CN103655527A - Application of benzoquinone compound in inhibiting Wnt/beta-catenin signal pathway of melanoma cells - Google Patents
Application of benzoquinone compound in inhibiting Wnt/beta-catenin signal pathway of melanoma cells Download PDFInfo
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- CN103655527A CN103655527A CN201210428306.8A CN201210428306A CN103655527A CN 103655527 A CN103655527 A CN 103655527A CN 201210428306 A CN201210428306 A CN 201210428306A CN 103655527 A CN103655527 A CN 103655527A
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Abstract
The invention relates to application of a benzoquinone compound in inhibiting a Wnt/beta-catenin signal pathway of a melanoma cell, and a pharmaceutical composition or a supplement prepared by applying the benzoquinone compound. The benzoquinone compound comprises a structure shown in a compound :
the compound (I) wherein R1Is hydrogen radical or [ 4H4)CH3]nN is 1-an integer of 5; r2Is hydrogen radical or methoxy radical.
Description
Technical field
The invention relates to a kind of purposes of benzoquinone compound, particularly relevant for a kind of benzoquinone compound and medical composition thereof of check melanin oncocyte hypertrophy.
Background technology
What melanoma (melanoma) was malignant tumour of skin is a kind of, and grade of malignancy is the highest, easily shift, and for westerner, be the cause of death that in dermatosis, sickness rate holds pride of place.
Not yet perfect for the research for the treatment of melanoma medicine at present.Due to the poor effect of melanoma to chemotherapy and radiation cure, be accompanied by again the transitivity of height, if therefore can search out the natural food materials of auxiliary treatment of cancer, be current important problem.
Wnt/ β-catenin signal pathway can regulate and control the process of many cells, comprises the transfer (motility) of hypertrophy (proliferation), differentiation (differentiation), survival (survival), apoptosis (apoptosis) and cell.Approximately have 30% melanoma to can be observed the phenomenon of Wnt/ β-catenin signal pathway abnormal activation, therefore suppressing Wnt/ β-catenin signal pathway also becomes one of melanomatous strategy for the treatment of.
Summary of the invention
Therefore, one aspect of the present invention is to provide a kind of purposes of benzoquinone compound, is the Wnt/ β-catenin signal pathway that is applied to check melanin oncocyte.Structure shown in benzoquinone compound inclusion compound (I) or its reduction-state:
Compound (I)
Wherein, R
1for hydrogen base or [(C
4h
4) CH
3]
n, the integer that n is 1-5; R
2for hydrogen base or methoxyl group.
According to embodiments of the present invention, the configuration isomeric compound of benzoquinone compound inclusion compound (I).
In one embodiment of embodiment of the present invention, the configuration isomeric compound of compound (I) comprises structure as follows:
Wherein, R
1for hydrogen base or [(C
4h
4) CH
3]
n, the integer that n is 1-5.
In one embodiment of embodiment of the present invention, benzoquinone compound can be 2,3-dimethoxy-5-methyl isophthalic acid, 4-benzoquinone (2,3-Dimethoxy-5-methyl-1,4-benzoquinone).
In another embodiment of embodiment of the present invention, benzoquinone compound can be 2-methoxyl group-6-methyl isophthalic acid, 4-benzoquinone (2-methoxy-6-methyl-1,4-benzoquinone).
In an embodiment again of embodiment of the present invention, benzoquinone compound can be 5-methyl isophthalic acid-benzo [1,3] dioxane-4,7-diketone (5-methyl-1-benzo[1,3] dioxole-4,7-dione).
Another aspect of the present invention is to provide the purposes of a kind of benzoquinone compound (I), is the medicine that is applied to prepare check melanin tumor cell growth.
According to embodiments of the present invention, the pharmaceutical pack of preparing check melanin tumor cell growth containing the medicine of check melanin oncocyte hypertrophy, medicine, the medicine that check melanin oncocyte shifts of the medicine of check melanin Cell differentiation, the survival of check melanin oncocyte or facilitate the medicine of melanoma cell apoptosis.
In the embodiment of embodiment of the present invention, the medicine of check melanin tumor cell growth is oral agents, injection or skin preparations for extenal use.
Another aspect of the present invention is to provide a kind of β-catenin signal pathway of the Wnt/ for check melanin oncocyte and improves the medical composition of the skin carcinoma patient's condition, comprises with benzoquinone compound (I), machining object that the benzoquinone compound (I) of take is effective ingredient or the extract of natural goods, the pharmaceutically acceptable salt class of benzoquinone compound (I), the pharmaceutically acceptable solvate of benzoquinone compound (I) or above-mentioned combination in any.
In the embodiment of embodiment of the present invention, the dried powder that the machining object that the benzoquinone compound (I) of take is effective ingredient is Antrodia camphorata fermentation liquid or Antrodia camphorata fermentation liquid.
Another aspect of the present invention is to provide a kind of supplement, comprises that to take benzoquinone compound (I) be effective ingredient, and wherein the source of benzoquinone compound (I) is synthetic, Antrodia camphorata fermentation liquid or Antrodia camphorata fermentation liquid extract.
In the embodiment of embodiment of the present invention, supplement are liquid state, powder, lozenge, capsule or injection.
Of the present inventionly be to provide on the one hand more a kind of ubiquinone-0(Ubiquinone-0) purposes, be the Wnt/ β-catenin signal pathway that is applied to check melanin oncocyte.
According to above-mentioned, Wnt/ β-catenin signal pathway of benzoquinone compound (I) the energy check melanin oncocyte of this bright embodiment, is applicable to prepare check melanin oncocyte hypertrophy, differentiation, survival, the medicine that shifts or facilitate melanoma cell apoptosis, medical composition or supplement.
