CN115337306A - Application of dihydroergotoxine mesylate targeting STAT3 in preparation of anti-inflammatory disease and anti-tumor drugs - Google Patents

Abstract

The invention discloses an application of dihydroergotoxine mesilate targeting STAT3 in preparing medicines for treating breast cancer, liver cancer, colon cancer, gastric cancer and the like. The drug is dihydroergotoxine mesylate, and the expression of inflammatory cytokines such as IL-1, IL-6, IL-8, IL-10, IL-12, IL-23, TNF-alpha and the like is reduced by directly reducing the expression and activity of STAT 3; and the effects of resisting the proliferation of tumor cells and inducing the apoptosis of the tumor cells are exerted by inhibiting the expression of target genes such as Cyclin D1, bcl-XL and the like. The medicament has less toxic and side effects than the traditional chemotherapeutic medicaments such as adriamycin, epirubicin, vincristine and the like, has low price than the targeted medicaments such as sorafenib, anti-IL-6 antibodies and the like, can also prevent or reverse drug resistance, and has good anti-tumor application prospect and development value.

Description

Application of dihydroergotoxine mesilate targeting STAT3 in preparation of anti-inflammatory diseases and antitumor drugs

Technical Field

The invention relates to the field of biological medicine, in particular to application of dihydroergotoxine mesylate targeting STAT3 in preparing anti-inflammatory diseases and antitumor drugs.

Background

STAT3 (Signal Transducer and Activator of Transcription 3 ), is one of the best studied transcriptional regulators of tumor-associated signals. Studies have shown that abnormal activation of STAT3 is associated with poor clinical prognosis in breast, liver, stomach, and colon cancers. Constitutive activation of STAT3 signaling 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 all the processes involved in regulating tumorigenesis and development such as proliferation, anti-apoptosis, drug resistance and metastasis (Leslie Kenneth, cynthia Lang, geeta Degan, et al. Cyclin D1 is a translocated regulated by and reconstructed for transformation by activated signal transduction and activator of 3.Cancer Res,2006,66 (5): 2544-2552; grad J.M., X.R.Zeng and L.H.Boise.Regulation of BCL-XL: a lite bit of this and a lite bit of STAT. Curr Opin Oncol,2000,12 (6): 543-549 Soleymani Absyaneh, gupta N, radziwon-Ballicka A, et al.STAT3 but not HIF-1alpha is immobilized Chemoresistance in mediating hypo-231, a triple negative barrier Cancer lines, cameras (base). 2017 (10): kamran MZcan, patil P, guide RP.roll of STAT3 in Cancer assays and related variables. Biomed Res. 2013. 421 Daniel E, gradient A, mutation R.2013. Origin and conversion, JAK-3, nature IL-3, clone R.1821, clone R.3, clone R.2013, mastig 3, nature IL-3, USA, JAK-7. In addition, STAT3 is one of the core transcription factors of "inflammatory cancer transformation". Factors such as smoking, alcohol abuse, UV irradiation, chronic stress and infection can activate STAT3 in a number of ways, and activated STAT3 in turn aggravates local inflammatory responses and even induces inflammatory-cancerous transformations by activating the positive feedback signaling pathway for inflammatory cytokines such as IL-1, IL-6, IL-8, IL-10, IL-12, IL-23 and TNF- α (Hua Yu, drew Pardol and Richard Jove.STATs in cancer inflammation and immunity: a lead roll for STATT 3.Cancer. 2009 (9): 798-809.). The development of drugs targeting STAT3 has become a hotspot of international research. Some STAT3 signal inhibitors such as Siltuximab, AZD9150, WP1066, TTI101, OPB-31121 and OPB-51602 have entered the clinical testing stage (Orlowski, R.Z. et al. A Phase 2, randomised, double blind, placebo-controlled study of Siltuximab (anti-IL-6 mAb) and bortezomib vertubes bortezomib alkane in tissues with strained psor recombinant multiple organism. Am. J.Hematol.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 Long Cancer Transit, 2015,7 (314) 314ra185, sau, S., et al, combination of clinical Dexamethosone derivative and STAT3 inhibitor (WP 1066) for an analytical texture for a purification for a Phase I clinical trial molecular chemistry, 2017.436 (1-2) 119-136; bharaj, U.S., et al, small-molecular inhibition of STAT3 in a radioactive head and a negative square Cell carbonate, oncotarget,2016.7 (18): 26307-26330, hayakawa, f., et al, a novel STAT inhibitor, OPB-31121, ha a diagnostic inhibitor or efficacy infection with STAT-additive on surfactants, blood Cancer J,2013.3, 166 ogura, m.a., phase I test of OPB-51602, signal inhibitor of transducer and activator of transcription 3, tissue culture/reaction of transcription 3, cancer tissue sample/reaction of transcription 3, cancer tissue, 896.7). However, these compounds are often of limited development due to insignificant therapeutic advantages or poor drugability. Currently, no drug targeting STAT3 is successfully marketed.

