Application of dihydroartemisinin in preparation of medicine for treating and / or relieving osteosarcoma

Abstract

The invention discloses application of dihydroartemisinin in preparation of a medicine for treating and / or relieving osteosarcoma, belongs to the technical field of biomedicine, and particularly relates to application of dihydroartemisinin in preparation of a medicine for treating and / or relieving osteosarcoma. The dihydroartemisinin is found to be capable of inhibiting the activity of osteosarcoma cells, inhibiting the migration ability and invasion ability of the osteosarcoma cells, inhibiting the proliferation ability of the osteosarcoma cells and inhibiting the self-renewal ability of osteosarcoma stem cell-like cells, so that the osteosarcoma is treated and / or relieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedical technology, and particularly relates to application of dihydroartemisinin in preparation of a drug for treating and / or relieving osteosarcoma. BACKGROUND

[0002] Osteosarcoma is a malignant bone tumor and one of the most common primary malignant tumors of bone tissue. The overall incidence of bone tumors is not high, but osteosarcoma is a relatively common type of malignant bone tumor. This tumor has obvious age characteristics, and the incidence is highest in 10-20-year-old adolescents. The disease course is often accompanied by severe pain and high mortality.

[0003] The fundamental cause of osteosarcoma is currently unknown. The most common symptoms in patients are pain and swelling around the bone or joint. At present, the treatment of osteosarcoma can be divided into three stages: preoperative chemotherapy to control tumor growth, surgical resection of diseased tissue, postoperative radiotherapy and chemotherapy, and anti-vascular targeted therapy. Preoperative chemotherapy can change the surgical staging, surgical indications and the risk of local recurrence. Surgery is still the main means to remove osteosarcoma, and is mainly divided into limb salvage and amputation according to the severity of the disease. When there is tumor residue or non-surgical treatment, chemotherapy is the most commonly used treatment. Whether it is adjuvant chemotherapy or palliative chemotherapy, it has a certain survival benefit for osteosarcoma patients. The most commonly used classic chemotherapy drugs for osteosarcoma include doxorubicin, methotrexate, cisplatin and ifosfamide. Radiotherapy can be used as an adjunctive treatment to enhance basic local control. At the same time, with the development of molecular targeted therapy, anti-angiogenic drugs have also obtained certain evidence of efficacy in the clinical treatment of osteosarcoma. However, the genetic information of osteosarcoma is complex, and the existing treatment methods still have limited efficacy. Surgery is difficult to remove micro-metastases; chemotherapy drugs have first-order kinetic limitations; targeted and immunotherapy not only has high economic costs, but also has little benefit. Therefore, recurrence, metastasis and drug resistance are the main factors that cause death and disability in osteosarcoma patients, and are also the bottleneck problem of clinical treatment of osteosarcoma.

[0004] Dihydroartemisinin (DHA) is an important derivative of artemisinin, which is modified on the basis of artemisinin structure and is mainly used for antimalarial treatment, especially for malaria caused by malignant plasmodium. Its mechanism of action is similar to that of artemisinin, which destroys the membrane structure of plasmodium by generating free radicals, but has higher stability and bioavailability. Studies have reported that dihydroartemisinin contains a sesquiterpene lactone structure with a peroxide bond. The present application focuses on the urgent needs of clinical treatment, explores the mechanism of new use of old drugs, and is committed to providing a new way for the clinical comprehensive treatment of osteosarcoma. SUMMARY

[0005] To solve the above technical problems, the application provides application of dihydroartemisinin in preparation of a drug for treating and / or relieving osteosarcoma.

[0006] To achieve the above object, the application provides application of dihydroartemisinin in preparation of a drug for treating and / or relieving osteosarcoma.

[0007] Preferably, the dihydroartemisinin can inhibit osteosarcoma cell activity, migration ability and invasion ability of osteosarcoma cells, and can inhibit proliferation ability of osteosarcoma cells and self-renewal ability of osteosarcoma stem cell-like cells, so as to treat and / or relieve osteosarcoma.

