CN111467345B

CN111467345B - Application of dexamethasone, rosiglitazone and 3-isobutyl-1-methylxanthine composition in preparing medicine for inhibiting growth and metastasis of ovarian cancer cells induced by cobalt chloride

Info

Publication number: CN111467345B
Application number: CN202010337378.6A
Authority: CN
Inventors: 张诗武; 张可昕; 李玉玮
Original assignee: Tianjin People Hospital
Current assignee: Tianjin People Hospital
Priority date: 2020-04-26
Filing date: 2020-04-26
Publication date: 2022-02-18
Anticipated expiration: 2040-04-26
Also published as: CN111467345A

Abstract

The invention discloses a method for inhibiting growth and metastasis of solid tumor cells and a special pharmaceutical composition, and belongs to the field of proliferation and transformation of animal cells. According to the invention, when ovarian cancer HEY cells or breast cancer MDA-MB-231 cells are induced, the culture medium is high in concentration CoCl₂After the action, the culture medium is replaced, and after the cells are recovered, the cells are repeatedly treated for 2 times to obtain a sufficient number of cells with high invasion and transfer capacity; the cells were then CoCl-free₂The culture medium is cultured, then a pharmaceutical composition consisting of dimethyl sulfoxide, dexamethasone, rosiglitazone and 3-isobutyl-1-methylxanthine is added into the removed culture medium for in vitro induction, and when obvious lipid drops are formed in tumor cell plasma, the fat differentiation of the tumor cells is shown. The invention thus designed induces the adipose differentiation of tumor cells to achieve the purpose of inhibiting the growth of tumor cells. Animal experiments prove that the medicine can be used for clinical treatment and metastasis inhibition of human solid tumors.

Description

Application of dexamethasone, rosiglitazone and 3-isobutyl-1-methylxanthine composition in preparing medicine for inhibiting growth and metastasis of ovarian cancer cells induced by cobalt chloride

Technical Field

The invention relates to proliferation and transformation of animal cells, in particular to a method for inhibiting growth and metastasis of solid tumor cells and a special pharmaceutical composition.

Background

Tumors are systemic diseases, and are the diseases with the highest morbidity and mortality rate in human beings. Currently, the main methods for the prevention and treatment of tumors include: surgery, radiotherapy and chemotherapy. Compared with operation treatment and radiation treatment, the drug treatment belongs to a therapy with a systemic effect, the traditional chemotherapeutic drugs not only kill tumor cells, but also kill normal body cells, and the toxicity and drug resistance of the traditional chemotherapeutic drugs are not negligible. In addition, the chemotherapy drugs can also enhance the capacity of inducing the invasion and the metastasis of tumor cells, and enhance the tolerance capacity of the tumor cells to the chemotherapy drugs, thereby finally causing the relapse and the metastasis of tumors. The most remarkable biological characteristics of tumor cells are unlimited proliferation and poor differentiation, and the occurrence of tumors is generally considered to be related to abnormal differentiation, so that the induction of benign differentiation of tumor cells becomes a new field of tumor treatment. Under the action of a differentiation inducer, dedifferentiated tumor cells can also be induced to differentiate into benign cells, whose biological properties are close to those of normal cells, or even transformed into normal cells, a phenomenon known as re-differentiation or redifferentiation. Treatment of malignant tumors using this strategy is called induced differentiation therapy. The basic characteristic of tumor inducing differentiation is not to kill tumor cells, but to induce tumor cells to differentiate into normal or nearly normal cells, i.e. some tumor cells have phenotype similar to normal cells under the action of some chemical agents, and some functions of normal cells are recovered. The Classic method for inducing mesenchymal stem cell adipose differentiation in vitro is the cocktail induction method (Classic cocktails) which is widely reported, and the cocktail combination comprises a basic culture medium: DMEM basal medium, 10% fetal bovine serum, 1% double antibody, 1 mu M/L dexamethasone, 0.5mM/L IBMX, 200 mu M/L indomethacin, and 10 mu M/mL insulin. The basic components in the adult adipose-derived mesenchymal stem cell adipogenic induction differentiation medium (HUXMD-90031) comprise: the fat mesenchymal stem cell liposome is prepared from a basic culture medium, fetal bovine serum, glutamine, double antibodies, insulin, IBMX, rosiglitazone and dexamethasone and can be used for adipogenic induction and differentiation of the fat mesenchymal stem cell. Tumor stem cells with the multidirectional differentiation potential exist in normal tumor tissues, and under an in-vitro culture environment, common tumor cells are treated by high-concentration cobalt chloride (450nM) to obtain cells with high invasion and metastasis capacities, wherein the cells are the tumor stem cells with the multidirectional differentiation potential and have the potential of being induced to differentiate into benign cells. At present, the research work of inducing differentiation of solid tumor is still in the application stage, and immature malignant cells can be reversed and differentiated towards normal cells by applying certain chemical substances.

