Application of STAT3 CCD structural domain small-molecule inhibitor HB in preparation of medicine for treating brain glioma

Abstract

The invention belongs to the field of tumor treatment, and particularly relates to application of an STAT3CCD structural domain small-molecule inhibitor hygromycin B (HB) in preparation of a drug for treating brain glioma. Researches show that HB can form hydrogen bonds with T145, T152, T265 and T272, and HB shows a strong inhibitory effect on the survival rate of GBM cells in a dose-dependent manner, indicating that HB is a potential small molecule which may destroy the interaction of CHI3L1-STAT3; furthermore, the HB obviously reduces the phosphorylation of STAT3 in the CHI3L1 induced MES50 cells, the nuclear accumulation of p-STAT3 and the expression level of CD44 and SPP1. When injected in situ, the administration of HB significantly reduces the tumor size and increases the survival rate in a dose-dependent manner. The invention provides a new means and thought for treating glioblastoma.

Description

Technical Field

[0001] The present invention belongs to the field of tumor treatment, and specifically relates to the application of hygromycin B (HB), a small molecule inhibitor of STAT3 CCD domain, in the preparation of a drug for treating brain glioma. Background Art

[0002] Glioblastoma (GBM) is highly heterogeneous and can be divided into pre-neural (PN), mesenchymal (MES), and classical (CL) subtypes, which respond differently to intensive treatment. Evidence suggests that PN cells acquire more invasiveness and treatment resistance by shifting phenotypic characteristics to the MES state, a process known as PN to MES transition (PMT). During the PMT process, several transcription factors play key regulatory roles, including STAT3 and NF-kB.

[0003] GBM also acquires high immunosuppression and immunotherapy resistance by reprogramming the tumor immune microenvironment (TIM). Among the TIMs of GBM, macrophages derived from blood monocytes constitute the most abundant immune cell infiltrating population. Increasing evidence shows that M2 macrophages accumulate in large numbers in high-grade tumors. In contrast, the anti-tumor M1 macrophage phenotype leads to inhibition of tumor growth. Macrophages play a crucial role in PMT of glioma. However, how macrophages drive PMT remains largely unclear. Therefore, elucidating the potential crosstalk mechanism between glioma cells and macrophages is of great significance for predicting new effective molecular targets for GBM.

[0004] In fact, the high heterogeneity and immunosuppressive microenvironment of glioblastoma are key factors in its treatment resistance. In the past, traditional drugs were developed based on the characteristics of the tumor itself, ignoring the impact of the immunosuppressive microenvironment around the tumor on the tumor, which ultimately led to treatment failure.

[0005] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides an application of a STAT3 CCD domain small molecule inhibitor HB in the preparation of a drug for treating brain glioma.

[0007] Preferably, the drug is a drug that can form hydrogen bonds with T145, T152, T265 and T272.

[0008] Preferably, the drug is a drug that can inhibit the survival rate of GBM cells.

[0009] Preferably, the drug is a drug that can inhibit the survival rate of GBM cells in a dose-dependent manner.

[0010] Preferably, the drug is a drug that disrupts the CHI3L1-STAT3 interaction.

[0011] Preferably, the drug is a drug that reduces the phosphorylation of STAT3, the nuclear accumulation of p-STAT3, and the expression levels of CD44 and SPP1 in MES50 cells induced by CHI3L1.

[0012] Preferably, the drug is a drug that inhibits the expression levels of MES markers and SPP1+ macrophage markers.

[0013] Preferably, the drug is in the form of an injection.

[0014] The beneficial effects of the present invention are:

[0015] The present invention has shown through research that hygromycin B (HB) is a new type of STAT3 CCD screening inhibitor, which can effectively block the CHI3L1-STAT3 interaction, thereby inhibiting tumor growth in vivo and in vitro, and provides a new means and idea for the treatment of glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 In different treatments (vector, STAT3 ΔCCD 、CHI3L1 WT 、CHI3L11 WT +STAT3 ΔCCD ) Cell viability in LN229 cell group.

[0018] Figure 2 In different treatments (vector, STAT3 ΔCCD 、CHI3L1 WT 、CHI3L11 WT +STAT3 ΔCCD ) Cell viability in the SNB19 cell group.