Dihydroergotoxine mesylate (Dihydroergotoxine), an alpha receptor blocker, has high binding activity to GABAA receptors. The dihydroergotoxine mesylate has the functions of relaxing vascular smooth muscle and enhancing neurotransmitter transmission, can inhibit the activities of ATPase and adenylate cyclase, reduce ATP decomposition, improve the oxidative utilization of glucose by nerve cells, and improve the blood flow and energy supply of brain cells. It is mainly used for treating vascular dementia and cerebrovascular disease sequelae. At present, the application of dihydroergotoxine mesylate in treating STAT3 target-related inflammatory diseases, tumors and the like is not reported.

Disclosure of Invention

Aiming at the problems that the development of the STAT3 inhibitor is not successful and corresponding clinical drugs are lacked in the prior art, the invention provides dihydroergotoxine with known pharmacokinetic and toxicological effects, less side effects, low price and good development and application prospects, and the application of the targeted STAT3 in preparing the drugs for treating tumors aims at solving the technical problems that clinical chemotherapeutic drugs are easy to resist drugs, so that tumors relapse and the existing targeted STAT3 compound is poor in drug forming property.

In order to achieve the purpose, the invention discloses application of dihydroergotoxine mesilate targeting STAT3 in preparing medicines for treating inflammatory diseases and tumors; the medicine is dihydroergotoxine mesylate and derivatives thereof.

The following technical scheme is adopted: in the early stage of the invention, a reporter molecule carrier (purchased from Shanghai-Auyang biotechnology limited) based on STAT3 transcriptional activity is constructed

1321 monomeric compounds of Chinese herbs (purchased from MCE) were screened by the Luciferase Assay System kit (purchased from Promega) in high throughput. Dihydroergotoxine mesylate was found to inhibit STAT3 transcriptional activity.

Subsequently, we investigated the effects of dihydroergotoxine mesylate targeting the STAT3 protein and inhibiting tumor growth by both in vitro cell lines and in vivo mouse experiments. Experiments show that the dihydroergotoxine mesylate can reduce the expression of downstream target genes Cyclin D1, bcl-XL and Survivin of STAT3, p-STAT3 (Tyr 705) and STAT3, inhibit the in vitro proliferation of cell strains such as breast cancer, liver cancer, gastric cancer and colon cancer, and induce the cycle arrest and apoptosis of tumor cells; the antitumor cell proliferation effect of dihydroergotoxine mesylate is equivalent to the activity of STAT3 inhibitor Static, but the application of the selective competitive antagonist GABAzine of GABAA receptor has almost no antitumor activity, and further shows that dihydroergotoxine mesylate plays a tumor inhibition effect by targeting STAT3 instead of GABAA receptor.

As another invention point of the invention, the combination of dihydroergotoxine mesylate and chemotherapeutic drugs such as adriamycin, 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 drug-resistant cells of the liver cancer and play a stronger role in synergy anti-tumor in vivo.

The tumor cells inhibited by dihydroergotoxine mesylate can be 4T1, MDA-MB-231, MDA-MB-468, CT-26, HGC-27, hepG2, huH-7, hepG2-R and HuH-7-R. The dihydroergotoxine mesylate is applied to the preparation of antitumor drugs, and the drugs are 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. The ligand dihydroergotoxine mesylate is combined with the active 'pocket' of STAT 3; B. a 3D structural schematic diagram of combination of dihydroergotoxine mesylate and STAT 3; C. dihedral plot of the mode of action and affinity between dihydroergotoxine mesylate and STAT3.

FIG. 2. Dual luciferase Activity assay detects inhibition of STAT3 reporter transcription activity by dihydroergotoxine mesylate at different concentrations.

FIG. 3 shows the effect of dihydroergotoxine mesylate on the mRNA expression levels of STAT3 and its downstream target genes in 4T1 (A) and HuH-7-R (B) cells as detected by real-time fluorescent quantitative PCR.

FIG. 4 is a Western blot method for detecting the effect of dihydroergotoxine mesylate with different concentrations on the expression levels of mRNA of STAT3, p-STAT3, and STAT3 downstream target genes and apoptosis and drug resistance related proteins in 4T1 (A) and HuH-7-R (B) cells.