[0008] The application further provides a drug for treating and / or relieving osteosarcoma, which comprises the dihydroartemisinin.

[0009] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0010] The application further provides application of the dihydroartemisinin in preparation of an osteosarcoma cell inhibitor.

[0011] Preferably, the dihydroartemisinin can inhibit osteosarcoma cell activity, migration ability and invasion ability of osteosarcoma cells, and can inhibit proliferation ability of osteosarcoma cells.

[0012] The application further provides an osteosarcoma cell inhibitor, which comprises the dihydroartemisinin.

[0013] Preferably, the concentration of dihydroartemisinin in the osteosarcoma cell inhibitor is greater than or equal to 5 μM.

[0014] Compared with the prior art, the application has the following advantages and technical effects:

[0015] The application provides application of dihydroartemisinin in preparation of a drug for treating and / or relieving osteosarcoma. A treatment strategy targeting osteosarcoma-initiating cells (OS-ICs) with a stem cell-like phenotype is a key to radically cure osteosarcoma, and the osteosarcoma-initiating cells refer to a special cell subpopulation with tumor initiating ability in osteosarcoma (OS), and the core definition is that the cell population can maintain tumor heterogeneity through self-renewal and differentiation, and can re-form a transplanted tumor with histological characteristics similar to the primary tumor in an in-vivo experiment. The OSIC with a stem-like phenotype plays an important role in the malignant progression of osteosarcoma patients, and is a key to osteosarcoma recurrence, metastasis and drug resistance. The recurrence and metastasis of the OSIC, the self-renewal potential and the high motility are important factors for promoting the recurrence and metastasis of osteosarcoma. The OSIC has specific oxidative stress pressure and regulation mechanism. Therefore, oxidative stress damage with multiple targets and multiple anti-tumor signals is a treatment idea for osteosarcoma, and the dihydroartemisinin can inhibit the stem cell-like phenotype of the osteosarcoma-initiating cells by mediating autophagy-related ferroptosis of osteosarcoma cells, inhibit the activity of the osteosarcoma cells, inhibit the migration ability and the invasion ability of the osteosarcoma cells, and simultaneously inhibit the proliferation ability of the osteosarcoma cells, inhibit the self-renewal ability of the osteosarcoma stem cell-like cells, so that the dihydroartemisinin can be used for preparing an osteosarcoma treatment drug, and a new way is provided for the clinical comprehensive treatment of osteosarcoma. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0017] Figure 1 Chemical structural formula of DHA and statistical results of half-inhibitory concentration of DHA on MG-63 and U-2OS cell lines, wherein A is the chemical structural formula of DHA, B is the half-inhibitory concentration of DHA on the MG-63 cell line, and C is the half-inhibitory concentration of DHA on the U-2OS cell line;

[0018] Figure 2Results of determination of the migration and invasion abilities of MG-63 and U-2OS cells inhibited by DHA, wherein A is a microscope observation diagram of the migration abilities of MG-63 and U-2OS cells inhibited by different concentrations of DHA, the scale is 100 μm, CTRL in the diagram represents a blank control group, B is a statistical diagram of the migration abilities of MG-63 cells inhibited by different concentrations of DHA, *** in the diagram represents a significant difference analysis, CTRL in the diagram represents a blank control group, C is a statistical diagram of the migration abilities of U-2OS cells inhibited by different concentrations of DHA, *** in the diagram represents a significant difference analysis, CTRL in the diagram represents a blank control group, D is a microscope observation diagram of the invasion abilities of MG-63 and U-2OS cells inhibited by different concentrations of DHA, the scale is 100 μm, CTRL in the diagram represents a blank control group, E is a statistical diagram of the invasion abilities of MG-63 cells inhibited by different concentrations of DHA, *** in the diagram represents a significant difference analysis, CTRL in the diagram represents a blank control group, F is a statistical diagram of the invasion abilities of U-2OS cells inhibited by different concentrations of DHA, *** in the diagram represents a significant difference analysis, CTRL in the diagram represents a blank control group;

