CN115337306B

CN115337306B - Application of dihydroergotoxine mesylate targeted STAT3 in preparation of anti-inflammatory diseases and anti-tumor drugs

Info

Publication number: CN115337306B
Application number: CN202211141779.XA
Authority: CN
Inventors: 杨争艳; 任志广; 胡延忠; 薛静蕊; 赵祎炜
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2022-09-20
Filing date: 2022-09-20
Publication date: 2023-10-20
Anticipated expiration: 2042-09-20
Also published as: CN115337306A

Abstract

The invention discloses an application of dihydroergotoxine mesylate targeted STAT3 in preparing medicaments for treating breast cancer, liver cancer, colon cancer, gastric cancer and the like. The medicine is dihydroergotoxine mesylate, and reduces the expression of inflammatory cytokines such as IL-1, IL-6, IL-8, IL-10, IL-12, IL-23, TNF-alpha and the like by directly down regulating the expression and activity of STAT 3; and plays roles of resisting tumor cell proliferation and inducing tumor cell apoptosis by inhibiting the expression of target genes such as Cyclin D1, bcl-XL and the like. The drug has small toxic and side effects compared with the traditional chemotherapeutic drugs such as doxorubicin, epirubicin and vincristine, has low price compared with the targeted drugs such as sorafenib and anti-IL-6 antibody, can prevent or reverse drug resistance, and has good anti-tumor application prospect and development value.

Description

Application of dihydroergotoxine mesylate targeted STAT3 in preparation of anti-inflammatory diseases and anti-tumor drugs

Technical Field

The invention relates to the field of biological medicine, in particular to application of dihydroergotoxine mesylate targeted STAT3 in preparation of anti-inflammatory diseases and anti-tumor medicines.

Background

STAT3 (Signal Transducer and Activator of Transcription, signaling and transcriptional activator 3) is one of the most well studied transcriptional regulators of tumor-associated signals. Studies have shown that abnormal activation of STAT3 is associated with poor clinical prognosis for breast, liver, stomach, and colon cancers, etc. The constitutive activation of STAT3 signals can directly or indirectly up-regulate the expression of many oncogenes such as Cyclin D1 (Cyclin D1), B-cell lymphokine XL (BCL-XL), multidrug resistance-associated protein 1 (ABCB 1, MDR 1) and Matrix Metalloproteinases (MMPs), and is involved in all processes of regulating tumorigenesis and development such as proliferation, anti-apoptosis, drug resistance and metastasis (Leslie Kenneth, cynthia Lang, geeta Devgan, et al. Cyclin D1 is transcriptionally regulated by and required for transformation by activated signal transducer and activator of transcription 3.Cancer Res,2006,66 (5): 2544-2552; grad J.M., X.R.Zeng and L.H. Regulation of BCL-XL: a little bit of this and a little bit of STAT.Curr Opin Oncol,2000,12 (6): 543-549;Soleymani Abyaneh H,Gupta N,Radziwon-Balicka A, et al.STAT3 but not yet-1 alpha is important in mediating hypoxia induced Chemoresistance in MDA-MB-231,a triple negative breast Cancer cell line.Cancers (Basel): 2019 (10): 20183/6): STAT 3/378:15:15:. In addition, STAT3 is one of the core transcription factors of "inflammatory cancer transformation". Factors such as smoking, alcoholism, ultraviolet radiation, chronic stress and infection can activate STAT3 through a variety of pathways, and the activated STAT3 in turn exacerbates local inflammatory responses and even induces inflammatory-cancer transformations through positive feedback signaling pathways that activate inflammatory cytokines such as IL-1, IL-6, IL-8, IL-10, IL-12, IL-23 and TNF- α (Hua Yu, drew Pardoll and Richard jove. STATs in cancer inflammation and immunity: a leading role for STATs in cancer inflammation and immunity: cancer 2009 (9): 798-809.). The development of drugs targeting STAT3 has become a hotspot in international research. Some STAT3 signaling inhibitors such as 39965 9150, WP1066, TTI101, OPB-31121 and OPB-51602 have entered the clinical testing stage (Orlowski, r.z. et al a phase 2,randomized,double blind,placebo-controlled study of siltuximab (anti-IL-6 mAb) and bortezomib versus bortezomib alone in patients with relapsed or refractory multiple myeloma.am.j. Hemalol.2015 (90): 42-49; hong, D., et al, AZD9150, a next-generation antisense oligonucleotide inhibitor of STAT3 with early evidence of clinical activity in lymphoma and lung Cancer Sci Transl Med,2015,7 (314): 314ra185; sau, S., et al, combination of cationic dexamethasone derivative and STAT3 inhibitor (WP 1066) for aggressive melanoma: a strategy for repurposing a phase I clinical trial drug Cell Biochem,2017.436 (1-2): 119-136; bharadwaj, U., et al, small-molecule inhibition of STAT3 in radioresistant head and neck squamous Cell carb. Oncostat, 2016.7 (18): 26307-26330; hayakawa, F., et al, A novel STAT inhibitor, OPB-31121,has a significant antitumor effection leukemia with STAT-additive incokines, blood Cancer J,2013.3:e166; ogura, M., et al, phase I study of OPB-51602,an oral inhibitor of signal transducer and activator of transcription 3,in patients with relapsed/52magnetic acids CAr 897, 2015.106 (897). However, these compounds are often limited in development due to insignificant therapeutic advantages or poor patentability. At present, no drug targeting STAT3 has been successfully marketed.

