Pharmaceutical composition for treating osteosarcoma and application thereof

Abstract

The invention discloses a pharmaceutical composition for treating osteosarcoma and application of the pharmaceutical composition, and particularly belongs to the field of biological medicine. The pharmaceutical composition comprises cis-platinum and daidzin, the daidzin provided by the invention is combined with beta-catenin protein, so that the protein stability of the beta-catenin protein is reduced, and a Wnt signal channel is further inhibited, so that the activity of osteosarcoma cells is inhibited, cell apoptosis is induced, and pulmonary metastasis is inhibited; the pharmaceutical composition of cis-platinum and daidzin provided by the invention has a synergistic effect, the sensitivity of cells to cis-platinum is remarkably improved, and the daidzin and cis-platinum combined medication has a remarkable inhibition effect on the activity and migration invasion ability of osteosarcoma cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a pharmaceutical composition for treating osteosarcoma and application thereof. Background Art

[0002] Osteosarcoma (OS) is a malignant bone tumor that develops from the stromal cell line and is common in adolescents and children. It can occur in any bone in the body, but the most common site is the end of the long bones, such as the knee, femur or tibia. At present, the treatment strategies for osteosarcoma mainly include surgical resection, chemotherapy and radiotherapy. Due to the continuous innovation of medical technology, the 5-year survival rate of osteosarcoma is about 60%-70%. However, in clinical practice, a considerable number of patients still have obvious chemotherapy resistance, and it is difficult to achieve satisfactory treatment effects. Therefore, improving the effect of chemotherapy is crucial to improving the prognosis of osteosarcoma.

[0003] Daidzin is a natural plant compound mainly found in soybeans, which has multiple biological activities, including anti-inflammatory and protective effects on the bone, cardiovascular and nervous systems. Current studies have shown that daidzin has certain anti-tumor effects. For example, daidzin has been reported to inhibit the growth of cervical cancer through the JAK2 / STAT3 pathway. In addition, daidzin affects MnSOD and EMT processes in prostate cancer and colorectal cancer by regulating the PI3K / Akt signaling pathway. However, whether daidzin has the potential to treat OS remains unknown.

[0004] Cisplatin (DDP) is a commonly used chemotherapy drug that inhibits the viability and division of tumor cells by interfering with DNA synthesis and its repair. At the same time, cisplatin is a classic recommendation for osteosarcoma strategies from the 2021 NCCN Guidelines (2021.v1). However, cisplatin resistance occasionally occurs in the clinical treatment of osteosarcoma, which limits the efficiency of chemotherapy and thus reduces survival.

[0005] Therefore, exploring appropriate methods to improve the sensitivity of osteosarcoma patients to chemotherapy drugs will enable osteosarcoma patients to achieve better prognosis. Summary of the invention

[0006] Aiming at the deficiencies in existing technologies;

[0007] The purpose of the present invention is to provide a pharmaceutical composition that can play a synergistic and mutually promoting role in the treatment of osteosarcoma, can effectively inhibit the activity of osteosarcoma cells, induce tumor cell apoptosis, and inhibit tumor metastasis.

[0008] To achieve the above object, the present invention discloses the following technical solutions:

[0009] In a first aspect, the present invention provides a pharmaceutical composition for treating osteosarcoma, comprising cisplatin and daidzein.

[0010] Preferably, the mass ratio of cisplatin to daidzein in the pharmaceutical composition is 1:12.

[0011] In a second aspect, the present invention provides use of the pharmaceutical composition for treating osteosarcoma according to the first aspect in preparing a drug for treating osteosarcoma.

[0012] Furthermore, the dosage form of the drug is an injection.

[0013] Furthermore, the drug is administered by intravenous injection.

[0014] Osteosarcoma is a common bone malignancy in adolescents and children, accounting for more than half of all primary bone malignancies. The malignancy of osteosarcoma lies in the fact that it is prone to hematogenous metastasis in the early stage. Even if the primary lesion is surgically removed, it is easy to recur or metastasize to other parts. In addition, standard adjuvant / neoadjuvant chemotherapy has become one of the standard treatments for osteosarcoma, but due to resistance to multiple chemotherapy drugs, the chemotherapy effect is poor.

