CN103705505A - Novel application of simvastatin - Google Patents
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Abstract
The invention discloses novel application of simvastatin. The novel application of simvastatin disclosed by the invention includes: A, application of simvastatin in preparing a product, such as a reagent, for inhibiting the activity of YAP (yes-associated protein) in cells; B, application of simvastatin in preparing a product, such as a reagent, for reducing the expression level of RHAMM (receptor for hyaluronan mediated motility) in cells; C, application of simvastatin in preparing a product, such as a reagent, for inhibiting tumor migration and/or infiltration.
Description
Technical field
The present invention relates to the new purposes of simvastatin.
Background technology
Breast carcinoma (breast cancer) is developed country and the modal tumor of the women of developing country.The whole world has 1,200,000 women to suffer from breast cancer every year, and 500,000 women die from breast carcinoma.European and American developed countries, just have 1 people to suffer from breast carcinoma in every 8-9 people.In developing country, due to life, urbanization and take Western lifestyle, the sickness rate of breast carcinoma sharply raises.In China, the metropolitan statistics such as Beijing, Shanghai, Tianjin, Guangzhou, Shenzhen shows that breast carcinoma is the modal malignant tumor of China women equally, and sickness rate is ascendant trend year by year.Data show: China's female mammary gland cancer morbidity rises year by year, at present from 5 years, 17/100000ths are increased to 100,000 of last year/52, rise over three times; Mortality rate has increased by 38.91%.What wherein sickness rate was the highest is the economically developed big cities such as Beijing, Shanghai, Guangzhou, Shenzhen, and the height of living standard is directly proportional to the height of sickness rate.Treatment breast carcinoma traditional operation, radiation and chemotherapy curative effect that especially advanced breast cancer adopts is limited at present, and breast carcinoma is still in women's cancer killer's the umber one.Whether existing its prognosis is closely related with diseases range transfer, and especially the transfer of organ has at a distance appearred in the patient of part pathological changes in late period mostly, and within 5 years, survival rate is only 10～30%.Therefore, disclose the molecular mechanism of breast carcinoma progress and cancerometastasis, can be biomarker (biomarkers) and the molecular targeted agents based theoretical of the more effective clinical prognosis of research and development, curative effect etc., there is epochmaking clinical meaning.
For the research of the molecular mechanism of breast carcinoma progress and cancerometastasis (progression and metastasis), more and more become the focus of domestic and international research.The motility (motile) that breast carcinoma progress and cancerometastasis relate to cancerous cell characterizes, and the positive migration (migration) of cancerous cell motion be its further invasion and attack (invasion) surrounding tissue and transfer (metastasis) to lymphoid tissue and the prerequisite of other organs at a distance, these a series of complex processes are subject to multiple Effects of Factors, comprise the extracellular matrix (extracellular matrix, ECM) of multiple somatomedin (growth factors), cytokine (cytokines), chemotactic factor (chemokines) and tumor microenvironment etc.The hyaluronic acid (Hyaluronan, HA) of one of extracellular matrix components is the anionic polymerisation body that contains glucuronic acid and 2-Acetamido-2-deoxy-D-glucose, closely related with transfer with tumor invasion.And can promote in conjunction with mitosis spindle the unstability of interphase in cell division microtubule and the integrity of spindle in conjunction with the motion receptor (Receptor for hyaluronan-mediated motility, RHAMM) of the hyaluronic acid mediated of hyaluronic acid mediated cell motion.Unique, RHAMM has and can be output to surface of cell membrane with CD44 interacts by non-classical approach, thereby promotes passing through combination and activating ERK1 of CD44-mediation, and 2, and then the expression of active cell motility related gene.
The effector Yes associated protein (Yes-associated protein, YAP) in Hippo signal path and downstream thereof is from fruit bat to mammal high conservative, in aspect performance decisive actions such as regulation and control organ size, tissue regeneration and stem cell self renewals.YAP can enter nucleus, mutually combines with the transcription factor in core, starts transcribing of downstream target gene.Once Hippo signal path is activated, upstream tyrosine phosphorylation YAP, the YAP being phosphorylated can interact with cytoskeletal protein 14-3-3, rests in kytoplasm, can not enter nucleus activated gene and transcribe.TEAD is combined topmost transcription factor in nucleus with YAP, after YAP is combined with TEAD, and DNA combined function territory in dependence TEAD, startup downstream gene is transcribed.