FIG. 5 shows the effect of dihydroergotoxine mesylate on the mRNA expression level of inflammatory cytokines in HuH-7 cells as determined by real-time fluorescent quantitative PCR.

FIG. 6CCK-8 method for detecting IC of dihydroergotoxine mesylate for inhibiting proliferation in different tumor cell lines ₅₀ The value is obtained.

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

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

FIG. 9 is a diagram showing the effect of the CCK-8 method on 4T1 cell proliferation of dihydroergotoxine mesylate at various concentrations in combination with chemotherapeutic agents at various concentrations. A. The mixture ratio of dihydroergotoxine mesylate and adriamycin; B. the proportion of dihydroergotoxine mesylate and vincristine; c, the proportion of dihydroergotoxine mesylate and epirubicin.

FIG. 10 is a CCK-8 method for determining the effect of varying concentrations of dihydroergotoxine mesylate in combination with varying concentrations of chemotherapeutic agents on HuH-7 cell proliferation. A. The proportion of dihydroergotoxine mesylate and adriamycin; B. the proportion of dihydroergotoxine mesylate and epirubicin; C. the mixture ratio of dihydroergotoxine mesylate and sorafenib.

FIG. 11 is a CCK-8 method for determining the effect of varying concentrations of dihydroergotoxine mesylate in combination with varying concentrations of chemotherapeutic agents on HuH-7-R cell proliferation. A. The proportion of dihydroergotoxine mesylate and adriamycin; B. the proportion of dihydroergotoxine mesylate and epirubicin; C. the proportion of dihydroergotoxine mesylate and sorafenib is as follows.

FIG. 12 Combination Index (CI) for dihydroergotoxine mesylate in combination with a chemotherapeutic agent.

FIG. 13. Effect of dihydroergotoxine mesylate and doxorubicin, alone or in combination, on the growth of 4T1 mice subcutaneously transplanted tumors. A. Time-varying curves of transplanted tumor volumes for each group of mice; B. effect of dihydroergotoxine mesylate and doxorubicin alone or in combination on mouse engraftment weight compared to solvent control (p <0.05; C. gross morphology of tumors transplanted in each group of mice.

Detailed Description

In order to make the technical purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is further described with reference to specific examples, but the implementation is intended to explain the present invention and should not be construed as a limitation of the present invention, and those who do not specify specific techniques or conditions in the examples follow the techniques or conditions described in the literature in the field or follow the product specification.

Example 1 in silico structural analysis of the interaction of dihydroergotoxine mesylate with the STAT3 protein.

We first verified the efficient binding of dihydroergotoxine mesylate to the target STAT3 by molecular docking. The 3D structure of dihydroergotoxine mesylate was obtained from PubChem Substance database (https:// www.ncbi.nlm.nih.gov /), minimizing the structural energy in the ChemBioDraw 3D module. The crystal structure of STAT3 is from the PDB database (https:// www. Rcsb. Org. /). The acceptor structure was modified (dehydrogenated) by AutodockTools 1.5.6, leading to pdbqt format. After defining the grid on the active site of the receptor protein, the docking procedure was performed by AutoDock-Vina 1.1.2, with the output score shown as kcal/mol. PyMOL 2.3.0 and BIOVIA Discovery Studio 2016 were applied for result processing and visualization.

The results of virtual docking show that the ligand molecule dihydroergotoxine mesylate is able to bind to the active pocket of STAT3 and has a very low binding potential (fig. 1A). The ligand molecules are able to form stable hydrogen bonds with branched amino hydrogens CYS418 and ARG423 of STAT3 (fig. 1B). In addition, the ligands are capable of forming van der waals forces, carbon hydrogen bonds, and alkyl groups with the amino residues of STAT3. Therefore, dihydroergotoxine mesylate can form a strong interaction with STAT3 protein, and inhibit the expression and activation of STAT3.

Example 2 dihydroergotoxine mesylate inhibited the transcriptional activity of STAT3 reporter.