Accompanying drawing explanation
For above and other objects of the present invention, feature, advantage and embodiment can be become apparent, appended the description of the drawings is as follows:
Fig. 1 is the quantification figure of compound (I) on the impact of HaCaT, A2058, B16F1 and B16F10 cell strain survival rate that bestows various dose;
Tu2Wei Yi population of cells forms the impact that analytic process analysis of compounds (I) forms the group of high-transfer cell B16F10;
Fig. 3 is the impact of compound (I) on β-Catenin, p-β-catenin, GSK3 β and p-GSK3 beta protein content in B16F10 cell that utilizes west ink dot analytic process to detect to bestow various dose;
Fig. 4 utilizes west ink dot analytic process to detect the protein expression situation that can promote tumor development in melanin tumor cell;
Fig. 5, for utilizing cell immunofluorescent staining method, is used the one-level antibody of β-Catenin and the photo under 200 times of visuals field with fluorescence microscope;
Fig. 6 analyzes with the compound (I) of variable concentrations and processes, the impact on the β-catenin expression in Cytoplasm and core for utilizing with west ink dot method;
Fig. 7 is for detect the expression situation give β-catenin albumen after compound (I) and proteasome inhibitor MG132 simultaneously;
Fig. 8 is for detect the expression situation give β-catenin albumen after compound (I) and GSK3 beta inhibitor SB216763 simultaneously;
Fig. 9 gives the expression situation of β-catenin after compound (I) or protein synthesis inhibitor for being detected on different time points;
Figure 10 is for utilizing immuno-precipitation analysis under compound (I) exists, the GSK3 β in β-catenin and its degraded complex, the reciprocal action of Axin;
Figure 11 analyzes the quantification figure of the expression of β-catenin promoter with fluorescence reporter gene, wherein (a) part is β-catenin promoter Analysis result; (b) part is matched group;
Figure 12 analyzes under compound (I) exists with west ink dot method, the expression situation of β-catenin downstream target gene outcome;
Figure 13 utilizes the breach end-labelling of DNA end transferring enzyme to detect apoptotic result quantification figure;
Figure 14 is for utilizing the impact of flow cytometry analysis various dose compound (I) on cell kenel;
Figure 15 is for utilizing west ink dot method to detect the expression of cell death related protein matter under compound (I) exists;
Figure 16 is the impact of compound (I) on specific protein content in melanoma high-transfer cell that utilizes west ink dot analytic process to detect to bestow various dose;
Figure 17 is the effect that affects for the migration of melanoma high-transfer cell with the compound (I) of cell scratch analysis of experiments various dose, wherein (a) part is the photo of microscope under 200 times of visuals field, and (b) part is the quantized result of the migrating cell of (a) part counting;
Figure 18 analyzes the compound (I) of various dose for the effect that affects of melanoma high-transfer cell invasion and attack with cell Transfer Experiment, and wherein (a) part is the microphotograph after dyeing; (b) part is the quantification figure of the result of (a) part;
Figure 19 is for utilizing under west ink dot analytic process compound (I) existence, and the cell of the melanoma high-transfer cell of the siRNA transfection of detection β-catenin shifts relevant protein content to be changed;
Figure 20 is that matched group is transplanted the growth natural law of nude mice and the big or small comparative result of gross tumor volume with the B16F10 of injection compound (I) group, and wherein (a) part is the comparison photo of the gross tumor volume of tested nude mice; (b) part is the growth natural law of tested nude mice and the big or small comparison diagram of gross tumor volume; (c) part is the big or small comparison diagram of the tumor weight of tested nude mice;
Figure 21 is the microphotograph of the histopathological examination of tumour transplatation nude mice;
Figure 22 is the comparative analysis of compound (I) to the apoptosis rate of tumour transplatation nude mice;
Figure 23 is respectively the microphotograph of immunohistochemical staining of the intracellular various correlative protein expression of tumour transplatation nude mice;
Figure 24 is the west ink dot analysis of the relevant protein expression of the Wnt/ β-catenin signal path in transplanting nude mice tumor tissues;
Figure 25 is the quantitative analysis of the result of Figure 24.
The specific embodiment
According to the embodiment of the present invention, the source of benzoquinone compound (I), its reduction-state or its isomeric compound that can check melanin oncocyte Wnt/ β-catenin signal pathway can be chemosynthesis, or comes from the extract of natural goods, machining object or machining object.
Compound (I)
Wherein, R
1for hydrogen base or [(C
4h
4) CH
3]
n, the integer that n is 1-5; R
2for hydrogen base or methoxyl group.
It should be noted that, the benzoquinone compound (I) that comes from natural goods, machining object or machining object extraction, can produce the exchange of redox state, different structural isomerism thing (constitutional isomers) or configuration isomeric compound (configurational isomers) under its naturalness.And according to embodiments of the invention, no matter be synthetic compound (I), or obtaining natural goods or the machining object extract containing compound (I) by the fermentation liquid of Antrodia camphorata, the effect of its check melanin oncocyte Wnt/ β-catenin signal pathway all presents unanimously.Therefore the concrete structure of the mark compound (I) of the test described in following test example is R
1for hydrogen base, R
2for methoxyl group.Will be understood that, the effect being produced by pure compound (I) there will be equally take compound (I) as mainly containing in the mixture of effective constituent.
Following examples are for illustrating present disclosure particular aspects and helping prior art person to understand and implement present disclosure.But present disclosure category is not limited in these embodiment.
Embodiment
One, compound (I) check melanin oncocyte hypertrophy (Proliferation)
Test example 1-1: compound (I) check melanin oncocyte survival rate
The cell that test is used is mankind's normal skin cells (HaCaT), melanin tumor cell (A2058), mouse melanin tumor cell (B16F1) and murine melanoma high-transfer cell (B16F10).Each subject cell is incubated in appropriate culture medium, and incubator maintains 5%CO2,37 ℃ of constant temperature.Cell culture during to some, is removed culture fluid for centrifugal l0 minute with 900 revolutions (rpm), in experiment, within first 24 hours, changes culture fluid, and calculates cell number with cell counter (hemocytometer).
