1 on the surface of melanoma cells, reduce the immune escape phenomenon of the combination of PD-L1 and PD-1 on the surface of T cells, and promote the normal immune function of the T cells.
- Application of TRAF6 inhibitor as PD-L1 regulator in preparing tumor therapeutic medicine.
- 6. The use of any one of claims 3 to 5 wherein the TRAF6 inhibitor is bortezomib, a clinical agent.
- 7. The use of claim 6, wherein bortezomib down-regulates tumor cell surface PD-L1 expression at a concentration and for a time of 30nM and 12 h.
- 8. The use according to claim 6, wherein bortezomib is used as a PD-L1 modulator at a concentration of 0.2 mg/kg.
- 9. The use according to claim 3 or 5, wherein the tumour is melanoma.
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| JIZHONG LIU等: "Plasma cells from multiple myeloma patients express B7-H1 (PD-L1) and increase expression after stimulation with IFN- and TLR ligands via a MyD88-,TRAF6-, and MEK-dependent pathway", 《BLOOD》 * |
| 张克君等: "BH3-only促凋亡蛋白的研究进展", 《中华普通外科学文献(电子版)》 * |
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