4T1 cells were seeded in 12-well plates at 1.0X 10 cell counts per well ⁵ Putting the mixture into an incubator at 37 ℃ for incubation for 24 hours; the culture medium was discarded, and the cells were washed 2 times with 1ml of PBS. STAT 3-luciferase reporter plasmid (purchased from Shanghai Haoyang Biotech, inc.) and pRL-TK Renilla plasmid (purchased from Promega) were diluted with serum-free DMEM medium (purchased from Hyclone) and liposomes (Lipofectamine TM 2000, purchased from Thermo Fisher). Mu.g plasmid was diluted in 250. Mu.l serum-free DMEM medium per well and gently pipetted and mixed. Diluting 10 μ g of liposome in 250 μ l of serum-free medium per well, and gently blowingMixing, and standing at room temperature for 5min. The diluted plasmid is added into the diluted liposome, gently blown and kept stand at room temperature for 20min. The mixture was slowly dropped into the cell culture well, gently mixed, incubated at 37 ℃ for 4 hours, then 1ml of fresh medium containing 10% FBS was replaced per well, and the culture was continued in the 37 ℃ incubator 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 for 48h each. The supernatant was discarded and the cells were washed once with PBS. The cells were lysed with 200. Mu.L of 5 XPLB lysate for 20min at room temperature. The cell lysate was transferred to a 1.5ml Ep tube. According to the following steps: 50, adding a dual-luciferase reporter gene detection kit (purchased from Promega, US, E1910) detection buffer solution, and respectively detecting the activities of firefly luciferase and sea cucumber luciferase in a multifunctional enzyme-labeling instrument (purchased from Varioskan Flash, thermo Fisher, US). Relative fluorescence intensity (RF) = firefly fluorescence value/renilla fluorescence value was calculated according to the formula and compared with the fluorescence intensity of the blank control group. As shown in FIG. 2, the experimental results show that the RF value (p) of STAT3 reporter was significantly reduced after 48h of treatment with 2.5. Mu.M dihydroergotoxine mesylate compared to the control wells with DMSO only (1. Mu.l/ml)<0.05 ); whereas the STAT3 reporter activity was more significantly inhibited after 48h of treatment with either 5. Mu.M or 10. Mu.M dihydroergotoxine mesylate (p)<0.0001). Thus, dihydroergotoxine mesylate may inhibit STAT3 activity from the transcriptional level and is dose-gradient dependent.

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

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

Table 1 primer information of STAT3 used for quantitative PCR and its target gene.

Real-time quantitative PCR reaction system:

each set of samples was provided with 3 replicate wells.

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 15sec; annealing at 60 ℃ for 15sec, elongation at 72 ℃ for 30sec.

The amplification was performed for 40 cycles, and data analysis was performed with the CT value of ACTB as an initial value.

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

Example 4 dihydroergotoxine mesylate inhibits the 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, and preparing into DMEM medium containing 10% fetal calf serum with density of 3.5 × 10 ⁵ Single cell suspension per mL. 2ml per wellThe cell suspension was seeded in 6-well cell culture plates. At 37 ℃ and 5% CO ₂ Incubate overnight and add dihydroergotoxine mesylate at concentrations of 0, 2.5. Mu.M, 5. Mu.M and 10. Mu.M. After further culturing for 48h, the cells were lysed with RIPA lysate, the proteins were collected, quantified and Western blot analyzed.

1. Procedure for determining protein concentration (BCA method):

A. 1 x Phosphate Buffer (PBS) dilution of protein standards (table 2):

table 2 formulation system of protein standards.

B. Preparing a BCA working solution: and calculating the total required amount of the mixed working solution A and B according to the number of the standard substance and the sample to be detected. And (3) according to the volume ratio of the BCA reagent A to the B of 50:1, preparing the working solution, and carrying out vortex oscillation and uniform mixing for later use.

C. The protein standard and the supernatant (10-fold dilution) of the sample diluted with Phosphate Buffered Saline (PBS) were each added to a new 96-well plate in 25 μ l. Then respectively adding 200 mul of the prepared BCA working solution to fully mix. And cutting to avoid air bubbles generated by blowing, tightly covering a 96-well plate cover, and reacting in a 37 ℃ incubator for 30min.

D. And taking out the 96-well plate, recovering to room temperature for 3-5min, measuring the absorbance value at the wavelength of 562nm on a microplate reader, and making a standard curve to calculate the content of 1 mu l of protein of each sample so as to prepare protein loading.

2. Sodium dodecyl sulfate-Polyacrylamide gel (SDS-PAGE)

(1) The gel plates were fixed and 10% SDS-PAGE gels were prepared (Table 3).

The separation gel was formulated as in table 3 below: 10ml of

Table 3 separation gel formulation information.

(2) Adding the mixed separation gel into 2 rubber plates respectively, adding to a position 1.0cm away from the top, filling the rubber plates with anhydrous ethanol, and standing for 30-45min.

(3) After the separation and gelation are finished, the residual absolute ethyl alcohol is poured out and is completely absorbed by filter paper.

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

Table 4 gel formulation information.

(5) Slowly adding the prepared concentrated glue into the rubber plate to avoid generating bubbles, inserting a sample comb, and standing for 30-45min.