[0015] Soybean glycoside is a natural bioflavonoid compound found in legumes, especially soybeans. Soybean glycoside has a toxic effect on tumor cells, induces tumor cell apoptosis, blocks tumor angiogenesis, and thus exerts an anti-tumor effect, indicating that soybean glycoside is a potential adjuvant drug for cancer treatment. The present invention has found that soybean glycoside has a significant anti-osteosarcoma effect both in vivo and in vitro. Soybean glycoside inhibits osteosarcoma cell viability, induces cell apoptosis, and further inhibits cell migration and invasion ability. The inhibitory effect of soybean glycoside is dose- and time-dependent. In terms of mechanism, the present invention clarifies that soybean glycoside inhibits the Wnt pathway, especially reduces the stability of the core molecule β-catenin of the pathway, and downregulates downstream targets C-myc, CyclinD1 and MMP9.

[0016] β-catenin protein is a structural protein and an important component of the Wnt pathway. The balance between its intracellular accumulation and nuclear translocation regulates the Wnt signaling pathway. The Wnt signaling pathway plays a decisive role in the occurrence and development of tumors. Abnormal activation of the Wnt pathway is closely related to a variety of malignant tumors. In osteosarcoma cells, abnormal Wnt signals can lead to excessive accumulation of β-catenin and enhanced transcriptional activity, thereby promoting cell viability, migration and anti-apoptosis ability. At the same time, Wnt signals can also regulate the fate of stem and progenitor cells, thereby affecting the degree of differentiation of osteosarcoma cells. Therefore, targeting the Wnt pathway has become a promising new idea for the treatment of osteosarcoma. The present invention found the potential binding between daidzein and β-catenin protein, and also found that daidzein can significantly reduce the protein stability of β-catenin.

[0017] Cisplatin is a commonly used chemotherapy drug in the treatment of osteosarcoma. It is often combined with other chemotherapy drugs such as doxorubicin, cyclophosphamide, and methotrexate to form a multi-drug combination chemotherapy regimen. However, cisplatin resistance remains a common and difficult clinical problem in the treatment of osteosarcoma. It can be caused by the following factors: 1. DNA repair: some tumor cells acquire enhanced DNA repair ability, thereby reducing DNA damage caused by cisplatin; 2. Increased transport pumps: transport pumps on the cell surface (such as p-glycoprotein) can quickly expel cisplatin from cells, reducing its effective concentration and thus reducing the therapeutic effect; 3. Apoptosis escape: some tumor cells have abnormal apoptosis pathways, making them insensitive to cisplatin; 4. Signaling pathway dysregulation: cisplatin resistance also involves abnormal expression or mutation of the Wnt signaling pathway, DNA damage response pathway, and transcription factors, thereby affecting the cell's response to cisplatin; therefore, any adjuvant that can improve cisplatin resistance will be beneficial to the treatment of osteosarcoma.

[0018] The present invention finds that the combination of daidzein and cisplatin has a synergistic effect, which significantly increases the sensitivity of cells to cisplatin. The combined use of daidzein and cisplatin has a stronger inhibitory effect on cell viability and migration and invasion ability than the two drugs alone. In vivo experiments further show that the combined use of daidzein and cisplatin has a more obvious inhibitory effect on tumor growth and metastasis than the use of daidzein and cisplatin alone.

[0019] Beneficial effects of the present invention:

[0020] 1. The soybean glycin provided by the present invention binds to the β-catenin protein, reduces its protein stability, and then inhibits the Wnt signaling pathway, thereby inhibiting the vitality of osteosarcoma cells, inducing cell apoptosis, and inhibiting lung metastasis.

