CN101683331B - Application of alkannin derivant - Google Patents
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Abstract
The invention provides an application of alkannin derivant for preparing drug treating cancers, and the drug takes orphan receptor Nur77 as an effect target. The invention also provides the application of the orphan receptor Nur77 which serves as an effect target to screen alkannin derivant. The alkannin derivant of the invention clarifies the mechanism of the anti-tumour effect of alkannin derivant via a great discovery that tumour cells are induced to apoptosis by acting on the orphan receptor Nur77, and provides effective target spots for decorating and optimizing the structure.
Description
Technical field
The present invention relates to field of Chinese herbal, specifically, is the application about alkannin derivant.
Background technology
Orphan receptor Nur77 (being called again TR3 or NGFI-B), the important member of nuclear receptor superfamily, it also is a kind of at once early gene, its expression can be induced rapidly by serum, somatomedin, myristoyl Buddhist ripple acetate-13 (TPA) and calcium signal (Zhang XK.et.al., Expert Opin Ther Tar2007; 11:69-79; Moll UM, et.al., Oncogene2006; 25:4725-43.).
Nur77 is considered to a kind of liability factor at first, and it can mediate multiple survival signal path, comprises protein kinase A, Protein kinase C, MAPK and NF-κ B path.The growth promoting function that Nur77 expresses all is proved (Chen GQ, et.al., Int J Cancer2002 in kinds of tumors; 99:171-8; Wu Q, et.al., Carcinogenesis2002; 23:1583-92.), having been reported and show, the expression of the mRNA of Nur77 in cancerous tissue is apparently higher than cancer beside organism.
Recently, increasing evidence shows that Nur77 also is a kind of short apoptosis molecule simultaneously.Nur77 at first is in the news in the apoptosis by the T-cell receptors signal at immature thymocyte cell and T-quadroma to the effect of apoptosis.Nur77 is proved in polytype cancerous cell to apoptotic effect, it can be to multiple antiapoptotic factors, such as Calcium ionophore, etoposide (VP-16), Buddhist ripple ester, cadmium and 1, (3 '-indolyl)-1-(p-substituted phenyl) methanes makes and replys (Stasik I 1-Bis, et.al., BBA-Mol Cell Res2007; 1773:1483-90; Gennari A, et.al., ToxicolAppl Pharmacol2002; 181:27-31; Liu S, et.al., World J Gastroenterol2002; 8:446-50; Wilson AJ, et.al., Cancer Res2003; 63:5401-7; Chintharlapalli S, et.al., J Biol Chem2005; 280:24903-14.).
One of the regulation and control proliferation function of Nur77 and biological mechanism of apoptotic effect are that the Subcellular Localization by Nur77 realizes.Nur77 realizes that by its effect in nucleus growth promotes, this effect needs Nur77 to combine with target gene DNA response regulatory element, and namely Nur77 has the function of inducing cell propagation at endonuclear transcriptional activation.And the apoptotic effect of Nur77 does not rely on and transcribes, and when its DNA binding structural domain disappearance, this apoptosis-induced effect still can occur.
Recently, the inventor finds by a series of research, under the effect of some short apoptosis factor, Nur77 can be induced out nuclear, and navigates to mitochondrion, Golgi body or endoplasmic reticulum, cell death inducing, this apoptotic effect can be by inducing the interaction of Nur77 and Bcl-2, cause that the Bcl-2 conformation changes, expose the BH3 domain of containing among the Bcl-2, make Bcl-2 change into the cell pro apoptotic protein from a cell anti-apoptotic proteins.Prove, in many different tumors such as carcinoma of prostate, pulmonary carcinoma, colon cancer, ovarian cancer and gastric cancer, different antiapoptotic factors can be induced the mitochondrion location of Nur77.Because many tumour high-expression Nur77 and Bcl-2, thereby, a kind of method of very effective screening anti-tumor medicine become by regulation and control Nur77/Bcl-2 apoptosis pathway.