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

(7) Fixing the rubber plate into an electrophoresis tank, adding SDS-PAGE electrophoresis buffer solution, pulling out a sample comb, and adding the processed protein samples into the sample tank in sequence.

(8) 80V electrophoresis for 40min.

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

3. western-blot membrane conversion

(1) And (3) putting the SDS-PAGE gel after electrophoresis into a TBST buffer solution for rinsing once, and putting the albumin gel into a membrane transfer buffer solution for soaking.

(2) Soaking one layer of spongy cushion in a membrane transfer buffer solution, clamping the spongy cushion to a membrane rotating instrument by using tweezers, sequentially placing three layers of filter paper, protein glue, a polyvinylidene fluoride (PVDF) membrane, three layers of filter paper and the spongy cushion, aligning, clamping and placing the spongy cushion on the membrane rotating instrument, and soaking the filter paper and the spongy cushion in the membrane rotating buffer solution during operation. If there are air bubbles between each layer, the air bubbles are expelled by gently rolling the glass test tube.

(3) The membrane rotating instrument is opened, and the membrane is rotated for 75min at 300 mA.

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

(5) Blocking with 5% Bovine Serum Albumin (BSA) blocking solution (20ml), 60rpm horizontal shaking apparatus at room temperature for 2h.

(6) Incubate overnight at 4 ℃ with a 60rpm horizontal shaker using 3ml of antibody incubation to which 3. Mu.l of primary antibody (1.

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

(8) The PVDF membrane was incubated for 2 hours with 20ml of antibody incubation solution to which 2. Mu.l of secondary antibody was added, and with a horizontal shaker at 60rpm at room temperature.

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

(10) Taking 1ml of each of the chemiluminescence substrate reagent solution A and the chemiluminescence substrate reagent solution B, uniformly mixing, and coating on a filter membrane.

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

As shown in FIGS. 4A and 4B, western blotting revealed that as the concentration of dihydroergotoxine mesylate increased, the protein expression of GAPDH in 4T1 and HuH-7-R cells remained unchanged, while the expression of STAT3, p-STAT3 (Tyr 705), cyclin D1, BCL-XL, surviving, ABCB1, β -catenin and p- β -catenin (ser 552) all decreased, while the expression of clear-PARP increased. Therefore, the 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 assay to determine the effect of dihydroergotoxine mesylate on mRNA expression levels of inflammatory cytokines in HuH-7 cells.

Inoculating HuH-7 cells 1 × 10 in 6-well plate ⁶ And (4) respectively. 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 measured. cDNA was synthesized using the extracted total RNA as a template according to the reverse transcription kit of Promega. STAT3 and its target gene were detected by real-time quantitative PCR amplification, with ACTB as the internal reference. The primers used are shown in Table 5.

Table 5 primer information for inflammatory cytokines used for the quantitative PCR.

Real-time quantitative PCR reaction system:

each set of samples was provided with 3 replicate wells.

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

The amplification was performed for 40 cycles, and data analysis was performed with the CT value of ACTB as an initial value.

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

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

Counting and preparing single cell suspension, inoculating into 96-well plate at 5000 cells per well and 100. Mu.l per well, and 5% CO ₂ Incubated overnight at 37 ℃ in an incubator. 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 dihydroergotoxine mesylate, stattic or GABAzine were added and incubated for 48h, 3 replicate wells per assay well, with no compound added as control. Cell viability was determined for each culture well by the CCK-8 method. Adding 10. Mu.l of CCK-8 reagent per well, and% _2， Incubated at 37 ℃ for 1 hour in an incubator, and the absorbance at 450nm of each well was measured with a microplate reader. Static is a commercial STAT3 inhibitor available from MCE. GABAzine selection for GABAA receptorsCompetitive antagonist, available from MCE corporation.

The test results are shown in figure 6, and the dihydroergotoxine mesylate with different concentrations can inhibit the proliferation and IC of liver cancer cells, liver cancer drug-resistant cells, breast cancer cells, colon cancer cells and stomach cancer cells ₅₀ The value is the lowest in 4T1 cells, and is lower than that of a parent cell HepG2 in a liver cancer drug-resistant cell HepG2-R, so that the liver cancer cells resistant to chemotherapy are more sensitive to dihydroergotoxine mesylate. Dihydroergotoxine mesylate was comparable to Stattic in its activity against tumor cell proliferation, whereas the selective competitive antagonist GABAzine of the GABAA receptor had little anti-tumor activity, indicating that dihydroergotoxine mesylate exerts anti-tumor effects by targeting STAT3 rather than the GABAA receptor.