[0021] 2. The pharmaceutical composition of cisplatin and daidzein provided by the present invention has a synergistic effect, significantly increasing the sensitivity of cells to cisplatin. The combined use of daidzein and cisplatin has a significant inhibitory effect on the vitality and migration and invasion ability of osteosarcoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solution of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0023] Figure 1The figure shows that daidzein inhibits the activity of osteosarcoma cells; A: chemical structure of daidzein; B: cell viability assay shows that daidzein inhibits osteosarcoma cell lines (U2OS, U2OS / MTX300, HOS, MNNG / HOS, SJSA-1, 143B and MG63) in a dose- and time-dependent manner, and the cells were treated with corresponding concentrations of daidzein for 24, 48, and 72 hours; C: daidzein can inhibit the cloning ability of osteosarcoma cells, and the above cells were treated with different concentrations of daidzein for 14 days; data are expressed as mean ± standard deviation, *P < 0.05, **P < 0.01, compared with the control group;

[0024] Figure 2 Figure 1 shows the effect of soybean glycosides on osteosarcoma cell apoptosis. A: Flow cytometry analysis of Annexin V / PI staining in 143B, SJSA-1 and U2OS cells treated with soybean glycosides. Representative images and quantitative analysis of flow cytometry results are presented. BD: Western blot analysis of the expression levels of apoptosis-related proteins BAX, Bcl-xL, Caspase3 and Caspase9 in 143B, SJSA-1 and U2OS cells treated with soybean glycosides for 48 hours. Data are expressed as mean ± standard deviation. Compared with the control group, **P < 0.01.

[0025] Figure 3 Figure 1 shows that daidzein enhances the sensitivity of osteosarcoma cells to cisplatin; A: Cell viability test results after 48 hours of combined treatment with daidzein at a specified concentration and cisplatin; Partial effect combination index diagram shows the synergistic effect of daidzein and cisplatin in combined treatment; B: Cell colony formation experiment shows the number of colonies of 143B, SJSA-1 and U2OS cells after combined treatment with daidzein (40μM) and cisplatin (1μM) or alone; CE: Western blot detection of the expression levels of apoptosis-related proteins BAX, Bcl-xL, Caspase3 and Caspase9 in 143B, SJSA-1 and U2OS cells after combined treatment with daidzein (40μM) and cisplatin (1μM) or alone; Data are expressed as mean ± standard deviation, **P < 0.01;

[0026] Figure 4Figure 2 shows that daidzein reduces the stability of β-catenin protein and inhibits Wnt signaling pathway in osteosarcoma cells; AC: Western blot detection of the expression levels of β-catenin, C-Jun, CyclinD1 and C-myc in 143B, SJSA-1 and U2OS cells after daidzein (40 μM) and cisplatin (1 μM) combined or treated alone; DF: RT-qPCR detection showed that the mRNA level of CTNNB1 in 143B, SJSA-1 and U2OS cells increased with the increase of daidzein concentration; GI: Western blot analysis showed the stability of β-catenin protein in 143B, SJSA-1 and U2OS cells after daidzein (40 μM) treatment; J: Representative pictures of daidzein and β-catenin protein automatic docking; Data are expressed as mean ± standard deviation. **P < 0.01, ns, no significant difference compared with the control group;

[0027] Figure 5 Figure 1 shows the synergistic inhibition of osteosarcoma cell migration and invasion by daidzein and cisplatin; AB: Transwell assay showing the migration and invasion abilities of 143B (A) and SJSA-1 (B) cells after daidzein (40 μM) and cisplatin (1 μM) combined treatment or the two agents alone; CD: Western blotting analysis showing the protein levels of MMP2 and MMP9 in 143B (C) and SJSA-1 (D) cells after daidzein (40 μM) and cisplatin (1 μM) combined treatment or each agent alone; data are expressed as mean ± standard deviation, **P < 0.01;

[0028] Figure 6 Figure 2 shows the synergistic inhibition of osteosarcoma growth and metastasis by daidzein and cisplatin in vivo; AC: In the xenograft model treated with daidzein and cisplatin, the volume, gross view and tumor weight of tumor samples were examined, or each agent was used alone, and the tumor volume of each group was measured every 3 days; D: The body weight of rats in each group was statistically analyzed, and the body weight of rats in each group was recorded every 3 days; E: Ki-67 staining and statistical analysis of tumor samples were performed to detect the inhibitory effect of daidzein and cisplatin on osteosarcoma proliferation, scale bar: 20μm, and lung samples of mouse models treated with daidzein and cisplatin or each agent alone were stained with hematoxylin and sulphonate; F: Statistical analysis of lung metastatic nodules, scale bar: 20μm; Data are expressed as mean ± standard deviation, *P < 0.05; **P < 0.01. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0031] Example:

