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Application of shikonin in preparing medicine for inducing apoptosis

A technology of programmed death and induction of cells, which is applied in the field of active ingredients of traditional Chinese medicine, can solve the problems of morphological changes, cell membrane rupture, mitochondrial membrane potential drop, etc., and achieve low toxicity effects

Inactive Publication Date: 2007-03-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] 2. Overexpression of multidrug resistance-associated protein (MRP)
[0016] 3. Overexpression of anti-apoptotic proteins Bcl-2 and Bcl-xL leads to apoptosis resistance of tumor cells
[0021] In 2005, Professor Junying Yuan from Harvard Medical School and her colleagues identified a new form of programmed necrosis - Necroptosis, which has the following main characteristics: 1. Necrotic-like morphological changes and early cell membrane rupture; 2. Mitochondrial membrane potential decreased; 3, necrosis was accompanied by autophagy; 4, the increase of intracellular reactive oxygen species was not the key factor of cell death; 5, this programmed necrosis could be specifically suppressed by a small molecule (necrostatin-1 ), and none of the commonly used inhibitors can inhibit this death

Method used

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  • Application of shikonin in preparing medicine for inducing apoptosis
  • Application of shikonin in preparing medicine for inducing apoptosis
  • Application of shikonin in preparing medicine for inducing apoptosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Comfrey kills tumor cells in a non-apoptotic manner

[0049] Experimental Materials:

[0050] MCF-7, HL60, and Hela all come from American Type Culture Collection (ATCC) company. Shikonin was purchased from China National Institute of Pharmaceuticals and Biological Products. Propidium iodide (PI), hoechst33342, z-VAD-fmk, staurosporine (STS) were purchased from Sigma. Caspase 3 activity detection kit was purchased from Bio Vision Research Products. Anti-caspase 8 and 9 antibodies, rabbit anti-AIF antibodies were purchased from CellSignaling Technology. FITC and HRP-labeled secondary antibodies were purchased from Santa Cruz Biotechnology.

[0051] experimental method:

[0052] 1. Take the well-growing MCF-7 and HL60 cells. MCF-7 adheres to the wall overnight. After treatment with shikonin, collect the cells, wash with PBS, a, 30 μg / ml PI staining at room temperature for 10 minutes, and use flow cytometry The instrument detects the permeability of the ce...

Embodiment 2

[0061] Example 2: Comfrey's way of killing tumor cells is necroptosis

[0062] Experimental Materials:

[0063] Necrostatin-1 (Nec-1) was purchased from American biomol company, 3-Methyladenine (3-MA) ​​was purchased from Sigma, 6-carboxy-2'7'-dichlorodihydro-fluoresceindiacetate (H 2 DCFDA) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzmidazolylcarbocyanine (JC-1), purchased from Molecular Probe Company.

[0064] experimental method:

[0065] 1. Treat MCF-7 cells with Necrostatin-1 one hour in advance, and then treat the cells with shikonin. Use PI exclusion method to estimate cell death rate, or observe cell growth under an inverted microscope.

[0066] 2. MCF-7 cells were treated with 10mM 3-Methyladenine one hour in advance, and then treated with 20μM shikonin for 6 hours. The cells were collected and the death rate was measured by PI staining.

[0067] 3. MCF-7 cells were adhered to the wall overnight, and the cells were collected after being treated with drugs. ...

Embodiment 3

[0074] Example 3: Shikonin caused necroptosis in all tested cells.

[0075] Experimental Materials:

[0076] The cell lines used are all from American Type Culture Collection (ATCC) company. Drug-resistant cell lines MCF-7 / ADR, K562 / ADR, and HL60 / ADR were all obtained from drug-induced screening in our research group. Plasmids pSFFV-Neo, pSFFV-Bcl-2 and pSFFV-Bcl-xL were gifts from Dr. Steven Grant, University of Virginia. Eukaryotic transfection of pSFFV plasmid, G418 screening to obtain MCF-7 and HEK293 Bcl2, Bcl-xL high expression strains.

[0077] experimental method:

[0078] After the cells were plated (adherent cells adhered overnight), 60 μM Nec-1 was treated for one hour in advance, and then the cells were treated with shikonin for 6 hours, and the cells were collected, and the cell death rate was calculated by PI exclusion.

[0079] Experimental results:

[0080] Among the more than 10 kinds of cells tested, the death caused by shikonin without exception (includ...

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Abstract

The present invention provides the application of shikonin in preparing medicine for inducing apoptosis. Shikonin has the molecular formula of C16H16O5 and the chemical name of (+)-5, 8-dihydroxy-2-(1-hydroxy-4-methyl-3-pentenyl)-1, 4-naphthoquinone, possesses optical activity, and constitutes one pair of antimers together with isoshikonin. The present invention has clinical application foreground owing to that shikonin can kill several kinds of drug resisting tumor cells in the effect higher than other available clinical medicines and possesses relatively low toxicity.

Description

technical field [0001] The invention belongs to the use of active ingredients of traditional Chinese medicines, and mainly relates to the new use of shikonin in the preparation of anti-multidrug-resistant tumor drugs. Background technique [0002] Shikonin is one of the main components extracted from the root of the perennial herb of the family Boraginaceae, which is distributed in most provinces and regions of my country. It has anti-inflammatory, astringent and antipyretic effects. It is applied clinically and has definite curative effect. Comfrey was first recorded in "Shen Nong's Materia Medica", and it is a traditional Chinese medicine for preventing and treating measles. Shikonin is the main component of anti-inflammation. Comfrey is rich in shikonin and its derivatives, its content accounts for 3-6.5%, its color is deep red, and its chemical structure is naphthoquinones, which can transfer mesons in the oxidation-reduction reaction in organisms. It has hemostatic, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/122A61K9/00A61K36/30A61P35/00A61K125/00
Inventor 胡汛韩卫东
Owner ZHEJIANG UNIV
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