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

4T1 and HuH-7 cells in logarithmic growth phase are taken, digested, counted and inoculated into a 12-well plate, and each well is 2 multiplied by 10 ⁵ For each cell, 3 parallel control wells were provided. 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. The cells were digested with pancreatin, resuspended and counted to adjust the cell concentration to 5X 10 ⁵ And (4) respectively. After digestion was complete, the supernatant was centrifuged off, the cells were washed twice with PBS (2000 rpm, centrifugation 5 min), after which the supernatant was discarded, 980. Mu.l of 70% cold ethanol and 0.1% BSA were added per tube and fixed in a refrigerator at 4 ℃ overnight. The fixative was discarded and washed 3 times with PBS (6000 rpm, centrifugation 0.5 min). After the cell washing, the subsequent operations were performed according to the instruction of the DNA content detection kit (product of beijing solibao). Each sample was incubated with 100. Mu.l RNase A for 30min at 37 ℃ and then 500. Mu.l of the prepared PI (propidium iodide) working solution was added to each sample and incubated for 30min at room temperature in the absence of light. Finally, the cell cycle was determined by flow cytometry. The results of the experiment were analyzed using the ModFit software and the ratio of the cycle distribution of the two cells was further analyzed by Graphpad prism 8.0.

FIGS. 7A and 7C are the results of analysis of the cycle profiles of 4T1 and HuH-7 cells with ModFit software in the absence or treatment with dihydroergotoxine mesylate. 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 can induce a significant increase in the G1/S phase ratio and a corresponding decrease in the G2 phase ratio of breast and liver cancer cells in a dose-dependent manner compared to the solvent control (DMSO). Thus, dihydroergotoxine can induce cycle arrest in tumor cells.

Example 8 dihydroergotoxine mesylate can induce apoptosis in tumor cells.

4T1 and HuH-7 cells in logarithmic growth phase are taken, digested, counted and inoculated in a 6-well plate with 5 multiplied by 10 per well ⁵ For each cell, 3 parallel control wells were provided. After about 16h, dihydroergotoxine mesylate was added at concentrations of 0, 2.5. Mu.M, 5. Mu.M and 10. Mu.M and incubation was continued for 48h. The cells were digested with trypsin without EDTA, resuspended and then counted to adjust the cell concentration to 1X 10 ⁶ And (4) respectively. The subsequent operations were carried out according to the instructions of Annexin V FITC-PI kit for detecting apoptosis (Beijing Solebao). The method comprises the following specific steps: after washing the cells 2 times with 1 XPBS (6000 rpm, centrifugation 0.5 min), washing the cells 1 times with 1 XPbinding buffer (6000 rpm, centrifugation 0.5 min) the supernatant was discarded, the cells were resuspended in 300. Mu.l of 1 XPbinding buffer, 5. Mu.l of Annexin V-FITC was added to each tube and incubated for 10min in the dark. Subsequently, 5. Mu.l of PI were added to each tube and incubated for 5min in the dark. And (5) performing detection on the machine in a dark place.

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

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

Conventional digestion of 4T1 cells with pancreatin, counting and preparing single cell suspensions, plating into 96-well plates at 5000 cells per well, 100. Mu.l per well, 5% CO ₂ Incubated overnight at 37 ℃ in an incubator. Gradient doses of dihydroergotoxine mesylate (0, 2 μ M and 4 μ M) were added, alone and in combination with doxorubicin (0, 0.0625 μ M and 0.125 μ M), vincristine (0, 0.5 μ M and 1 μ M) and epirubicin (0, 0.0625 μ M and 0.125 μ M), respectively, and incubated for 48h with 3 replicate wells per experimental well. By CCThe K-8 method measures the cell viability of each culture well. Adding 10. Mu.l of CCK-8 reagent per well, and% _2， Incubation is carried out at 37 ℃ for 1 hour, and absorbance at 450nm of each well is measured with a microplate reader.

As shown in fig. 9, in contrast to cell culture wells without dihydroergotoxine mesylate and chemotherapeutic agents, 4T1 cell proliferation was significantly inhibited by either dihydroergotoxine mesylate alone (2 μ M and 4 μ M) or doxorubicin alone (0.0625 μ M and 0.125 μ M), vincristine (0.5 μ M and 1 μ M), and epirubicin (0.0625 μ M and 0.125 μ M); dihydroergotoxine mesylate (2 μ M and 4 μ M) in combination with doxorubicin (0.0625 μ M and 0.125 μ M), vincristine (0.5 μ M and 1 μ M) and epirubicin (0.0625 μ M and 0.125 μ M) respectively, further significantly inhibited cell viability ("×" stands for p < 0.0001). Thus, dihydroergotoxine mesylate may enhance the sensitivity of 4T1 cells to conventional chemotherapeutic drugs for breast cancer in vitro.