Simvastatin (simvastatin) (structural formula is suc as formula 1) is rate-limiting enzyme HMG-CoA reductase (3-hydroxy-methylglutaryl CoA reductase in mevalonic acid path (Mevalonate pathway), HMGCR) specific inhibitor is used to treat hypercholesterolemia and angiocardiopathy preventing always.The inhibition of statins to HMGCR, can hinder mevalonate pathway, thereby reduce the generation of Mevalonate and downstream product isoprenoid (Isoprenoids), and then affect multiple important cell function, comprise signal of interest albumen (as G-protein family members' such as RhoA, Ras, Rac, Rap, Rab) post translational modification (comprising geranylgeranylation/farnesylation), signal conduction, cholesterol biosynthesis, the integrity of cell membrane and the regulation and control of cell cycle.
Summary of the invention
Technical problem to be solved by this invention is to provide the new purposes of simvastatin.
The new purposes of simvastatin provided by the present invention is as follows:
Application in the product (as reagent) of A, simvastatin YAP activity in preparation inhibition cell.
Application in the product (as reagent) of B, simvastatin RHAMM expression in preparation reduction cell.
C, simvastatin are being prepared inhibition tumor cell migration and/or are infiltrating the application in product (as reagent).
In above-mentioned A, in described inhibition cell, YAP activity can be the core that goes out that improves the phosphorylation level of YAP in cell and/or promote YAP in cell.In described promotion cell, the core that goes out of YAP is presented as that the YAP in nucleus reduces, but in cell, the total amount of YAP is constant.
Described cell can be tumor cell.Described tumor cell can be breast cancer cell.Described breast cancer cell can be three negative breast cancer cells.Described three negative breast cancer cells can be MCF-7 MDA-MB-231.
In above-mentioned B, in described reduction cell, RHAMM expression can be the transcriptional level that reduces RHAMM in cell and/or the protein translation level that reduces RHAMM in cell.Described tumor cell can be breast cancer cell.Described breast cancer cell can be three negative breast cancer cells.Described three negative breast cancer cells can be MCF-7 MDA-MB-231.
In above-mentioned C, described tumor cell can be breast cancer cell.Described breast cancer cell can be three negative breast cancer cells.Described three negative breast cancer cells can be MCF-7 MDA-MB-231.
In above-mentioned A, B and C, the concentration of described simvastatin can be 5 * 10
-6m.
In the application, YAP is the protein of aminoacid sequence as shown in SEQ ID No.1; RHAMM is the protein of aminoacid sequence as shown in SEQ ID No.2.
The application's the simvastatin that experimental results show that can suppress by suppressing Mevalonate pathway the RHAMM protein expression of YAP mediation, thereby suppresses migration and the infiltration of tumor.Mevalonate pathway can activate the transcription factor YAP of Hippo pathway, and then the mRNA that activates RHAMM transcribes and protein expression, thereby promotes migration and the infiltration of tumor.
Accompanying drawing explanation
Fig. 1 is that simvastatin (simvastatin) suppresses the expression of RHAMM by suppressing the activity of YAP, thereby suppresses migration and the infiltration of human breast carcinoma MDA-MB-231 cell.
In Fig. 1, A is for detecting the Western blot immunoblot experiment result of phosphorylation YAP, wherein, p-YAP(S) be the result of the P-YAP that obtains of short time exposure, wherein S represents the short time (10s), p-YAP(L) be the result of the P-YAP that obtains of time exposure, wherein L representative long-time (30s), YAP is total YAP albumen in the cell detecting, and comprises the YAP of phosphorylation and the YAP not being phosphorylated.A in Fig. 1, the 1st classifies matched group (Meva-Sim-group) cell as from left to right, the 2nd classifies experimental group (Meva+Sim-group) cell as from left to right, the 3rd classifies experimental group (Meva-Sim+ group) cell as from left to right, the 4th classifies experimental group (Meva+Sim+ group) cell as from left to right.