[0019] Figure 3 Different treatments (vector, STAT3 ΔCCD 、CHI3L1 WT 、CHI3L11 WT +STAT3 ΔCCD) group in LN229 and SNB19 cells.

[0020] Figure 4 In different treatments (vector, STAT3 ΔCCD 、CHI3L1 WT 、CHI3L11 WT +STAT3 ΔCCD Wound healing assay was performed in LN229 cells with 5% paraformaldehyde (P < 0.05), 1% paraformaldehyde (P < 0.01), and 1% paraformaldehyde (P < 0.05).

[0021] Figure 5 In different treatments (vector, STAT3 ΔCCD 、CHI3L1 WT 、CHI3L11 WT +STAT3 ΔCCD Wound healing assay was performed in SNB19 cells with 5% paraformaldehyde (P < 0.05), 1% paraformaldehyde (P < 0.01), and 1% paraformaldehyde (P < 0.05).

[0022] Figure 6 In different treatments (vector, STAT3 ΔCCD 、CHI3L1 WT 、CHI3L11 WT +STAT3 ΔCCD ) Quantification of wound healing assay in LN229 and SNB19 cells.

[0023] Figure 7 In different treatments (vector, STAT3 ΔCCD 、CHI3L1 WT 、CHI3L11 WT +STAT3 ΔCCD Wound healing assay was performed in LN229 and SNB19 cells. All data are expressed as mean ± SD (n = 3) *P < 0.05, **P < 0.01 compared with the control group.

[0024] Figure 8 Bioluminescent images of mice bearing GL261-derived tumors treated with vector, CHI3L1 ΔWT or CHI3L1 ΔWT +STAT3 ΔCCD Transfected.

[0025] Fig. 9 is the quantification of bioluminescent imaging signal intensity at 28 days (n=6).

[0026] Fig.10Figure 2 is the Kaplan-Meier survival curve of mice bearing GL261 tumors, which were derived from mice transfected with Vector, CHI3L1 ΔWT or CHI3L1 ΔWT +STAT3 ΔCCD of mice.

[0027] Fig.11 Transfection Vector, CHI3L1 ΔWT or CHI3L1 ΔWT +STAT3 ΔCCD Representative H&E stained brain sections and IHC (CHI3L1) staining images in transfected GL261 mouse model. Scale bar: 40 μm.

[0028] Fig.12 Transfection Vector, CHI3L1 ΔWT or CHI3L1 ΔWT +STAT3 ΔCCD Representative IHC (p-STAT3, VIM and CD44) staining images in transfected GL261 mouse model. Scale bar: 40 μm.

[0029] Fig.13 Transfection Vector, CHI3L1 ΔWT or CHI3L1 ΔWT +STAT3 ΔCCD Representative IHC (OPN, CD163, and PD-L1) staining images in transfected GL261 mouse model. Scale bar: 40 μm.

[0030] Fig.14 It is the molecular docking model of hygromycin B (HB) and STAT3-CCD domain (macro).

[0031] Fig.15 It is the molecular docking model of hygromycin B (HB) and STAT3-CCD domain (microscopic).

[0032] Fig.16 It is the chemical structure of HB.

[0033] Fig.17 are the computer-predicted scores for the three top inhibitors.

[0034] Fig.18 The effect of hygromycin B on the interaction with STAT3 was detected by immunoprecipitation.

[0035] Fig.19 The effect of Verbascoside on the interaction between CHI3L1 and STAT3 was detected by immunoprecipitation.

[0036] Fig. 20 The effect of 8-Oxo-dG on the interaction between CHI3L1 and STAT3 was detected by immunoprecipitation.

[0037] Fig.21 Cell viability of LN229 and SNB-19 cells treated with different concentrations of HB for 24 hours.

[0038] Fig. 22 is the cell viability of MES50 and MES52 cells treated with different concentrations of HB for 24 hours.

[0039] Fig.23 The effects of HB treatment (10 μM) for 24 h on the levels of pSTAT3 (Tyr705), STAT3, CD44, and SPP1 in MES50 cells transfected with vector or CHI3L1.

[0040] Fig.24 Immunofluorescence analysis of the subcellular localization of CHI3L1, pSTAT3 (Tyr705), CD44 and SPP1 in MES50 cells.