[0032] 1 Materials and Methods

[0033] 1.1 Cell culture

[0034] Osteosarcoma cells such as U2OS, HOS, MNNG / HOS, SJSA-1, 143B, and MG63 were obtained from ATCC tissue. U2OS / MTX300 cells were provided by Dr. M. Serra (Istituti Ortopedici Rizzoli, Bologna, Italy). The cells were placed in high-glucose DMEM (Gibco) containing 10% fetal bovine serum (FBS) and 1% streptomycin / penicillin (Thermo Fisher) at 37°C and 5% CO. 2 Culture in an incubator.

[0035] 1.2 Cell viability assay

[0036] Labeled osteosarcoma cells were evenly added to a 96-well plate and grown under appropriate conditions. Cells were treated under ideal experimental conditions. The culture medium was then aspirated and replaced with CCK-8 reagent for 2 to 4 hours, and the absorbance of the formaldehyde dye was calculated using a spectrophotometer at a wavelength of around 450nm. The absorbance values ​​were then normalized to the control conditions to calculate relative cell viability. The combination index was determined using the Chou-Talalay method. The interaction between daidzein and cisplatin was divided into antagonism (CI value greater than 1), synergy (CI value less than 1), and addition (CI value equal to 1).

[0037] 1.3 Flow cytometry apoptosis assay

[0038] With increasing concentrations of daidzein, osteosarcoma cells were gently scraped and collected and then resuspended in binding buffer. Annexin V-FITC / PI apoptosis kit (KeyGEN) was used in the indicated cells according to standard instructions. The cells were incubated at room temperature in the dark for 20 min, and the stained samples were detected by Gallios flow cytometer and the apoptosis rate was measured.

[0039] 1.4 Colony formation assay

[0040] The indicated cells were evenly seeded at a density of 500 cells / well on a 6-well plate containing 2 mL of culture medium. The culture plate was placed in an incubator at 37°C for 2 weeks. The cells were then fixed with paraformaldehyde and stained with crystal violet to visualize and quantify the cells. The stained colonies were counted and analyzed to determine the colony-forming ability.

[0041] 1.5 Western blot analysis

[0042] Total cell protein was obtained using a lysis buffer containing protease inhibitors. The blood drug concentration was then determined using the Bradford method. The samples were separated using SDS-PAGE gels and subsequently transferred to PVDF membranes at 350 mA for 1 hour. The membranes were then blocked with skim milk for 1 hour at room temperature and soaked overnight in a primary antibody diluted at 4°C. The membranes were then soaked in a suitable secondary antibody at room temperature for 1 hour and determined using ECL reagents. The antibodies used were rabbit anti-BAX antibody (ab32503), rabbit anti-caspase3 antibody (ab32351), rabbit anti-Bcl-xL antibody (ab32370), rabbit anti-β-catenin antibody (ab223075), rabbit anti-caspase9 antibody (ab32539), rabbit anti-MMP2 antibody (ab92536), rabbit anti-MMP9 antibody (ab76003) and rabbit anti-ACTB antibody (ab8227) from Abcam, rabbit anti-c-Jun antibody (AF6090) and rabbit anti-CyclinD1 antibody (AF0931) from Affinity, and rabbit anti-c-Myc antibody (18583) from Cell Signaling Technology.

[0043] 1.6 Transwell assay

[0044] Cells were seeded in Boyden chambers containing 8 μm pore membranes. The upper chamber was pre-coated with diluted Matrigel for cell invasion analysis or uncoated for cell migration analysis. 150 μL of cell suspension without FBS was seeded in the upper chamber, and 600 μL of culture medium containing 10% FBS was added to the lower chamber. After appropriate incubation, cells attached to the upper chamber were washed with a swab, and cells invading the lower chamber were fixed and stained with crystal violet for visualization. Three fields of view were randomly selected from each well, and the stained cells were photographed and counted.