In Fig. 1, B is that laser confocal microscope detects the positioning result of YAP in cell, wherein, Con is matched group (Con) cell, and Sim is experimental group (Sim) cell, Meva is experimental group (Meva) cell, and Meva+Sim is experimental group (Meva+Sim) cell; DAPI is nucleus, and YAP is total YAP albumen in cell, comprises the YAP albumen that is not phosphorylated in nucleus and the P-YAP albumen in Cytoplasm, and Merge be the result that two pictures are superposeed.
In Fig. 1, C is the result of variations of the mRNA level of fluorescence quantitative PCR detection RHAMM, wherein, the 1st post is matched group (Meva-Sim-group) cell from left to right, the 2nd post is experimental group (Meva+Sim-group) cell from left to right, the 3rd post is experimental group (Meva-Sim+ group) cell from left to right, and the 4th post is experimental group (Meva+Sim+ group) cell from left to right.
In Fig. 1, D is for detecting the Western blot immunoblot experiment result of RHAMM, wherein, the 1st classify matched group (Meva-Sim-group) cell as from left to right, the 2nd classify experimental group (Meva+Sim-group) cell as from left to right, the 3rd classify experimental group (Meva-Sim+ group) cell as from left to right, the 4th classify experimental group (Meva+Sim+ group) cell as from left to right.
In Fig. 1, E and G are the migration that simvastatin can suppress MDA-MB-231 cell, and mevalonate can have the inhibition of rescue simvastatin on cell migration.E in Fig. 1, Con represents matched group (Con) cell, and Sim represents Sim group cell, and Meva represents Meva group cell, and Meva+Sim represents Meva+Sim group cell.G in Fig. 1, the 1st post is matched group (Con) cell from left to right, and the 2nd post is Meva group cell from left to right, and the 3rd post is Sim group cell from left to right, and the 4th post is Meva+Sim group cell from left to right.
In Fig. 1, F and H are the infiltration that simvastatin can suppress MDA-MB-231 cell, and mevalonate can have the inhibition of rescue simvastatin to cellular infiltration.F in Fig. 1, Con represents matched group (Con) cell, and Sim represents Sim group cell, and Meva represents Meva group cell, and Meva+Sim represents Meva+Sim group cell.H in Fig. 1, the 1st post is matched group (Con) cell from left to right, and the 2nd post is Meva group cell from left to right, and the 3rd post is Sim group cell from left to right, and the 4th post is Meva+Sim group cell from left to right.
Fig. 2 be simvastatin (simvastatin) in zoopery level to the active of YAP and the inhibitory action of transcribing with translating to RHAMM.
In Fig. 2, A is for detecting the Western blot immunoblot experiment result of phosphorylation YAP, p-YAP detects phosphorylation YAP albumen, YAP is total YAP albumen in the cell detecting, comprise the YAP of phosphorylation and the YAP not being phosphorylated, wherein YAP is internal reference, in the situation that intracellular total YAP is consistent, the comparison that the amount of P-YAP is carried out.
In Fig. 2, B is the variation of the transcriptional level of RHAMM in fluorescence quantitative PCR detection tumor tissues.
In Fig. 2, C is that Western blot immunoblot experiment detects RHAMM protein expression level in tumor tissues
In Fig. 2, Saline is matched group, and Sim is treatment group.In Fig. 1 and Fig. 2, with t-Test, carry out significance of difference analysis, * represents that there were significant differences (p < 0.05).
The specific embodiment
Below in conjunction with the specific embodiment, the present invention is further described in detail, the embodiment providing is only in order to illustrate the present invention, rather than in order to limit the scope of the invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Quantitative test in following examples, if no special instructions, all arranges three repetitions.In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
People three negative breast cancer cell line MDA-MB-231 in following embodiment buy from ATCC.The estrogen receptor of MDA-MB-231 (ER), progesterone receptor (PR) and human epidermal growth factor acceptor (HER2) are all negative.
In following embodiment is that in L-15 culture medium (Gibco), to add hyclone (Hyclone) to the volumetric concentration of hyclone be 10% culture medium obtaining containing the L-15 culture medium of 10% hyclone.