The mensuration of cell survival rate (viability) is with cell survival rate analysis (Method of transcriptional and translational assay; MTT assay) carry out.The step that the test method that cell survival detects proposes with reference to people such as Parada-Turska.
Subject cell kind is in 24 porose discs (2.5 * 10
5cells/well), after cell attachment, with PBS, clean every other day.Add the DMEM culture fluid containing 1% hyclone (FBS), allow after cell synchronization, add every other day the compound (I) of variable concentrations, be placed in 37 ℃, 5%CO
2cultivate 24 hours, with PBS, clean once, respectively add the MTT reagent of 400 μ l, react after 4 hours and add again the 10%SDS of 400 μ l, react 12 hours, extract the supernatant of 200 μ l out in 96 hole trace dishes, with 570 nm wavelength, survey light absorption value.
Please refer to Fig. 1, for the compound (I) of bestowing various dose is on suppressing the quantification figure of the impact of mankind's normal skin cells (HaCaT), melanin tumor cell (A2058), mouse melanin tumor cell (B16F1) and murine melanoma high-transfer cell (B16F10) survival rate, data result is with mean ± SD value representation, n=3, p<0.05.
The result demonstration of Fig. 1, experimental concentration is that the compound (I) of 0-20 μ M acted on subject cell after 24 hours, and to normal skin cells, HaCaT does not exert an influence, and the survival rate of A2058 has 70%; IC to B1 6F1
50concentration is between 15-20 μ M; High-transfer cell B16F10 is had to obvious poisoning, IC
50concentration is about 10 μ M.
The effect according to the known compound of the above results (I) with potent inhibition murine melanoma high-transfer cell strain activity.In following test example, " tumor cell " of use, " melanoma cell " term, as in situation about not specializing, all refer to murine melanoma high-transfer cell strain B16F10.
Test example 1-2: compound (I) inhibition tumor cell group forms
The impact group of melanoma high-transfer cell B16F10 being formed in order to understand compound (I), forms analytic process (Colony formation assay) with population of cells and observes it.
(a) part of Fig. 2 is for to be attached at cell after cellular matrix, with compound (I) reaction of variable concentrations 24 hours, then changes fresh containing after 10%FBS cell culture fluid into, cultivates the colony growth situation of 5 days; Result shows that compound (I) has the effect of group's formation of significant check melanin oncocyte.
(b) part of Fig. 2 is by the upper computer of population of cells's scanning shown in (a) part and with 70% ethanol, group is dissolved, uses the numerical value obtaining after light absorption value 540 nm measurements.Result can obviously find out, the melanoma cell group number form of test becomes to present the dosage correlation with compound (I).
Two, compound (I) check melanin oncocyte Wnt/ β-catenin control path
Start the approach that growth of cancer cells has many signals to transmit, involve the activation of multiple protein.Have 30% melanoma to can be observed the phenomenon of Wnt/ β-catenin signal pathway abnormal activation, therefore suppressing Wnt/ β-catenin signal pathway also becomes one of melanomatous strategy for the treatment of.In Wnt/ β-catenin signal pathway, relevant protein expression or activation can regulate and control the process of tumor cell, comprise the transfer (motility) of hypertrophy (proliferation), differentiation (differentiation), survival (survival), apoptosis (apoptosis) and cell.
Test example 2-1: compound (I) promotes β-Catenin phosphorylation
Glycogen synthesizes kinases-3 β (Glycogen synthase kinase-3 beta; GSK3 β) be a multi-functional kinase enzyme, in cell, not only participate in glycogen synthesis path also regulate and control many signal transmission, gene expression, cells survival or deathward etc. important machine turn.β-catenin is a kind of tumor correlated albumen matter that regulates and controls degraded, current studies show that β-catenin can be by GSK3 β phosphorylation, and the phosphorylation of β-catenin and canceration are closely related.
The compound (I) of analyzing (Western blotting) observation variable concentrations with west ink dot is below on the β-catenin in melanoma cell and the protein content of GSK3 β and the impact of phosphorylation reaction thereof.
Fig. 3 will cultivate the melanoma cell B16F10 of 24 hours after the compound (I) of concentration 0,5,10,15 and 20 μ M is processed, with β-catenin(p-β-catenin of β-catenin and phosphorylation in the ink dot analyzing and testing melanoma cell of west), the protein content result of the GSK3 β (p-GSK3 β) of GSK3 β and phosphorylation.β-actin is interior control group.
The step of west ink dot analysis is cell with the PBS washing secondary of 4 ℃, add and dissolve buffer solution (lysis buffer), with cell scraping blade (rubbr policeman), scrape broken and dissolved cell, this cytolysate is taken in the microcentrifugal tube of ml l.5, concussion l minute, in 12000 revolution centrifugal l0 minute, get supernatant again, with Bio-Rad protein assay kit, measure the concentration of protein, and remaining protein extraction liquid is placed in to-70 ℃ of preservations.Get the protein solution of l00 μ g, add the SDS/protein loading buffer of approximately two times of concentration of equal-volume to boil 10 minutes in 100 ℃, after cooling, protein example is put in each, voltage is adjusted to l00 volt, the transfer printing of protein electrophorese is run through and carried out to protein example by the time.Get a polyvinylidene fluoride film (poly (vinylidene fluoride); PVDF), after methanol soaks, put into transfer groove in 4 ℃ of refrigerator-freezers, with 600 microamperes of current, carry out the transfer of protein, after 4 hours, PVDF is taken out, be positioned in the PBS blocking solution of l%BSA, rock 2 hours, put again in the blocking solution of an antibody, in room temperature, slowly rock l hour, with PBST, wash pvdf membrane 3 times, each 3-5 minute, revolution is 100 rpm.According to different one-level antibody, extension rate, be placed on horizontal rotator in room temperature vibration 2 hours, with PBST, wash three times, then use cold light image instrument, pvdf membrane is inserted on ferrous metal dish with plastic grip, protein powder upward, the Chemiluminesent Substrate mixing with 1:1 is incorporated on film, then determines time of exposure according to the fluorescence power of different antibodies.
