CN107198688A - Application of the traditional Chinese medicine monomer lycorine in treatment breast cancer medicines are prepared - Google Patents
Application of the traditional Chinese medicine monomer lycorine in treatment breast cancer medicines are prepared Download PDFInfo
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Abstract
The present invention discloses application of the traditional Chinese medicine monomer lycorine (Lycorine) shown in a kind of formula (I) in the medicine for preparing treatment breast cancer relevant disease.The invention discloses growth, Clone formation, invasion and attack and the migration that lycorine shown in formula (I) can significantly inhibit a variety of breast cancer cells in vitro and in vivo, and Apoptosis can be caused and suppress the related signal path of migration, there is certain prospect in breast cancer treatment.
Description
Technical field
The present invention relates to pharmaceutical technology field, and in particular to application of the traditional Chinese medicine monomer lycorine in the medicine for preparing treatment breast cancer disease.
Background technology
Breast cancer (Breast cancer) is the frequently-occurring cancer of women, and its incidence of disease ranks the first in all malignant tumours, seriously endangers women's health.The morbidity and mortality of breast cancer still rose day by day now, according to U.S. 2015《Journal of Clinical Oncology》Newest report, in the U.S., breast cancer incidence ranks the first in female cancer, and about 232,670 women suffer from breast cancer, and the incidence of disease is about 29%, and fatal rate is 15% or so, is number two in all kinds of cancers of women.Summarized according to Chinese tumour annual report in 2015, breast cancer is in women in 2014, and the incidence of disease, which is still, to rank first place, and fatal rate is number four；Newly it is published according to 2014 medical colleges of Fudan University《Lancet》On data display, from nineteen nineties, the Female Breast Cancer in China incidence of disease is just with the increase of twice of trend, particularly flourishing urban area, and in the world as one of fertility-rate very low city Shanghai and Beijing, its incidence of disease is also high.
According to it evolution occurs for breast cancer, can be divided into DCIS, invasive ductal carcinoma and ILC etc..Research shows that breast cancer orthotopic tumour is that the growth of DCIS is not the main cause of tumor lethal, and the reason for more than 90% patient with breast cancer is final dead is to there occurs metastases, such as breast cancer Lung metastases, Bone tumour and hepatic metastases.Along with breast cancer so rate occurred frequently, the means for the treatment of breast cancer also constantly tend to be ripe and perfect always.For the type and developing stage of different breast cancer, main treatment means have operative treatment, radiotherapy, chemotherapy, hormone therapy and targeted therapy etc..So in these treatment means, chemotherapy plays highly important effect always.The development experience of the breast cancer clinical chemotherapy scheme course of single medicine chemotherapy and combined chemotherapy.US National is integrated in the therapeutic scheme of breast cancer that cancer net was delivered in 2015, anthracycline, yew alkanes, and this kind of wide spectrum chemotherapeutics such as alkylating agent is still as front-line chemotherapeutic agents, and drug combination in different ways treats the breast cancer patients of different phase.But this kind of medicine has two very big defect, one is that toxic side effect is big, and long-term use can cause patient is different degrees of to obtain cardiac toxic, hepatotoxicity wind agitation and neurotoxicity etc.；Two be to be also easy to produce resistance, Treated with Chemotherapeutic Drugs thing is produced resistance to be unable to reach drug effect, so limiting the long-term use of such medicine.Therefore, the good chemotherapeutics of new safe and curative effect is developed very urgent.Traditional Chinese medicine ingredients are increasingly taken seriously in anti-tumor aspect, and Chinese medicine resource enriches, and with low cost, toxic side effect is small and the curative effect feature such as substantially, have had many Chinese herbal medicine monomers to be used in oncotherapy now, and obtain good therapeutic effect.So small molecule monomer of the exploitation with treatment breast cancer disease is significant in Chinese herbal medicine.
The content of the invention
The present invention proposes the application of lycorine monomeric compound or its hydrate or pharmaceutically acceptable salt or pharmaceutically acceptable carrier in treatment breast cancer malignant tumor medicine is prepared shown in a kind of formula (I), shown in the structure such as formula (I) of the lycorine monomeric compound；
Short-tube lycoris alkali cpd shown in formula (I) is a kind of Chinese herbal medicine monomer, English name：Lycorine, molecular formula：C16H17NO4, molecular weight：287.3, CAS accession number：476-28-8.