[0041] Fig.25 It is the process of establishing an orthotopic GBM mouse model and drug administration.

[0042] Fig.26 are bioluminescent images of mice bearing GL261CHI3L1-WT tumors treated with different concentrations of HB for the indicated times.

[0043] Fig. 27 The bioluminescence imaging signal intensity was quantified on day 28 (n=6).

[0044] Fig.28 GL261 treated with different concentrations of HB at the specified time CHI3L1-WT Kaplan-Meier survival curves of mice with derived tumors.

[0045] Fig.29 GL261 cells were transfected with HB at the indicated concentrations. CHI3L1-WT Representative H&E-stained brain sections and IHC staining images in mouse models (CHI3L1).

[0046] Fig.30 GL261 cells were transfected with HB at the indicated concentrations. CHI3L1-WT Representative IHC staining images (p-STAT3, VIM, and CD44) in mouse models.

[0047] Fig.31 GL261 cells were transfected with HB at the indicated concentrations.CHI3L1-WT Representative IHC staining images (OPN, CD206, and PD-L1) in mouse models. DETAILED DESCRIPTION

[0048] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0049] Example

[0050] (I) In vitro experiments

[0051] (1) Cell culture

[0052] Human GBM cell lines (LN229 and SNB-19), mouse glioma cell line (GL261), and normal human astrocytes (NHAs) were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) containing 10% FBS (HyClone) and 1% penicillin-streptomycin (P / s).

[0053] (2) CCK-8 assay

[0054] Cell Counting Kit 8 was used to assess the viability of GBM cells according to the manufacturer's instructions. Briefly, 1 × 10 4 Pretreated cells were seeded into 96-well plates, and the volume of medium added to each well was 100 μL. The next day, after adding 10 μL of CCK8 solution to each well, the cells were incubated at 37°C for 2 hours. Finally, the absorbance (450 nm) was measured by SpectraMax i3x reader.

[0055] (3) Co-immunoprecipitation (Co-IP)

[0056] Total protein of the indicated cells was extracted with RIPA buffer on ice, and a small amount of lysate was removed as input. The remaining cell extract was incubated with anti-rabbit IgG (0.5 μg) or corresponding antibody (0.5 μg) on ​​a rotator at 4 ° C overnight. Subsequently, magnetic beads were added to the protein lysate and incubated at 4 ° C for 2 hours. The next morning, the magnetic bead mixture was washed with PBS and then subjected to 10% SDS-PAGE for further western blot detection.

[0057] (4) Immunofluorescence

[0058] For IF of cells, slides with cultured cells were fixed in 4% paraformaldehyde for 20 min, permeabilized, and blocked with 0.3% Triton X-100 and 10% donkey serum in PBS for 30 min at 37°C. Next, coverslips were incubated overnight at 4°C with primary antibodies diluted in 5% goat serum in PBS, followed by incubation with Alexa-Fluor-488- or 674-conjugated secondary antibodies diluted in 5% goat serum in PBS for 2 h in the dark. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Immunofluorescence images were recorded by laser scanning confocal microscopy (Leica, TCS SPE).

[0059] (5) Colony formation test

[0060] In the colony formation assay, a total of 3000 cells were seeded in a 6-well plate and incubated for 14 days. The resulting colonies were washed twice with PBS, fixed with 4% formaldehyde for 10 minutes, and stained with 0.1% crystal violet for 30 minutes. The colonies were photographed and counted.

[0061] (6) Migration experiment

[0062] To evaluate cell migration ability, equal amounts (1×10 6 ) were seeded into the upper chamber and incubated overnight. Subsequently, 500 μL of complete medium supplemented with 10% FBS was added to the lower chamber of the Transwell insert to promote cell migration. After another 24 h of incubation, cells that migrated through the Transwell insert membrane were stained with crystal violet and photographed by microscopy.

[0063] (7) Wound healing test

[0064] Wound healing assay was used to evaluate the migration ability of different cells. Briefly, glioma cells were seeded in 6-well plates and incubated overnight. The next day, the inserts were removed and serum-free medium was added. After 36 hours, bright-field images were acquired using a microscope.