[0019] Figure 3 Results of determination of the proliferation abilities of MG-63 and U-2OS cells inhibited by DHA in a cell plate cloning experiment, wherein A is a microscope observation diagram of the proliferation abilities of MG-63 and U-2OS cells inhibited by different concentrations of DHA, the scale is 100 μm, CTRL in the diagram represents a blank control group, B is a statistical diagram of the proliferation abilities of MG-63 cells inhibited by different concentrations of DHA, ** and *** in the diagram represent significant difference analyses, CTRL in the diagram represents a blank control group, C is a statistical diagram of the proliferation abilities of U-2OS cells inhibited by different concentrations of DHA, ** and *** in the diagram represent significant difference analyses, CTRL in the diagram represents a blank control group;

[0020] Figure 4 A comparison diagram of effects of the scratch experiments of MG-63 and U-2OS cells treated by DHA, wherein A is the effects of the scratch experiments of MG-63 cells treated by different concentrations of DHA at different times, the scale is 100 μm, CTRL in the diagram represents a blank control group, B is the effects of the scratch experiments of U-2OS cells treated by different concentrations of DHA at different times, the scale is 100 μm, CTRL in the diagram represents a blank control group;

[0021] Figure 5 Results of enrichment and culture of osteosarcoma stem cells by using a low-adhesion culture method, CTRL in the diagram represents a blank control group, and DHA represents a DHA drug treatment group;

[0022] Figure 6Figure for DHA effect on ROS level in MG-63 and U-2OS cells, wherein A is ROS flow peak figure of MG-63 and U-2OS osteosarcoma cells after DHA treatment, B is ROS fluorescence intensity value figure of MG-63 cell strain after DHA treatment, C is ROS fluorescence intensity value figure of U-2OS cell strain after DHA treatment, CTRL in the figure represents blank control group, and “***” in the figure represents significant difference analysis;

[0023] Figure 7 Figure for DHA effect on LPO level in MG-63 and U-2OS cells, wherein A is LPO flow peak figure of MG-63 and U-2OS osteosarcoma cells after DHA treatment, B is LPO fluorescence intensity value figure of MG-63 cell strain after DHA treatment, C is LPO fluorescence intensity value figure of U-2OS cell strain after DHA treatment, CTRL in the figure represents blank control group, and “**” and “***” in the figure represent significant difference analysis. DETAILED DESCRIPTION

[0024] The detailed description set forth below describes various illustrative embodiments of the application. The detailed description is not intended to limit the scope of the application, but rather to provide a more detailed description of certain aspects, features and embodiments of the application.

[0025] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term “about” in relation to a value or a range of values is intended to include each individual value or range of values that falls within the range of the upper and lower limits of the range. Each individual value and range of values within the range of the upper and lower limits of the range is included within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0027] Many modifications and variations of this application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0028] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0029] Sources of materials used in the present application: Human osteosarcoma cells MG-63 and U-2OS were purchased from Wuhan Punsai Life Science and Technology Co., Ltd., and were routinely cultured in the laboratory of Xuzhou Medical University. The culture conditions were 37℃, 5% CO2, and humidity 95%. The osteosarcoma stem cell special culture solution (20 ng / mL EGF, 10 ng / mL bFGF, DMEM / F12 serum-free culture medium) was used. The basic culture medium DMEM was purchased from Kaygen Biotech, fetal bovine serum was purchased from Biosharp, PBS was purchased from Biosharp, 0.1% crystal violet staining solution was purchased from Biyun Tian, 4% paraformaldehyde was purchased from Biosharp, 0.25% Trypsin-EDTA was purchased from Gibco, 0.25% Trypsin-EDTA Free was purchased from Biosharp, Matrigel was purchased from Corning, Inc., 6-well plates, 24-well plates and 96-well plates were purchased from Anhui Zhenxuan, ROS detection kit and LPO detection kit were purchased from Wuhan Elirette, CCK-8 kit was purchased from Shanghai Puyou, and Transwell 8μm chamber was purchased from Corning, Inc.