Dihydroergotoxine mesylate, an alpha receptor blocker, has high binding activity to the GABAA receptor. The dihydroergotoxine mesylate can relax vascular smooth muscle and strengthen neurotransmitter transmission, inhibit the activity of ATPase and adenylate cyclase, reduce ATP decomposition, raise the oxidation and utilization of glucose by nerve cells, and improve the blood flow and energy supply of brain cells. Is mainly used for treating vascular dementia and cerebrovascular disease sequelae. At present, the application of dihydroergotoxine mesylate in treating inflammatory diseases, tumors and the like related to STAT3 targets has not been reported.

Disclosure of Invention

Aiming at the problems that the development of STAT3 inhibitors in the prior art is not successful and corresponding clinical medicines are lacking, the invention provides dihydroergotoxine which has the advantages of known drug generation and toxicological effects, less side effects, low price and good development and application prospect, and the application of targeting STAT3 in preparing medicines for treating tumors, and aims to solve the technical problems that clinical chemotherapeutic medicines are easy to resist, tumor recurrence is caused and the existing targeted STAT3 compound has poor patentability.

In order to achieve the aim, the invention discloses application of dihydroergotoxine mesylate targeted STAT3 in preparing medicaments for treating inflammatory diseases and tumors; the medicine is dihydroergotoxine mesylate and derivatives thereof.

The following technical scheme is adopted in detail: the invention is realized in the early stage by constructing a report molecule carrier (purchased from Shanghai right-yang biotechnology Co., ltd.) based on STAT3 transcriptional activityLuciferase Assay System kit (from Promega corporation) 1321 traditional Chinese medicine monomer compounds (from MCE) were subjected to high throughput screening. Dihydroergotamine mesylate was found to inhibit the transcriptional activity of STAT3.

Subsequently, we studied the effects of dihydroergotoxine mesylate on targeting STAT3 protein and inhibiting tumor growth by both in vitro cell lines and in vivo experiments in mice. Experiments show that the dihydroergotoxine mesylate can down regulate the expression of STAT3, p-STAT3 (Tyr 705) and downstream target genes of STAT3, cyclin D1, bcl-XL and Survivin, inhibit the proliferation of cell lines such as breast cancer, liver cancer, gastric cancer and colon cancer in vitro, and induce tumor cell cycle arrest and apoptosis; the anti-tumor cell proliferation effect of the dihydroergotoxine mesylate is equivalent to that of STAT3 inhibitor Static, and the selective competitive antagonist GABAzine applied to the GABAA receptor has almost no tumor inhibition activity, so that the dihydroergotoxine mesylate has tumor inhibition effect by targeting STAT3 instead of the GABAA receptor.

As another invention point, the dihydroergotoxine mesylate is combined with chemotherapeutic drugs such as doxorubicin, sorafenib and the like, can down regulate the expression of P-gp, enhance the sensitivity of breast cancer and liver cancer to the chemotherapeutic drugs, reverse the chemotherapeutic resistance of liver cancer resistant cells and exert stronger synergistic anti-tumor effect in vivo.

The tumor cells inhibited by dihydroergotoxine mesylate can be specifically 4T1, MDA-MB-231, MDA-MB-468, CT-26, HGC-27, hepG2, huH-7, hepG2-R and HuH-7-R. The application of the dihydroergotoxine mesylate in preparing antitumor drugs is that the drug is one of dihydroergotoxine mesylate, dihydroergotoxine mesylate derivatives, compositions containing dihydroergotoxine mesylate or compositions containing dihydroergotoxine mesylate derivatives; the administration mode of the medicine is oral administration or injection.

Drawings

FIG. 1 computer simulated structural analysis of the interaction of dihydroergotoxine mesylate with STAT3 protein. A. Binding of the ligand dihydroergotoxine mesylate to the active "pocket" of STAT 3; B. 3D structure schematic diagram of combination of dihydroergotoxine mesylate and STAT 3; C. dihedral patterns of mode of action and affinity between dihydroergotoxine mesylate and STAT3.

FIG. 2 shows the inhibition of STAT3 reporter transcriptional activity by dihydroergotoxine mesylate at various concentrations using a dual luciferase activity assay.

FIG. 3. Real-time fluorescent quantitative PCR method for detecting the effect of dihydroergotoxine mesylate on mRNA expression levels of STAT3 and its downstream target genes in 4T1 (A) and HuH-7-R (B) cells.

FIG. 4 Western blot analysis of mRNA expression levels of STAT3, p-STAT3, downstream target genes of STAT3 and apoptosis and drug resistance related proteins in 4T1 (A) and HuH-7-R (B) cells with varying concentrations of dihydroergotoxine mesylate.

FIG. 5. Real-time fluorescent quantitative PCR method to detect the effect of dihydroergotoxine mesylate on mRNA expression levels of inflammatory cytokines in HuH-7 cells.