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

Conventionally digesting HuH-7 cells with pancreatin, counting and preparing a single cell suspension, inoculating into a 96-well plate at 5000 cells per well, 100. Mu.l per well, 5% CO ₂ Incubated overnight at 37 ℃ in an incubator. Gradient doses of dihydroergotoxine mesylate (0, 2 μ M and 4 μ M) were added, alone and in combination with doxorubicin (0, 0.25 μ M and 0.5 μ M), epirubicin (0, 0.25 μ M and 0.5 μ M) and sorafenib (0, 1 μ M,2 μ M and 4 μ M), respectively, and incubated for 48h with 3 replicate wells per experimental well. Cell viability was determined for each culture well by the CCK-8 method. Adding 10. Mu.l of CCK-8 reagent per well, and% _2， Incubated at 37 ℃ for 1 hour in an incubator, and the absorbance at 450nm of each well was measured with a microplate reader.

As shown in fig. 10, in contrast to cell culture wells without dihydroergotoxine mesylate and chemotherapeutic agents, dihydroergotoxine mesylate alone (2 μ M and 4 μ M) or doxorubicin alone (0.25 μ M and 0.5 μ M), epirubicin (0.25 μ M and 0.5 μ M), and sorafenib (1 μ M,2 μ M, and 4 μ M) all significantly inhibited HuH-7 cell proliferation; dihydroergotoxine mesylate (2 μ M and 4 μ M) in combination with doxorubicin (0.25 μ M), epirubicin (0.25 μ M) and sorafenib (1 μ M,2 μ M and 4 μ M), respectively, further significantly inhibited cell viability ("x" for p <0.01, "x" for p <0.001, "x" for p < 0.0001). The inhibition rate of the single use of adriamycin (0.5 mu M) and epiadriamycin (0.5 mu M) on HuH-7 cells reaches 80%, so that the effect is not stronger but is not weakened after the combined use of dihydroergotoxine. Thus, dihydroergotoxine mesylate may enhance HuH-7 cell sensitivity to low doses of conventional chemotherapeutic agents for liver cancer in vitro.

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

Conventional digestion of HuH-7-R cells with pancreatin, counting and preparing single cell suspensions, plating into 96-well plates at 5000 cells per well and 100. Mu.l per well, and determining the content of CO 5% ₂ Incubated overnight at 37 ℃ in an incubator. Gradient doses of dihydroergotoxine mesylate (0, 2 μ M and 4 μ M) were added, alone and in combination with doxorubicin (0, 0.25 μ M and 0.5 μ M), epirubicin (0, 0.25 μ M and 0.5 μ M) and sorafenib (0, 1 μ M,2 μ M and 4 μ M), respectively, and incubated for 48h with 3 replicate wells per experimental well. Cell viability was determined for each culture well by the CCK-8 method. Add 10. Mu.l of CCK-8 reagent per well and put 5% CO _2， Incubation is carried out at 37 ℃ for 1 hour, and absorbance at 450nm of each well is measured with a microplate reader.

As shown in FIG. 11, in contrast to cell culture wells without dihydroergotoxine mesylate and chemotherapeutic agents, dihydroergotoxine mesylate alone (2 μ M and 4 μ M) or doxorubicin alone (1 μ M and 2 μ M), epirubicin (1 μ M and 2 μ M), and sorafenib (1 μ M,2 μ M, and 4 μ M) all significantly inhibited HuH-7 cell proliferation; dihydroergotoxine mesylate (2 μ M and 4 μ M) in combination with doxorubicin (1 μ M and 2 μ M), epirubicin (1 μ M and 2 μ M) and sorafenib (1 μ M,2 μ M and 4 μ M) respectively, further significantly inhibited cell viability ("×" for p <0.05, "×" for p <0.001, "×" for p < 0.0001). Therefore, dihydroergotoxine mesylate can enhance the sensitivity of chemotherapy-resistant HuH-7-R cells to conventional chemotherapeutic liver cancer drugs in vitro.

Example 12 Combination Index (CI) analysis of dihydroergotoxine mesylate in combination with chemotherapeutic agents

The results of the CCK-8 assay in FIGS. 9 and 10 were analyzed using CompuSyn software. Fa represents the fraction of cell viability. When 0.3-straw-woven CI(s) are 0.7, strong synergistic effect is shown; when 0.7 were constructed with CI-s of 0.9, slight synergy was indicated; when 0.9-woven fabric (CI) woven fabric (1.1) shows that the overlapping effect exists; when CI is greater than 1.1, antagonism is indicated.