Dexamethasone (Dexamethasone synnyms: BRL 49653) is a glucocorticoid and is mainly used for resisting inflammation, toxicity, allergy and rheumatism clinically and inhibiting the uptake and utilization of glucose by peripheral tissues. The high-concentration glucocorticoid inhibits the combination of insulin and a receptor thereof and damages a glucose transport system of the insulin receptor of peripheral tissues, so that insulin resistance is caused, and the concentration of dexamethasone is different and has a relation with the differentiation rate and the differentiation time of induced differentiation. Dexamethasone has been reported in the literature to reverse cAMP-induced glial cell differentiation by inhibiting CREB phosphorylation. In addition, the blood sugar-reducing medicine rosiglitazone combined with MEK inhibitor tramitinib can differentiate breast cancer cells into fat cells, reduce the invasiveness of tumors and inhibit tumor metastasis.

Rosiglitazone, belonging to the family of thiazolidinediones insulin sensitizers, is a ligand for the gamma receptor (PPAR γ) of specific peroxisome proliferator activator. Activation of PPAR γ is a key step in the fat differentiation process.

3-isobutyl-1-methylxanthine (IBMX), a broad-spectrum phosphodiesterase inhibitor, inhibits degradation of cyclic adenosine phosphates by phosphodiesterases, and promotes lipolysis by accumulation of cyclic adenosine phosphates in cells, and IBMX enhances insulin release and increases glycogenolysis and lipolysis in hepatocytes and adipocytes.

Dimethyl sulfoxide (DMSO) is used as reaction solvent for synthesis of medicine and intermediate, medicine solvent and medicine carrier, and can increase chemical reaction speed and reactant yield.

Disclosure of Invention

The invention provides a method for inhibiting the growth and the metastasis of solid tumor cells and a special pharmaceutical composition (DRI) for solving the problem of inducing and differentiating malignant tumor cells into normal cells.

The invention is realized according to the following technical scheme.

A pharmaceutical composition for inhibiting the growth and metastasis of solid tumor cells is prepared by combining the following raw material medicines in dimethyl sulfoxide:

dexamethasone, 0.0001-0.001 μ g/ml

Rosiglitazone, 0.001-0.005. mu.g/ml

3-isobutyl-1-methylxanthine 0.08-0.15 μ g/ml.

The pharmaceutical composition for inhibiting the growth and the metastasis of solid tumor cells is prepared by combining the following raw material medicines in dimethyl sulfoxide according to the concentration:

dexamethasone 0.0003-0.0008 μ g/ml

0.002-0.004 mu g/ml rosiglitazone

3-isobutyl-1-methylxanthine 0.1-0.13. mu.g/ml.

dexamethasone 0.00039 μ g/ml

Rosiglitazone 0.00357. mu.g/ml

3-isobutyl-1-methylxanthine 0.11. mu.g/ml.

A method for inhibiting growth and metastasis of solid tumor cells, comprising the steps of:

[ when HEY was induced in the ovarian cancer cell line, 5mL of RPMI-1640 medium was used in the presence of 535.5. mu.L of CoCl₂Replacing RPMI-1640 culture medium after 48h, and repeatedly treating for 2 times when the fusion degree of the cells is 80% -90% after the cells recover for 10-14 days to obtain a sufficient number of cells with high invasion and transfer capacity;

② the obtained HEY cells with high invasive and metastatic capacities do not contain CoCl₂When the fusion degree reaches 60%, adding a pharmaceutical composition containing dexamethasone 0.0001-0.001 μ g/ml, rosiglitazone 0.001-0.005 μ g/ml and 3-isobutyl-1-methylxanthine 0.08-0.15 μ g/ml into the removed culture medium, inducing in vitro, and obtaining the lipodifferentiated tumor cells when obvious lipid drops are formed in the tumor cell plasma.

The method for inhibiting the growth and the metastasis of the solid tumor cells comprises the following steps:

[ when MDA-MB-231 cells, which are breast cancer cell lines, were induced, 5mL of DMEM medium plus 535.5. mu.L of CoCl was used₂(ii) a After 33h of action, the DMEM medium is replaced;

② the obtained adenocarcinoma MDA-MB-2311 cell with high invasion and metastasis ability is treated by CoCl-free₂The DMEM medium of (1).

The invention designed in this way induces tumor cells to differentiate into fat cells in vitro by treating the cells of the ovarian cancer cell line HEY or the cells of the breast cancer cell line MDA-MB-231 with cobalt chloride in an in vitro culture environment and in a drug composition environment of small molecular compounds, and is verified by in vivo experiments of animals. Can be used for in vitro and in vivo induced differentiation treatment of malignant tumor cells, and inhibiting growth and metastasis of highly malignant solid tumor. The small molecular compound has the prospect of being used for clinical treatment of human solid tumors, and the medicinal composition of the small molecular compound has small toxic or side effect on human bodies and cannot cause tolerance of organisms after treatment.