In present invention application, formula (I) short-tube lycoris alkali cpd has following effect in vitro and in vivo：The propagation that breast carcinoma cell strain can be suppressed, the Clone formation that breast cancer cell can be suppressed, the invasion and attack for suppressing the migration of breast cancer cell, suppressing breast cancer cell, the transfer for suppressing breast cancer cell, the apoptosis of a variety of breast cancer cells of promotion, the expression for suppressing breast cancer cell migration GAP-associated protein GAP and then the signal path for suppressing migration correlation.
In present invention application, formula (I) short-tube lycoris alkali cpd can suppress the propagation of a variety of breast carcinoma cell strains in vitro and in vivo, including suppress breast carcinoma cell strain MDA-MB-231,4T1, MCF-7 and T47D propagation.
In present invention application, formula (I) short-tube lycoris alkali cpd can suppress the Clone formation of breast cancer cell in vitro and/or in vivo.
In present invention application, formula (I) short-tube lycoris alkali cpd can suppress breast cancer cell MDA-MB-231 and 4T1 migration in vitro and/or in vivo.
In present invention application, formula (I) short-tube lycoris alkali cpd can suppress breast cancer cell MDA-MB-231 and 4T1 invasion and attack in vitro and/or in vivo.
In present invention application, formula (I) short-tube lycoris alkali cpd can suppress breast cancer cell MDA-MB-231 transfer in vitro and/or in vivo.
In present invention application, formula (I) short-tube lycoris alkali cpd can promote the apoptosis of a variety of breast cancer cells in vitro and/or in vivo, it is preferable that can promote the apoptosis of MDA-MB-231,4T1, MCF-7 and T47D breast cancer cell in vitro and/or in vivo.
In present invention application, formula (I) short-tube lycoris alkali cpd can significantly inhibit the expression that breast cancer cell migrates GAP-associated protein GAP in vitro and/or in vivo, and then suppress the related signal path of migration.Specifically, it is suppression Src/FAK signal paths.
In present invention application, hydrate or pharmaceutically acceptable salt or pharmaceutically acceptable carrier of formula (I) short-tube lycoris alkali cpd etc. equally have and formula (I) lycorine identical inhibition.
In present invention application, formula (I) short-tube lycoris alkali cpd can be used alone or is used in combination with other drugs.
Suppress the method for Cells Proliferation of Human Breast Cancer in vitro and/or in vivo the invention also provides formula (I) short-tube lycoris alkali cpd.As formula (I) lycorine can suppress a variety of breast carcinoma cell strain propagation and Clone formation, and the growth of suppression MDA-MB-231 and 4T1 cells in vivo.
The method that short-tube lycoris alkali cpd shown in proposition formula (I) of the present invention suppresses breast cancer cell transfer in vitro and/or in vivo.As formula (I) lycorine can suppress the Lung metastases of breast cancer cell line MDA-MB-231 and 4T1 cell migration and MDA-MB-231 cells in vitro.
Proposition formula (I) short-tube lycoris alkali cpd induced breast cancer Apoptosis method of the present invention.Formula (I) lycorine is by apoptosis-induced correlative protein expression such as CL.Caspase3, CL.PARP etc., so as to promote a variety of breast carcinoma cell strain apoptosis.
The present invention obtains monomer formula (I) lycorine by cytotoxicity screening, and the monomer can substantially suppress the migration of breast cancer cell, and the signal path that can effectively block Src/FAK to mediate in vitro and in vivo.