[0065] (II) Animal experiments

[0066] (1) Mouse tumor model

[0067] Female C57BL / 6 mice (6-8 weeks old, 18-20 g) were purchased from the Animal Center of SPF (Beijing) Biotechnology Co., Ltd. and were raised under special pathogen-free conditions. Luciferase-induced GL261 cells (1×10 6 ) Overexpression vector CHI3L1 WT or CHI3L1 WT +STAT3 ΔCCDStereotactic injection was performed into the brain of C57BL / 6 mice at a depth of 4 mm in the right hemisphere, 1 mm relative to the anterior horn and 2 mm lateral to the brain. Intracranial tumors were observed by BLI at the designated time. WT Mice were randomly divided into 3 groups (n=3 in each group) and treatment was started 7 days after tumor implantation. We prepared HB in sodium chloride solution for intraperitoneal injection. Mice received 100 μL of sodium chloride solution (control group) or HB (5 or 10 mg / kg) in sodium chloride solution five times a week for four weeks by intraperitoneal injection. At 7, 14 and 28 days, intracranial tumors were measured with BLI by intraperitoneal injection of D-luciferin. After all mice died, the survival time of the mice was recorded, and the mouse tissues were carefully extracted and fixed with 10% formalin for H&E and IHC staining. All animal experiments were approved by the Institutional Animal Care Committee.

[0068] (2) Plasmid construction and lentiviral packaging

[0069] Mouse CHI3L1 WT and STAT3 ΔCCD Overexpression lentivirus was synthesized and purchased from Gene Chemical Company (Shanghai, China). All transfections were performed using Lipo3000 transfection reagent.

[0070] (III) Results and Discussion

[0071] (1) Given the important role of the CHI3L1-STAT3 axis in SPP1 production in tumor cells and macrophages, leading to the occurrence of PMT, we compared different treatments (vector, STAT3 ΔCCD 、CHI3L1 WT 、CHI3L1 WT +STAT3 ΔCCD ) were transfected into LN229 and SNB-19 cells.

[0072] The results showed that STAT3 ΔCCD Can significantly counteract CHI3L1 WT Overexpression-mediated tumor cell survival ( Figure 1 , Figure 2 ), colony formation ( Figure 3 ) and Migration( Figure 4 to Figure 7 ), more specifically:

[0073] Depend on Figure 1 , Figure 2 It can be seen that: in different treatments (vector, STAT3 ΔCCD 、CHI3L1 WT 、CHI3L11 WT +STAT3 ΔCCD) Cell viability was measured in LN229 and SNB19 cells, and the results showed that STAT3 ΔCCD Can significantly counteract CHI3L1 WT Overexpression-mediated tumorigenicity on cell survival.

[0074] Depend on Figure 3 It can be seen that colony formation assays showed that STAT3 ΔCCD Can significantly counteract CHI3L1 WT Overexpression mediates tumor colony formation.

[0075] Depend on Figure 4 to Figure 7 (All data are expressed as mean ± standard deviation (n = 3), compared with the control group, *P < 0.05, **P < 0.01) It can be seen that the migration test results showed that STAT3 ΔCCD Can significantly counteract CHI3L1 WT Overexpression-mediated tumor effects on migration.

[0076] In addition, this example uses different treatments (carrier, CHI3L1 WT or CHI3L1 WT +STAT3 ΔCCD ) to establish an orthotopic mouse model and express luciferase to facilitate volume measurement. WT In comparison, CHI3L1 WT +STAT3 ΔCCD Overexpression of Figure 8 , Fig. 9 ) and prolonged survival ( Fig.10 ), more specifically:

[0077] Depend on Figure 8 It can be seen that at the specified time, the carriers were derived from the transfected vector, CHI3L1 ΔWT or CHI3L1 △WT +STAT3 ΔCCD Bioluminescent images of GL261 tumor-bearing mice, showing that STAT3 ΔCCD Significantly reduced tumor growth volume.

[0078] Depend on Fig. 9 It can be seen that: is the quantification of the bioluminescent imaging signal intensity of a at 28 days (n=6), the results show that STAT3 ΔCCD Significantly reduced bioluminescence intensity within the tumor growth volume.

[0079] Depend on Fig.10 It can be seen that: Kaplan-Meier survival curves of mice carrying GL261 tumors, these tumors are derived from mice transfected with Vector, CHI3L1ΔWT or CHI3L1 ΔWT +STAT3 ΔCCD GL261.