The results are shown in FIG. 12, where 2 μ M dihydroergotoxine mesylate in combination with doxorubicin or sorafenib had CI values of about 0.78, 0.66 and 0.84, 0.79, respectively, in 4T1 cells and HuH-7 cells, indicating that low doses of dihydroergotoxine mesylate in combination with doxorubicin and sorafenib, respectively, had a slight synergistic effect; and 4 mu.M dihydroergotoxine mesylate is respectively used together with adriamycin or sorafenib, and CI values are respectively about 0.49, 0.34, 0.42 and 0.37, which shows that higher doses of dihydroergotoxine mesylate respectively have stronger synergistic effect with adriamycin and sorafenib.

Example 13 animal experiments

The experimental animals were female Balb/c mice of 6 weeks old, purchased from Beijing Wintolite Biotechnology Ltd, and bred in SPF animal laboratories. Will be 1 × 10 ⁴ Individual 4T1 cells were seeded in the mouse underarm fat pad. Approximately one week later, the mice developed distinct opalescent nodules in the axilla, and the mice were randomly divided by weight into solvent control groups (10% DMSO +90% corn oil), dihydroergotoxine mesylate (20 mg/kg), doxorubicin hydrochloride (1 mg/kg), and dihydroergotoxine mesylate (20 mg/kg) + doxorubicin hydrochloride (1 mg/kg), with 4 mice per group. The preparation is administered by intraperitoneal injection 1 time per day. The 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 transplanted tumors were taken, weighed and photographed.

The results in 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 the short drug-induced period of dihydroergotoxine mesylate, the need for optimization of dosing frequency and dose, etc.) compared to the solvent control. The positive control drug doxorubicin hydrochloride (1 mg/kg) group had significant inhibitory effects on the volume, tumor weight and morphology of the transplanted tumors ("+" stands for p < 0.05). In the combination of dihydroergotoxine mesylate (20 mg/kg) and doxorubicin hydrochloride (1 mg/kg), the tumor volume, tumor weight and morphology size of the mice were further inhibited (". About." indicates that 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 property, can reduce the gene and protein expression of STAT3, and reduce phosphorylation of STAT3 and 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; inhibiting proliferation of breast cancer, liver cancer, chemotherapy-resistant liver cancer, colon cancer and gastric cancer cells; inducing tumor cell cycle arrest and apoptosis; enhancing the in vitro sensitivity of a plurality of tumor cells to doxorubicin, epirubicin, vincristine, and sorafenib; and can enhance the in vivo activity of the adriamycin, and has good application prospect in the aspect of tumor resistance.

Claims (8)

1. Application of dihydroergotoxine mesylate in preparing antitumor drugs is provided.

2. The use of dihydroergotoxine mesylate according to claim 1 in the preparation of a medicament for the treatment of tumors, wherein dihydroergotoxine mesylate is dihydroergotoxine mesylate and its derivatives.

3. Use of dihydroergotoxine mesylate for the preparation of a medicament against tumors according to claim 1, wherein the medicament is for the treatment or prevention of breast cancer, stomach cancer, colon cancer, liver cancer and drug-resistant liver cancer.

4. The use of dihydroergotoxine mesylate for the preparation of a medicament for the treatment of tumors as claimed in claim 1, wherein the tumor cells are 4T1, MDA-MB-231, MDA-MB-468, CT-26, HGC-27, hepG2, huH-7, hepG2-R and HuH-7-R.

5. Use of dihydroergotoxine mesylate for the preparation of a medicament against tumors according to claim 1, wherein the medicament is administered orally or by injection.

6. Use of dihydroergotoxine mesylate according to claim 1 for the preparation of a medicament against tumors, wherein the medicament is one of dihydroergotoxine mesylate, a dihydroergotoxine mesylate derivative, a composition comprising dihydroergotoxine mesylate or a composition comprising a dihydroergotoxine mesylate derivative.

7. The use of dihydroergotoxine mesylate for the preparation of a medicament against tumors as claimed in claim 1, wherein said medicament inhibits tumor cell proliferation and survival by targeting STAT3.

8. The application of dihydroergotoxine mesylate in preparing antitumor drugs according to claim 1, wherein the dihydroergotoxine mesylate is combined with adriamycin and sorafenib drugs to down-regulate the expression of P-gp, enhance the sensitivity of breast cancer and liver cancer to chemotherapeutic drugs, reverse the chemotherapeutic resistance of drug-resistant cells of liver cancer, and play a stronger role in vivo in synergy of antitumor.

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