Radix Arnebiae (Radix Lithospermi) is comfrey (Boraginaceae), and several classes such as Arnebia euchroma Amebia euchroma (Royle) Johnst, Liaoning gromwell root (Lithospermum erythrorhizon Sieb.et Zucc.) or arnebia guttata Bunge (Amebia guttataBunge) are arranged in China.Radix Arnebiae (Radix Lithospermi) property is sweet, salty, cold, GUIXIN, Liver Channel, and bitter in the mouth, cold in nature, clearing away heat and cooling blood is arranged, the function of rash detoxifcation.
Radix Arnebiae (Radix Lithospermi) contains multiple naphthoquinone class pigment such as shikonin, acetyl alkannin etc.Lithospermum euchromum Royle contains β, beta-dimethyl-acryloyl shikonin (β, (β-Hydroxyixovalerylshikonin), deoxyshikonin (Deoxyshikonin) etc., they have multiple biological activity for β-Dimethyl-acryl-shikonin), shikonin (shikonin), acetylshikonin (Acetyl shikonin), isobutyryl shikonin (Isobutyryl shikonin), IVS (Isovalerylshikonin), beta-hydroxyisovalerylshiderivative.
In recent years, tcm clinical practice studies have shown that Radix Arnebiae (Radix Lithospermi) has preferably effect for the treatment kinds cancer, and has proved that its main active component is shikonin and derivant thereof.At present the research of shikonin and derivant antitumor action thereof is reported, comprise that inhibition tumor cell growth, cell death inducing, inhibition DNA topoisomerase, Profilin tyrosine kinase examine (the Chen X such as anti-angiogenic proliferative activity, etal., Phytother Res.2002May; 16:199-209.; Shen CC, et al., J Nat Prod.2002Dec; 65:1857-62).But, the mechanism of shikonin and derivant antitumor action thereof does not still understand so far, and never find a clear and definite action target spot, thereby be difficult to this structure is effectively modified and optimized, this be restriction its be developed further into main reason into cancer therapy drug.
Summary of the invention
The present inventor finds in the research process of alkannin derivant for the mechanism of action of tumor cell, and some alkannin derivant can be induced the apoptosis of kinds of tumor cells by inducing the Nur77/Bcl-2 apoptosis pathway.
Therefore, first purpose of the present invention is, the application of a kind of alkannin derivant for the preparation of the treatment cancer drug is provided.
Second purpose of the present invention is, provides a kind of orphan receptor Nur77 to be used for the application of screening alkannin derivant as action target spot.
According to the present invention, alkannin derivant is for the preparation of the application of medicine for the treatment of cancer, described medicine with orphan receptor Nur77 as action target spot.
According to the present invention, alkannin derivant can inducing tumor cell apoptosis, concrete, alkannin derivant can induce orphan receptor Nur77 expression, induce orphan receptor Nur77 to go out nuclear and be positioned on the mitochondrion, induce Bc1-2 conformation change, activate Bax, and cause that cytochrome C discharges and apoptosis from mitochondrion.
According to the present invention, described alkannin derivant comprises acetylshikonin, 5,8-diacetoxy-2-(1 ' acetoxyl group-4 ' methyl-3 ' pentenyl)-1,4-naphthoquinone and 5,8-diacetoxy-6-(1 ' acetoxyl group-4 ' methyl-3 ' pentenyl)-1,4-naphthoquinone.
According to the present invention, described cancer is preferably pulmonary carcinoma, cervical cancer, breast carcinoma, hepatocarcinoma, gastric cancer.
According to the present invention, as action target spot, can be used for the screening alkannin derivant with orphan receptor Nur77, be used for the treatment of various cancers with further exploitation.
The orphan receptor Nur77 inducing apoptosis of tumour cell that acts on provided by the invention can be used for instructing the screening alkannin derivant, therefrom obtain to have the chemical compound of anti-tumor activity, and medicine and then the preparation that obtains can be able to be acted on the activity of orphan receptor Nur77 inducing apoptosis of tumour cell and the medicine of function.
Description of drawings
Figure 1A shows that alkannin derivant is to Nur77 protein expression amount regulation and control result in the NIH-460 cell; Figure 1B shows that alkannin derivant is to Nur77 protein expression amount regulation and control result in the HeLa cell; Fig. 1 C shows that SK07 increases the expression of inducing Nur77 and has good timeliness and dose-effect relationship; The fluorescence staining that shows Fig. 1 D detects alkannin derivant and causes Nur77 to move out from nucleus, and is positioned on the mitochondrion after going out nuclear.
