CN114028399A

CN114028399A - Application of MIF inhibitor 4-IPP in preparation of medicine for treating brain glioma

Info

Publication number: CN114028399A
Application number: CN202111549537.XA
Authority: CN
Inventors: 杜延茹; 郑琳
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-12-18
Filing date: 2021-12-18
Publication date: 2022-02-11

Abstract

The invention discloses an application of an MIF inhibitor 4-IPP in preparing a medicament for treating brain glioma. The invention establishes that the MIF inhibitor 4-IPP can inhibit the proliferation, clone formation and transfer capability of glioma cells, and simultaneously the micromolecule compound can inhibit the growth of subcutaneous ectopic transplantation tumors and has the effect of treating glioma. The micromolecule compound has application value in glioma treatment according to the tumor inhibition effect of the micromolecule compound on glioma.

Description

Application of MIF inhibitor 4-IPP in preparation of medicine for treating brain glioma

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of an MIF inhibitor 4-IPP in preparation of a medicine for treating brain glioma.

Background

Brain Gliomas (Gliomas) are the most common primary malignancies of the central nervous system, with invasive growth, high recurrence, poor prognosis, and are one of the most challenging diseases in cancer treatment [1 ]. The annual incidence rate of the glioma is about 6/10 ten thousand, and the average survival rate in 5 years is lower than 10% [2 ]; glioblastoma (glioblastomas) are WHO grade iv gliomas with extremely high malignancy, with an average time from diagnosis to death of less than 18 months [3 ].

Currently, surgical resection, chemotherapy in combination with radiotherapy and targeted immunotherapy are the main approaches to the treatment of glioma. However, the mean survival time of glioma patients has not been substantially improved due to postoperative tumor residues, decreased sensitivity to radiation therapy, chemotherapy resistance, and the like. Macrophage Migration Inhibitory Factor (MIF) is a pluripotent cytokine with properties of enzymes, chemokines, hormones and the like [4], and MIF directly participates in the regulation of tumor cells to promote tumor growth, metastasis and angiogenesis [5 ]. The research shows that MIF is highly expressed in a plurality of tumors such as prostate cancer, pancreatic cancer, melanoma, colon cancer, glioma and the like [6-10 ]. RT-PCR quantitatively detected that MIF mRNA expression in malignant glioma cells was 800-fold higher than in normal brain tissue and this was associated with high levels of total cell lysate and secreted MIF protein [11 ]. It has also been found that overexpression of MIF induces angiogenesis, associated with stage and metastatic spread of glioma [4-5 ]; elevated MIF expression is also associated with tumor recurrence and poor prognosis in patients with glioblastoma [12 ]. Thus, MIF may be a novel target for glioma therapy.

Disclosure of Invention

The invention aims to provide an application of an MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP) in preparation of a glioma treatment drug for preparation of a glioma treatment drug.

The second purpose of the invention is to provide an application of MIF inhibitor 4-IPP in preparing glioma cell proliferation inhibition drugs.

The third purpose of the invention is to provide an application of MIF inhibitor 4-IPP in preparing a brain glioma cell metastasis inhibition drug.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses the application of MIF inhibitor 4-IPP in preparing a medicament for treating brain glioma for the first time, wherein in vitro experiments are carried out to treat U87 and U251 glioma cells by adopting 4-IPP with different concentrations, and CCK-8 and clone formation experiments are used for detecting the proliferation capacity and clone formation capacity of U87 and U251 glioma cells after 4-IPP treatment; the inhibition of U87 and U251 cell Migration/Invasion by 4-IPP was examined by Migration/Invasion experiments using a Transwell chamber. Further in vivo experiments, a male nude mouse subcutaneous xenograft tumor model is constructed by U87, the treatment effect of MIF inhibitor 4-IPP on glioma is observed by injecting PBS group (Control group) and 20mg/kg 4-IPP group (treatment group) into nude mice intraperitoneally, the tumor size is calculated, the tumor mass is weighed, and the tumor volume is calculated. Research results show that 4-IPP can effectively inhibit glioma cell proliferation, clone formation and metastasis and treat glioma.