The result of Fig. 3 shows that the protein expression of β-catenin can be subject to concentration to be increased and reduce along with compound (I) concentration for the treatment of increases, but the expression of its phosphorylated protein (p-β-catenin) increases with concentration, presents dosage correlation; Otherwise, the situation that the amount of GSK3 β has combined thing (I) to suppress, its phosphorylated protein (p-GSK3 β) also increases and reduces with compound (I) concentration.According to above-mentioned, confirm that compound (I) can promote to contribute to suppress protein p-β-Catenin phosphorylation of tumor development, and regulate and control the degraded of non-phosphorylating β-Catenin and GSK3 β.
Test example 2-2: compound (I) suppresses Wnt/ β-catenin and regulates and controls correlative protein expression
Below with regard to thering is the protein of pointer in Wnt/ β-catenin control path, carry out west ink dot analysis again, to illustrate that compound (I) turns the effect machine of check melanin tumor high-transfer cell.
Please refer to Fig. 4, for promoting the protein expression situation of tumor development in melanin tumor cell, comprise Wnt-5a, Low-density lipoprotein receptor-related protein 6(LPR6), Axin, Dvl.β-actin is interior control group, and the step that west ink dot is analyzed is with shown in test example 2-1.
The result demonstration of Fig. 4, along with the dosage increase of compound (I), in melanin tumor high-transfer cell, Wnt-5a, LPR6, Axin, the Dvl protein expression of Wnt/ β-catenin control path are all suppressed, and present dosage correlation.
Test example 2-3: compound (I) suppresses β-Catenin and enters nuclear expression
According to above-mentioned, compound (I) can promote to contribute to suppress protein p-β-Catenin phosphorylation of tumor development, and regulates and controls the degraded of non-phosphorylating β-Catenin and GSK3 β.At present the unphosphorylated β-catenin of the known major part of research is positioned in core, below under existing at compound (I), the β-catenin position in cell is analyzed.
Fig. 5, for utilizing immunofluorescent staining (Immunofluenrence staining) method, is used the one-level antibody of β-Catenin and the photo under 200 times of visuals field with fluorescence microscope.
Immunofluorescent staining analysis is by after the compound of variable concentrations (I) irritation cell, adds the one-level antibody (1:250 dilution) of β-Catenin, is placed in 37 ℃, 5%CO
2 middle cultivation 2 hours.Add again fluorescence secondary antibody (1:200 dilution), finally add DAPI(1:1000 dilution) lucifuge is placed in room temperature 5 minutes, fluorescence microscopy Microscopic observation take pictures (200x) re-use mounting gel mounting.
The result demonstration of Fig. 5, compound (I) can suppress β-catenin and enter core, and reduces the protein expression of caryoplasm β-catenin, and evidence compound (I) can promote p-β-Catenin phosphorylation to suppress tumor development.
Test example 2-4: compound (I) promotes in core and Cytoplasm β-catenin degraded
Owing to being subject to the β-catenin of phosphorylation, can in Cytoplasm, not accumulate and be indexed into nucleus, more further analyze under compound (I) exists, whether there is the effect of promotion β-catenin degraded.
Fig. 6 analyzes with the compound (I) of variable concentrations and processes, the impact on the expression in core and in Cytoplasm of the β-catenin in Cytoplasm and core for utilizing with west ink dot method.Histone and β-actin distinguishes in core and cytoplasmic interior control group.
The result demonstration of Fig. 6, along with the concentration for the treatment of raising of compound (I), the β in Cytoplasm and core-catenin amount all significantly reduces.
Test example 3-1: compound (I) promotes the degraded of β-catenin
According to aforementioned known, compound (I) can suppress β-catenin and enter nuclear expression, and reduces the protein expression of caryoplasm β-catenin.For deterministic compound (I) turns the machine that affects of β-catenin protein content in Wnt/ β-catenin control path, to give the mode of given the test agent proteasome inhibitor (proteasome inhibitor), GSK3 beta inhibitor or protein synthesis inhibitor (protein synthesis inhibitor), with the variation of albumen quality in the ink dot method analysis and observation melanin tumor high-transfer cell of west.
Please refer to Fig. 7, for detecting, give after compound (I) and proteasome inhibitor MG132 the expression situation of β-catenin albumen simultaneously.β-actin is interior control group.The step that west ink dot is analyzed is with shown in test example 2-2.
As seen from Figure 7, under compound (I) individualism, the amount of β-catenin albumen in cell obviously reduces, and when proteasome inhibitor MG1 32 individualism, the amount of β-catenin albumen in cell is very high; Yet when giving compound (I) and proteasome inhibitor MG1 32, can make the protein content of β-catenin reply as small amount simultaneously.
Fig. 8 gives after compound (I) and GSK3 beta inhibitor SB216763 for detecting simultaneously, the expression situation of β-catenin albumen.
As seen from Figure 8, under compound (I) individualism, the amount of β-catenin albumen in cell obviously reduces, and when GSK3 beta inhibitor SB216763 individualism, the amount of β-catenin albumen in cell remains unchanged; Yet when giving compound (I) and GSK3 beta inhibitor SB216763, still can reduce the protein content of β-catenin simultaneously.
Fig. 9 gives compound (I) or protein synthesis inhibitor (Cycloheximide for being detected on different time points; CHX) after, the expression situation of β-catenin.
The result of Fig. 9 shows, with protein synthesis inhibitor CHX, processes cell 0,6,12,24 hours, and the amount of β-catenin in cell reduces along with increasing between processing; And also present similar trend with the test group that the compound (I) of 15 μ M is processed cell, infer that compound (I) is relevant with the degraded of promotion β-catenin.