FIG. 6 IC for detecting proliferation inhibition of dihydroergotoxine mesylate in various tumor cell lines by CCK-8 method ₅₀ Values.

FIG. 7 flow cytometry examined the effect and quantification of varying concentrations of dihydroergotoxine mesylate on the periodic progression of 4T1 (A and B) and HuH-7 (C and D) cells.

FIG. 8 flow cytometry examined the effect of varying concentrations of dihydroergotoxine mesylate on apoptosis of 4T1 (A) and HuH-7 (B) cells.

FIG. 9. Effect of CCK-8 assay on proliferation of 4T1 cells using various concentrations of dihydroergotoxine mesylate in combination with various concentrations of chemotherapeutic agents. A. Proportioning dihydroergotoxine mesylate and doxorubicin; B. proportioning dihydroergotoxine mesylate and vincristine; c, proportioning dihydroergotoxine mesylate and epirubicin.

FIG. 10. Effect of CCK-8 assay on HuH-7 cell proliferation of various concentrations of dihydroergotoxine mesylate in combination with various concentrations of chemotherapeutic agents. A. Proportioning dihydroergotoxine mesylate and doxorubicin; B. proportioning dihydroergotoxine mesylate and epirubicin; C. the proportion of the dihydroergotamine mesylate and the sorafenib.

FIG. 11. Effect of CCK-8 assay on proliferation of HuH-7-R cells using various concentrations of dihydroergoline mesylate in combination with various concentrations of chemotherapeutic agents. A. Proportioning dihydroergotoxine mesylate and doxorubicin; B. proportioning dihydroergotoxine mesylate and epirubicin; C. the proportion of the dihydroergotamine mesylate and the sorafenib.

Figure 12 Combination Index (CI) of dihydroergoline mesylate and chemotherapeutic agent combination.

FIG. 13 effect of dihydroergotoxine mesylate and doxorubicin alone and in combination on growth of 4T1 mice subcutaneously transplanted tumors. A. Time change curves of transplanted tumor volumes of mice in each group; B. the effect of dihydroergotoxine mesylate and doxorubicin alone or in combination on the weight of mouse engrafted tumors compared to the solvent control group (p <0.05; p < 0.01); C. the mice of each group were transplanted with the general morphology of the tumor.

Detailed Description

In order to make the technical objects, technical solutions and advantageous effects of the present invention more apparent, the technical solutions of the present invention will be further described with reference to specific examples, but the implementation is intended to explain the present invention and should not be construed as limiting the present invention, and specific techniques or conditions are not specified in the examples, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications.

Example 1, computer-simulated structural analysis of the interaction of dihydroergotoxine mesylate with STAT3 protein.

We first validated the effective binding of dihydroergotoxine mesylate to the target STAT3 by molecular docking. The 3D structure of dihydroergoline mesylate is from the PubChem Substance database (https:// www.ncbi.nlm.nih.gov /), which minimizes structure in the ChemBioDraw 3D module. The crystal structure of STAT3 is from the PDB database (https:// www.rcsb.org /). The acceptor structure was modified by autodock tools 1.5.6 (hydrohydrogenation) to derive pdbqt format. After a grid is defined on the active site of the receptor protein, the docking procedure is performed by AutoDock-Vina 1.1.2, with the output fraction shown as kcal/mol. PyMOL 2.3.0 and BIOVIA Discovery Studio 2016 are applied to result processing and visualization.

The results of virtual docking showed that the ligand molecule dihydroergotoxine mesylate was able to bind to the active pocket of STAT3 and had very low binding potential (fig. 1A). The ligand molecule is able to form stable hydrogen bonds with the branched aminohydrogens 418 and ARG423 of STAT3 (fig. 1B). Furthermore, the ligand is able to form van der waals forces, carbon hydrogen bonds and alkyl groups with amino residues of STAT3. Thus, dihydroergotoxine mesylate can form a strong interaction with STAT3 protein, inhibiting STAT3 expression and activation.

Example 2, dihydroergotoxine mesylate inhibits the transcriptional activity of STAT3 reporter.