Fig. 2 A shows that fluorescence staining detects the apoptosis of alkannin derivant inducing tumor cell; Fig. 2 B shows the percentage result of apoptosis; Fig. 2 C shows the PARP protein degradation in the alkannin derivant inducing tumor cell.
Fig. 3 A is that the two detection alkannin derivants that dye of flow cytometry analysis Annexin-V/PI are induced the apoptosis result of NIH-H460 lung carcinoma cell; Fig. 3 B be flow cytometry analysis Annexin-V/PI two dye detect alkannin derivant inducing mouse embryonic fibroblast MEF and MEF Nur77-/-the apoptosis result of cell.
The fluorescence staining that shows Fig. 4 A, B detects the expression of the apoptosis-induced Nur77 of depending on of alkannin derivant and goes out nuclear; Fig. 4 C showed cell apoptosis rate result.
Fig. 5 A shows that fluorescence staining detects the common location of Nur77 and Bcl-2; The fluorescence staining that shows Fig. 5 B detects SK07 and has activated Bax; Fig. 5 C shows that the activation of fluorescence staining detection Bax is accompanied by the release of cytochrome c; The fluorescence staining that shows Fig. 5 D detects SK07 and has induced the Bcl-2 conformation change.
The specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples are only for explanation the present invention but not for limiting scope of the present invention.
The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition such as " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The reagent that uses among the present invention:
Liposome 2000, Trizol LS are available from Invitrogen company; ECL, goat antirabbit, mountain sheep anti mouse horseradish peroxidase two resist available from Thermo company; The mountain sheep anti mouse two of Cy3 labelling is anti-available from Chenicon international; Nur77 multi-resistance (sc-5569), Hsp60 (sc-7150), Bcl-2 (sc-509), Bax (sc-493), Bax (6A7) (sc-23959) goat antirabbit two of antibody, PARP (sc-556494) and FITC labelling resist available from Santa Cruz; Cytochrome C (556433) antibody is available from BD company; β-actin antibody is available from Sigma company.The Nur77 monoclonal antibody is available from R﹠amp; D; Bcl2-BH3 (AP1303a) antibody is available from Abgent, and pvdf membrane is available from Millipore.Outside indicating especially, chemical reagent used among other the present invention is all available from Sigma.
Take by weighing 20kg Liaoning gromwell root (commercially available), grind rear mistake 80 mesh sieves, collect the Arnebia Root of the rear acquisition of sieving, carry out the room temperature diafiltration with 95% ethanol and extract, to extracting solution be closely colourless till.
Merge extractive liquid, is evaporated to solution and becomes aubergine, obtains approximately 250g of puce grease crude product.
After removing by filter insoluble matter, add dense HCl souring soln, become aubergine by blueness to solution, have this moment a large amount of precipitations to generate standing over night.
Crude product is dissolved with chloroform, and per 50 milliliters add 17 the gram 200～300 purpose silica gel, upper prop stirs.Eluant is used respectively petroleum ether (60～90 ℃)-acetone and petroleum ether (1:1)-acetone-chloroform (1:1), and eluting is also collected respectively eluent successively.