Test example 3-2: compound (I) promotes that β-catenin is degraded to non-GSK3 β dependent form
Figure 10 is for utilizing immuno-precipitation (Immunoprecipitation; IP) analyze under compound (I) exists the GSK3 β in β-catenin and its degraded complex, the reciprocal action of Axin.
By Figure 10, can be learnt, under compound (I) exists, β-catenin is combined with Axin, and with the combination of GSK3 β not obvious, be illustrated under compound (I) existence, β-catenin is degraded to non-GSK3 β dependent form.
Test example 4-1: compound (I) suppresses β-catenin transcriptional activity
Because compound (I) has the effect that promotion β-catenin degrades, carry out β-catenin promoter Analysis (TCF/ β-catenin reporter assay), can learn the gene expression situation in β-catenin downstream.
(a) part of Figure 11 is with fluorescence reporter gene, to analyze the quantification figure of the expression of β-catenin promoter.Result demonstration, along with the dosage increase of compound (I), the β-catenin promoter expression in B16F10 cell decreases, and compound (I) can suppress β-catenin transcriptional activity.
(b) part of Figure 11 is for adding GSK3 beta inhibitor LiCL(Lithim Chroride) as a control group, with fluorescence reporter gene, analyze the quantification figure of the expression of β-catenin promoter.Result shows, only add GSK3 beta inhibitor LiCl can increase β-catenin promoter intensity, yet the dosage of processing along with compound (I) increases, and adds LiCl and compound (I) simultaneously, and compound (I) still shows the effect that can suppress β-catenin transcriptional activity.
Test example 4-2: compound (I) suppresses β-catenin downstream gene expression
According to above-mentioned, compound (I) can reduce the expression of β-catenin promoter, the gene that can know its lower cursor by inference as promoted c-myc albumen, the cyclin cyclin D1 of tumor proliferation, the albumen of inhibited apoptosis should be affected and reduce as the expression of survivin (survivin).
Figure 12 analyzes under compound (I) exists with west ink dot method, the expression situation of β-catenin downstream target gene outcome.Result demonstration, the concentration of processing along with compound (I) increases, and the expression of c-myc, cyclinD1 and survivin all reduces thereupon.
Two, compound (I) promotes melanoma cell apoptosis (Apoptosis)
Test example 5-1: compound (I) promotes melanoma cell apoptosis
Known from test example 1-1 and test example 1-2, compound (I) has the effect that reduces melanoma cell survival rate, below will understand compound (I) with DNA specificity fluorescent staining and the analysis of cell kenel and whether promote apoptosis.
Figure 13 is breach end-labelling (the terminal-deoxynucleotidyl transferase mediated nick end labeling that utilizes DNA end transferring enzyme; TUNEL) chromosome breakage situation in analyzing and testing cell, to detect apoptotic result quantification figure.Result by Figure 13 shows, along with the concentration for the treatment of of compound (I) improves, apoptotic degree is higher.
Refer again to Figure 14, for utilizing flow cytometer (flow cytometry) to analyze the variation of the lower cell kenel of compound (I) existence of various dose, to observe, whether have the situation of cell death to produce.Result by Figure 14 shows, along with the concentration for the treatment of of compound (I) improves, it is higher that cell presents dead ratio.
Test example 5-2: compound (I) promotes the content that promotes melanoma cell apoptin in cell
Apoptotic pathways involves a series of related protein and regulates.For example, Procaspase-9, Procaspase-3 are the protein caspase-9 of cell death inducing and the predecessor of caspase-3, in tumor cell, the activate mechanism of Procaspase-9 and Procaspase-3 is destroyed, so the Procaspase-9 finding in most of cancerous cell, Procaspase-3 amount is all higher than normal cell.
The physiological function of Bcl-2 is inhibited apoptosis, and Bax participates in cells apoptosis.In addition,, when cytotoxic drug inducing apoptosis, the rising of nuclear membrane Bax amount and the degraded of nucleoprotein PARP are proportionate.P53 is apoptosis activating gene, and its expression by regulation and control Bc1-2 and Bax gene promotes apoptosis.
Previous test example has confirmed that compound (I) has the apoptotic effect of promotion, therefore can, by the relevant protein content of discussion apoptotic pathways and the dependency of compound (I) dosage, further differentiate the related mechanism that it suppresses tumor development.
Figure 15 is for utilizing west ink dot method to detect the expression of cell death related protein matter under compound (I) exists.
The result demonstration of Figure 15, the concentration of processing along with compound (I) increases, and in tumor cell, the protein of inhibited apoptosis comprises that the content of Procaspase-9, Procaspase-3, ProPARP and Bcl-2 all declines thereupon, presents dosage correlation; On the contrary, the PROTEIN B ax and the p53 that participate in cells apoptosis improve and increase with compound (I) concentration for the treatment of.
The above results confirms that compound (I) has and brings out apoptotic ability, and follow the protein expression of p53 to increase, ratio (ratio) raising of Bax/Bcl-2 and the cracking of caspase-3, caspase-9 and PARP.By the protein expression that promote to participate in cells apoptosis, and the protein expression that reduces inhibited apoptosis in cell, reach the effect of inhibition tumor cell growing multiplication.
Three, compound (I) check melanin oncocyte shifts (Metastasis)
Neoplasm metastasis is that tumor cell is diffused into long-range organ-tissue from original position via a succession of step, forms the process of another new tumor mass.Cancer metastasis comprises the process that starts infiltration capability (invasion), oozes out (extravasation) and transfer (metastasis).At invasion procedure, tumor cell lost cell and intercellular cohesiveness, obtain mobility; Then the cell oozing out enters blood and lymph is system, and last cancer cell metastasis becomes new tumor cell to target location.