4T1 cells were seeded in 12-well plates with a cell number of 1.0X10 cells per well ⁵ Incubating in a 37 ℃ incubator for 24 hours; the culture was discarded and the cells were washed 2 times with 1ml of PBS. STAT 3-luciferase reporter plasmid (from Shanghai right Biotechnology Co., ltd.) and pRL-TK Renilla plasmid (from Promega) plasmid and liposomes (Lipofectamine TM 2000 from Thermo Fisher) were diluted with serum-free DMEM medium (from Hyclone). Mu.g of plasmid was diluted with 250. Mu.l serum-free DMEM medium per well and gently swirled. Mu.l of serum-free medium is taken for diluting 10 mu g of liposome per hole, gently beaten and mixed evenly, and the mixture is kept stand for 5min at room temperature. Adding the diluted plasmid into the diluted liposome, lightly blowing, and standing at room temperature for 20min. The mixture was slowly dropped into the cell culture wells, gently mixed, incubated at 37℃for 4 hours, and then 1ml of fresh medium containing 10% FBS was replaced per well, and the culture was continued in an incubator at 37℃for 60 hours. 4T1 cells were treated with different concentrations (0, 2.5. Mu.M, 5. Mu.M and 10. Mu.M) of dihydroergotoxine mesylate, respectively, for 48h. The supernatant was discarded and the cells were washed once with PBS. 200. Mu.L of 5 XPLB lysate was used for 20min at room temperature. Cell lysates were transferred to 1.5ml Ep tubes. According to the following steps of 1: 50 in a ratio of two luciferase reporter assay kit (from Promega, US, E1910) assay buffer was added to a multifunctional microplate reader (from Varioska)n Flash, thermo Fisher, US) to detect the activity of firefly and sea cucumber luciferases, respectively. The relative fluorescence intensity (Relative fluorescence, RF) =firefly fluorescence value/renilla fluorescence value was calculated according to the formula and compared with the fluorescence intensity of the blank group. As shown in FIG. 2, the experimental results demonstrate that the RF value (p) of STAT3 reporter can be significantly down-regulated after 48h treatment with 2.5. Mu.M dihydroergotoxine mesylate compared to control wells with DMSO only (1. Mu.l/ml)<0.05 A) is provided; whereas the activity of STAT3 reporter was more significantly inhibited after 48h treatment with dihydroergotoxine mesylate at 5 μm or 10 μm (p<0.0001). Thus, dihydroergotoxine mesylate can inhibit STAT3 activity from the transcriptional level and has a dose-gradient dependence.

Example 3, dihydroergotoxine mesylate inhibits the mRNA expression level of STAT3 and its downstream target genes.

6-well plates were taken, each well was seeded with 4T1 and HuH-7-R cells 1X 10, respectively ⁶ And each. After overnight cell culture, dihydroergotoxine mesylate (0 and 5. Mu.M) was treated for 48h. Total cellular RNA was extracted according to the TRIzol one-step method and the concentration was determined. cDNA was synthesized using the extracted total RNA as a template according to the instructions of the reverse transcription kit of Promega company. STAT3 and its target gene were detected by real-time quantitative PCR amplification, with ACTB as an internal reference. The primers used are shown in Table 1.

Table 1 primer information of STAT3 and target genes thereof for quantitative PCR.

Real-time quantitative PCR reaction system:

each set of samples was provided with 3 duplicate wells.

Reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation, 95 ℃,15sec; annealing at 60 ℃,15sec, extension, 72 ℃ for 30sec.

The data analysis was performed by amplifying for 40 cycles with CT values of ACTB as initial values.

FIG. 3A and FIG. 3B show the effect of Q-PCR assay on the mRNA expression levels of STAT3 and its downstream target gene in 4T1 and HuH-7-R cells after 48h of dihydroergotoxine mesylate (5. Mu.M) treatment. Statistical analysis by Two-Annova showed that dihydroergotoxine mesylate significantly down-regulates mRNA expression levels of STAT3, SOX2, BC-XL, CTNNB1, CCND1 and MMP2 genes in 4T1 cells compared to solvent control ("x" means P <0.01, "x" means P < 0.0001). The results of fig. 3B demonstrate that dihydroergotoxine mesylate can significantly down-regulate mRNA expression levels of STAT3, SOX2, BC-XL, CTNNB1, NANOG, CCND1, MMP2, MMP7, and MMP9 genes in HuH-7-R cells ("x" represents P <0.05, "x" represents P <0.01, "x" represents P < 0.0001). Thus, dihydroergotoxine mesylate can inhibit the expression of STAT3 and its downstream target genes from mRNA levels.

Example 4, dihydroergotoxine mesylate inhibits protein expression levels of STAT3, p-STAT3 and downstream target genes.

Taking 4T1 and HuH-7-R cells in logarithmic growth phase, digesting with pancreatin, preparing into density 3.5X10 with DMEM medium containing 10% foetal calf serum ⁵ Single cell suspensions per mL. 2ml of cell suspension per well was inoculated into 6-well cell culture plates. Placing at 37deg.C 5% CO ₂ Incubator cultures overnight, with addition of dihydroergotoxine mesylate at concentrations of 0, 2.5. Mu.M, 5. Mu.M and 10. Mu.M. After further culturing for 48 hours, cells were lysed with RIPA lysate, and protein was collected, quantified and Western blot analysis was performed.

1. A step of measuring the protein concentration (BCA method):

A. protein standards (table 2) were diluted in 1 x Phosphate Buffered Saline (PBS):

table 2 formulation system of protein standards.

B. BCA working solution preparation: the total required amount of A and B mixed working fluid is calculated according to the number of the standard substance and the sample to be tested. Volume ratio of BCA reagent a to B50: 1, preparing working solution, and uniformly mixing by vortex oscillation for standby.

C. Protein standards and sample supernatants diluted with Phosphate Buffered Saline (PBS) (10-fold dilution) were each 25 μl added to a new 96-well plate. Then 200. Mu.l of BCA working solution prepared in advance was added respectively and mixed well. The bubbles are not generated by blowing, the cover of the 96-well plate is tightly covered, and the reaction is carried out for 30min in a constant temperature box at 37 ℃.

D. The 96-well plate was removed and returned to room temperature for 3-5min, absorbance at 562nm was measured on an enzyme-labeled instrument, and a standard curve was made to calculate 1 μl protein content per sample for protein loading.