In following embodiment is that in L-15 culture medium (Gibco), to add hyclone (Hyclone) to the volumetric concentration of hyclone be 20% culture medium obtaining containing the L-15 culture medium of 20% hyclone.
In the application, YAP is the protein of aminoacid sequence as shown in SEQ ID No.1; RHAMM is the protein of aminoacid sequence as shown in SEQ ID No.2.
Embodiment 1, simvastatin decline and come migration and the infiltration of inhibition tumor cell by suppressing the protein level of RHAMM of the activity mediation of YAP
Simvastatin is dissolved in and in dimethyl sulfoxide (DMSO), makes simvastatin solution and carry out following experiment.
1.1 are divided into two groups by MDA-MB-231 cell, and one group of cell is 5 * 10
-6m culture medium (is being 5 * 10 containing adding simvastatin solution to the concentration of simvastatin in the L-15 culture medium of 10% hyclone
-6the culture medium that M obtains) at 37 ℃, cultivate 24 hours, obtain 5 * 10
-6the MDA-MB-231 cell that M simvastatin is processed, as experimental group (Meva-Sim+ group) cell; One group of cell DMSO culture medium (containing in the L-15 culture medium of 10% hyclone, add with experimental group (Meva-Sim+ group) in the culture medium that obtains of the isopyknic DMSO of simvastatin solution that adds) at 37 ℃, cultivate 24 hours, obtain the MDA-MB-231 cell that 0M simvastatin is processed, as a control group (Meva-Sim-group) cell.
1.1.1 collect respectively experimental group (Meva-Sim+ group) and matched group (Meva-Sim-group) cell, cell lysis extracts protein, then uses the primary antibodie (buying from CST) of anti-phosphorylation YAP and the antibody (buying Zi Yibaikang biotech inc) of anti-RHAMM to carry out Western blot immunoblot experiment and detects protein level variation.In this Western blot immunoblot experiment, with YAP albumen total in YAP(cell, comprise the YAP of phosphorylation and the YAP not being phosphorylated) be internal reference, by the primary antibodie (buying from Abnova) of anti-YAP, carry out Western blot immunoblot experiment.
Result show simvastatin significance lifting the phosphorylation level of YAP (seeing A in Fig. 1), suppressed the protein level (D in Fig. 1) of RHAMM.The experimental result of protein quantification shows, the p-YAP(S of experimental group (Meva-Sim+ group) cell) content is the p-YAP(S of matched group (Meva-Sim-group) cell) 1.8 times of content, the p-YAP(L of experimental group (Meva-Sim+ group) cell) content is the p-YAP(L of matched group (Meva-Sim-group) cell) 1.8 times of content, the YAP content of experimental group (Meva-Sim+ group) cell is 1 times of YAP content of matched group (Meva-Sim-group) cell; The RHAMM content of experimental group (Meva-Sim+ group) cell is 0.4 times of RHAMM content of matched group (Meva-Sim-group) cell.
1.1.2 extract total RNA of experimental group (Meva-Sim+ group) and matched group (Meva-Sim-group) cell simultaneously, with PrimeScript RT-PCR Kit (TaKaRa), carry out reverse transcription experiment and obtain cDNA.By the method for fluorescent quantitation, detect the transcriptional level of RHAMM, i.e. the variation of the mRNA level of RHAMM.Wherein, fluorescence quantitative PCR detection be take GAPDH as reference gene, and the PCR primer sequence of fluorescence quantitative PCR detection reference gene is: GAPDH:sense,
5’-CCAGAACATCATCCCTGCCTCTACT-3’;
Anti-sense, 5 '-GGTTTTTCTAGACGGCAGGTCAGGT-3 '; The primer of the transcriptional level of fluorescence quantitative PCR detection RHAMM is: RHAMM:sense,
5’-AGAACCAACTCAAGCAACAGG-3’;anti-sense:
5’-AGGAGACGCCACTTGTTAATTTC-3’。
Fluorescent quantitative PCR result shows, the mRNA level of the RHAMM of experimental group (Meva-Sim+ group) cell is 0.55 times of mRNA level of the RHAMM of matched group (Meva-Sim-group) cell.Simvastatin (simvastatin) suppresses the mRNA level (C in Fig. 1) of RHAMM.