[0030] Example 1

[0031] I. CCK-8 cell activity experiment

[0032] To investigate the effect of DHA on the activity of osteosarcoma cells, two osteosarcoma cell lines U-2OS and MG-63 were exposed to different concentration gradients of DHA for 72 hours, and then the cell activity was evaluated using CCK-8 reagent. Specifically, the U-2OS and MG-63 cells after three generations of recovery were digested with trypsin, and after complete digestion, the supernatant was discarded by centrifugation, and the resuspended pellet was resuspended U-2OS and MG-63 cells, and then counted. By calculating 2000 cells per 150 μL, 2000 cells per 150 μL were added to the 96-well plate, and the blank group was DMSO, and the control group was only added 2000 cells per 150 μL of U-2OS cells or MG-63 cells, and the experimental group used different drug concentrations of dihydroartemisinin (DHA) (drug gradient concentrations were 0, 2.5, 5, 10, 20, 40, 60, 80, 100 μM) respectively set 6 replicates. The 96-well plate was placed in the cell incubator for culture, and 10 μL of CCK8 reagent was added to the test wells of each group at 12h (note that there should be no bubbles at this time), and the absorbance value at 450nm was detected in the enzyme marker 1h later. Record the data, cell activity (%) = (control group OD value - experimental group OD value) / (control group OD value - blank group OD value) x 100%, and draw the concentration gradient of the drug and the cell activity or inhibition rate curve.

[0033] DHA is a derivative of artemisinin, and its chemical structure is as shown in Figure 1 A, as shown in Figure 1 B and Figure 1 C, 24h, DHA for MG-63 cells IC 50 10.03 μM, for U-2OS cells IC 50 5.07 μM, therefore, the subsequent experiment on MG-63 adopts the concentration of DHA as 10 μM, and for U-2OS adopts the concentration of DHA as 5 μM.

[0034] II. Transwell experiment

[0035] Transwell experiment is an important tool for in vitro cell behavior research, which can deeply understand various biological processes, including cancer metastasis, immune cell migration and tissue development. By calculating the number of cells that migrate or invade the bottom of the chamber, the migration and invasion ability of cells can be quantitatively evaluated.

[0036] Transwell cell invasion experiment: After DMSO and different concentrations of DHA were used to treat U-2OS and MG-63 cells for 72 h in a 6-well plate, the cells were washed with PBS, digested, and centrifuged to prepare the cells. 4 h before the experiment, Matrigel glue was mixed with 4°C pre-cooled serum-free DMEM medium (containing high glucose) (purchased from Kaygen Biotech) at a volume ratio of 1:10. Immediately, 50 μL of the prepared Matrigel glue was evenly added to the upper chamber, and care was taken to avoid air bubbles. The cells were digested with trypsin, the supernatant was discarded after centrifugation, and the precipitate was collected. Then, the precipitate (cells) was resuspended with 1 mL of serum-free DMEM medium (containing high glucose). After counting the cells, the U-2OS cell suspension and the MG-63 cell suspension were both diluted to a cell density of 50,000 cells / mL. The chamber was placed in a 24-well plate, and 800 μL of serum-containing DMEM medium (containing high glucose) (purchased from Kaygen Biotech) was added to the lower chamber. 200 μL of the diluted U-2OS cell suspension or MG-63 cell suspension was evenly dropped into the upper chamber, and the culture was continued for 48 h. The chamber was removed, washed with PBS for 3 times to remove the residual medium and non-invasive cells, and the cells invaded into the lower chamber were fixed with paraformaldehyde for 20 min. After washing with PBS for 3 times again, the cells were stained with 1% crystal violet for 20 min to color the cells invaded into the lower chamber, which facilitated observation and counting. The cells in the upper chamber were gently wiped off with a cotton swab, leaving only the cells that had passed through the Matrigel glue and invaded into the lower chamber. The chamber membrane was carefully cut off, placed on a glass slide with the lower chamber facing up, and sealed with neutral balsam. The fields were randomly selected under a microscope and photographed for counting.