2. Sodium dodecyl sulfonate-polyacrylamide gel (SDS-PAGE)

(1) The gel plate was fixed and 10% SDS-PAGE separating gel was prepared (Table 3).

The release gel was formulated as follows in table 3: 10ml

Table 3 isolates the glue formulation information.

(2) Adding the mixed separating glue into 2 glue plates, respectively, adding into the position 1.0cm away from the top, filling the glue plates with absolute ethyl alcohol, and standing for 30-45min.

(3) After the gel is separated, the residual absolute ethyl alcohol is poured out, and the residual absolute ethyl alcohol is sucked clean by filter paper.

(4) 5ml of 5% concentrated gel was prepared according to Table 4

Table 4 concentrate formulation information.

(5) Slowly adding the prepared concentrated gel into a gel plate to avoid bubble generation, inserting a sample comb, and standing for 30-45min.

(6) Taking out the protein sample, heating in water bath at 100deg.C for 5min, rotating at 10000rpm, and centrifuging for 10min.

(7) The gel plate is fixed in an electrophoresis tank, SDS-PAGE electrophoresis buffer is added, a sample comb is pulled out, and the processed protein samples are added into the sample tank in sequence.

(8) Electrophoresis at 80V for 40min.

(9) Changing the voltage to 120V for electrophoresis for about 1.5 hours until bromophenol blue goes out of the colloid;

3. western-blot transfer film

(1) And (3) placing the SDS-PAGE gel subjected to electrophoresis into TBST buffer solution for rinsing once, and placing the protein gel into transfer buffer solution for soaking.

(2) Soaking a layer of foam cushion in a film transfer buffer solution, clamping the foam cushion onto a film transfer device by using tweezers, and soaking the foam cushion, the three layers of filter paper, the albumin glue, the polyvinylidene fluoride (PVDF) film, the three layers of filter paper and the foam cushion in sequence, aligning the foam cushion, clamping the foam cushion onto the film transfer device, and during operation, soaking the filter paper and the foam cushion in the film transfer buffer solution. If bubbles exist between each two layers, the bubbles are removed by using a glass test tube to gently roll.

(3) The film transfer instrument was turned on, and 300mA was transferred for 75min.

(4) The membrane was removed and placed in TBST buffer and rinsed 3 times with a 60rpm horizontal shaker for 8min each.

(5) Blocking was performed with 20ml of 5% Bovine Serum Albumin (BSA) blocking solution at 60rpm with a horizontal shaker at room temperature for 2h.

(6) Incubation was performed overnight with a 3ml antibody incubation with 3. Mu.l primary antibody (1:1000) at 4℃with a 60rpm horizontal shaker.

(7) The PVDF membrane was washed three times, 10min each, with 10ml TBST, at room temperature with a 60rpm horizontal shaker.

(8) PVDF membrane was incubated with 20ml of antibody incubation with 2. Mu.l of secondary antibody at room temperature with a 60rpm horizontal shaker for 2h.

(9) The PVDF membrane was washed three times, 10min each, with 10ml TBST, at room temperature with a 60rpm horizontal shaker.

(10) 1ml each of chemiluminescent substrate reagent solution A and solution B was taken, mixed well and coated on a filter membrane.

(11) The liquid on the membrane was blotted dry with filter paper and developed with a developer.

As shown in FIGS. 4A and 4B, the results of Western blotting showed that the protein expression levels of GAPDH in 4T1 and HuH-7-R cells were unchanged as the concentration of dihydroergotamine mesylate was increased, while the expression levels of STAT3, p-STAT3 (Tyr 705), cyclin D1, BCL-XL, surviving, ABCB1, β -catenin and p- β -catenin (ser 552) were all in a decreasing trend, while the expression of clear-PARP was increased. Thus, dihydroergotoxine mesylate can inhibit protein expression and activation of STAT3 and protein expression of downstream target genes related to cell proliferation, drug resistance and apoptosis resistance.

Example 5 real-time fluorescent quantitative PCR method the effect of dihydroergotoxine mesylate on mRNA expression levels of inflammatory cytokines in HuH-7 cells was examined.

6-well plates were taken and seeded with HuH-7 cells 1X 10 per well ⁶ And each. After overnight cell culture, dihydroergotoxine mesylate (0 and 5. Mu.M) was treated for 48h. Total cellular RNA was extracted according to the TRIzol one-step method and the concentration was determined. cDNA was synthesized using the extracted total RNA as a template according to the instructions of the reverse transcription kit of Promega company. STAT3 and its target gene were detected by real-time quantitative PCR amplification, with ACTB as an internal reference. The primers used are shown in Table 5.

Table 5 primer information for quantitative PCR of inflammatory cytokines.

Real-time quantitative PCR reaction system:

each set of samples was provided with 3 duplicate wells.

FIG. 5 is a graph showing the effect of Q-PCR assay on mRNA expression levels of inflammatory cytokines in HuH-7 cells after 48h of dihydroergotoxine mesylate (5. Mu.M) treatment. Statistical analysis by T-test showed that dihydroergotoxine mesylate significantly down-regulates the mRNA expression levels of IL-1, IL-6, IL-8, IL-10, IL-12, IL-23 and TNF- α in HuH-7 cells compared to solvent control ("" indicates P < 0.0001). Thus, dihydroergotoxine mesylate can inhibit the expression of these inflammatory cytokines from mRNA levels.