Drawings

FIG. 1 is a cell morphology before and after induction of differentiation (. times.200) in HEY cell adipose differentiation medium;

FIG. 2 is a diagram of cell morphology before and after induction of differentiation in MDA-MB-231 cell adipocyte differentiation medium;

FIG. 3 is a Western blot to detect expression in HEY cells and MDA-MB-231 cells;

FIG. 4 is a graph showing the gross morphology and growth of transplanted tumor tissue in4 groups of nude mice in HEY cell line;

FIG. 5 is a model of nude mouse transplantation tumor of 4 groups of cells in HEY cell line;

FIG. 6 is a graph of tumor cell morphology (x 400) in H & E stained mouse graft tumor tissue;

FIG. 7 shows the detection of FABP4 and vimentin expression in mouse transplanted tumor tissue by immunohistochemical staining (X400);

FIG. 8 shows the Western blot assay for detecting expression of proteins of interest in tissues of animal transplantable tumors in the HEY cell line;

FIG. 9 is a gross morphology of tumor tissue in the DRI-treated group of the HEY mouse graft tumor model;

FIG. 10 is a gross morphology of tumor tissue in the DRI-treated group of the HEY mouse graft tumor model;

FIG. 11 is a gross morphology of tumor tissues of HEY mouse graft tumor model DMSO control group;

FIG. 12 is a gross morphology of tumor tissue from HEY mouse graft tumor model DMSO control group;

FIG. 13 is a gross morphology of tumor tissues from a blank group of the HEY mouse graft tumor model;

FIG. 14 is a gross morphology of tumor tissues from a blank group of the HEY mouse graft tumor model;

FIG. 15 is CoCl₂The gross morphology of tumor tissues of the treated cell mouse transplanted tumor model DRI treatment group;

FIG. 16 is CoCl₂The gross morphology of tumor tissues of the treated cell mouse transplanted tumor model DRI treatment group;

FIG. 17 is CoCl₂The treated cell mouse transplanted tumor model DMSO control group tumor tissue gross morphogram;

FIG. 18 is CoCl₂The treated cell mouse transplanted tumor model DMSO control group tumor tissue gross morphogram;

FIG. 19 is CoCl₂The treated cell mouse transplanted tumor model blank tumor tissue gross morphogram;

FIG. 20 is CoCl₂The treated cell mouse transplanted tumor model blank tumor tissue gross morphogram;

FIG. 21 is a graph of growth of three groups of tumor tissues in a HEY mouse graft tumor model;

FIG. 22 is CoCl₂Three groups of tumor tissue growth curves in the treated cell mouse transplantation tumor model.

Detailed description of the invention

The present invention will be described in detail below with reference to the accompanying drawings and examples.

② the obtained HEY cells with high invasive and metastatic capacities do not contain CoCl₂Culturing in RPMI-1640 mediumWhen the fusion degree reaches 60%, adding a pharmaceutical composition containing dexamethasone 0.0001-0.001 μ g/ml, rosiglitazone 0.001-0.005 μ g/ml and 3-isobutyl-1-methylxanthine 0.08-0.15 μ g/ml into the removed culture medium, and inducing in vitro to obtain fat-differentiated tumor cells when obvious lipid droplets are formed in the tumor cell plasma.

② the obtained MDA-MB-2311 cell with high invasion and metastasis ability for breast cancer, and then CoCl-free cells₂The DMEM medium of (1).

dexamethasone, 0.0001-0.001 μ g/ml

Rosiglitazone, 0.001-0.005. mu.g/ml

3-isobutyl-1-methylxanthine 0.08-0.15 μ g/ml.

dexamethasone 0.0003-0.0008 μ g/ml

0.002-0.004 mu g/ml rosiglitazone

3-isobutyl-1-methylxanthine 0.1-0.13. mu.g/ml.

dexamethasone 0.00039 μ g/ml

Rosiglitazone 0.00357. mu.g/ml

3-isobutyl-1-methylxanthine 0.11. mu.g/ml.

Example 1:

firstly, malignant tumor cells are an ovarian cancer HEY cell line and a breast cancer MDA-MB-231 cell line; the method for inducing HEY cells and MDA-MB-231 cells to differentiate fat in vitro comprises the following steps:

respectively using high-concentration CoCl₂Two cell lines were induced to obtain cells with high invasive metastatic capacity: wherein the ovarian cancer cell line HEY is prepared by adding 535.5 μ L CoCl into 5mL of RPMI-1640 culture medium₂Replacing RPMI-1640 culture medium after 48 hours of action; mammary cancer cell line MDA-MB-231 was cultured in DMEM medium at 5mL + 535.5. mu.L CoCl₂After 33 hours of action, the DMEM medium is replaced; observing cell state, replacing fresh culture medium, recovering cells for 10-14 days, and allowing CoCl to pass₂When the treated cells recover 80% -90% of fusion degree, the CoCl with the same concentration is reused₂Treating for the same time with high concentration CoCl₂After 2 repeated treatments, a sufficient number of cells were obtained.

② control group cells after in vitro induced differentiation and CoCl₂Cells after induction treatment, using CoCl-free cells₂When the RPMI-1640 medium was cultured until the confluency reached 60%, the culture solution was removed. Adding medicinal composition containing dexamethasone 0.0001-0.001 μ g/ml, rosiglitazone 0.001-0.005 μ g/ml, and 3-isobutyl-1-methylxanthine (IBMX) 0.08-0.15 μ g/ml into culture medium in dimethyl sulfoxide, and inducing control cells and CoCl in vitro₂After the induction treatment, the tumor cell plasm is observed under a microscope to have obvious lipid drop formation, and the cell state is continuously maintained by using the cell containing 10% FBS and 1 mug/mL insulin, so that the tumor cell with fat differentiation can be obtained.

The pharmaceutical composition containing dexamethasone 0.0003-0.0008 μ g/ml, rosiglitazone 0.002-0.004 μ g/ml and IBMX0.1-0.13 μ g/ml in dimethyl sulfoxide is added into culture medium to induce fat differentiation of HEY cell line and MDA-MB-231 cell line in vitro.

