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Toosendanin, a traditional Chinese medicine monomer, as a stat3 inhibitor and its application in the preparation of anti-osteosarcoma drugs

A technology of toosendanin and osteosarcoma, applied in the field of medicine, can solve problems such as unclear molecular targets of Chinese herbal medicine monomers

Active Publication Date: 2020-09-01
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The anti-tumor effect of some Chinese herbal medicine monomers has also attracted people's attention, but the molecular targets of Chinese herbal medicine monomers are not clear, which is also the biggest obstacle to the anti-tumor research and development based on Chinese herbal medicine monomers. Molecular targets that act directly and are applied to the treatment of metastasis and recurrence of osteosarcoma are of great significance for prolonging the life of osteosarcoma patients, and have always been the main direction of research and development of new anti-osteosarcoma drugs based on Chinese herbal medicine monomers

Method used

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  • Toosendanin, a traditional Chinese medicine monomer, as a stat3 inhibitor and its application in the preparation of anti-osteosarcoma drugs
  • Toosendanin, a traditional Chinese medicine monomer, as a stat3 inhibitor and its application in the preparation of anti-osteosarcoma drugs
  • Toosendanin, a traditional Chinese medicine monomer, as a stat3 inhibitor and its application in the preparation of anti-osteosarcoma drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: toosendanin (TSN) suppresses the activity of STAT3

[0038] 1. Technical methods

[0039] 1. Western Blot experiment

[0040] After the cells were treated with different concentrations of drugs for 48 hours, the cells were lysed with the lysate to extract the protein, and the protein samples separated by polyacrylamide gel PAGE electrophoresis after boiling and denaturation were transferred to PVDF, incubated overnight with the corresponding primary antibody, and washed three times with PBST , 10 minutes each time. Incubate with HPR-labeled secondary antibody for 1 hour, and then wash with PBST three times, 10 minutes each time. ECL color development.

[0041]2. Immunofluorescence

[0042] 143B cells were inoculated on the pre-coated cell "climbing sheet". After 24 hours, basic medium containing different concentrations of toosendanin was added. After 24 hours, they were stimulated with 50ng / mL IL6 for 30 minutes. Cells were fixed with 4% paraformalde...

Embodiment 2

[0047] Example 2: Toosendanin (TSN) and STAT3-SH2 domain binding

[0048] 1. Technical method

[0049] 1. Molecular docking experiment

[0050] For the preliminary research on the combination of TSN and STAT3, we used the structure STAT3 (PDB Id: 1BG1) in the database for computer simulation. Artificially mimicking TSN small molecules into the STAT3 pocket. Find potential binding sites of TSN and STAT3.

[0051] 2. SH2 domain protein purification

[0052] The prokaryotic expression plasmid of STAT3-SH2 domain was constructed, and the vector was pGEX-4T-1. The constructed expression plasmid was transformed into E.coli BL21(DE3) expression strain. When the OD value of the bacterial solution was around 0.6, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce the expression of the target protein at 37°C, and the soluble protein was obtained by ultrasound. The target protein was purified by GST bead, and identified by SDS gel electrophoresis and Coomassie stai...

Embodiment 3

[0060] Embodiment 3: Toosendanin (TSN) inhibits the proliferation, clone formation and induces apoptosis of osteosarcoma cells

[0061] 1. Technical method

[0062] 1. Cell culture and cell survival experiments

[0063] Osteosarcoma cells 143B, HOS, MG63.2, MG63, DUNN, LM8 and human osteoblasts used in the present invention are cultivated in a 37°C constant temperature incubator (humidity 95%, CO 2 Concentration 5%). Cell viability was determined by MTS method. Cells at 5 x 10 3 Inoculate to a 96-well plate at a density per well, add different concentrations of the monomer compound after 24 hours, add the same amount of DMSO to the control group, and set 6 replicate wells in each group. After continuing to culture for 48 hours, add 20 μl MTS and incubate at 37° C. for 1-4 hours, and detect the absorbance at 490 nm with a microplate reader. Experiments were repeated 3 times independently. Cell viability (%) = OD value of drug added / OD value of control group × 100%.

[00...

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Abstract

The invention discloses application of traditional Chinese medicine monomer toosendanin as an STAT3 inhibitor in preparation of osteosarcoma resisting drug. The anti-tumor activity of the toosendanin and a specific action mechanism thereof are discussed from various aspects of external, internal and biochemical molecular level. Mechanism research shows that the activity of STAT3 is inhibited through the toosendanin by directly targeting STAT3; an external experiment result shows that the toosendanin can inhibit proliferation of various osteosarcoma cells lines, induce cell apoptosis, stop cell invasion and reverse epithelium-mesenchymal cell transformation of osteosarcoma; in vivo, the toosendanin can inhibit growth of osteosarcoma and transfer and prolong the lifetime of tumor-bearing mice; and finally, by a human-derived osteosarcoma model, it discovers that the toosendanin remarkably inhibits growth of the human-derived osteosarcoma. The result shows that the toosendanin can be used as the STAT3 inhibitor to prepare the drug for resisting the osteosarcoma.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to the traditional Chinese medicine monomer toosendanin as a STAT3 inhibitor and its application in the preparation of anti-osteosarcoma drugs. Background technique [0002] Osteosarcoma is a primary bone malignancy originating from the mesenchymal tissue, which mostly occurs in children and adolescents. The incidence of osteosarcoma occupies the first place among primary malignant tumors. The tumor has a high degree of malignancy and a very poor prognosis. It can metastasize to the lungs within a few months, and the survival rate within 3 to 5 years after amputation is only 5 to 20%. About 80% to 90% of osteosarcomas occur in the metaphysis of the long tubular bones of the extremities, especially the distal femur, proximal tibia, and proximal humerus. Clinically, surgical resection is still the first choice for the treatment of osteosarcoma, and chemotherapy and radiotherapy are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/58A61P35/00A23L33/105
CPCA23V2002/00A61K31/58A23V2200/308A23V2250/21
Inventor 张涛李静婕尹飞华莹奇蔡郑东
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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