[0045] 1.7 RNA extraction and RT-qPCR experiments

[0046] According to the instructions, TRIzol was used to lyse cell RNA, and cDNA was synthesized using the Vazyme kit. RT-qPCR was performed using the Vazyme fluorescent quantitative PCR kit. ACTB was selected as an internal control. The Ct (2-△△Ct) method was used to normalize the mRNA expression levels shown.

[0047] Primers for CTNNB1 and ACTB are as follows:

[0048]

[0049]

[0050] 1.8 Molecular docking

[0051] The computational docking method was used to analyze the interaction between daidzein and β-catenin protein. The crystal structure of β-catenin protein was obtained from the UniProt website. The potential binding sites of β-catenin protein involved in daidzein-induced coordination docking were analyzed using the official Autodock software. During the docking combination process, the interaction type, potential conformation, best matching docking configuration, binding energy value and bond distance were calculated. Finally, the configuration with the lowest binding energy was used as a representative binding energy model.

[0052] 1.9 Animal experiments

[0053] Four-week-old male BALB / c nude mice were provided by GemPharmatech. The in vivo experiments were approved by the Animal Research Committee. In the subcutaneous tumor formation experiment, osteosarcoma cell line 143B (7×10 cells / 100 μl saline) was injected subcutaneously. 5 cells) were injected into the mouse axillary skin. After 10 days, the tumor grew to about 50 mm 3 At the same time, BALB / c nude mice were randomly divided into four groups: control group, cisplatin treatment group (injected once every 3 days, 5 mg / kg), daidzin treatment group (injected once a day, 20 mg / kg), cisplatin combined with daidzin treatment group (injected once a day, cisplatin 5 / 3 mg / kg and daidzin 20 mg / kg were mixed and injected), and given corresponding treatments respectively. Records were taken every 3 days, and mice were killed after 27 days. Tumor volume was calculated according to the formula: width 2×length×π / 6. Tumor weight and body weight were recorded. 143B cells (density 1×10 6 The lung metastasis experiment was performed with 100 μl of saline solution (100 μl of saline solution). All samples were collected one month after injection. Lung tissues were obtained by surgery and fixed for hematoxylin (HE) staining. Mice with other causes of death (such as fighting infection) were excluded. In the outcome analysis, the study adopted a blind method for the allocation of each group.

[0054] 1.10 Immunohistochemistry (IHC)

[0055] Tumor tissue samples were first dewaxed and rehydrated, and then antigen retrieval was performed. Then, tumor samples were blocked with goat serum and then incubated with anti-Ki-67 antibody (Abcam, ab15580, 1:500) at 4°C overnight. Immunohistochemical staining was performed by DAB reagent detecting horseradish peroxidase conjugate. The IHC staining level was evaluated by two independent pathologists. The degree of immunostaining was measured and calculated as the staining index.

[0056] 1.11 Statistical analysis

[0057] Each experiment was performed with 3 biological replicates, and each biological replicate contained 3 technical replicates. GraphPad Prism9.0 software was used for statistical analysis. Data were expressed as mean ± standard deviation. Two-sided t-test was used for comparison of quantitative data. Two-way analysis of variance with Tukey's multiple comparisons was used for the second variable. P < 0.05 was considered statistically significant.

[0058] 2 Results

[0059] 2.1 Soybean glycosides inhibit osteosarcoma cell viability

[0060] Soybean glycoside is a natural product with biological activity. Its molecular formula is C 21 H 20 O 9 , molecular weight is 416.38. Chemical structure Figure 1 As shown in A.

[0061] In order to study whether daidzin has an anti-tumor effect on osteosarcoma cells, the present invention first treated several osteosarcoma cells, U2OS, U2OS / MTX300, HOS, SJSA-1, MNNG / HOS, 143B and MG63, with different concentrations of daidzin. CCK-8 detection showed that daidzin had obvious toxic effects on the above cells, such as Figure 1 As shown in B. The present invention observed that daidzein inhibited the viability of these osteosarcoma cells in a time- and dose-dependent manner.