Drawings

FIG. 1 is a graph showing the effect of the experiment on proliferation of CCK-8 of U87 cells treated with 4-IPP of example 1;

FIG. 2 is a graph showing the effect of the experiment on proliferation of CCK-8 in U251 cells treated with 4-IPP of example 1;

FIG. 3 is a graph showing the effect of the 4-IPP treatment of U87 and U251 cells in the cell clonogenic assay of example 1;

FIG. 4 is a graph showing the effect of the 4-IPP treatment of U87 and U251 cells in the Migration experiment and Invasion experiment of example 2;

FIG. 5 is a graph showing the effect of the subcutaneous xenograft tumor models in the PBS group and the 20mg/kg 4-IPP group of example 3;

Detailed Description

The specific experimental methods and results are as follows:

example 1

The first experiment method comprises the following steps:

1. cell proliferation assay CCK-8:

a generation of U87 and U251 cells was seeded at 2000 cells/well in a 96-well plate, and 4-IPP solutions at concentrations of 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, 100. mu.M and 200. mu.M were added after 12 hours, respectively. After 24 hours of culture, 20. mu.L of CCK-8 kit was added and incubated at 37 ℃ for 2 hours, and then OD value was measured with a fluorescence microplate reader.

2. Clone formation experiments:

taking monolayer cultured cells in logarithmic phase, digesting with pancreatin, blowing into single cells, counting the cells after resuspension, paving the cells into a six-hole plate according to the density of 200 cells/hole, shaking the plate to disperse the cells, and placing the cells in an incubator overnight. The following day, 12.5. mu.M, 25. mu.M and 50. mu.M of 4-IPP were added, and the solution was changed 2 times per week for 2-3 weeks. The culture was terminated when macroscopic colonies appeared in the culture dish, observed every other day. The supernatant was discarded, fixed and stained with crystal violet, and the clones were counted directly with the naked eye or the number of clones larger than 10 cells was counted under a microscope (low power mirror).

II, experimental results:

FIG. 1 shows that 4-IPP can significantly inhibit the proliferation capacity of U87 by detecting the proliferation of U87 cells by using a CCK-8 reagent after the U87 cells are treated for 24 hours in vitro by setting a concentration gradient of 4-IPP. FIG. 2 shows that after U251 cells are treated for 24h by setting concentration gradient 4-IPP in vitro, the cell proliferation condition is detected by using CCK-8 reagent, and the result shows that the 4-IPP can obviously inhibit the proliferation capacity of U251. Based on the results obtained from the CCK-8 experiment, further cloning experiments were performed using concentration gradients, and the results in FIG. 3 show that 4-IPP can effectively inhibit the clonogenic capacity of U87/U251. The above results indicate that 4-IPP can effectively inhibit the proliferation and clonogenic capacity of glioma cell U87/U251.

Example 2

The first experiment method comprises the following steps:

1. migration and Invasion experiments:

transwell cells (24-well plates) were used for the Migration experiments and the matrigel-coated Invasion experiments. For the Migration experiment, 200ul of cell suspension (density 2X 10) was taken⁵) Transwell chambers were added and 500. mu.l of complete medium containing different concentrations of 4-IPP was placed in the lower chamber and samples were collected after 24 hours of culture. For the invasion assay, 200ul of cell suspension (density 2X 10) was placed after Matrigel coating the upper chamber⁵) At the same time, 500. mu.l of complete medium containing 4-IPP at various concentrations was added to the lower chamber and the samples were collected after 24 hours of culture. After the sample is collected, the crystal violet is stained, and a picture is taken by a microscope.

II, experimental results:

FIG. 4 shows the validation of the inhibitory effect of MIF inhibitor 4-IPP on U87 and U251 cell Migration by Migration and Invasion experiments in vitro using Transwell chambers; the results show that 4-IPP treatment can significantly reduce the number of U87/U251 cell metastases; further laying Matrigel to simulate the matrix environment for Invasion experiments, the 4-IPP treatment also significantly reduced the number of U87/U251 cells invading the lower part of the chamber through the matrix.

Example 3

The first experiment method comprises the following steps:

1. establishment of a male nude mouse subcutaneous ectopic transplantation tumor model:

male nude mice for 4 weeks were housed in an SPF-rated animal house with 5 mice per cage, and given sufficient food and water with appropriate temperature, humidity and light cycle control. Make a total of 510⁷One U87 stable cell was injected into the lower right dorsal side (each group n-5).