Test example 6-1: compound (I) reduces MMPs in melanoma cell expresses
In the process of cancer cell metastasis, be conventionally accompanied by extracellular matrix (Extracellular matrix; ECM) decomposition is matrix metalloproteinase (Matrix metalloproteinase and mainly play the part of the role who decomposes ECM; MMPs).Tumor cell can be secreted MMPs in a large number, and much research points out that MMP-2 and MMP-9 play an important role in neoplasm metastasis process.In addition the expression of matrix metallo-proteinase inhibitor TIMP-1, TIMP-2 can suppress the activity of MMPs, reaches inhibition tumor cell invasion and the effect of shifting.
Therefore, below with regard to the above-mentioned cell protein relevant to neoplasm metastasis, carry out west ink dot analysis, with the mechanism of action that illustrates that compound (I) shifts check melanin oncocyte.
Please refer to Figure 16, for utilize compound (I) that west ink dot analytic process detects compound (I) various dose of bestowing concentration 0,2.5,5,7.5 μ M in melanoma high-transfer cell to the impact of shifting relevant protein content.β-actin is interior control group.
The result of Figure 16 shows, along with the dosage of compound (I) increases, in melanoma high-transfer cell the expression of MMP-2, MMP-9 suppressed, and present dosage correlation; On the contrary, along with the dosage increase of compound (I), in melanoma high-transfer cell, the expression of TIMP-1, TIMP-2 all increases thereupon.
According to the known compound of the above results (I), can, by the activity that promotes MMP inhibitor TIMP-1, TIMP-2 expression inhibiting MMPs, and reach inhibition tumor cell, invade and the effect of shifting.
Test example 6-2: compound (I) check melanin oncocyte migration (migration)
During cancer cell metastasis, if high-transfer cell strain, when iuntercellular is damaged, adds that cell itself discharges a large amount of proteases and promotes extracellular matrix degradation, can increase the migration situation of cancerous cell, cause the transfer of cancerous cell.
(a) part of Figure 17, for analyzing the compound (I) of various dose for the effect that affects of melanoma high-transfer cell migration with cell scratch test (Wound scratching assay).
The method of observation of cell migration (Wound migration) is by B16F10(3 * 10
5) plant in 12 porose discs, with PBS, clean 2 times every other day, change into containing 1%FBS cell culture fluid, every other day to scrape behind a space in porose disc bottom, remove cell culture fluid and wash 2 times with PBS, add containing the cell culture fluid of 1%FBS and the compound of variable concentrations (I), the situation of the observation of cell of taking pictures respectively at 0 and 24 hour migration, after reaction finishes, with 75% ice alcohol fixation 30 minutes, after ethanol volatilizees completely, with GiemsaStain, dye, under microscope bottom, (200X) selects the cell concentration (Lin et al., 2008) of three visual field counting migrations at random.Cell scratch test take do not add compound (I) for matched group, add 2.5,5,7.5 μ M compound (I) as test group processing cell 24, hour after, observe the quantized result of compound (I) to melanoma high-transfer cell B1 6F1 0 migration, data result is with mean ± SD value representation, n=3.
As shown in (b) part of Figure 17, the cell concentration of counting migration is under the compound (I) of low concentration (2.5 μ M) exists, after 24 hours, still there is cell migration, and at 5 μ M, when above, can significantly suppress the transfer ability of cell when the concentration of compound (I).
Test example 6-3: compound (I) check melanin oncocyte is invaded (invasion)
When Evil tumor is prepared to shift, tumor cell is by destroying extracellular matrix(ECM) and then invade the basement membrane (basement membrane) of organizing, be called intrusion (invasion), be the participation by metalloprotein enzyme group MMPs, promote the ability that cancer cell is invaded.
Please refer to Figure 18, for analyzing the compound (I) of various dose with cell Transfer Experiment (Transwell invasion assay) for the effect that affects of melanoma high-transfer cell invasion and attack, wherein (a) is partly the microphotograph after the dyeing of cell Transfer Experiment; (b) part is the quantification figure of the result of (a) part, and data result is with mean ± SD value representation, n=3.
Cell Transfer Experiment is the composition that utilizes Matrigel to be extracellular matrix, environment between can analog cell, cell in BD MatrigelTM Invasion Chamber is subject to the factors stimulated growth in Matrigel matrix, cell can be secreted some substrate degradation enzymes, Matrigel is decomposed, the transfer of cell can be attracted, if cell transfer ability is strong, after dyeing, cell can be on filter membrane, observed.
Test method is that the DMEM that adds 800 μ l to contain 10%FBS in transfer table (Transwell) works as chemoattractant, again the cell of 500 μ l (1 * 105) is planted into transfer table, add respectively the DMEM culture fluid and 0.5,1 without FBS, the compound (I) of 2 μ M to move into 37 ℃, 5%CO
2incubator cultivate 20 hours, with Giemsa dyeing, after 15 minutes, with optical microscope, under 200 times of visuals field, observe and take pictures.It is representative that each group in each experiment all be take the meansigma methods of transfer table of 3 repetitions.
The method that cell Transfer Experiment quantizes is to choose at random 9 visuals field that are arranged in transfer table film lower surface, and calculates the total cell number in each visual field; The index that the meansigma methods of 9 visual field cell number moves for the cell chemotaxis of each transfer table, it is representative (Rose et al., 2005) that each group in each experiment all be take the meansigma methods of transfer table of 3 repetitions.
By the microphotograph observation of cell transfer scenario of Figure 18 (a) part, can obviously see along with compound (I) concentration increases, can significantly reduce the transfer of cancerous cell.B16F10 is a kind of high transfer melanoma cell strain, therefore in photo, can observe matched group has many cells to be subject to the attraction of chemoattractant (10%FBS cell culture fluid), transfer is passed through the Matrigel of analog cell epimatrix to filter membrane, and in the test group of compound (I) that adds 2.5,5,7.5 μ M, along with the increase of compound (I) concentration reduces the transfer of melanoma cell B16F10 significantly.
In addition, (b) in part, show that the cell concentration of branch on count significantly reduces along with the concentration increase of compound (I) ability shifting.
According to the above results, can learn that compound (I) can make MMP-9, MMP-2 expression reduce and reduce the expression that cancerous cell is invaded.