In HEY cells, a pharmaceutical composition containing dexamethasone 0.00039 μ g/mL, rosiglitazone 0.00357 μ g/mL and IBMX 0.11 μ g/mL in dimethyl sulfoxide was added to a culture medium and maintained for 48 hours, then 10% FBS and 1 μ g/mL insulin were replaced and maintained for 24 hours, the cycle was repeated 1 time, and 10% FBS and 1 μ g/mL insulin were replaced and maintained until 168 hours from the initiation of induction, and adipocytes with good differentiated and matured states were obtained.

FIG. 1 shows the detection of HEY cell lineage differentiation induced in vitro into adipocytes by oil red staining, and the induction of HEY cells before and after cobalt chloride treatment using a fat-induced differentiation medium, the oil red staining showing that the cobalt chloride-treated cells and their progeny differentiate into mature adipocytes containing large lipid droplets.

FIG. 1 is a cell morphology before and after induction of differentiation (. times.200) in the HEY cell adipose differentiation medium. Wherein a is subjected to CoCl₂Oil red staining of the treated HEY cells; b. by CoCl₂Oil red staining after fat differentiation of the treated HEY cells; c. not subjected to CoCl₂Treating HEY cell oil red staining; d. not subjected to CoCl₂Oil red staining after fat differentiation of HEY cells after treatment.

For MDA-MB-231 cells, a pharmaceutical composition containing dexamethasone 0.00039 mu g/mL, rosiglitazone 0.00357 mu g/mL and IBMX 0.11 mu g/mL in dimethyl sulfoxide is added into a culture medium for 24 hours (33 hours), then the culture medium is maintained for 24 hours by replacing 10% FBS and 1 mu g/mL insulin, the circulation is repeated for 2 times, and the culture medium is maintained for 168 hours from the beginning of induction by replacing 10% FBS and 1 mu g/mL insulin, so that the tumor cells with fat differentiation can be obtained. CoCl-free Induction Using the same protocol₂Treated HEY cells and MDA-MB-231 cells served as the corresponding control groups.

FIG. 2 shows that in vitro induction differentiation of MDA-MB-231 cell line into adipocytes is detected by oil red staining, MDA-MB-231 cells before and after cobalt chloride treatment are induced by using an adipose-induced differentiation medium, and the oil red staining shows that the cells with high invasion and transfer capacity after cobalt chloride treatment are mature adipocytes containing large granular lipid droplets.

FIG. 2 is a diagram showing the morphology of cells before and after induction of differentiation (. times.200) in MDA-MB-231 cell adipocyte differentiation medium. Wherein a is subjected to CoCl₂MDA-MB-231 cells after treatment are stained with oil red; b. by CoCl₂Oil red staining after fat differentiation of the treated MDA-MB-231 cells; c. not subjected to CoCl₂Treatment of MDA-MB-231 cellsDyeing with oil red; d. not subjected to CoCl₂And performing Western blotting on MDA-MB-231 cells after treatment to detect the expression changes of CREB, P-CREB, CEBP beta, CEBP alpha and PPAR gamma before and after inducing differentiation in HEY cells and MDA-MB-231 cells. C-C: not subjected to CoCl₂Treated tumor cells; C-A: not subjected to CoCl₂Induction of adipose differentiation of the treated tumor cells; P-C: CoCl₂Treating the cells; P-A: CoCl₂After treatment, the cell fat differentiation is induced.

Extracting cell protein to be used for detecting the expression condition of fat differentiation related protein by a protein immunoblotting experiment,

FIG. 3 is a Western blot to detect expression in HEY cells and MDA-MB-231 cells; western blotting was performed to detect the expression changes of CREB, P-CREB, CEBP beta, CEBP alpha and PPAR gamma before and after inducing differentiation in HEY cells and MDA-MB-231 cells. C-C: not subjected to CoCl₂Treated tumor cells; C-A: not subjected to CoCl₂Induction of adipose differentiation of the treated tumor cells; P-C: CoCl₂Treating the cells; P-A: CoCl₂After treatment, the cell fat differentiation is induced.

The results are shown in FIG. 3. After the cells treated by the cobalt chloride are induced by in vitro fat differentiation, the expression of PPARy is induced by the transcription factor CREB, and the expression of the transcription factors C/EBP beta and C/EBP alpha and the adipocyte marker gene PPAR gamma prompt the cells to differentiate to mature fat cells.

Example 2:

construction of nude mouse transplantation tumor model

The control group cells which are induced to differentiate in vitro and CoCl₂The cells after induction treatment are injected subcutaneously into the left rat groin of a 6-8-week old Balb/c immunodeficiency female mouse to construct a mouse transplantation tumor model. Mouse transplantation tumor experiments prove that adipocytes obtained by in vitro induction of an ovarian cancer HEY cell line and a breast cancer MDA-MB-231 cell line have a normal adipocyte specific molecular label and stably exist in a mouse transplantation tumor model.