Eluent is concentrated into Precipitation, then recrystallization (the recrystallization method referenced patent number is 02114973.9 Chinese patent), purification Radix Arnebiae (Radix Lithospermi) compounds, product is defined as acetylshikonin through MNR, MSUV and elementary analysis data, and its structural formula is as follows:
Acetylshikonin
(SK03)
200710046917.5 and the method for 200710041978.2 Chinese patent application, synthesis of derivatives 5,8-diacetoxy-2-(1 '-acetoxyl group-4 '-methyl-3 '-pentenyl)-1,4-naphthoquinone (SK06) and 5,8-diacetoxy-6-(1 '-acetoxyl group-4 '-methyl-3 '-pentenyl)-1,4-naphthoquinone (SK07), the structural formula of SK06 and SK07 is as follows respectively:
2 '-diacetyl shikonin 6 '-the diacetyl shikonin
(SK06) (SK07)
2.1, cell culture
Select lung carcinoma cell NIH-H460 (ATCC, HTB-177) and cervical cancer cell HeLa (ATCC, CCL-2), with RPM1640 culture medium (available from Hyclone), be incubated in the 24 orifice plate tissue culturing plates 37 ℃ and 5% CO2 gas incubator, change liquid after 24 hours, carry out dosing (not containing serum) after hungry 16 hours and process.Alkannin derivant is dissolved among the DMSO (DMSO final concentration＜0.1%), and matched group is processed with the DMSO of same concentration, specifically processes as follows:
(1) matched group (DMSO)
(2) SK03 group
(3) SK06 group
(4) SK07 group
The concentration of SK03, SK06, SK07 is 5 or 10 μ M, and the processing time is 12 or 24 hours, with without the NIH-H460 cell of any processing and HeLa cell in contrast.In order to prove conclusively timeliness and dose-effect relationship, process the NIH-H460 cell with SK07, concentration for the treatment of is 1,5 or 10 μ M, the processing time is 6,12 or 24 hours, with without the NIH-H460 cell of any processing in contrast.
2.2, Western blotting analyzes the expression of Nur77
Cell is with improved RIPA lysis buffer (50mM Tris-Hcl, pH7.4; 150mM NaCl; 5mM EDTA; 1%NP-40,0.5% NaTDC is 0.1%SDS) in cracking on ice 30 minutes.With identical applied sample amount, the 8%SDS-PAGE electrophoresis, transferring film, TBST (50mM Tris-HCl (pH7.4), 150mM NaCl and0.1%Tween20) room temperature with 5% defatted milk powder was sealed 1 hour, in room temperature 2 hours or 4 ℃ of night incubation primary antibodies, incubated at room two resists one hour, the ECL colour developing, exposure, the result is shown in Figure 1A, 1B, 1C.
By the result of Figure 1A as can be known, in the NIH-H460 lung carcinoma cell, alkannin derivant SK03 can effectively induce the expression of Nur77.Concentration with SK035-10 μ M was processed cell 12-24 hour, and its Nur77 protein expression amount of inducing is about 3-5 times of contrast.Process the NIH-H460 cell with SK07 and have the effect that similar regulation and control Nur77 expresses, but SK06 is significantly suppressed the expressional function of Nur77.
By the result of Figure 1B as can be known, in the HeLa cervical cancer cell, alkannin derivant SK03 can effectively induce the expression of Nur77.Concentration with SK035-10 μ M was processed cell 12-24 hour, and its Nur77 protein expression amount of inducing significantly improves.Process the HeLa cell with SK07 and also can cause significantly improving of Nur77 expression, and SK06 is suppressed to the inducing action of Nur77.
By the result of Fig. 1 C as can be known, along with the increase of SK07 concentration for the treatment of and the prolongation in processing time, the expression of Nur77 significantly improves, and has good timeliness and dose-effect relationship.
2.3, alkannin derivant induces Nur77 to go out nuclear
Select lung carcinoma cell NIH-H460 (ATCC, HTB-177), with RPM1640 culture medium (available from Hyclone), be incubated in the 24 orifice plate tissue culturing plates that are covered with coverslip 37 ℃ and 5% CO2 gas incubator, change liquid after 24 hours and carry out dosing (not containing serum) and process.Alkannin derivant is dissolved among the DMSO (DMSO final concentration＜0.1%), and matched group is processed with the DMSO of same concentration, specifically processes as follows:
(1) matched group (DMSO)
(2) TPA group
(3) SK03 group
(4) SK06 group
(5) SK07 group
(6) with general mycin A (LMB) (10ng/mL) after the pretreatment, add the SK07 group.
Wherein, organize in contrast with (1), (2) positive matched group.
The cumulative volume of each hole solution is 1ml in the Sptting plate, and the concentration of SK03, SK06 and SK07 is 5 μ M.