Example 6, dihydroergotoxine mesylate can inhibit proliferation of a variety of tumor cell lines.

Single cell suspensions were counted and prepared, inoculated into 96 well plates with 5000 cells per well, 100 μl per well, and placed in 5% CO ₂ Incubated overnight at 37 ℃. Gradient doses (0.1952125. Mu.M, 0.390625. Mu.M, 0.78125. Mu.M, 1.5625. Mu.M, 3.125. Mu.M, 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, and 100. Mu.M) of dihydroergotamine mesylate, static, or GABAzine were added, respectively, and incubated for 48h with 3 multiplex wells per experimental well, with cell culture wells without compound added as controls. Cell viability was determined for each culture well by the CCK-8 method. Mu.l of CCK-8 reagent was added to each well and 5% CO was placed in the well _2， Incubator at 37℃for 1 hour, absorbance at 450nm was measured for each well with a microplate reader. Static is a commercially available STAT3 inhibitor, available from MCE company. GABAzine is a selective competitive antagonist of the GABAA receptor and is available from MCE corporation.

The test results are shown in FIG. 6, and the dihydroergotoxine mesylate with different concentrations can inhibit proliferation of liver cancer cells, liver cancer drug-resistant cells, breast cancer cells, colon cancer cells and gastric cancer cells, and has IC ₅₀ The value is lowest in 4T1 cells, and is lower than that of parent cell HepG2 in liver cancer drug-resistant cells HepG2-R, which suggests that chemotherapy-resistant liver cancer cells are more sensitive to dihydroergotoxine mesylate. Dihydroergotamine mesylate has activity comparable to that of STAT against tumor cell proliferation, while GABAzine, a selective competitive antagonist of GABAA receptor, has little tumor-inhibiting activity, suggesting that dihydroergotamine mesylate exerts tumor-inhibiting effects by targeting STAT3 rather than GABAA receptor.

Example 7, dihydroergotoxine mesylate can induce cycle arrest in tumor cells.

Taking 4T1 and HuH-7 cells in logarithmic growth phase, digesting and counting, inoculating into 12-well plate, and inoculating 2×10 each well ⁵ Cells were plated with 3 parallel control wells. After about 16h, dihydroergotoxine mesylate was added at concentrations of 0, 2.5. Mu.M, 5. Mu.M and 10. Mu.M and incubation continued for 48h. Cells were digested with pancreatin, counted after resuspension, and cell concentration was adjusted to 5×10 ⁵ And each. After completion of the digestion, the supernatant was centrifuged off, the cells were washed twice with PBS (2000 rpm, 5 min), after which the supernatant was discarded, 980. Mu.l of 70% cold ethanol and 0.1% BSA were added to each tube, and the mixture was frozen overnight at 4 ℃. The fixative was discarded and washed 3 times with PBS (6000 rpm, centrifuged for 0.5 min). After the completion of the cell washing, the subsequent operations were performed according to the instructions of the DNA content detection kit (product of Beijing Suolibao). Each sample was incubated with 100. Mu.l of RNase A at 37℃for 30min, and then 500. Mu.l of the prepared PI (propidium iodide) working solution was added to each sample, and incubated at room temperature for 30min in the absence of light. Finally, cell cycle was determined by flow cytometry. The experimental results were analyzed using ModFit software and the proportion of the periodic distribution of the two cells was further analyzed by Graphpad prism 8.0.

FIGS. 7A and 7C are results of analysis of periodic distribution of 4T1 and HuH-7 cells with ModFit software for the presence or absence of dihydroergotoxine mesylate treatment. Fig. 7B and 7D are further quantitative analyses of the results of fig. 7A and 7C by Graphpad prism 8.0. These results indicate that dihydroergotoxine mesylate induces a significant increase in the G1/S phase ratio and a corresponding decrease in the G2 phase ratio in breast and liver cancer cells in a dose dependent manner compared to the solvent control (DMSO). Thus, dihydroergoline can induce tumor cell cycle arrest.

Example 8, dihydroergotoxine mesylate can induce apoptosis in tumor cells.

Taking 4T1 and HuH-7 cells in logarithmic growth phase, digesting and counting, inoculating into 6-well plate, and inoculating 5×10 each well ⁵ Cells were plated with 3 parallel control wells. After about 16h, dihydroergotoxine mesylate was added at concentrations of 0, 2.5. Mu.M, 5. Mu.M and 10. Mu.M and incubation continued for 48h. Cells were digested with EDTA-free pancreatin, counted after resuspension, and cell concentration was adjusted to 1X 10 ⁶ And each. According to Annexin V FITC-PIInstructions for the apoptosis detection kit (product of beijing solebao corporation) were followed. The method comprises the following steps: cells were washed 2 times with 1 XPBS (6000 rpm, 0.5min by centrifugation), 1 times with 1 Xbinding buffer (6000 rpm, 0.5min by centrifugation) and the supernatant was discarded, and cells were resuspended in 300. Mu.l of 1 Xbinding buffer, 5. Mu.l of Annexin V-FITC was added to each tube and incubated in the dark for 10min. Subsequently, 5. Mu.l of PI was added to each tube and incubated for 5min in the dark. And (5) detecting in a light-shielding machine.