2. Grouping: after one week, we randomly divided the mice into two groups. The PBS group and the 20mg/kg 4-IPP group were injected intraperitoneally, respectively. After 3 weeks of injection every other day, mice were euthanized and tumors were removed and photographed.

3. Preparation of injection: 4-IPP was first dissolved in DMSO, and then the DMSO stock solution was dissolved in PBS for preparation of 4-IPP injections at different concentrations.

II, experimental results:

FIG. 5 is a graph showing the effect of the subcutaneous xenograft tumor models in PBS and 20mg/kg 4-IPP groups. As can be seen, compared with the Control group, the glioma tissue volume and weight of the 4-IPP group with 20mg/kg are obviously reduced, which indicates that the 4-IPP can effectively inhibit the growth of glioma cells in vivo and has the function of treating brain glioma.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

The references referred to in this specification are as follows:

[1]Lapointe S,Perry A,Butowski NA.Primary brain tumours in adults.Lancet,2018,392(10145):432-446.

[2]Hiroko Ohgaki,Paul Kleihues.Population-based studies on incidence,survivalrates,and genetic alterations in astrocytic and oligodendroglial gliomas.JNeuropathol Exp Neurol,2005,64(6):479-489.

[3]Stupp R,Hegi ME,Mason WP,et al.Effects of radiotherapy with concomitantand adjuvant temozolomide versus radiotherapy alone on survival in glioblastomain a randomised phase III study:5-year analysis of the EORTC-NCIC trial.Lancet Oncol,2009,10(5):459–466.

[4]Bach JP,Deuster O,Balzer-Geldsetzer M,et al.The Role of MacrophageInhibitory Factor in Tumorigenesis and Central Nervous System Tumors.Cancer,2009,115(10):2031-2040.

[5]Beatriz E Rendon,Sharon S Willer,Wayne Zundel,et al.Mechanisms ofmacrophage migration inhibitory factor(MIF)-dependent tumormicroenvironmental adaptation.Experimental and Molecular Pathology,2009,86(3):180-185.

[6]Hussain F,Freissmuth M,

D,et al.Human anti-macrophage migrationinhibitory factor antibodies inhibit growth of human prostate cancer cells in vitroand in vivo.Mol Cancer Ther,2013,12(7):1223-1234.

[7]Shouhui Yang,Peijun He,Jian Wang,et al.ANovel MIF Signaling PathwayDrives the Malignant Character of Pancreatic Cancer by Targeting NR3C2.Cancer Res,2016,76(13):3838-3850.

[8]Masako Imaoka,Keiji Tanese,Yohei Masugi,et al.Macrophage migrationinhibitory factor-CD74 interaction regulates the expression of programmed celldeath ligand 1 in melanoma cells.Cancer Sci,2019,110(7):2273-2283.

[9]Shugang Xu,Xing Guo,Xiao Gao,et al.Macrophage migration inhibitoryfactor enhances autophagy by regulating ROCK1 activity and contributes to theescape of dendritic cell surveillance in glioblastoma.Int J Oncol,2016,49(5):2105-2115.

[10]Wendy Ha,Hatice Sevim-Nalkiran,Ashraf M Zaman,et al.Ibudilast sensitizesglioblastoma to temozolomide by targeting Macrophage Migration InhibitoryFactor(MIF).Sci Rep,2019,9(1):2905.

[11]Mittelbronn M,Platten M,Zeiner P,et al.Macrophage migration inhibitory factor(MIF)expression in human malignant gliomas contributes to immune escape andtumour progression.Acta Neuropathol,2011,122(3):353-65.

[12]Xiao-Bing Wang,Xiao-Ying Tian,Yang Li,et al.Elevated expression ofmacrophage migration inhibitory factor correlates with tumor recurrence and poorprognosis of patients with gliomas.J Neurooncol,2012,106(1):43-51.

Claims

1. an application of a small molecular compound macrophage migration inhibitory factor MIF inhibitor 4-IPP in preparing a medicament for treating brain glioma is shown in the following figure.

2. Use of the small molecule compound 4-IPP of claim 1 in the manufacture of a medicament for inhibiting glioma cell proliferation.

3. Use of the small molecule compound 4-IPP of claim 1 in the manufacture of a medicament for inhibiting glioma cell metastasis.