Application of dioxin in inhibiting cell migration
阅读说明：本技术 二恶英在抑制细胞迁移中的应用 (Application of dioxin in inhibiting cell migration ) 是由 赵斌 刘奕耘 谢群慧 徐丽 陈旸升 于 2020-12-31 设计创作，主要内容包括：本发明涉及二恶英在抑制细胞迁移中的应用,具体地,本发明涉及二恶英或含有二恶英的药物组合物在制备抑制胶质瘤细胞迁移的药物中的用途。本发明首次发现特定浓度的TCDD可抑制胶质母细胞瘤细胞系U87的迁移,为胶质母细胞瘤的治疗提供了一种新的选择。(The invention relates to application of dioxin in inhibition of cell migration, in particular to application of dioxin or a pharmaceutical composition containing dioxin in preparation of drugs for inhibiting glioma cell migration. The invention discovers that TCDD with a specific concentration can inhibit the migration of a glioblastoma cell line U87 for the first time, and provides a new choice for the treatment of glioblastoma.)
1. Use of dioxin and pharmaceutical compositions containing dioxin in the preparation of a medicament for inhibiting glioma cell migration.
2. The use of claim 1, wherein the glioma cell is an astrocytoma cell;
preferably, the glioma cell is a glioblastoma cell; preferably, the glioblastoma cell is a cell from the subject or a glioblastoma cell line; preferably, the subject has glioblastoma; preferably, the glioma cell line is the glioblastoma cell line U87.
3. The use of claim 1 or 2, wherein the concentration of dioxin is 10-12-10-9M。
4. Use of dioxin and pharmaceutical compositions containing dioxin in the preparation of anti-glioma drugs.
5. The use of claim 4, wherein the medicament is for inhibiting glioma growth, metastasis and/or invasion.
6. The use of claim 4 or 5, wherein the glioma is an astrocytoma; preferably, the glioma is a glioblastoma.
7. The use of any one of claims 1-6, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and optionally other therapeutic agents.
8. The use of claim 7, wherein the other therapeutic agent is selected from the group consisting of temozolomide, bevacizumab, lomustine, carmustine, carboplatin, procarbazine, and vincristine.
9. The use according to any one of claims 1 to 8, wherein the dioxins are selected from the group consisting of chlorinated dibenzo-p-dioxins, chlorinated dibenzofurans, nonadjacent polychlorinated biphenyls and monoadjacent polychlorinated biphenyls.
10. The use of claim 9, wherein the chlorinated dibenzo-p-dioxin-like compound is 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-HpCDD or OCDD;
preferably, the dibenzofuran chloride compound is 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,4,6,7,8-HpCDF, 1,2,3,4,7,8,9-HpCDF or OCDF;
preferably, the non-ortho-substituted polychlorinated biphenyls are 3,3',4,4' -tetraCB (PCB 77), 3,4,4',5-tetraCB (PCB 81), 3',4,4',5-pentaCB (PCB 126) or 3,3',4,4',5,5' -hexaCB (PCB 169);
preferably, the mono-ortho-substituted polychlorinated biphenyl is 2,3,3',4,4' -pentaCB (PCB 105), 2,3,4,4',5-pentaCB (PCB 114), 2,3',4,4',5-pentaCB (PCB 118), 2',3,4,4',5-pentaCB (PCB 123), 2,3,3',4,4',5-hexaCB (PCB 156), 2,3,3',4,4',5' -hexaCB (PCB 157), 2,3',4,4',5,5'-hexaCB (PCB 167) or 2,3,3',4,4',5,5' -hexaCB (PCB 189);
preferably, the dioxin is TCDD.
Technical Field
The present invention relates to the field of molecular biology, more specifically, the present invention relates to techniques related to cancer therapy. The invention mainly comprises the application of dioxin in inhibiting cell migration, and particularly discloses the application of TCDD in inhibiting cell migration of glioblastoma.
Background
In all cancer-related case studies, brain and central nervous system tumors are the third leading cause of cancer-related death. In brain tumors, the incidence of astrocytoma is high, accounting for 15.1% of primary central nervous system tumors and 46.1% of primary central nervous system malignant tumors. Among them, glioblastoma is the most common, belonging to a very aggressive primary glioma, and is also the IV grade tumor in the malignancy classification of World Health Organization (WHO) tumors.
The comprehensive treatment of glioblastoma includes surgery, radiotherapy, systemic treatment (chemotherapy, targeted therapy) and supportive treatment, but the overall prognosis is still poor and the long-term survival rate is low. There remains a need in the art for more therapeutic approaches for glioblastoma.
Disclosure of Invention
Studies have shown that there is diffuse growth of glioblastoma and, in addition to intracranial short-range migration, extracranial metastasis of tumor cells is also found. Therefore, inhibiting the migratory motor of glioblastoma may be beneficial for treating tumors, reducing the risk of postoperative recurrence and improving disease prognosis.