Test example 7: the invasion and attack of compound (I) check melanin oncocyte are expressed with metastasis related protein
In melanoma high-transfer cell, have β-catenin accumulation and downstream protein cmyc, cyclin D1, survivin etc. and express the phenomenon increasing, siRNA (small interfering RNA by β-catenin, siRNA) make the gene delection of β-catenin, cell invasion and transferance that the associated protein of observable compound (I) regulation and control Wnt/ β-catenin signal path participates in.
Figure 19 is for utilizing under west ink dot analytic process compound (I) existence, and the protein content that the transfer of the melanoma high-transfer cell of the siRNA transfection of detection β-catenin is relevant changes.β-actin is interior control group.Result shows, compares with the cell of untransfected, less with the accumulation of the melanoma high-transfer cell of β-catenin siRNA transfection, and the expression that adds compound (I) to process more for its downstream metastasis related protein has inhibition.
Four, compound (I) suppresses nude mice melanin tumor growth
According to the above results, known compound (I) is via the effect that suppresses the growth of Wnt/ β-catenin signal path with check melanin tumor high-transfer cell, transfer, intrusion, below will further verify its effect in vivo test (in vivo) mode.
Test example 7-1: compound (I) suppresses the melanin tumor growth of tumour transplatation nude mice
In vivo test is carried out with nude mice heterotopic transplantation tumor pattern (nude mice xenograft tumor model).Laboratory animal is purchased from the BALB/c-nu germline nude mice of TaiWan, China country animal center, raises in sterile board, and light application time is 7:00-19:00, and the air of filtration and water and the feedstuff of sterilizing were provided, and makes it edible voluntarily.Adopt the maternal instinct nude mice of 8-10 week maturation to test.
By melanoma high-transfer cell B16F10(2x10
6/ 100 μ l) in hypodermic mode, implant 60 nude mice back one sides, approximately 10 days nude mice backs are divided into arbitrarily 4 groups after growing the tumor of 1-3 mm, again by 3 groups of compounds (I) of giving with various dose, observe situation the preservation of taking a picture that its tumor generates every day, gross tumor volume of every three staggering amount, and the formula that the account form of volume proposes according to people such as Osborne in 1987.
Experimental animal is divided into matched group (without any processing), group of solvents (an injection normal saline solution) and injection compound (I) group (dosage is 2 mg/Kg), injection every day, by animal under normal conditions, bring up, film recording, respectively to organize the situation of laboratory animal tumor growth.
Please refer to Figure 20, (a) part is the photo that matched group and the B16F10 of injection compound (I) group transplant the gross tumor volume of nude mice; (b) part is that matched group is transplanted the growth natural law of nude mice and the big or small comparison diagram of gross tumor volume with the B16F10 of injection compound (I) group; (c) part is the comparison diagram that matched group and the B16F10 of injection compound (I) group transplant the tumor weight of nude mice.
(a) part by Figure 20 can be found out, the transplanting nude mice of injection compound (I), and its gross tumor volume is obviously little compared with matched group.
Also the transplanting nude mice that can obviously find out injection compound (I) in the comparison diagram of (b) part of Figure 20, its gross tumor volume is little compared with matched group.
In the comparison diagram of (c) part of Figure 20, show that the tumor weight between matched group and injection group has significant difference.
According to the above results, compound (I) has the direct effect that suppresses tumor tissue growth.
Test example 7-2: the histopathological examination comparison of tumour transplatation nude mice
The histopathological examination of tumour transplatation nude mice is to carry out with pathology H & E staining.By tumor and internal organs with paraffin-embedded tissue, and microtome is cut into the thickness slide of 5 mm, again through de-cured, backwater, renaturation etc., with intermediate water, clean and dye 30 to 50 seconds with Hematoxylin solution, again to react 20 seconds in 0.1% acetic acid water, and rinse with intermediate water, finally with Eosin, dye 100 seconds again, and with Histoclad mounting.
Figure 21 is the microphotograph of histopathological examination.Do not inject as can be seen from FIG. the matched group of compound (I) and transplant nude mice, and its tumor cell presents the mitosis state (enlargement ratio 400X) of height; On the contrary, within 2 mg/Kg/ days, to inject the injection group of compound (I), transplant nude mice, its tumor cell presents low mitosis state (enlargement ratio 400X).The above results shows the effectively division of inhibition tumor cell of compound (I) of the suitable dosage of direct injection.
Test example 7-3: injection compound (I) is on the apoptotic impact of tumour transplatation nude mice
Figure 22 is for bestowing the apoptotic comparative analysis of compound (I) to tumour transplatation nude mice, with the breach end-labelling (TUNEL) of DNA end transferring enzyme, carries out.
Result by Figure 22 shows, injects the nude mice of compounds (I) with the dosage of 2 mg/kg/ days, and the degree of its apoptosis of tumor cells is apparently higher than the control group of not injecting, and the difference of two groups has statistical meaning.
Test example 7-4: injection compound (I) transmits the impact of the expression of associated protein on signal in tumour transplatation nude mice cell
The expression of the intracellular signal transferrin of tumour transplatation nude mice carries out with immunohistochemical staining analysis (Immunochemistry Staining) method.This test detects for the correlative protein expression of Wnt/ β-catenin signal path.
Shown in Figure 23 (a)-(d), be respectively the microphotograph of immunohistochemical staining of the intracellular various correlative protein expression of tumour transplatation nude mice, wherein (a) is that the β-catenin of control group and injection group expresses situation; (b) be that cyclin D 1 expresses situation; (c) for survivin expresses situation; (d) for MMP-9 expresses situation; The right be the comparison after the result quantification shown in (a)-(d).
Result by Figure 24 shows, compare with control group, within 2 mg/Kg/ days, to inject the injection group of compound (I), transplant nude mice, in its tumor tissues, promote that Wnt/ β-catenin signal path protein expression of tumor development is all suppressed, and the difference between two groups all has statistical meaning.