When constructing a nude mouse transplantation tumor model, 20 BALB/cNU/NU nude mice (Beijing Wittingli laboratory animal technology Co., Ltd.) were all 6-8 weeks old and female, and in SThe breeding room of the PF-level nude mice is adapted to about 1 week, 5 mice/cage, and 6 cages in total, the mouse cage is replaced for 1 time/week in the breeding period, and the sterilization of the feed, padding, water source and the mouse cage is noticed; preparation of plain HEY tumor cells, CoCl, without any treatment₂Treated cells, HEY in vitro-induced adipocyte differentiation cells obtained in example 1, and CoCl₂Four groups of cells are treated and subjected to in vitro induction of fat differentiated cells. Culturing sufficient amount of cells in T75 culture flask, washing with buffer solution, performing trypsinization, washing with cold buffer solution once, preparing into tumor cell suspension, and inoculating 100 μ L of tumor cell suspension (cell amount of about 10) into the skin of the left groin of each nude mouse⁶) Each group was inoculated with 5. Normally feeding, observing the tumor formation condition of the nude mice every day, measuring the size of the nude mice subcutaneous tumor (measuring longest diameter x shortest diameter) every other day when the nude mice subcutaneous tumor formation is carried out, marking the number of the left and right ear cuts of the nude mice in each cage, and preventing repeated measurement. The same neck was sacrificed at day 30 of the construction of the transplanted tumor model (3 groups of tumor-bearing mice to be compared in two tumor cell lines were guaranteed to be sacrificed at the same time); immediately after the mice are sacrificed, tumor tissues are taken down to measure the size and take pictures, the liver, the lung and part of the tumor tissues of the mice are taken and fixed by 10 percent neutral formalin for 24 hours, and the mice are processed by material taking, dehydration, wax dipping, embedding, slicing and the like and then processed by subsequent H&E staining to observe the tumor cell morphology and the presence or absence of metastasis.

FIG. 6 is H&E staining of tumor cell morphology in mouse transplanted tumor tissue (H)&E, x 400) at a.H&E staining for CoCl for in vitro fat-induced differentiation₂After the treatment, the tumor cell morphology is that a large amount of fat vacuoles can be seen in the cytoplasm of the tumor cell cultured by fat-induced differentiation. B.H&E staining for CoCl that did not undergo fat-induced differentiation₂Tumor cell morphology after treatment.

The results are shown in fig. 6, which shows that the morphology of tumor cells in transplanted tumor tissues is observed by H & E staining, and in two groups of cell transplanted tumor tissues, the tumor tissues of untreated groups are compact and have a large number of necrotic foci; there are a large number of lipid droplet vacuoles in the cytoplasm of the tumor tissue cells of the induced differentiation group.

ImmunohistologyChemical staining method for detecting FABP4 and its related protein expression, and FIG. 7 is a graph (x 400) of immunohistochemical staining for detecting FABP4 and vimentin expression in mouse transplanted tumor tissue, wherein A. CoCl subjected to in vitro induced fat differentiation₂Expression of FABP4 in tumor cells after treatment. B. CoCl without in vitro induced lipodifferentiation₂Expression of FABP44 in tumor cells after treatment. C. CoCl for inducing adipose differentiation in vitro₂Expression of vimentin in tumor cells after treatment. D. CoCl without in vitro induced lipodifferentiation₂Expression of vimentin4 in tumor cells after treatment.

FIG. 7 shows the observation of the expression of the adipocyte marker protein FABP4 and human vimentin antibody for immunohistochemical staining, the results are shown in FIG. 7; in the induced differentiation group of the two groups of cells, FABP4 and vimentin are remarkably expressed in the cytoplasm of tumor tissues, which indicates that the fat cells in the transplanted tumor tissues in the treatment group are fat cells which are successfully induced and differentiated in vitro and are stable and irreversible in mice. The tumor tissues of the untreated groups are compact, FABP4 is weakly positive in expression, and vimentin is not expressed. The HEY ovarian cancer cell line is proved to be induced to differentiate into irreversible fat cells in vitro and stably exist in a nude mouse body.

In addition, a part of fresh tumor tissues are retained and immediately stored in liquid nitrogen, and tissue protein is extracted and used for detecting protein expression conditions in a protein immunoblotting experiment.

Fig. 8 is a western blot assay to detect relevant protein expression in animal transplanted tumor tissue in HEY cell lines, where HPC groups: CoCl without in vitro induced lipodifferentiation₂Expressing the tumor tissue protein after treatment; HPA group: CoCl for inducing adipose differentiation in vitro₂Tumor tissue expression after treatment.

The results are shown in fig. 8, which shows that in order to detect the expression of the protein related to tissue protein fat differentiation by extracting tissue protein western blot experiment, after the cells treated by cobalt chloride are induced by in vitro fat differentiation, the transcription factors CREB induce the expression of PPARy, and the transcription factors C/EBP β, C/EBP α and the adipocyte marker gene PPAR γ cooperatively maintain the cells to be differentiated into mature adipocytes. The results show that in the two groups of cell transplanted tumor tissues, the fat differentiation related protein expression of the in vitro induction group and the fat differentiation related protein expression of the control group are obviously different.