FIGS. 8A and 8B are flow cytometry assays for the effect of dihydroergotoxine mesylate on apoptosis of 4T1 and HuH-7 cells. The results show that dihydroergotoxine can induce an increase in the rate of apoptosis in a dose-dependent manner compared to control wells.

Example 9 low doses of dihydroergoline mesylate may enhance the sensitivity of 4T1 cells to chemotherapeutic agents.

4T1 cells were routinely digested with pancreatin, single cell suspensions were counted and prepared, inoculated into 96 well plates, 5000 cells per well, 100 μl per well, and placed in 5% CO ₂ Incubated overnight at 37 ℃. Gradient doses of dihydroergotoxine mesylate (0, 2. Mu.M and 4. Mu.M) were added and incubated for 48h with doxorubicin (0, 0.0625. Mu.M and 0.125. Mu.M), vincristine (0, 0.5. Mu.M and 1. Mu.M) and epirubicin (0, 0.0625. Mu.M and 0.125. Mu.M), respectively, with 3 multiplex wells per experimental well. Cell viability was determined for each culture well by the CCK-8 method. Mu.l of CCK-8 reagent was added to each well and 5% CO was placed in the well _2， Incubator at 37℃for 1 hour, absorbance at 450nm was measured for each well with a microplate reader.

As shown in fig. 9, in contrast to cell culture wells without dihydroergotoxine mesylate and chemotherapeutic agent, dihydroergotoxine monomethacrylate (2 μm and 4 μm) or doxorubicin monomethazine (0.0625 μm and 0.125 μm), vincristine (0.5 μm and 1 μm) and epirubicin (0.0625 μm and 0.125 μm) all significantly inhibited 4T1 cell proliferation; dihydroergotamine mesylate (2. Mu.M and 4. Mu.M) in combination with doxorubicin (0.0625. Mu.M and 0.125. Mu.M), vincristine (0.5. Mu.M and 1. Mu.M) and epirubicin (0.0625. Mu.M and 0.125. Mu.M), respectively, can further significantly inhibit cell viability ("/x" stands for p < 0.0001). Thus, dihydroergoline mesylate may enhance the sensitivity of 4T1 cells to conventional chemotherapeutics for breast cancer in vitro.

Example 10 low doses of dihydroergoline mesylate may enhance the sensitivity of HuH-7 cells to chemotherapeutic agents.

HuH-7 cells were routinely digested with pancreatin, single cell suspensions were counted and prepared, inoculated into 96-well plates, 5000 cells per well, 100 μl per well, and 5% CO was placed ₂ Incubated overnight at 37 ℃. Gradient doses of dihydroergotamine mesylate (0, 2. Mu.M and 4. Mu.M) were added and incubated for 48h with doxorubicin (0, 0.25. Mu.M and 0.5. Mu.M), epirubicin (0, 0.25. Mu.M and 0.5. Mu.M) and sorafenib (0, 1. Mu.M, 2. Mu.M and 4. Mu.M), respectively, with 3 multiplex wells per experimental well. Cell viability was determined for each culture well by the CCK-8 method. Mu.l of CCK-8 reagent was added to each well and 5% CO was placed in the well _2， Incubator at 37℃for 1 hour, absorbance at 450nm was measured for each well with a microplate reader.

As shown in fig. 10, dihydroergotoxine monomethacrylate (2 μm and 4 μm) or monogamrubicin (0.25 μm and 0.5 μm), epirubicin (0.25 μm and 0.5 μm) and sorafenib (1 μm,2 μm and 4 μm) can significantly inhibit HuH-7 cell proliferation compared to cell culture wells without dihydroergotoxine mesylate and chemotherapeutic; dihydroergotamine mesylate (2 μm and 4 μm) in combination with doxorubicin (0.25 μm), epirubicin (0.25 μm) and sorafenib (1 μm,2 μm and 4 μm), respectively, can further significantly inhibit cell viability ("" represents p <0.01, "" represents p <0.001, "" represents p < 0.0001). When doxorubicin (0.5. Mu.M) and epirubicin (0.5. Mu.M) were used alone, the inhibition rate on HuH-7 cells was 80%, so that the effect was not stronger but not diminished after dihydroergotoxine was used in combination. Thus, dihydroergotoxine mesylate can enhance the sensitivity of HuH-7 cells to low doses of conventional chemotherapeutics for liver cancer in vitro.

Example 11 low doses of dihydroergoline mesylate may enhance the sensitivity of HuH-7-R cells to chemotherapeutic agents.

HuH-7-R cells were routinely digested with pancreatin, single cell suspensions were counted and prepared, inoculated into 96-well plates, 5000 cells per well, 100 μl per well, and 5% CO was placed ₂ Incubated overnight at 37 ℃. Adding gradient dosage of dihydroergotoxine mesylate (0),2. Mu.M and 4. Mu.M) were used singly and in combination with doxorubicin (0, 0.25. Mu.M and 0.5. Mu.M), epirubicin (0, 0.25. Mu.M and 0.5. Mu.M) and sorafenib (0, 1. Mu.M, 2. Mu.M and 4. Mu.M), respectively, and incubated for 48h with 3 multiplex wells per experimental well. Cell viability was determined for each culture well by the CCK-8 method. Mu.l of CCK-8 reagent was added to each well and 5% CO was placed in the well _2， Incubator at 37℃for 1 hour, absorbance at 450nm was measured for each well with a microplate reader.