Behind treated 24 hours of the cell, with 0.1M phosphate-buffered salt PBS washing 3 times, fix with 4% paraformaldehyde solution 40ul, again to contain the 0.1M PBS solution perforation 5min of 1%Triton; Then, with contain 5%BSA 0.1M PBS solution, 37 ℃ the sealing half an hour, then add the anti-Nur77 polyclonal antibody of rabbit (1:200 dilution) (Santa Cruz, sc-5569) as primary antibodie, 4 ℃ are spent the night.
Get spend the night cell solution after processing of step, after room temperature is placed 15min, sopped up primary antibodie, with the PBS washing of 0.1M 3 times, every all over 5min; Add the sheep anti mouse two anti-(the 1:500 dilution is available from Chemicon) that cy3 is coupled, behind 37 ℃ of placement 30min, sop up two and resist, with 0.1M PBS washing 3 times, every all over 5min.
DAPI (available from Sigma) dyeing is for detection of nucleus.Then use fluorescence microscope (Carl Zeiss) to observe the Subcellular Localization (BioRad) of endogenous Nur77, the result is shown in Fig. 1 D.
According to the result of Fig. 1 D as can be known, process 24 hours with alkannin derivant after, Nur77 moves out from nucleus, and is positioned on the mitochondrion after going out nuclear; And the Nur77 in the matched group still stays in the nucleus; LMB be known depend on CRM1 go out nuclear translocation path and inhibitor, behind LMB and SK07 co-treatment cell, the accumulation of the Nur77 that SK07 induces in Cytoplasm can be suppressed by LMB significantly, illustrate SK07 strengthened Nur77 go out to examine.
The above results shows: alkannin derivant can induce Nur77 from nucleus to cytoplasmic migration and be positioned on the mitochondrion.
Embodiment 3, the alkannin derivant inducing tumor cell apoptosis
3.1 alkannin derivant is induced the NIH-H460 apoptosis
Select lung carcinoma cell NIH-H460 (ATCC, HTB-177), with RPM1640 culture medium (available from Hyclone), be incubated in the 24 orifice plate tissue culturing plates 37 ℃ and 5% CO2 gas incubator, change liquid after 24 hours and carry out dosing (not containing serum) and process.Alkannin derivant is dissolved among the DMSO (DMSO final concentration＜0.1%), and matched group is processed with the DMSO of same concentration, specifically processes as follows:
(1) matched group (DMSO)
(2) SK03 group
(3) SK06 group
(4) SK07 group
The cumulative volume of each hole solution is 1ml in the Sptting plate, and the concentration of SK03, SK06 and SK07 is 5 μ M.
After processing 24h, cell 0.5% trypsinization after 0.1M PBS washing, is fixed with 4% paraformaldehyde, dyes with 1 μ g/ml DAPI, and with the form of fluorescence microscope (Carl Zeiss) observation of cell nuclear, the result is shown in Fig. 2 A.Select at random 5 visuals field, calculate the apoptosis number of 300 cells in every visual field, get 5 apoptotic averages in the visual field, calculate the percentage ratio of apoptosis, the result is shown in Fig. 2 B.
By Fig. 2 A as seen, process 24 hours with alkannin derivant after, the obvious apoptosis of cell.The cell of apoptosis shows typical morphologic feature, and, film concentrated such as Cytoplasm is blister, and nuclear concentrates and produce fragment etc.By the result of Fig. 2 B as can be known, the cell of not processing with alkannin derivant seldom has the apoptosis phenomenon that (7%) occurs, can cause the obvious apoptosis of cell (22%) with the SK03 processing, the apoptosis-promoting effect of SK07 significantly strengthens (33%), and SK06 is to the effect of apoptosis weak (14%).This shows, the alkannin derivant cancerous cell has apoptosis-promoting effect in various degree.