The present invention is screened by high-throughout cytotoxicity, and screening has obtained traditional Chinese medicine monomer lycorine (Lycorine) from a variety of Chinese herbal medicine monomers and micromolecular compound.Lycorine (Lycorine can be abbreviated as Lyc) is a kind of alkaloid that separation is extracted from the bulb of amrallid short-tube lycoris, and wherein short-tube lycoris is a kind of herbaceos perennial for being distributed widely in China.Many researchers have found that lycorine has many pharmacy functions in recent years, including anti-inflammatory sterilization, anti-malarial, antiviral etc., study less in anti-tumor aspect, available data shows lycorine in vitro to there is preferable Inhibit proliferaton effect in the cells such as leukaemia, prostate cancer and osteosarcoma, but lycorine in vivo do not study always by anti-breast cancer propagation and transfer effect.Because it contains representational Fourth Ring skeleton, and with good biological safety, increasing chemist is also transformed on lycorine parent, and synthesizes a variety of antineoplastic compounds.Therefore, all there is far-reaching medical research to be worth in pharmaceutical chemistry and biological study.Although breast cancer has had the therapeutic scheme of comparative maturity at this stage, still there is more defect.Compound lycorine employed in present invention application, experiment proves it by inducing mammary cancer cell-apoptosis to suppress the growth of tumor of breast in vivo and in vitro；By blocking the associated signal paths of Src/FAK mediations and then hindering the Lung metastases of breast cancer.Therefore, short-tube lycoris alkali cpd is in terms of breast cancer is treated, with very big DEVELOPMENT PROSPECT.
The invention also provides a kind of pharmaceutical composition, it includes formula (I) short-tube lycoris alkali cpd or its hydrate or pharmaceutically acceptable salt or pharmaceutically acceptable carrier.
The invention also provides the application of formula (I) short-tube lycoris alkali cpd or its hydrate or pharmaceutically acceptable salt or pharmaceutically acceptable carrier in treatment propagation and the stronger malignancy disease medicine of transfer ability is prepared.
Wherein, the malignant tumour is the malignant tumours such as lung cancer, colon cancer, the cancer of the brain, cutaneum carcinoma, carcinoma of urinary bladder, kidney.
Brief description of the drawings
Fig. 1 show inhibition of formula (I) lycorine to various breast carcinoma cell strain proliferation activities；Fig. 1 (A) represents the molecular structure of lycorine；Fig. 1 (B) represents that formula (I) lycorine suppresses a variety of breast carcinoma cell strain MDA-MB-231,4T1, MCF-7, T47D and normal cell strain MCF-10A (human mammary epithelial cell strain), HAF (human adrenal gland fibroblast) and the L-02 (Human normal hepatocyte) in people source cultivation effect；Fig. 1 (C) represents that formula (I) lycorine suppresses breast cancer cell 4T1, MDA-MB-231, MCF-7 and T47D Clone formation and corresponding statistical chart.
Fig. 2 show the inhibition that formula (I) lycorine is migrated and attacked to breast carcinoma cell strain；Fig. 2 (A) represents that formula (I) lycorine suppresses breast cancer cell 4T1 and MDA-MB-231 lateral transfer and statistical chart；Fig. 2 (B) represents that formula (I) lycorine suppresses breast cancer cell 4T1 and MDA-MB-231 vertical migration and statistical chart；Fig. 2 (C) represents that formula (I) lycorine suppresses breast cancer cell 4T1 and MDA-MB-231 invasion and attack and statistical chart.
Fig. 3 show the inhibition of formula (I) lycorine in vivo to breast cancer cell MDA-MB-231 subcutaneous growths；Fig. 3 (A, B and C) represents that formula (I) lycorine and taxol suppress breast cancer cell MDA-MB-231 subcutaneous growth effects in mouse model；Fig. 3 (D) represents the influence of formula (I) lycorine and taxol to mouse weight；Fig. 3 (E) represents the inhibition of formula (I) lycorine on tissue sections to the growth of tumour.
Fig. 4 show the inhibition of formula (I) lycorine in vivo to breast cancer cell 4T1 growth in situ；Fig. 4 (A and B) represents that formula (I) lycorine and taxol suppress breast cancer cell 4T1 growth in situ effects in mouse model；Fig. 4 (C) represents the inhibition of formula (I) lycorine on tissue sections to the growth of tumour；Fig. 4 (D) represents the toxic effect of formula (I) lycorine on tissue sections to mouse main organs.