[0037] The experimental results are shown in Figure 2 A, Figure 2 B, Figure 2 C, Figure 2 D, Figure 2 E, and Figure 2 F, and the results show that the migration ability and invasion ability of MG-63 and U-2OS cells are inhibited in a dose-dependent manner after DHA treatment.

[0038] III. Cell plate cloning experiment

[0039] The cell plate cloning experiment is used to evaluate the sensitivity of different killing factors (drugs, genes, etc.) on the cell proliferation ability or population dependence of tumors, and can also be used to evaluate the tumorigenicity of cells in vivo. Not all tumor cells can form tumors in vivo, but if the cloning ability of cells in vitro is stronger, it indicates that the tumorigenicity of cells in vivo is stronger.

[0040] U-2OS and MG-63 cells were digested and resuspended for standby. It is appropriate to add 600 cells per group, and after adding the cells, gently rotate to ensure that the cells are evenly dispersed, so that each cell has the opportunity to form a clone. The IC50 Values for reference, set DHA concentration gradient 1.25 μM, 2.5 μM, 5 μM and 10 μM, respectively, on MG-63, U-2OS two cell lines treated continuously for 72 h, 1-2 weeks in cell incubator, interval every 3d change liquid once, to maintain the nutrients required for cell growth and suitable environment. Change liquid need to pay attention to gentle operation, avoid damage to the cells. When the culture dish visible cell mass, discard the culture medium, washed with PBS 2-3 times, to remove residual medium and dead cells. Add 4% paraformaldehyde, fixed 15 min to keep the cell morphology stable. Washed with PBS 2 times with fixative, add 100 μL of 1% crystal violet staining, apply dyeing fluid 20 min. After washing the dyeing fluid with PBS, the culture dish was placed in room temperature air dry, take pictures.

[0041] The experimental results are shown in Figure 3 A, Figure 3 B and Figure 3 C, showing that MG-63 and U-2OS two cell lines treated with DHA, the growth of clone groups were inhibited, and with the increase of drug concentration, DHA on the formation of clone groups more obvious inhibitory effect.

[0042] Four, cell scratch test

[0043] Take logarithmic growth period of U-2OS and MG-63 cells were inoculated in six-well plates, cell fusion degree 100%, 200 μL gun head vertical scratch, PBS, basic medium culture, DHA treatment on MG-63 and U-2OS two cell lines (MG-63 treatment concentration were 2.5 μM, 5 μM and 10 μM, U-2OS treatment concentration was 1.25 μM, 2.5 μM and 5 μM, control group CTRL for DMSO treatment, concentration was the corresponding DMSO content in each DHA treatment group highest concentration), take pictures regularly. Cells migrate after intracellular and extracellular stimulation, participate in tumor deterioration and metastasis. Cell migration is an important means of evaluating the efficacy of tumor treatment.

[0044] The experimental results are shown in Figure 4 A and Figure 4 B, showing that MG-63 and U-2OS two cell lines treated with DHA, the migration ability is weakened.

[0045] Five, low adhesion culture method to enrich tumor stem cell experiment

[0046] MG-63 and U-2OS cells were inoculated in low-adhesion 6-well plates at 3000 cells per well, and 1 mL of DMEM / F12 serum-free medium containing 20 ng / mL EGF and 10 ng / mL bFGF was added. Every 3 days, 500 μL of DMEM / F12 serum-free medium containing 20 ng / mL EGF and 10 ng / mL bFGF was supplemented. After 10 days of culture, the medium was replaced with 2 mL of the above medium containing 10 μM DHA (2 mL of the above medium without DHA was added to the control group), and the culture was continued for another 3 days. Under a microscope, 10 different fields were randomly selected, and the number of serum-free culture suspension spheroids with a diameter greater than 100 μM was counted.