Dioxins are common names for polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), including 2,3,7, 8-tetrachlorodibenzo-p-dioxins (TCDD) and the like. TCDD has been considered carcinogenic and is classified as a carcinogen by the international health organization (WHO). However, in recent years, the carcinogenicity of TCDD is questioned, and the TCDD has been found to have a complex regulation effect on the occurrence and development of tumors.
The invention discovers that the low-concentration TCDD can inhibit the migration of a glioblastoma cell line U87, and provides a new choice for the treatment of glioblastoma.
Thus, in one aspect, the present invention provides the use of dioxin and pharmaceutical compositions containing dioxin in the preparation of a medicament for inhibiting glioma cell migration.
In some embodiments, the glioma cell is an astrocytoma cell.
In some embodiments, the glioma cell is a glioblastoma cell. In some embodiments, the glioblastoma cell is a cell from a subject or a glioblastoma cell line. In some embodiments, the subject has a glioblastoma. In some embodiments, the glioblastoma cell line is glioblastoma cell line U87.
In some embodiments, the concentration of dioxin is 10-12～10-9M, e.g. 10-12M～10-11M、10-12M～10-10M、10-12～10-9M、10-11M～10-10M、10-11M～10-9M or 10-10M～10-9M。
In another aspect, the present invention provides the use of dioxin and pharmaceutical compositions containing dioxin in the preparation of anti-glioma drugs.
In some embodiments, the medicament is for inhibiting glioma growth, metastasis and/or invasion.
In some embodiments, the glioma is an astrocytoma. In some embodiments, the glioma is a glioblastoma.
In another aspect, the present invention provides a method of inhibiting glioma cell migration comprising the step of contacting the cell with an effective amount of dioxin and dioxin-containing pharmaceutical compositions.
In some embodiments, the glioma cell is an astrocytoma cell.
In some embodiments, the glioma cell is a glioblastoma cell. In some embodiments, the glioblastoma cell is a cell from a subject or a glioblastoma cell line. In some embodiments, the subject has a glioblastoma. In some embodiments, the glioblastoma cell line is glioblastoma cell line U87.
In some embodiments, the concentration of dioxin is 10-12～10-9M, e.g. 10-12M～10-11M、10-12M～10-10M、10-12～10-9M、10-11M～10-10M、10-11M～10-9M or 10-10M～10-9M。
In some embodiments, the method is performed in vivo.
In some embodiments, the method is performed in vitro.
In another aspect, the present invention provides a method of treating a glioma, comprising the step of administering to a subject in need thereof a therapeutically effective amount of dioxin and dioxin-containing pharmaceutical compositions.
In some embodiments, the medicament is for inhibiting glioma growth, metastasis and/or invasion.
In some embodiments, the glioma is an astrocytoma. In some embodiments, the glioma is a glioblastoma.
The pharmaceutical compositions of the present invention also contain a pharmaceutically acceptable carrier and optionally other therapeutic agents.
In some embodiments, the additional therapeutic agent is selected from temozolomide, bevacizumab, lomustine, carmustine, carboplatin, procarbazine, and vincristine.
In any aspect of the invention the dioxin is selected from the group consisting of chlorinated dibenzo-p-dioxins, chlorinated dibenzofurans, nonadjacent polychlorinated biphenyls and monoadjacent polychlorinated biphenyls.
In some embodiments, the chlorinated dibenzo-p-dioxin-like compound is 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-HpCDD, or OCDD.
In some embodiments, the dibenzofuran chloride is 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,4,6,7,8-HpCDF, 1,2,3,4,7,8,9-HpCDF or OCDF.
In some embodiments, the non-ortho-substituted polychlorinated biphenyls are 3,3',4,4' -tetraCB (PCB 77), 3,4,4',5-tetraCB (PCB 81), 3',4,4',5-pentaCB (PCB 126), or 3,3',4,4',5,5' -hexaCB (PCB 169).
In some embodiments, the mono-ortho substituted polychlorinated biphenyl is 2,3,3',4,4' -pentaCB (PCB 105), 2,3,4,4',5-pentaCB (PCB 114), 2,3',4,4',5-pentaCB (PCB 118), 2',3,4,4',5-pentaCB (PCB 123), 2,3,3',4,4',5-hexaCB (PCB 156), 2,3,3',4,4',5' -hexaCB (PCB 157), 2,3',4,4',5,5'-hexaCB (PCB 167), or 2,3,3',4,4',5,5' -hexaCB (PCB 189).
In some embodiments, the dioxin is TCDD.
Definition of terms
Molecular biology, microbiology, and recombinant DNA techniques that may be used in the present invention are within the skill of the art. These techniques have been explained fully in the literature. See, for example: sambrook, Fritsch & Maniatis, Molecular cloning: a Laboratory Manual, (1982); DNA Cloning, A Practical Approach Volumes I & II, D.N. Glover. 1985; oligonucleotide Synthesis, m.j.gait ed.1984; nucleic Acid Hybridization, B.D.Hames & S.J.Higgins eds.1985; transformation and transformation, B.D.Hames & S.J.Higgins eds.1984; animal Cell Culture, r.i. freshney, ed.1986; immobilized Cells And Enzymes, IRL Press, 1986; BPerbal, A Practical Guide To Molecular Cloning,1984 based thereon, the terms appearing herein are defined as follows.