[0080] IHC analysis showed that mice carrying CHI3L1WT expressed more MES markers (CHI3L1, p-STAT3, VIM, and CD44) and SPP1+ macrophage markers (OPN, CD206, and PD-L1), while CHI3L1 WT +STAT3 ΔCCD The opposite results were shown in the Figure 11 to Figure 13 ), more specifically:

[0081] Depend on Figure 11 to Figure 13 It can be seen that it is a transfection vector, CHI3L1 ΔWT or CHI3L1 ΔWT +STAT3 ΔCCD Representative h&E-stained brain sections and IHC staining images of CHI3L1, p-STAT3, VIM, CD44, and SPP1+ macrophage markers (OPN, CD163, and PD-L1) in the transfected GL261 mouse model. The results show that: WT In contrast, IHC analysis showed that CHI3L1 WT Mice with CHI3L1 expressed more MES markers (CHI3L1, p-STAT3, VIM, and CD44) and SPP1+ macrophage markers (OPN, CD206, and PD-L1), while CHI3L1 WT +STAT3 ΔCCD The group showed the opposite results.

[0082] These results suggest that targeting the STAT3-CCD domain may be beneficial for the treatment of GBM.

[0083] (2) Through virtual screening of the TargetMol database, three results were obtained, namely, hygromycin B (HB), Verbascoside (VB) and 8-Oxo-dG (8D) ( Figure 14 to Figure 17 ), more specifically:

[0084] Figure 14 to Figure 17 The molecular docking model of hygromycin B (HB) and STAT3-CCD domain, as well as the chemical structure of HB, are shown, and the computer-predicted scores of the three top inhibitors are shown.

[0085] Notably, HB, but VB and 8D, interfered with the CHI3L1-STAT3 interaction ( Figures 18 to 20 ).

[0086] At the same time, the docking results showed that hygromycin B (HB) could form hydrogen bonds with T145, T152, T265 and T272 ( Figure 14 to Figure 16 ), HB showed a strong inhibitory effect on the survival rate of GBM cells in a dose-dependent manner ( Figure 21-22 ), indicating that HB is a potential small molecule that may disrupt CHI3L1-STAT3 interaction. Then, we continued to study whether HB would interfere with CHI3L1-STAT3 interaction and inhibit SPP1 expression. The results showed that HB significantly reduced the phosphorylation of STAT3, nuclear accumulation of p-STAT3, and expression levels of CD44 and SPP1 in MES50 cells induced by CHI3L1 ( Figure 23-24 ).

[0087] (3) In a mouse model injected orthotopically with GL261CHI3L1-WT cells ( Fig.25 ). Administration of HB significantly reduced tumor size in a dose-dependent manner ( Fig.26 , Fig. 27 ) and improved survival ( Fig.28 ). IHC results showed that HB significantly inhibited the expression of MES markers ( Fig.29 , Fig.30 ) and the expression levels of SPP1+ macrophage markers ( Fig.31 ). These data suggest that STAT3-CCD domain inhibitor HB can be used as a therapeutic agent for GBM.

[0088] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Application of a STAT3 CCD domain small molecule inhibitor HB in the preparation of drugs for treating brain glioma.

2. The use according to claim 1, characterized in that: The drug is a drug that can form hydrogen bonds with T145, T152, T265 and T272.

3. The use according to claim 1, characterized in that: The drug is a drug that can inhibit the survival rate of GBM cells.

4. The use according to claim 3, characterized in that: The drug is a drug that can inhibit the survival rate of GBM cells in a dose-dependent manner.

5. The use according to claim 1, characterized in that: The drug is a drug that destroys the CHI3L1-STAT3 interaction.

6. The use according to claim 1, characterized in that: The drug is a drug that reduces the phosphorylation of STAT3, the nuclear accumulation of p-STAT3 and the expression levels of CD44 and SPP1 in MES50 cells induced by CHI3L1.

7. The use according to claim 1, characterized in that: The drug is a drug that inhibits the expression levels of MES markers and SPP1+ macrophage markers.

8. The use according to claim 1, characterized in that: The medicine is in the form of an injection.

Images