Fig. 5 show the inhibition of formula (I) lycorine in vivo to breast cancer cell MDA-MB-231 Lung metastases；Fig. 5 (A) represents that formula (I) lycorine suppresses the fluorescent effect figure and corresponding statistical chart of breast cancer cell MDA-MB-231 Lung metastases in mouse model；Fig. 5 (B) represents that formula (I) lycorine suppresses the effect and corresponding statistical chart of the formation of breast cancer cell MDA-MB-231 pulmonary nodules in mouse model；Fig. 5 (C) represents inhibition and corresponding statistical chart of the formula (I) lycorine on tissue sections to the formation of mouse lung tubercle.
Fig. 6 show facilitation effect of formula (I) lycorine to many plants of Apoptosis of Breast Cancer；Fig. 6 (A) represents that formula (I) lycorine promotes the design sketch of breast cancer cell 4T1 and MDA-MB-231 apoptosis；Fig. 6 (B) represents that formula (I) lycorine promotes the statistical chart of many plants of Apoptosis of Breast Cancer；Fig. 6 (C) represents the expression of formula (I) lycorine inducing mammary cancer cell 4T1 and MDA-MB-231 apoptosis-related protein.
Fig. 7 show inhibition of formula (I) lycorine to migration associated signal paths, represents that formula (I) lycorine lowers the expression that breast cancer cell 4T1 and MDA-MB-231 migrate GAP-associated protein GAP.
Embodiment
With reference to specific examples below and accompanying drawing, the present invention is described in further detail, and protection content of the invention is not limited to following examples.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art it is conceivable that change and advantage be all included in the present invention, and using appended claims as protection domain.Implement process, condition, reagent, experimental method of the present invention etc., be the universal knowledege and common knowledge of this area in addition to the following content specially referred to, content is not particularly limited in the present invention.
Embodiment 1：Lycorine is to the propagation of a variety of breast cancer cells and the inhibition of Clone formation
Technical method
1. cell culture
Human breast cancer cell line Bcap-37 used, T47D, MDA-MB-231, Mouse mammary cells 4T1 in the present invention, people normal mammary epithelial MCF-10A, human adrenal gland fibroblast HAF and Human normal hepatocyte L-02 are both from ATCC cell banks.Breast cancer cell MDA-MB-231-Luc with firefly luciferase reporter gene comes from Medical College, Shanghai Communication Univ..MCF-7, T47D, 4T1, MCF-10A and L-02 culture medium are the RPMI1640 containing 10% hyclone, and HAF culture mediums are the DMEM containing 10% hyclone and 1% glutamine, and MDA-MB-231 culture mediums are the MEM containing 10% hyclone and nonessential amino acid.Cell culture is in 37 DEG C of constant incubators (humidity 95%, CO2Concentration 5%) in.
2.SRB (Sulforhodamine) method determines cell propagation
Different cell lines are with 5 × 103-8×103Individual/hole density is seeded to 96 orifice plates (Corning), after 24h, add various concentrations lycorine monomeric compound, control group adds the DMSO of equivalent, each group sets 3 multiple holes, continues to cultivate after 48h, plus the TCA (trichloroacetic acids precooled, 50%, w/V) 4 DEG C be incubated more than 60min and fix cell.After fixation, flowing water is rushed 5 times, is air-dried.50 μ l SRB dye liquors (4%, w/V), incubation at room temperature 10min dyeing are added per hole.Dye liquor is suctioned out afterwards, the μ l of 1% acetic acid 100 are added per hole and are washed 5 times, uncombined dyestuff is removed.After air-drying, it is the μ l of 10mM Tris solution 100, the SRB dyestuffs that concussion dissolving is combined that concentration is added per hole.96 orifice plates are placed in ELIASA (SPECTRA MAX 190), OD values are determined under 515nm wavelength.Experiment is independently repeated 3 times.Cell survival rate (%)=dosing thing OD values/control group OD value × 100%.
3. colony formation
With every hole 1 × 103Breast cancer cell is inoculated in six orifice plates by the density of individual cell, after adding formula (I) lycorine without concentration after cell attachment, cell is fixed with paraformaldehyde after one week, afterwards with 2 ‰ violet staining 3min, gently cleaned with the running water slowly flowed afterwards, to wash uncombined crystal violet dye liquor, natural drying at room temperature off.Taken pictures under microscope and count clone's number, every group of experiment is in triplicate.Clone formation is to detect one of effective ways of Cells Proliferation of Human Breast Cancer ability.