As shown in fig. 11, dihydroergotoxine monomethacrylate (2 μm and 4 μm) or monogamrubicin (1 μm and 2 μm), epirubicin (1 μm and 2 μm) and sorafenib (1 μm,2 μm and 4 μm) can significantly inhibit HuH-7 cell proliferation compared to cell culture wells without dihydroergotoxine mesylate and chemotherapeutic; dihydroergotamine mesylate (2 μm and 4 μm) in combination with doxorubicin (1 μm and 2 μm), epirubicin (1 μm and 2 μm) and sorafenib (1 μm,2 μm and 4 μm), respectively, can further significantly inhibit cell viability ("" represents p <0.05, "" represents p <0.001, "" represents p < 0.0001). Thus, in vitro, dihydroergotoxine mesylate can enhance the sensitivity of chemotherapy-resistant HuH-7-R cells to conventional chemotherapeutics for liver cancer.

Example 12 Combination Index (CI) analysis of dihydroergoline mesylate and chemotherapeutic agent combination

The CCK-8 detection results in FIGS. 9 and 10 were analyzed using CompuSyn software. Fa represents the fraction of cell viability. When 0.3< CI <0.7, a stronger synergistic effect is shown; when 0.7< ci <0.9, a slight synergy is indicated; when 0.9< CI <1.1, the superposition is shown; antagonism is indicated when CI > 1.1.

As shown in fig. 12,2 μm dihydroergotamine mesylate used in combination with doxorubicin or sorafenib in 4T1 cells and HuH-7 cells had CI values of about 0.78, 0.66 and 0.84, 0.79, respectively, indicating that low doses of dihydroergotamine mesylate had slight synergy with doxorubicin and sorafenib, respectively; whereas 4 μm dihydroergotamine mesylate was used in combination with doxorubicin or sorafenib, respectively, with CI values of about 0.49, 0.34 and 0.42, 0.37, respectively, indicating a stronger synergy with doxorubicin and sorafenib, respectively, at higher doses.

Example 13 animal experiments

The experimental animals were female Balb/c mice 6 weeks old purchased from Biotechnology Inc. of Lerio, beijing and kept in SPF-grade animal laboratory. Will be 1X 10 ⁴ The individual 4T1 cells were seeded on the mouse axillary fat pads. After about one week, the mice showed obvious milky white nodules on the axilla, and the mice were randomly divided into a solvent control group (10% DMSO+90% corn oil), a dihydroergotoxine mesylate (20 mg/kg) group, a doxorubicin hydrochloride (1 mg/kg) group, and a dihydroergotoxine mesylate (20 mg/kg) +doxorubicin hydrochloride (1 mg/kg) group, each of 4 mice by weight. The administration was carried out 1 time per day by intraperitoneal injection. The body weight and tumor size of each group of mice were monitored every other day. 10 days after administration, each group of mice was sacrificed, subcutaneous tumors were transplanted, weighed and photographed.

The results of FIGS. 13A-C show that dihydroergotoxine mesylate (20 mg/kg) alone has no significant inhibitory effect on tumor volume, tumor weight and morphology (possibly related to short drug generation cycle, frequency and dose of dosing, etc. of dihydroergotoxine mesylate, which requires optimization) compared to the solvent control group. The positive control drug doxorubicin hydrochloride (1 mg/kg) has obvious inhibition effect on the volume, tumor weight and morphology of transplanted tumor ("x" represents p < 0.05). Whereas dihydroergotamine mesylate (20 mg/kg) in combination with doxorubicin hydrochloride (1 mg/kg) in mice was further inhibited in tumor volume, tumor weight and morphology size ("x" represents p < 0.01). These results demonstrate that dihydroergotoxine mesylate can significantly enhance the growth inhibitory effect of doxorubicin on breast cancer cells in vivo.

The results show that the dihydroergotoxine mesylate has good STAT3 targeting, can down regulate the gene and protein expression of STAT3, and reduce the phosphorylation of STAT3 and the expression of various target genes; inhibiting the expression of inflammatory cytokines such as IL-1, IL-6, IL-8, IL-10, IL-12, IL-23, and TNF-alpha; proliferation of liver cancer, colon cancer and gastric cancer cells which obstruct the drug resistance of breast cancer, liver cancer and chemotherapy; inducing tumor cell cycle arrest and apoptosis; enhancing the in vitro sensitivity of various tumor cells to doxorubicin, epirubicin, vincristine and sorafenib; and can enhance the in vivo activity of the doxorubicin, and has good application prospect in the aspect of resisting tumors.

Claims

Application of 1.2 mu M dihydroergotoxine mesylate and 0.25 mu M doxorubicin in preparing anti-liver cancer medicine is provided.
2.5 mu M dihydroergotoxine mesylate and 1 mu M doxorubicin are used in preparing anti-liver cancer medicine.