3.2SK07 induce the PARP protein degradation of NIH-H460 cell and HeLa cell
Select lung carcinoma cell NIH-H460 (ATCC, HTB-177) and cervical cancer cell HeLa (ATCC, CCL-2), with RPM1640 culture medium (available from Hyclone), be incubated in the 24 orifice plate tissue culturing plates 37 ℃ and 5% CO2 gas incubator, after 24 hours, the RPMI1640 with the serum-free that is added with 5 μ M SK07 processed 3,6,12,24 hours respectively.Cell is with improved RIPA lysis buffer (50mM Tris-Hcl, pH7.4; 150mM NaCl; 5mM EDTA; 1%NP-40,0.5% NaTDC is 0.1%SDS) in cracking on ice 30 minutes.With identical applied sample amount, the 8%SDS-PAGE electrophoresis, transferring film, TBST (50mM Tris-HCl (pH7.4), 150mM NaCl and0.1%Tween20) room temperature with 5% defatted milk powder was sealed 1 hour, in room temperature 2 hours or 4 ℃ of night incubation primary antibodie anti-PARP (1:1000 dilution), incubated at room two resists one hour, the ECL colour developing, exposure, the result is shown in Fig. 2 C.
Shown in Fig. 2 C, it is the specificity catabolite of 89kD that SK07 can induce the PARP protein degradation of 113kD.Amount of degradation products with the prolongation 89kD of SK07 action time increases gradually, particularly in the HeLa cell after effect 12 hours and 24 hours, the PARP albumen of 113kD almost completely disappears, and all is degraded.
In the situation that have and lack processing NIH-H460 lung carcinoma cell 24 hours with 5 μ M SK07 at general mycin A (LMB) in the serum-free RPM1640 culture medium (available from Hyclone), with the cell that do not add SK07 in contrast.Cell is analyzed with flow cytometer (BeckmanCoulter EPICS ALTRA) according to Vybrant Apoptosis Assay Kit# 2 workbook dyeing PI and AnnexinV.Analyze with EXPO32ADC software (Beckman Coulter EPICS ALTRA), the result as shown in Figure 3A.
Can be found out by Fig. 3 A, the SK07 of 5 μ M causes 37.9% NIH-H460 early apoptosis of cells in the situation that lack LMB, and in the situation that exist LMB substantially can not induce NIH-H460 cell apoptosis.Showing that SK07 processes can induce the early apoptosis of NIH-H460 cell, and does not cause necrocytosis (right lower quadrant represents early apoptosis among the figure, and right upper quadrant represents necrosis); And inducing of this early apoptosis can be suppressed by LMB.
Similarly, the MEFNur77-that mouse embryo fibroblasts MEF (ATCC, SCRC-1046) and Nur77 are knocked out/-cell (Carcinogenesis, vol28, pp1653-1658,2007) processed 24 hours with 1 μ M and 10 μ M SK07 or 10 μ MSK03, use the flow cytometry analysis apoptosis, the result is shown in Fig. 3 B.
Can be found out the MEF Nur77 that Nur77 knocks out by Fig. 3 B
-/-Cell, the apoptosis-promoting effect of SK07 is significantly suppressed.Processed 24 hours with 1 μ M SK07, the apoptosis rate of MEF cell is 14.6%, and same processing can not cause MEFNur77
-/-The apoptosis of cell.The SK07 of high concentration (10 μ M) is although can cause MEF Nur77
-/-The apoptosis rate of cell 23.7%, but be significantly less than the apoptosis rate of MEF cell 57%.This result shows, the apoptosis that SK07 induces depends on the expression of Nur77 dramatically.When processing cell with SK03 (10 μ M), find that it can cause MEF cell 54.6% and MEF Nur77
-/-The necrosis rate of cell 66.8%, the effect of this explanation SK03 does not rely on the expression of Nur77, and has non-specific toxicity for cell.
For transposition role in the apoptosis that alkannin derivant is induced of studying Nur77, with the antibody of Nur77 and the DAPI NIH-H460 cell (cell culture with embodiment 2.3) of SK07 after processing that jointly dye, the fluorescence microscope result is shown in Fig. 4 A.Result according to Fig. 4 A can find out, in the cell that kytoplasm Nur77 distributes, nucleus presents the representative configuration of apoptosis more, and Nur77 does not express or is distributed in the nuclear in the intact cell of nucleus.
For the transposition of further proof Nur77 with the relation between the apoptosis-inducing, with the SK07 processing of 10 μ M 24 hours, then DAPI staining examine apoptosis was used fluorescence microscope with the NIH-H460 cell of GFP-transfected-Nur77, the result is shown in Fig. 4 B.