Shown in experimental result such as Fig. 1 (B), formula (I) lycorine has significant inhibition to each strain breast cancer cell.It is 2 μM -8 μM that it, which handles breast carcinoma cell strain MDA-MB-231,4T1, MCF-7 and T47D the half-inhibition concentration after 48h,.And lycorine is to people Normal breast epithelial cell MCF-10A, the influence of the propagation of human adrenal gland fibroblast HAF and human normal liver cell L 02 is smaller.
Experimental result as shown in Fig. 1 (C), formula (I) lycorine also significantly inhibits breast carcinoma cell strain MDA-MB-231,4T1, MCF-7 and T47D Clone formation simultaneously.
Experiment shows that formula (I) lycorine can significantly inhibit the propagation of breast carcinoma cell strain above.
Embodiment 2：The inhibition that lycorine is migrated and attacked to breast cancer cell
Technical method
1. line migration experiment
MDA-MB-231 and 4T1 cells are seeded to 6 orifice plates, in 37 DEG C, 5%CO2Cellar culture 24h in incubator, the density to cell length to 90% just changes serum free medium, carries out the Nature enemy of appropriateness.After the completion of Nature enemy, the cell in 6 orifice plates is rule with 100 μ l sterilizing pipette tips, cell is washed twice with PBS after line, remove dead cell.Respectively toward the lycorine of addition various concentrations in different holes, 37 DEG C of continuation cellar culture appropriate times.The situation that micro- Microscopic observation cell is moved to dashed part, takes pictures.Statistical analysis various dose medicine group migrates into the cell quantity of scribe area, determines the influence of medicine cell migration ability.
2.Transwell cells are migrated and Matrigel
Digest and count MDA-MB-231 the and 4T1 cells in exponential phase, cell is resuspended in serum-free and dissolved with the basal medium of various concentrations lycorine, and cell is with 6 × 104The MDA-MB-231 cells in individual/hole or 1 × 105The 4T1 cells (each 200 μ l) in individual/hole are seeded in the upper chamber of Transwell cells.The complete medium that 600 μ l contain corresponding concentration lycorine is then added in lower room.It is placed in cell culture incubator and cultivates 12h-24h.Transwell cells are taken out gently to remove cell that cell upper surface do not migrate with cotton swab and fix small chamber lower surface cell 15 minutes with 4% paraformaldehyde, then cell is handled 3 minutes with 2 ‰ crystal violet dye liquors, clean cell, remove the crystal violet being not associated with cell, the upside of cell is gently wiped with cotton swab, the dyestuff for being non-specifically bound in cell upper surface is wiped, so as to follow-up microscopy.Cell after natural drying is placed under microscope and taken pictures, the cell number in multiple visuals field is counted.It need to shift to an earlier date in cell barrier film upper surface layer overlay matrigel for cell invasion experiment, and inoculating cell number can suitably increase.Cell migration rate (%)=dosing thing cell migration number/cellular control unit transport number × 100%.
Shown in experimental result such as Fig. 2 (A) (B) (C), lycorine just can effectively suppress breast cancer cell MDA-MB-231 and 4T1 migration and invasion and attack at lower doses, it is seen that lycorine can be used as the transcellular potential drug of anti-breast cancer.
Embodiment 3：The inhibition of lycorine lotus knurl growth subcutaneous to mouse breast cancer and in situ
Technical method
1. the subcutaneous lotus knurl model of mouse
By 2 × 106Individual breast cancer cell MDA-MB-231 is subcutaneously injected into immune deficiency female mice (BLAB/c-nude, nude mice) dorsal sc, treats that hypodermic tumour grows to 50-100mm3During left and right, mouse is divided into 4 groups (every group 7).The lycorine that 5mg/kg is dissolved in DMSO is injected intraperitoneally in low dose group mouse daily, the lycorine that 10mg/kg is dissolved in DMSO is injected intraperitoneally in high dose group mouse daily, every 2 days injection 5mg/kg of positive group are dissolved in DMSO taxol, control group injects isometric DMSO daily, measure within every 3 days and record mouse weight and the length of tumour with it is wide, according to formula volume=length × wide2× 0.52 calculates, and counts gross tumor volume, and continuous dispenser puts to death mouse after 30 days.Tumour is peeled off, is taken pictures, and carry out H＆E dyeing and immunohistochemical experiment.