As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and the like.
As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than an active ingredient that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, preservatives, and the like.
As used herein, the term "individual" or "subject" is a mammal, e.g., a bovine, equine, porcine, canine, feline, rodent, primate; among these, particularly preferred subjects are humans.
As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.
The amount of the drug administered to the subject depends on the type and severity of the disease or condition and the characteristics of the subject, such as general health, age, sex, body weight and tolerance to the drug, as well as on the type of formulation and mode of administration of the drug, and the period or interval of administration. One skilled in the art will be able to determine the appropriate dosage based on these and other factors.
As used herein, the terms "glioma", "glioma" refer to tumors derived from neuroepithelium and include primarily astrocytomas, oligogliomas, ependymomas and the internodal (malignant) ependymomas, mixed gliomas, choroid plexus tumors, and the like, by pathological classification. "glioblastoma" is the most malignant glioma among astrocytomas.
Drawings
FIG. 1 shows 10-12～10-9And detecting the change of the absorbance value of the cell sample by an enzyme label instrument in 24h and 48h of M TCDD treated U87 cells.
FIG. 2 shows the results of scoring experiments to evaluate the effect of TCDD on the migration ability of U87 cells. Wherein FIG. 2A shows a warp beam 10-10After the M TCDD treats the cells for 36 hours, the area of scratches among the cells is larger than that of a control group, which shows that the migration distance of the cells is reduced and the migration capacity of the cells is reduced; FIG. 2B shows a warp 10-10The migration distance of U87 cells decreased by 18.5% after M TCDD treatment, indicating that TCDD can effectively inhibit cell migration.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
U87 cells were purchased from the cell resource center of the Chinese academy of medical sciences.
TCDD is available from Wellington Laboratory.
The CCK8 kit was purchased from Sangon Biotech (shanghai).
Microscope: CKX41(Olympus Japan).
A camera: 600D (Canon Japan).
NanoDrop spectrophotometer: 2000/2000c (Thermo usa).
An enzyme-labeling instrument: tecan 2000pro (Tecan Switzerland).
Example 1 investigation of the toxic Effect of TCDD on glioblastoma
U87 cells were cultured at 1X 105one/mL was seeded into 96-well plates at 100mL cell suspension per well. Waiting for overnight cultureThe cells adhere to the wall. The 96-well plate was removed, blotted clean of the original medium, and washed once with Phosphate Buffered Saline (PBS). The experiment was set up with a background control group, a solvent control group and a drug treatment group. Correspondingly, low serum medium (serum concentration 1%, no cells in the background control wells), solvent-containing dimethyl sulfoxide (DMSO) or various concentrations of TCDD were added to each well, maintaining the volume of solvent at 0.1%. And putting the medicated 96-well plate into an incubator to continuously culture for 24 hours or 48 hours. After the end of incubation time was reached, 10. mu.L of CCK8 reagent was added to each well as written in the kit instructions, and the well plate was again placed in the incubator and incubated for 1h before reading the absorbance with the microplate reader.
As can be seen in FIG. 1, use 10-12～10-9M TCDD treated U87 cells did not cause obvious change of absorbance value within 48h, and had no influence on cell proliferation and viability. Therefore, the TCDD has no cytotoxicity under the concentration and the treatment time, and can be used for subsequent experiments.
Example 2 TCDD inhibits migration of U87 cells
The migration ability of the cells was evaluated using a scratch test. The scratch was located by drawing 3 lines transversely across the bottom of the 6 well plate with a marker pen. Cells were plated at 4X 1052mL of cells were seeded at density per mL in 6-well plates and cultured overnight to wait for the cells to adhere. After the cells completely covered the bottom of the well plate, the cell monolayer was scratched with the tip of a 1mL pipette tip to form a scratch. And selecting the junction of the scratch and the mark drawn by the mark pen as a photographing position. The floating cells were removed by washing with phosphate buffer and then a low serum medium containing the solvent DMSO or TCDD was added. And collecting the image by a digital camera under an inverted microscope 0-48h after scratching. Three independent replicates were performed, each concentration group in each independent experiment had 3 replicate wells, and each well contained 3 scratches, and 12 images were taken for counting and quantification. Cell migration distance was calculated by dividing the scratch reduction area by the scratch length and analyzed using Image Pro Plus 6.0 software. The scale bar on the representative scratch image is 0.05 mm.
As shown in FIG. 2A, after TCDD treated cells for 36h, the area of the scratches between cells was larger than that of the control group, indicating that the migration distance of the cells was decreased and the cells wereThe migration ability is decreased. The statistical data according to FIG. 2B show that-10Migration distance of U87 cells was reduced by 18.5% after M TCDD treatment. It was shown that TCDD can effectively inhibit cell migration.
Although specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that, based upon the overall teachings of the disclosure, various modifications and alternatives to those details could be developed and still be encompassed by the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.