[0062] Among them, the IC50 values ​​of daidzein treated for 48 and 72 hours are shown in the following table:

[0063]

[0064] Subsequently, clone formation experiments were performed to further explore the biological effects of daidzein on 143B, SJSA-1 and U2OS cells. The present invention found that after 2 weeks of daidzein treatment, the colony formation ability of 143B, SJSA-1 and U2OS cells decreased significantly with increasing concentrations, and in a dose-dependent manner ( Figure 1 C). Taken together, these observations suggest that daidzein can effectively inhibit osteosarcoma cell viability.

[0065] 2.2 Soybean glucoside promotes apoptosis of osteosarcoma cells

[0066] The present invention performed flow cytometry analysis of cell apoptosis and found that compared with the control group, the Annexin V / PI-stained cell ratio of 143B, SJSA-1 and U2OS cells gradually increased with the increase of daidzein concentration ( Figure 2 A). Soybean glucoside promotes apoptosis of osteosarcoma cells in a dose-dependent manner. Further, in order to explain this phenomenon at the molecular level, the present invention detected the levels of some apoptosis-related proteins, including BAX, Bcl-xL, Caspase3 and Caspase9. After 143B, SJSA-1 and U2OS were treated with soybean glucoside for 48 hours, the present invention found that with the increase of soybean glucoside concentration, the expression level of pro-apoptotic protein BAX was up-regulated in a dose-dependent manner, while the expression level of anti-apoptotic protein Bcl-xL was down-regulated in a dose-dependent manner. In addition, compared with the control group, with the increase of soybean glucoside treatment, the levels of Caspase3 and Caspase9 also increased ( Figure 2 BD). Taken together, these data suggest that daidzein can promote apoptosis of osteosarcoma cells.

[0067] 2.3 Daidzein enhances the sensitivity of osteosarcoma cells to cisplatin

[0068] Cisplatin resistance is an obstacle to the treatment of osteosarcoma patients. Therefore, it is particularly urgent to increase the sensitivity of osteosarcoma cells to cisplatin. In order to explore whether daidzein can increase the efficacy of cisplatin on OS cells, the present invention conducted CCK-8 experiments in 143B, SJSA-1 and U2OS cells. As the concentration of cisplatin and / or daidzein increased, after 48 hours, the present invention found that the inhibitory effect of daidzein combined with cisplatin was stronger than the inhibitory effect of cisplatin or daidzein alone. The present invention also calculated the CI value to evaluate the antagonistic, additive or synergistic effects of the combined treatment. The results showed that the combination of daidzein and cisplatin (CI value less than 1) showed a synergistic effect in inhibiting the OS cells shown ( Figure 3 A). The colony formation assay showed that compared with cisplatin or daidzin alone, the combination of cisplatin and daidzin showed a stronger inhibitory effect on the colony formation ability of OS ( Figure 3 B). Subsequently, the present invention found that under the combined action of cisplatin and daidzin, the protein expression of pro-apoptotic proteins BAX, Caspase3 and Caspase9 was significantly upregulated. In addition, when cisplatin and daidzin were treated together, the level of anti-apoptotic protein Bcl-xL in the cells was significantly reduced compared with the use of either agent alone ( Figure 3CE). In conclusion, daidzein increased the sensitivity of osteosarcoma cells to cisplatin.

[0069] 2.4 Soybean glycosides reduce the stability of β-catenin protein in osteosarcoma cells and inhibit the Wnt signaling pathway

[0070] Abnormal activation of the Wnt pathway can lead to the occurrence and development of osteosarcoma and further regulate its progression. Western blot results showed that the expression levels of β-catenin, a key node of the Wnt signaling pathway, and its downstream target proteins C-Jun, CyclinD1, and C-myc were significantly downregulated in 143B, SJSA-1, and U2OS cells after daidzin treatment. In addition, the inhibitory effect of combined treatment with cisplatin and daidzin on the Wnt signaling pathway was stronger than that of daidzin and cisplatin alone ( Figure 4 AC). The present invention found that after treatment with different concentrations of daidzein, there was no difference in the mRNA expression level of CTNNB1, which indicates that daidzein does not regulate the Wnt signaling pathway by affecting the transcriptional events of CTNNB1 ( Figure 4 DF). In order to further clarify this problem, the present invention measured the stability of β-catenin protein after daidzein treatment. Figure 4 As shown in GI, the stability of β-catenin protein was significantly reduced after daidzein treatment.