2. mouse original position lotus knurl model
By 1 × 105Individual breast cancer cell 4T1 is expelled on the 4th pair of breast pad of immune deficiency female mice (BLAB/c-nude, nude mice), is divided into mouse 4 groups (every group 7) after 3 days.The lycorine that 5mg/kg is dissolved in DMSO is injected intraperitoneally in low dose group mouse daily, the lycorine that 10mg/kg is dissolved in DMSO is injected intraperitoneally in high dose group mouse daily, every 2 days injection 5mg/kg of positive group are dissolved in DMSO taxol, control group injects isometric DMSO daily, measure within every 3 days and record mouse weight and the length of tumour with it is wide, according to formula volume=length × wide2× 0.52 calculates, and counts gross tumor volume, and continuous dispenser puts to death mouse after 30 days.Tumour is peeled off, is taken pictures, and carry out H＆E dyeing and immunohistochemical experiment.Dissect out the typical metastatic target organ heart, liver, spleen, lung, kidney and carry out H＆E dyeing.
Experimental result such as Fig. 3 A, 3B, shown in 3C, lycorine can significantly inhibit the growth of mouse hypodermic tumour, and high dose group effect is suitable with positive group taxol effect.Fig. 3 D are mouse weight change curve, and low dose group, high dose group are suitable with control group body weight, and positive group taxol treatment group mouse weight substantially mitigates, and illustrates that lycorine is small compared with taxol to the toxic effect of mouse.ImmunohistochemistryResults Results (Fig. 3 E) show that proliferating cell nuclear antigen (PCNA) expression quantity is reduced in administration group, illustrate that lycorine and taxol can suppress tumour growth.
Shown in Fig. 4 A and 4B, lycorine can suppress the growth of mouse in situ tumor.H＆E is dyed and ImmunohistochemistryResults Results (Fig. 4 C and 4D) show, lycorine can lower PCNA expression quantity, and to the no neurotoxic injury of major organs such as conscience spleen and lung kidney etc. of mouse.
Result above shows that lycorine can effectively suppress the growth of mouse hypodermic tumour and in situ tumor in non-toxic.
Embodiment 4：Inhibition of the lycorine to mouse breast cancer tail vein metastasis model
Technical method
Mouse tail vein metastasis model
By 1.5 × 106Individual MDA-MB-231-luc cells pass through in tail vein injection to immune deficiency nude mouse, detected every other day with living imaging system Xenogen IVIS 2000Luminal Imager in Mice Body and fluorescence content and mouse is randomly divided into by three groups (every group 6) according to the experimental program formulated, be negative control group (DMSO), positive controls (taxol) and experimental group (lycorine) respectively.Three groups of mouse are administered by being injected intraperitoneally, the lycorine that 5mg/kg is dissolved in DMSO is injected intraperitoneally in dosing group daily, every 2 days injection 5mg/kg of positive group are dissolved in DMSO taxol, and control group injects isometric DMSO daily.Carry out fluoroscopic examination and record data to mouse week about afterwards, every 3 days measurement mouse weights, successive administration puts to death mouse after three weeks.Mouse lung tissue is peeled off, tumor nodule is calculated, fixing organization is used for follow-up histologic analysis.
Shown in experimental result such as Fig. 5 (A) (B) and (C).The design sketch suppressed after medication 21 days as shown in Fig. 5 (A) to mouse cancer metastasis, photo is shot by living animal imaging system, due to carrying luciferase in tumour cell, after the injected fluorescein substrate into Mice Body, tumour cell can send fluorescence at transfer, so neoplasm metastasis position and metastasis degree are assured that using living animal imaging system, shade represents fluorescence signal in figure, show that there is tumour cell aggregation in the region, fluorescence intensity shows that more by force metastases degree is deeper.Fig. 5 (B) is the in vitro lung tissue of mouse, illustrates that experimental group and medicine used in positive controls can suppress the Lung metastases of breast cancer cell.Fig. 5 (C) is the H＆E coloration results of mouse lung tissue, it is seen that lycorine and taxol can suppress the formation of lung tumors tubercle.But pass through the measurement of mouse weight, it has been found that there is the phenomenon that mouse weight is decreased obviously to taxol treatment group in the phase upon administration, and experimental group does not occur the phenomenon then, illustrates that toxic side effect of the lycorine to mouse under the dosage is smaller.