Calculating the volume of the tumor according to the maximum diameter and the shortest diameter of the tumor measurement, wherein the volume formula is as follows: major diameter x minor diameter²And/2, drawing the tumor growth curves of 3 groups of mice to be compared in two tumor cell lines, and performing statistical analysis. All experimental data in this study were statistically processed using statistical analysis software SPSS 17.0(IBM corporation), the t-test of independent samples was used to compare the difference between the two groups of nude mice subcutaneous inoculation tumorigenic growth volumes, and the P value < 0.05 was considered statistically different. See tables 1 and 2,

TABLE 1 absence of CoCl₂Statistical analysis results of tumor tissue volumes of fat-induced group and control group in treated HEY cell mouse transplantation tumor model

Table 1 shows that in the HEY mouse graft tumor model, the tumor tissue volume of the induction group and the control group was statistically analyzed, and the t-test of the independent samples showed that the two groups of variances were aligned, so that the "induction group" and the "control group" P were 0.045 less than 0.05, and the difference in tumor volume size was statistically significant. The results show that the tumorigenic capacity of the cells subjected to in vitro induced adipose differentiation is obviously reduced compared with that of the cells not subjected to adipose induced differentiation.

TABLE 2 passage through CoCl₂Statistical analysis of tumor tissue volumes in the adipose-induced and control groups in the treated mouse transplanted tumor model

Table 2 shows that the tumor tissue volume of the induction group and the control group in the HEY high-invasion metastatic capacity mouse transplantation tumor model is statistically analyzed, and the t test of independent samples shows that the two groups of variances are different, so that the P of the induction group and the P of the control group are 0.042 and 0.05, and the tumor volume size difference is systematically differentThe meaning of the study. Indicating CoCl that undergoes in vitro induced lipodifferentiation₂The cell tumorigenicity ability after the induction treatment is obviously reduced compared with that of the ordinary differentiated cell without fat induction.

Fig. 4 and 5 show the volume of the transplanted tumor, the gross morphology of the tumor tissue and the growth curve. The results show that the volume of the transplanted tumor formed by inoculating the tumor cells subjected to fat-induced differentiation into the nude mice is obviously smaller than that of the transplanted tumor formed by the tumor cells not subjected to fat-induced differentiation in the nude mice, and the compound can induce the differentiation of the tumor cells and inhibit the growth of the tumor cells.

FIG. 4 is a graph showing the gross morphology and growth of transplanted tumor tissue in4 groups of nude mice in HEY cell line, wherein A. the transplanted tumor tissue is treated with CoCl ₂2 groups of cells in the treated cells were seeded into the gross morphology of the tumorigenic tissue. The Control group is cells which are not subjected to fat-induced differentiation, the Treatment group is cells which are subjected to fat-induced differentiation in vitro, and each group comprises 5 nude mice. B. In vitro induction of CoCl before and after lipodifferentiation₂The cell nodulation growth curve was processed. C. CoCl-free in vitro before and after induction of adipose differentiation₂The treated cell animal transplants the gross morphology of the tumor tissue. The Control group is HEY cells without fat differentiation, the Treatment group is HEY cells subjected to in vitro induced fat differentiation, and each group comprises 5 nude mice. D. CoCl-free in vitro induction of adipose before and after differentiation₂The cell nodulation growth curve was processed.

FIG. 5 is a model of nude mouse transplantation tumor of 4 groups of cells in HEY cell line; cocl in the figure₂A cell nude mouse transplantation tumor model after being treated and subjected to in vitro induced fat differentiation; CoCl₂The treated nude mouse has no in vitro fat induced differentiated cell transplantation tumor model; C. not subjected to CoCl₂The treated in vitro induced adipose differentiation HEY cell nude mouse transplantation tumor model; D. not subjected to CoCl₂Treating and in vitro fat-induced differentiation cell nude mouse transplantation tumor model.

Example 3:

final concentration and therapeutic dose of pharmaceutical composition (DRI) for animal experiments:

(1) on the 21 st day of the tumor forming experiment of each group of nude mice, the tumor volume of each group exceeds 100mm³Then, HEY cells were made into tumor groups, CoCl₂The treated cell groups were randomly divided into a drug composition (DRI) treatment group, a DMSO control group, and a blank control group, respectively.

(2) Exploration of different drug treatment doses in DRI treatment group: according to LD50, IC50, EC50 of the three small molecule compounds, the three drug therapeutic doses are preferably: dexamethasone (10mM × 1mL in DMSO) therapeutic dose: 1.77-4.17 mug/25 g mouse weight; therapeutic dose of rosiglitazone (10mM × 1mL in DMSO): 400-625 mu g/25g mouse weight; therapeutic doses of 3-isobutyl-1-methylxanthine (10mM X1 mL in DMSO): 180.57-275. mu.g/25 g mouse body weight.

The three drugs with different treatment doses are selected to be combined to obtain 18 treatment groups, and an in vivo induced differentiation experiment is carried out on the HEY tumor cell group, the experimental steps are as described in the following (4), after the in vivo induction is finished, the volume of the transplanted tumor of the treatment groups is measured, and the result is shown in the table 5.

TABLE 5 therapeutic dose screening results for small molecule compounds

Table 5 shows the sizes of the tissues of the treated groups transplanted with tumor after the induction of the HEY tumor cell groups in vivo induced differentiation experiments of the pharmaceutical compositions with different therapeutic doses.