[0047] The experimental results are shown in Table A, Table B and Table C. Figure 5 As shown in Table A, Table B and Table C, after DHA treatment, the tumor spheres of both MG-63 and U-2OS cells became loose and smaller.

[0048] Six, Cell Reactive Oxygen Species Experiment

[0049] Flow cytometry was used to detect cell reactive oxygen species. MG-63 and U-2OS cells in the logarithmic growth phase were inoculated in six-well plates, and after adhesion, they were divided into groups and treated with DHA (the concentrations were set at 2.5 μM and 5 μM) and a blank control (CTRL, no drug treatment). When the cell confluence was 50-70%, 1 mL of ROS (10 μM) probe was added to each well, and the cells were incubated at 37°C in the dark for 30 min. After washing with the basic medium for 3 times, the cells were induced to incubate for 2 h. The cells were collected by trypsinization and centrifugation, and the flow cytometry was performed within 1 h.

[0050] The experimental results are shown in Table A, Table B and Table C. Figure 6 Table A, Table B and Table C. Figure 6 Table A, Table B and Table C. Figure 6 As shown in Table A, Table B and Table C, after DHA treatment, the ROS content of both MG-63 and U-2OS cells increased, indicating that the active oxygen content in the osteosarcoma cells was enhanced after DHA treatment.

[0051] Seven, Cell Lipid Peroxidation Experiment

[0052] Flow cytometry was used to detect cell lipid peroxidation. MG-63 and U-2OS cells in the logarithmic growth phase were inoculated in six-well plates, and after adhesion, they were divided into groups and treated with DMSO (CTRL, blank control group) and DHA (the concentrations were set at 2.5 μM and 5 μM). When the cell confluence was 50-70%, 1 mL of LPO probe was added to each well, and the cells were incubated at 37°C in the dark for 30 min. After washing with the basic medium for 3 times, the cells were induced to incubate for 2 h. The cells were collected by trypsinization and centrifugation, and the flow cytometry was performed within 1 h.

[0053] The experimental results are shown in Table A, Table B and Table C. Figure 7 Table A, Table B and Table C.Figure 7 Medium B and Figure 7 As shown in Medium C, it is indicated that the LPO content of both MG-63 and U-2OS cells is increased after DHA treatment, which shows that the lipid peroxidation level in the osteosarcoma cells is enhanced after DHA treatment.

[0054] The above-described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope defined by the claims of the present application.

Claims

1. The use of dihydroartemisinin in the preparation of drugs for the treatment and / or relief of osteosarcoma.

2. The application according to claim 1, characterized in that, The dihydroartemisinin can inhibit osteosarcoma cell activity, inhibit the migration and invasion of osteosarcoma cells, and inhibit the proliferation of osteosarcoma cells and the self-renewal ability of osteosarcoma stem cell-like cells, thereby treating and / or alleviating osteosarcoma.

3. A drug for treating and / or alleviating osteosarcoma, characterized in that, The drug includes dihydroartemisinin as described in claim 1.

4. The drug according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The use of dihydroartemisinin as described in claim 1 in the preparation of osteosarcoma cell inhibitors.

6. The application according to claim 5, characterized in that, The dihydroartemisinin can inhibit osteosarcoma cell activity, inhibit the migration and invasion abilities of osteosarcoma cells, and also inhibit the proliferation ability of osteosarcoma cells.

7. An osteosarcoma cell inhibitor, characterized in that, The osteosarcoma cell inhibitor includes dihydroartemisinin as described in claim 1.

8. The osteosarcoma cell inhibitor according to claim 7, characterized in that, The concentration of dihydroartemisinin in the osteosarcoma cell inhibitor is ≥5 μM.

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