[0071] Previous studies have shown that drug monomers can not only bind to specific sites of proteins to regulate protein folding, stability or degradation, but also affect the structure and stability of proteins by changing the charge, hydrogen bonding or hydrophobic properties of the protein surface. In order to predict the potential interaction between daidzin and β-catenin protein, the present invention used Autodock software to perform molecular docking and determined the binding configuration between them. The predicted binding configuration of daidzin and β-catenin protein showed an appropriate shape match, and the residues Glu267 and Lys270 of β-catenin were responsible for forming hydrogen bonds with daidzin ( Figure 4 J). The predicted docking energy is -5.77 kcal / M, which has good binding ability. In summary, daidzein can bind to β-catenin protein, reduce its protein stability, and thus inhibit the Wnt pathway in osteosarcoma.

[0072] 2.5 Daidzein and cisplatin synergistically inhibit the migration and invasion of osteosarcoma cells

[0073] The malignancy of osteosarcoma cells is not only reflected in their rapid proliferation rate, but also in their ability to metastasize. The results of the transwell experiment showed that daidzin can significantly reduce the migration and invasion ability of 143B and SJSA-1 cells. In addition, compared with the use of daidzin alone or cisplatin alone, the combined use of cisplatin and daidzin has a stronger inhibitory effect on cell migration and invasion ability ( Figure 5 A and B). The present invention found that compared with the use of any one agent alone, the combined use of cisplatin and daidzein significantly downregulated the expression levels of MMP2 and MMP9 proteins in cells after 48 hours ( Figure 5 C and D). The combination of daidzin and cisplatin synergistically inhibits the metastatic ability of osteosarcoma by reducing the expression of MMP2 and MMP9.

[0074] 2.6 Cisplatin and daidzein synergistically inhibit osteosarcoma viability and metastasis in vivo

[0075] Previous studies of the present invention have shown that daidzin and cisplatin can synergistically inhibit cell viability and metastasis in vitro. In order to further verify this conclusion in vivo, the present invention established a nude mouse subcutaneous transplant tumor model. The specific experimental procedures are detailed in 1.9 Animal Experiment. Compared with the control group, the tumor volume and weight of the cisplatin group and the daidzin group were significantly reduced. In addition, compared with the use of either drug alone, the combined treatment of cisplatin and daidzin showed further tumor inhibition ( Figure 6 AC). The present invention found that daidzein had no effect on the body weight of each group ( Figure 6 D). Immunohistochemical staining of tumor tissues showed that the proportion of Ki-67 positive cells in the cisplatin and daidzein combined treatment group was significantly reduced compared with either agent alone ( Figure 6 E). In addition, the present invention establishes a mouse osteosarcoma lung metastasis model to determine the effect of cisplatin and daidzin combined therapy on osteosarcoma metastasis. HE staining of lung samples confirmed that cisplatin and daidzin combined therapy showed a lower metastasis incidence rate than either drug alone ( Figure 6 F). In conclusion, cisplatin and daidzein have a synergistic inhibitory effect on the activity and metastasis of osteosarcoma in vivo.

[0076] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating osteosarcoma, characterized in that: The pharmaceutical composition comprises cisplatin and daidzein.

2. The pharmaceutical composition for treating osteosarcoma according to claim 1, characterized in that: The mass ratio of cisplatin to daidzein in the pharmaceutical composition is 1:

12.

3. Use of the pharmaceutical composition for treating osteosarcoma according to claim 1 or 2 in preparing a drug for treating osteosarcoma.

4. The use according to claim 3, characterized in that The dosage form of the drug is injection.

5. The use according to claim 3, characterized in that: The drug is administered by intravenous injection.

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