(3) As shown by the results in table 5,

most preferably, the dosages of each component are selected as follows: dexamethasone (10mM × 1mL in DMSO) at therapeutic doses: 3.25 mu g/25g mouse weight, molecular weight 392.46, 0.828 mu L to obtain therapeutic dose concentration of 3.25 mu g; rosiglitazone (10mM × 1mL in DMSO) at therapeutic doses: 625 mug of the composition per 25g of mouse body weight, 357.43 molecular weight, 174.859 mug of the composition, and 625 mug of the composition in therapeutic dose. IBMX (10 mM. times.1 mL in DMSO) at therapeutic doses: 212.5 μ g/25g mouse weight, molecular weight 222.24, 95.617 μ L was taken to obtain a therapeutic dose concentration of 212.5 μ g.

(4) In vivo induced differentiation experiment: the method comprises injecting the composition around tumor tissue subcutaneously according to the concentration of the purchased medicine under the condition of ensuring the therapeutic dose concentration of the medicineThe dose, the molecular mass of the drug, the mass of the needed drug and the mass of a nude mouse are uniformly calculated as 25g, 271 mu L of the most preferable therapeutic dose of the drug composition is configured at room temperature, a control group is treated by DMSO with a certain volume, injection is completed at three positions around a tumor tissue, each point is injected with 90.4 mu L, injection is performed once every other day, three injections are completed, observation is performed for 5 days, and the same neck is killed at the same time at the 30 th day of constructing a transplantation tumor model (3 groups of tumor-bearing mice to be compared in two groups of tumor cell lines are guaranteed to be killed at the same time); immediately after the mice are sacrificed, tumor tissues are taken down to measure the size and take pictures, the liver, the lung and part of the tumor tissues of the mice are taken and fixed by 10 percent neutral formalin for 24 hours, and the mice are processed by material taking, dehydration, wax dipping, embedding, slicing and the like and then processed by subsequent H&E staining for observing the morphology of tumor cells and the existence of metastasis, and detecting the expression conditions of FABP4 and related proteins by an immunohistochemical staining method. In addition, a part of fresh tumor tissues are reserved and immediately placed in liquid nitrogen for preservation, and tissue protein is extracted for detecting protein expression conditions in a subsequent protein immunoblotting experiment; calculating the volume of the tumor according to the maximum diameter and the shortest diameter of the tumor measurement, wherein the volume formula is as follows: major diameter x minor diameter²And/2, drawing the tumor growth curves of 3 groups of mice to be compared in two tumor cell lines, and performing statistical analysis. All experimental data in this study were statistically processed using statistical analysis software SPSS 17.0(IBM corporation), and the analysis of variance of ANVOA was used to compare the difference between the two groups of subcutaneous inoculation tumor growth volumes of nude mice, and the P value < 0.05 was considered as having statistical differences. Tables 3 and 4,

TABLE 3 statistical analysis of tumor tissue volume in three groups, treatment, control and blank, in the HEY mouse transplantation tumor model

Table 3 shows the results of statistical analysis of tumor tissue volumes in the treatment, control and blank groups in the HEY cell line mouse transplantation tumor model for ovarian cancer. The homogeneous variance test indicates that the variances are uniform, the blank group and the control group have a P value of 0.288 which is more than 0.05 through pairwise comparison among the blank group, the control group and the DRI treatment group, and the difference of the tumor volumes has no statistical significance; the "blank group" and the "DRI treatment group" P ═ 0.031 < 0.05, and the difference in tumor volume size had statistical significance; the 'control group' and the 'DRI treatment group' P is 0.476 & gt 0.05, and the difference of the tumor volumes has no statistical significance.

TABLE 4 passage through CoCl₂In the treated cell mouse transplantation tumor model, the tumor tissue volume statistical analysis results of three groups of a treatment group, a control group and a blank group

Table 4 shows the statistical analysis results of the tumor tissue volumes of the treated group, the control group and the blank group in the ovarian cancer HEY cell line cobalt chloride-induced high invasive metastatic capacity mouse transplantation tumor model. The homogeneous variance test indicates that the variances are uniform, and the P of a blank group and the P of a contrast group are 0.51 & gt 0.05 through pairwise comparison among the blank group, the contrast group and the DRI treatment group, so that the difference of the tumor volumes has no statistical significance; the 'blank group' and the 'DRI treatment group' P < 0.001 < 0.05, the difference in tumor volume size has statistical significance; the "control group" and the "DRI-treated group" P ═ 0.062>0.05, and the tumor volume size differences were not statistically significant.

FIG. 9 is a general morphology of tumor tissue of a DRI-treated group of a HEY mouse graft tumor model, and FIG. 10 is a general morphology of tumor tissue of a DRI-treated group of a HEY mouse graft tumor model. Shows the HEY cell line cultured in vitro, injected subcutaneously into the left groin of 5 Balb/c immunodeficient female mice aged 6-8 weeks, and the number of injected cells is 10⁶One week later, a mouse graft tumor model was successfully constructed and treated with a peritumoral injection of pharmaceutical composition (DRI) to tumor tissue volume size per 100 μ L.

Fig. 11 is a gross morphology of tumor tissues of HEY mouse transplanted tumor model DMSO control group, and fig. 12 is a gross morphology of tumor tissues of HEY mouse transplanted tumor model DMSO control group; indicated as HEY cell line cultured in vitro, injected subcutaneously into the left groin of 5 Balb/c immunodeficient female mice aged 6-8 weeks, injected finelyThe number of cells is 10⁶One week later, a mouse graft tumor model was successfully constructed, and tumor tissue volume size was equal after the same time treatment with the same volume of DMSO.