Figure 24 is the west ink dot analysis of the relevant protein expression of the Wnt/ β-catenin signal path in transplanting nude mice tumor tissues.As can be seen from FIG., do not give compound (I) and transplant nude mice matched group, in its tumor cell, promote that the albumen quality of tumor development is all higher, for example β-catenin, c-myc, cyclin D1, survivin, ProPARP and Bcl-2, promote apoptotic protein to measure less as p53, Bax; On the contrary, give to inject for 2 mg/Kg/ days the injection group transplanting nude mice of compound (I), in its tumor tissues, promote that the albumen quality of tumor development is all suppressed, promote apoptotic albumen quality relatively to promote.
Figure 25 is the quantitative analysis of Figure 24, and the expression of control group of take is 100%, analyzes the degree that the expression of injection group is suppressed or promotes.Wherein, compare each albumen content in the tissue of control group and injection group, no matter which kind of albumen, the difference between its two groups all has statistical meaning.In addition, from Bax/Bcl2 ratio, the ratio of injection group is significantly higher than control group, and representation compound (I) can promote tumor tissue cell's apoptosis.
According to above-mentioned, the compound of the embodiment of the present invention (I) and medical composition thereof, can be in order to be applied to hyperplasia, melanoma cell transfer, the promotion melanoma cancer cell-apoptosis of the check melanin tumor via the signal transmission of adjusting Wnt/ β-catenin signal path or to be applied to the preparation medicine relevant with above-mentioned functions.
Therefore, the mixture energy inhibition tumor cell hypertrophy that compound (I) or the compound (I) of take are active ingredient, so the mixture that compound (I) or the compound (I) of take are active ingredient can be provided as with medical composition form the medicine for the treatment of.In addition, can also supplement form be provided as health product.
For example, in the medical composition that contains compound (I), compound (I) is to exist with free state form or with pharmaceutically acceptable salt form.In addition, the medical composition that contains compound (I), can more comprise one or more pharmaceutically acceptable supporting agent, for example excipient, solvent, emulsifying agent, suspending agent, distintegrant, binding agent, stabilizing agent, antiseptic, lubricant, absorption delay agent and liposome.The medical composition that contains compound (I), can be made into an applicable types of administration according to actual needs, and the form of oral administration medicine supplying, injection or externally used coating agent for example, in order to inhibition tumor cell hypertrophy.
The benzoquinone compound of the embodiment of the present invention (I) for effective ingredient make medical composition or supplement, the source of its benzoquinone compound can be synthetic, Antrodia camphorata fermentation liquid or Antrodia camphorata fermentation liquid extract, and supplement can be liquid state, powder, lozenge, capsule or injection form.
Although the present invention discloses as above with embodiment; so it is not in order to limit the present invention; anyly have the knack of this skill person; without departing from the spirit and scope of the present invention; when being used for a variety of modifications and variations, so the scope that protection scope of the present invention ought define depending on accompanying claims is as the criterion.
Claims (10)
1. benzoquinone compound is applied to a purposes for Wnt/ β-catenin signal pathway of check melanin oncocyte, the reduction-state of the structure shown in wherein said benzoquinone compound inclusion compound (I) or compound (I):
Compound (I)
Wherein, R
1for hydrogen base or [(C
4h
4) CH
3]
n, the integer that n is 1-5; R
2for hydrogen base or methoxyl group.
2. benzoquinone compound is applied to the purposes of Wnt/ β-catenin signal pathway of check melanin oncocyte as claimed in claim 1, and the configuration isomeric compound of wherein said benzoquinone compound inclusion compound (I), has structure as follows:
Wherein, R
1for hydrogen base or [(C
4h
4) CH
3]
n, the integer that n is 1-5.
3. described benzoquinone compound is applied to the purposes of Wnt/ β-catenin signal pathway of check melanin oncocyte as claimed in claim 1, wherein said benzoquinone compound is 2,3-dimethoxy-5-methyl isophthalic acid, 4-benzoquinone, 2-methoxyl group-6-methyl isophthalic acid, 4-benzoquinone or 5-methyl isophthalic acid-benzo [1,3] dioxane-4,7-diketone.
4. a purposes for benzoquinone compound as claimed in claim 1, is the medicine that is applied to prepare check melanin tumor cell growth.
5. the purposes of benzoquinone compound as claimed in claim 4, the wherein said pharmaceutical pack of preparing check melanin tumor cell growth containing the medicine of check melanin oncocyte hypertrophy, medicine, the medicine that check melanin oncocyte shifts of the medicine of check melanin Cell differentiation, the survival of check melanin oncocyte or facilitate the medicine of melanoma cell apoptosis.
6. the purposes of benzoquinone compound as claimed in claim 4, the medicine of wherein said check melanin tumor cell growth is oral agents, injection or skin preparations for extenal use.
7. for Wnt/ β-catenin signal pathway of check melanin oncocyte, improve a medical composition for the skin carcinoma patient's condition, machining object or the extract of natural goods, the pharmaceutically acceptable salt class of described benzoquinone compound, the pharmaceutically acceptable solvate of described benzoquinone compound or above-mentioned combination in any that to comprise with benzoquinone compound claimed in claim 1, the described benzoquinone compound of take be effective ingredient.
8. β-catenin the signal pathway of the Wnt/ for check melanin oncocyte as claimed in claim 7 and improve the medical composition of the skin carcinoma patient's condition, wherein said machining object is the dried powder of an Antrodia camphorata fermentation liquid or Antrodia camphorata fermentation liquid.
9. supplement, comprise and take benzoquinone compound claimed in claim 1 as effective ingredient, and the source of wherein said benzoquinone compound is synthetic, Antrodia camphorata fermentation liquid or Antrodia camphorata fermentation liquid extract.
10. supplement as claimed in claim 9, wherein said supplement are liquid state, powder, lozenge, capsule or injection.
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