Embodiment 5：Lycorine suppresses breast cancer cell growth by inducing cell apoptosis
Technical method
1. Apoptosis (Apoptosis) is tested
Cell is inoculated in 6 orifice plates, added after 24h after the complete medium containing various concentrations short-tube lycoris alkali cpd, 48 or 72h, apoptosis detection is carried out using apoptosis detection kit.When fluidic cell apoptosis is detected, cell, which is divided into, does not contaminate group, single dye PI groups, single dye Annexin V groups and the double dye groups of PI, Annexin V.Culture medium supernatant is collected, 1ml PBS washing cells are simultaneously reclaimed.Vitellophag, terminates and cell is collected after digestion into corresponding centrifuge tube, centrifugation.4 × Binding Buffer are diluted to 1 × solution with Milli-Q water.Cell after centrifugation is washed 2 times with PBS, often pipe add 100 μ l be diluted to 1 × combination buffer, gently blown and beaten with liquid-transfering gun, cell is resuspended, group is not contaminated and is not added with PI and Annexin V, and single dye PI groups add the μ l of PI 5, and single dye Annexin V groups add the μ l of Annexin V 5, the double dye groups of Annexin-V-PI add each 5 μ l of PI and Annexin V, mix.Room temperature lucifuge is incubated 15 minutes, is added 400 μ l combination buffers and is mixed and be transferred in 5ml streaming pipes, machine testing on flow cytometer.
2. Western blotting (Western Blot) is tested
Cell is after various concentrations short-tube lycoris alkali process, albumen is extracted in cracking, protein example is separated by electrophoresis with polyacrylamide gel (SDS-PAGE) after denatured by boiling in albumen, it is then transferred on cellulose nitrate film, it is incubated with corresponding protein antibodies, it is incubated again with two antibody of fluorescence labeling, finally with the expression for sweeping film instrument Odyssey and detecting the albumen.
Experimental result picture 6A, 6B show, lycorine energy inducing mammary JEG-3 apoptosis.Under 50 μM of short-tube lycoris alkali process, breast cancer cell line mcf-7 apoptosis rate reaches 26.49% (48h), breast carcinoma cell strain 4T1 apoptosis rates reach 55.61% (48h), breast carcinoma cell strain MDA-MB-231 reaches 60.51% (72h), and the apoptosis rate of breast cancer T47D is especially apparent, up to 81.67% (48h).
In immunoblot experiment, the change of apoptosis protein expression quantity also indicates that lycorine can be with inducing mammary cancer cell-apoptosis.As shown in Figure 6 C, apoptotic proteins PARP and the expression quantity of Caspase 3 increase and reduced with short-tube lycoris alkali concn, and CL.PARP and the increase of the expression quantity of CL.Caspase 3.Pro apoptotic protein Bax and anti-apoptotic proteins Bcl-2 ratio (Bax/Bcl-2) are that Bax/Bcl-2 does not change in the mark of Apoptosis, our result, show that lycorine may be by extrinsic pathway and carry out induced breast cancer apoptosis.
Embodiment 6：Lycorine suppresses Src/FAK signal paths
Technical method
Experimental method is as previously described for Western blotting (Western Blot).
Experimental result is as shown in Figure 7.Lycorine is capable of the expression of the downward Src/FAK signal path GAP-associated protein GAPs of dose dependent, includes p-FAK, p-Src, p-JNK, p-c-Jun and MMP-2 expression, so as to suppress the transfer of breast cancer cell.
The protection content of the present invention is not limited to above example.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art it is conceivable that change and advantage be all included in the present invention, and using appended claims as protection domain.