Can find out from the result of Fig. 4 B, in cellular control unit, GFP-Nur77 is positioned in the nucleus, and karyomorphism is normal.Cell GFP-Nur77 after SK07 processes then is positioned in the Cytoplasm, and nucleus is condensing, fragmentation.
If the nuclear translocation that goes out of Nur77 works, suppress going out nuclear and can reducing apoptosis of Nur77 in the apoptosis that SK07 induces.Therefore, the NIH-H460 cell of GFP-transfected-Nur77 is processed with SK07 in the situation that LMB exists or lacks.Then DAPI staining examine apoptosis is drawn bar diagram with fractographic result, the results are shown in Figure 4C.
Can find out from the result of Fig. 4 C, under the condition without the GFP-Nur77 transfection, process the H460 cell with SK07, apoptosis rate is nearly 35%, and matched group is 7%.In the cell of GFP-Nur77 transfection, after SK07 processed, apoptosis rate reached 88%.And process simultaneously through LMB when cell, the apoptosis rate that SK07 induces significantly reduces, and non-transfected cells is 10%, and transfectional cell is 16%.
The expression that the apoptosis that above presentation of results SK07 induces not only depends on Nur77 also depends on its Cytoplasm location.
The NIH-H460 cell utilizes immunostaining to pressing down the location of apoptotic proteins Nur77, Bcl-2 after processing 24 hours with SK07, the activation of Bax, and the release of cytochrome C and the conformation change of Bcl-2 are analyzed, and the dilution ratio of following antibody is 1:500.
6.1, the common location of Nur77 and Bcl-2
SK07 (5 μ M) acts on cell after 24 hours in the situation that LMB exists and lacks, with jointly dye cell after the processing of the antibody of the antibody of Nur77 and Bcl-2.Fluorescence microscope is analyzed the distribution of Nur77 and Bcl-2, and the result is shown in Fig. 5 A.Fig. 5 A has shown the common location of Nur77 and Bcl-2.
6.2, SK07 activated Bax
Cell dyes the antibody of Bax (6A7) and the antibody of Hsp60 after processing 24 hours with 5 μ M SK07 altogether.The fluorescence microscope analysis result is shown in Fig. 5 B.According to the result of Fig. 5 B as can be known, in control cells, do not observe the Bax of activation, but in the cell that SK07 processes, but show clearly, Nur77 and mitochondrion differential protein Hsp60 locate jointly.The dyeing that activates the Bax cell shows karyopycnosis usually, has shown that the apoptosis that SK07 induces is relevant with the activation of Bax, and SK07 has activated Bax, and the common location of Bax (6A7) and Hsp60 is associated with apoptosis.
6.3Bax activation be accompanied by the release of cytochrome C
SK07 (5 μ M) acted on cell 24 hours in the situation that LMB exists and lacks, and the antibody of Bax (6A7) and the antibody of cytochrome C and DAPI be transfect cell altogether, and the fluorescence microscope analysis result is shown in Fig. 5 C.According to the result of Fig. 5 C as can be known, cytochrome C is the same with its mitochondrion location, shows little dye speck.But cytochrome C is disperse and distributes in the cell of processing with the Bax immunostaining that activates.This phenomenon shows that SK07 has induced the activation of Bax relevant with the release of cytochrome C.LMB co-treatment cell, what stoped Nur77 goes out nuclear and Bcl-2 conformation change, almost completely suppresses Bax activation that SK07 induces and the release of cytochrome C.
6.4SK07 induced the Bcl-2 conformation change
SK07 (5 μ M) processed the NIH-H460 cell after 24 hours, and with antibody and the DAPI dyeing of Bcl2 (BH3) and BAX (6a7), the fluorescence microscope analysis result is shown in Fig. 5 D.According to the result of Fig. 5 D as can be known, the antibody of Bcl-2 (BH3) control cells that can not dye, but by force dyeing in can the cell after SK07 processes, and mostly and the dyeing of Bax (6A7) consistent.Copolymerization Jiao the analysis showed that, these the two kinds of albumen of most cells after SK07 processes are common location.Thereby illustrate, the Bcl-2 conformation change that SK07 induces and activation and the apoptosis of Bax are closely related.