FIG. 13 is a gross morphology of tumor tissues of a blank group of a HEY mouse graft tumor model, and FIG. 14 is a gross morphology of tumor tissues of a blank group of a HEY mouse graft tumor model; shows the HEY cell line cultured in vitro, injected subcutaneously into the left groin of 5 Balb/c immunodeficient female mice aged 6-8 weeks, and the number of injected cells is 10⁶And (4) successfully constructing a mouse transplantation tumor model by using 100 mu L of the tumor cells, wherein the volume of the tumor tissue of the blank control group is large.

FIG. 15 is CoCl₂Gross morphology of tumor tissue in mice transplanted with treated cells model DRI treated groups, FIG. 16 is CoCl₂The gross morphology of tumor tissues of the treated cell mouse transplanted tumor model DRI treatment group; the cells after cobalt chloride treatment, which were induced in vitro, were injected subcutaneously into the left groin of 5 6-8 weeks old Balb/c immunodeficient female mice, and the number of injected cells was 10⁶One week later, a mouse graft tumor model was successfully constructed, and tumor tissue volume size was post-treatment with a first pharmaceutical composition (DRI) injection peritumorally.

FIG. 17 is CoCl₂After treatment, the tumor model of the cell mouse transplanted tumor is DMSO control tumor tissue gross morphology, and FIG. 18 is CoCl₂The treated cell mouse transplanted tumor model DMSO control group tumor tissue gross morphogram; it is shown that the cells after cobalt chloride treatment induced in vitro were injected subcutaneously into the left rat groin of 5 6-8 weeks old Balb/c immunodeficient female mice, and the number of injected cells was 10⁶One week later, a mouse graft tumor model was successfully constructed, and tumor tissue volume size was equal after the same time treatment with the same volume of DMSO.

FIG. 19 is CoCl₂Gross morphology of blank tumor tissue in post-treatment cellular mouse graft tumor model, FIG. 20 is CoCl₂The treated cell mouse transplanted tumor model blank tumor tissue gross morphogram; it is shown that the cells after cobalt chloride treatment induced in vitro were injected subcutaneously into the left rat groin of 5 6-8 weeks old Balb/c immunodeficient female mice, and the number of injected cells was 10⁶One week later, mice were successfully constructed at a dose of 100. mu.LTumor model was transplanted, and tumor volume size was blank control group.

Fig. 21 is a graph showing the growth curves of three groups of tumor tissues in HEY mouse graft tumor model, which are the tumor tissue growth curves of three groups of mice in the treatment group, the control group and the blank group in the HEY cell line mouse graft tumor model for ovarian cancer.

FIG. 22 is CoCl₂The growth curve graphs of three groups of mouse tumor tissues in the treated cell mouse transplanted tumor model are shown as the growth curves of the tumor tissues of three groups of a treated group, a control group and a blank group in the cell mouse transplanted tumor model induced by the HEY cell line of ovarian cancer through cobalt chloride.

Fig. 9-22 show the volume of transplanted tumors, gross tumor morphology and growth curves in the treated, control and blank groups, most preferably at the drug dose combination. The result shows that the small molecular compound combination can obviously inhibit the growth of tumor cells and induce the adipose differentiation of the tumor cells. In addition, the small molecular compound can also obviously inhibit the growth of the tumor cells with high metastatic capacity and induce the adipose differentiation of the tumor cells with high metastatic capacity.

Claims

1. The application of dexamethasone, rosiglitazone and 3-isobutyl-1-methylxanthine composition in preparing the medicine for inhibiting the growth and the metastasis of ovarian cancer cells induced by cobalt chloride is characterized in that: the composition of dexamethasone, rosiglitazone and 3-isobutyl-1-methylxanthine is prepared by combining the following raw material medicines in dimethyl sulfoxide:

dexamethasone 0.0001-0.001 μ g/ml

0.001-0.005 mu g/ml rosiglitazone

3-isobutyl-1-methylxanthine 0.08-0.15 μ g/ml.

2. Use of dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine composition according to claim 1 in the manufacture of a medicament for inhibiting cobalt chloride-induced growth metastasis of ovarian cancer cells, wherein: the composition of dexamethasone, rosiglitazone and 3-isobutyl-1-methylxanthine is prepared by combining the following raw material medicines in dimethyl sulfoxide:

dexamethasone 0.0003-0.0008 μ g/ml

0.002-0.004 mu g/ml rosiglitazone

3-isobutyl-1-methylxanthine 0.1-0.13. mu.g/ml.

3. Use of dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine composition according to claim 2 in the manufacture of a medicament for inhibiting cobalt chloride-induced growth metastasis of ovarian cancer cells, wherein: the composition of dexamethasone, rosiglitazone and 3-isobutyl-1-methylxanthine is prepared by combining the following raw material medicines in dimethyl sulfoxide:

dexamethasone 0.00039 μ g/ml

Rosiglitazone 0.00357. mu.g/ml

3-isobutyl-1-methylxanthine 0.11. mu.g/ml.