Claims (15)
1. Chinese herbal medicine monomeric small molecule compound lycorine or its hydrate or pharmaceutically acceptable salt shown in formula (I) or pharmaceutically
Application of the acceptable carrier in the medicine for preparing treatment breast cancer disease, the lycorine has with following formula (I) structure：
2. application as claimed in claim 1, it is characterised in that formula (I) the short-tube lycoris alkali cpd is in vitro and/or in vivo
Propagation, the Clone formation for suppressing breast cancer cell, the migration for suppressing breast cancer cell, the suppression breast cancer for suppressing breast cancer cell
The invasion and attack of cell, the transfer for suppressing breast cancer cell, the apoptosis for promoting a variety of breast cancer cells, suppression breast cancer cell migration phase
Close the expression of albumen and then suppress the related signal path of migration.
3. application as claimed in claim 1 or 2, it is characterised in that formula (I) the short-tube lycoris alkali cpd is used to suppress in vitro
Breast carcinoma cell strain is bred.
4. application as claimed in claim 3, it is characterised in that the breast carcinoma cell strain include MDA-MB-231,4T1,
MCF-7 and T47D.
5. application as claimed in claim 1 or 2, it is characterised in that formula (I) the short-tube lycoris alkali cpd is used to suppress in vitro
Breast cancer cell growth, migration and invasion and attack and promotion Apoptosis of Breast Cancer.
6. application as claimed in claim 5, it is characterised in that formula (I) lycorine is used to suppress a variety of breast carcinoma cell strains
MDA-MB-231,4T1, MCF-7 and T47D growth.
7. application as claimed in claim 5, it is characterised in that formula (I) lycorine is used to suppress breast cancer cell
MDA-MB-231 and 4T1 migration and invasion and attack.
8. application as claimed in claim 5, it is characterised in that formula (I) lycorine is used to promote a variety of breast cancer cells
Apoptosis.
9. application as claimed in claim 1 or 2, it is characterised in that formula (I) the short-tube lycoris alkali cpd is used to suppress in vivo
The growth of breast cancer cell.
10. application as claimed in claim 9, it is characterised in that formula (I) lycorine is used to suppress breast cancer cell in vivo
MDA-MB-231 and 4T1 growth.
11. application as claimed in claim 1 or 2, it is characterised in that formula (I) the short-tube lycoris alkali cpd is used to suppress in vivo
The transfer of breast cancer cell.
12. application as claimed in claim 11, it is characterised in that formula (I) lycorine is used to suppress breast cancer cell in vivo
MDA-MB-231 transfer.
13. a kind of pharmaceutical composition, it is characterised in that it includes formula (I) short-tube lycoris alkali cpd or its hydrate or pharmaceutically acceptable
Salt or pharmaceutically acceptable carrier.
14. formula (I) short-tube lycoris alkali cpd as claimed in claim 1 or its hydrate or pharmaceutically acceptable salt can pharmaceutically connect
Application of the carrier received in treatment propagation and the stronger malignant tumor medicine of transfer ability is prepared.
15. application as claimed in claim 13, it is characterised in that the malignant tumour be lung cancer, colon cancer, the cancer of the brain, cutaneum carcinoma,
The malignant tumours such as carcinoma of urinary bladder, kidney.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610156676.9A CN107198688A (en) | 2016-03-18 | 2016-03-18 | Application of the traditional Chinese medicine monomer lycorine in treatment breast cancer medicines are prepared |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610156676.9A CN107198688A (en) | 2016-03-18 | 2016-03-18 | Application of the traditional Chinese medicine monomer lycorine in treatment breast cancer medicines are prepared |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114042154A (en) * | 2021-12-14 | 2022-02-15 | 澳门大学 | Application of medicine composition in preparing anti-tumor combination therapy medicine |
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| CN104800217A (en) * | 2015-04-17 | 2015-07-29 | 华东师范大学 | Application of Chinese medicine monomer (lycorine) to preparation of drugs for treating prostate tumors |
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| CN104800217A (en) * | 2015-04-17 | 2015-07-29 | 华东师范大学 | Application of Chinese medicine monomer (lycorine) to preparation of drugs for treating prostate tumors |
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Application publication date: 20170926 |