As can be known from the above results, SK03 MEF and MEF Nur77-/-all induced obvious early apoptosis and non-viable non-apoptotic cell (Fig. 3 B) in the cell, therefore illustrate that the biological activity of SK03 depends on the non-Dependent of Nur-77-largely; And the apoptosis effect of SK07 with induce Nur77 to express and go out to examine relevant, by inducing Nur77 to express and going out nuclear translocation, mitochondrion is located, and Bcl-2 conformation change and activation Bax effectively activate the Nur77 approach, cause cytochrome C to discharge from mitochondrion, apoptosis-induced; And SK06 does not have above-mentioned effect.
In sum, alkannin derivant of the present invention navigates to Cytoplasm by inducing the Nur77 expression to reach from nucleus, thus the apoptosis of inducing tumor cell.Therefore alkannin derivant of the present invention can for the preparation of the treatment cancer with the medicine of orphan receptor Nur77 as action target spot.In addition, orphan receptor Nur77 can be used as action target spot for the screening alkannin derivant.
Claims (7)
1. an alkannin derivant is characterized in that for the preparation of the application of the medicine for the treatment of pulmonary carcinoma or cervical cancer, and described alkannin derivant is 5,8-diacetoxy-6-(1 ' acetoxyl group-4 ' methyl-3 ' pentenyl)-1,4-naphthoquinone; Described medicine with orphan receptor Nur77 as action target spot.
2. application as claimed in claim 1 is characterized in that, described medicine is used for the apoptosis of inducing tumor cell.
3. application as claimed in claim 2 is characterized in that, the apoptosis of described inducing tumor cell is by inducing the expression of orphan receptor Nur77.
4. application as claimed in claim 2 is characterized in that, the apoptosis of described inducing tumor cell is by inducing orphan receptor Nur77 to go out nuclear and being positioned on the mitochondrion.
5. application as claimed in claim 2 is characterized in that, the apoptosis of described inducing tumor cell is by inducing the conformation change of Bcl-2.
6. application as claimed in claim 2 is characterized in that, the apoptosis of described inducing tumor cell is by activating Bax.
7. application as claimed in claim 2 is characterized in that, the apoptosis of described inducing tumor cell is by causing that cytochrome C discharges from mitochondrion.
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| CN103494860A (en) * | 2013-10-10 | 2014-01-08 | 王喜军 | Method for preparing lithospermum extract and application of lithospermum extract |
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| CN102211997A (en) * | 2011-03-28 | 2011-10-12 | 上海交通大学 | Alkannin naphthazarine mother nucleus acetoxylation derivative as well as preparation method and application thereof |
| CN102424696A (en) * | 2011-09-05 | 2012-04-25 | 江西农业大学 | Shikonin amino deoxy glycosides and application thereof in preparation of antitumor medicines |
| CN113855661A (en) * | 2021-09-27 | 2021-12-31 | 温州医科大学附属第一医院 | Application of acetylshikonin in preparation of anti-cancer drugs |
| CN114933522B (en) * | 2022-06-17 | 2023-10-17 | 石河子大学 | Preparation method and application of shikonin compound |
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| CN1374288A (en) * | 2002-03-19 | 2002-10-16 | 中山大学 | Gronwell naphthaquinone derivative and its application in preparing anticancer medicine |
| CN101066919A (en) * | 2007-06-14 | 2007-11-07 | 上海交通大学 | Process of synthesizing shikolin dimethyl ether derivative |
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| CN1374288A (en) * | 2002-03-19 | 2002-10-16 | 中山大学 | Gronwell naphthaquinone derivative and its application in preparing anticancer medicine |
| CN101066919A (en) * | 2007-06-14 | 2007-11-07 | 上海交通大学 | Process of synthesizing shikolin dimethyl ether derivative |
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| CN103494860A (en) * | 2013-10-10 | 2014-01-08 | 王喜军 | Method for preparing lithospermum extract and application of lithospermum extract |
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