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Novel application of gossypol

A kind of gossypol and functional technology, applied in the field of medicine, can solve the problems that have not yet been seen, and achieve the effect of improving the sensitivity of chemotherapy

Inactive Publication Date: 2014-03-26
THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no reports about the ability of gossypol to inhibit the redox and DNA damage repair functions of APE1.

Method used

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  • Novel application of gossypol
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  • Novel application of gossypol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: The effect of gossypol on the proliferation of Hela and HepG2 cells

[0033] Inoculate Hela cells in a 96-well plate to make the cell density about 5000~10000 / well, and fill the peripheral holes with 100μL of PBS buffer at 37℃, CO 2 Cultivate for 24 hours under the condition of 5% volume fraction and saturated humidity.

[0034] Then take the adherent Hela cells and add 6 drug concentration gradient gossypol solutions to make the final concentration in molar concentration sequentially 10nmol / mL, 20nmol / mL, 40nmol / mL, 60nmol / mL, 80nmol / mL , Each concentration has 5 replicate wells, and take 5 wells containing Hela cells but no gossypol solution as a control, at a temperature of 37℃, CO 2 The volume fraction is 5%, and the culture is 48 hours under saturated humidity.

[0035] Then, add 10μL of CCK8 solution (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) with a volume concentration of 10% to each well, continue to incubate for 2 hours, terminate the incub...

Embodiment 2

[0038] Example 2: CCK8 method to investigate the effect of APE1 stable knockdown cell line and APE1 wild-type cell line on the tumor-killing effect of gossypol

[0039] APE1 wt , APE1 shRNA Hela cells were seeded in 96-well plates with a cell density of about 5000~10000 / well; the peripheral holes were filled with 100μL of PBS; at 37℃, CO 2 The volume fraction is 5%, cultured for 24 hours under saturated humidity;

[0040] After the cells adhere to the wall, in APE1 wt , APE1 shRNA Add 5 drug concentration gradient gossypol solutions to the cells (to make the final concentration in molar concentration 10nmol / mL, 20nmol / mL, 40nmol / mL, 60nmol / mL, 80nmol / mL), each concentration is set to 5 Multiple holes. At 37℃, CO 2 The volume fraction is 5%, cultured for 48 hours under saturated humidity.

[0041] Add 10 μL of MTT solution with a volume concentration of 10% to each well, and continue to incubate for 2 hours; 450 Measure the absorbance of each hole at the wavelength. See the res...

Embodiment 3

[0043] Example 3: Western Blot verifies the endogenous APE1 protein knockdown and exogenous APE1 protein expression of the two Hela cell models

[0044] First, extract Hela cells and APE1 shRNA , APE1 wt And APE1 C65S For total cell protein, the specific operation is as follows: aspirate the cell culture supernatant, then wash twice with ice PBS; add 150μL of 1× protein loading buffer, hang down adherent cells with a cell brush until the liquid is viscous; Pipette the viscous sample liquid into a clean 1.5mL centrifuge tube, sonicate 30 pulses to lyse the nucleic acid until a white bubble is generated; place the lysed liquid on a dry thermostat for 5 minutes at a temperature of 100°C; the extracted sample is at -80°C Store at ℃.

[0045] Then, the Western Blot electrophoresis gel was prepared, and the lower separation gel formula was:

[0046]

[0047] After the lower layer is gelled, prepare the upper layer of concentrated glue. The concentrated glue formula is:

[0048]

[0049]...

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Abstract

The invention relates to the field of medicines, and discloses novel application of gossypol. The gossypol can be used for effectively inhibiting the activity of NF-kappa B, AP-1 and HIF-1 alpha which are induced by reduction-oxidation of APE1 (apurinic / apyrimidinic endonuclease-1) and combined with DNA (deoxyribonucleic acid), and also remarkably inhibiting the DNA damage repairing function of APE1, so that the invention provides the application of gossypol in preparing an APE1 reduction-oxidation inhibitor or a DNA damage repairing function inhibitor of APE1. Furthermore, gossypol can be used for improving the sensitivity of tumor cells to etoposide chemotherapy, and can also reverse drug resistance of the tumor cells to chemotherapeutic medicines caused by high expression of APE1 in the tumor cells, so that the invention also provides application of gossypol in preparing adjuvants or chemosensitizer for treating cancers.

Description

Technical field [0001] The invention relates to the field of medicine, in particular to the new application of gossypol, in particular to the application of gossypol as an APE1 inhibitor and its application in the preparation of adjuvants for the treatment of cancer or chemotherapy sensitizers. Background technique [0002] With the improvement of living standards and the aggravation of environmental pollution, the incidence of malignant tumors has been increasing year by year. It has been clinically defined as common and frequently-occurring diseases, which seriously threaten human health. Apurinic aprimidinic endonuclease / Redox factor-1 (Apurinic aprimidinic endonuclease1 / Redox factor-1, APE1 / Ref-1) is an example of biological macromolecule functional complexes. On the one hand, APE1 has DNA repair activity and it acts as an AP nucleic acid Endonuclease is one of the main rate-limiting enzymes in DNA base excision repair (BER) pathway; on the other hand, APE1 can also regulate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/11A61P35/00
Inventor 钱程远王东隋江东李梦侠
Owner THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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