1.2 are inoculated in MDA-MB-231 cell in six orifice plates that the L-15 culture medium that contains 10% hyclone is housed that are covered with 22 * 22mm coverslip, are divided into two groups.One group is that to add simvastatin solution to the concentration of simvastatin be 5 * 10 in experimental group (Sim) every hole
-6m; Another group is matched group (Con), and every hole adds isopyknic DMSO.These two groups are all carried out immunofluorescence experiment in 37 ℃ of cultivations after 24 hours.Wherein primary antibodie is YAP antibody (buying from Abnova).4 ℃ of overnight incubation of primary antibodie, after PBS washing, two anti-incubated at room 1 hour, two resist for FITC(green) IgG(of the mountain sheep anti mouse of labelling buys from Santa Cruz); DAPI(is blue, buys from Sigma) for transfect cell core, after two anti-washings, with DAPI, dye core 8 minutes, after PBS washing, add mountant mounting, laser confocal microscope is taken pictures.Result shows, what in matched group (Con) cell, DAPI dyed is nucleus, and YAP is distributed in nucleus and Cytoplasm, is wherein mainly distributed in nucleus, and after two pictures stacks, the dyeing site of the location of YAP in nucleus and DAPI is basically identical; What in experimental group (Sim) cell, DAPI dyed is nucleus, and the distribution of YAP in nucleus reduces in a large number, is that in matched group (Con) nucleus, 20%, the two pictures stack of YAP only has a small amount of YAP to distribute at the dyeing site of DAPI afterwards; In the nucleus of all cells of matched group (Con) and Cytoplasm, YAP all detected; In the nucleus of all cells of experimental group (Sim), the distribution of YAP seldom, the YAP major part of all cells of experimental group (Sim) is present in Cytoplasm, illustrate that simvastatin has reduced the content of YAP in nucleus, illustrate that simvastatin (simvastatin) processes the core that goes out that promotes YAP after cell, enter (B in Fig. 1) in Cytoplasm.YAP is activity form in nucleus, go out after core enters Cytoplasm and become inactivation form, so simvastatin (simvastatin) suppresses the activity of YAP.
1.3 are inoculated in MDA-MB-231 cell to be covered with 22 * 22mm(or 24 * 24mm) being equipped with in six orifice plates containing the L-15 culture medium of 10% hyclone of coverslip, be divided into two groups.One group be the every hole of experimental group (Meva+Sim+ group) add mevalonic acid (mevalonate) (Sigma) to the concentration of mevalonic acid be 250 * 10
-6m, it is 5 * 10 that 37 ℃ of cultivations add simvastatin solution to the concentration of simvastatin after 4 hours again
-6m; Another group for the every hole of experimental group (Meva+Sim-group) add mevalonic acid (mevalonate) (Sigma) to the concentration of mevalonic acid be 250 * 10
-6m, 37 ℃ cultivate after 4 hours, add again with experimental group (Meva+Sim+ group) in the isopyknic DMSO of simvastatin solution adding.These two groups are all carried out Western blot immunoblot experiment and carry out fluorescence quantitative PCR detection according to the method for 1.1.2 according to the method for 1.1.1 after 24 hours in 37 ℃ of cultivations, result shows: compare with experimental group (Meva+Sim-group), P-YAP's does not have a significant change (A in Fig. 1) in experimental group (Meva+Sim+ group), is 1 times of P-YAP in experimental group (Meva+Sim-group); The rna level of RHAMM does not have significance to change (C in Fig. 1) in experimental group (Meva+Sim+ group), be RHAMM in experimental group (Meva+Sim-group) RNA amount 87%; In experimental group (Meva+Sim+ group), the protein content of RHAMM does not have significance to change (D in Fig. 1), is 80% of the middle RHAMM protein content of experimental group (Meva+Sim-group).The result (A, C and D in Fig. 1) that adds mevalonate can overcome simvastatin (simvastatin) to cause is described.
1.4 are inoculated in MDA-MB-231 cell in six orifice plates that the L-15 culture medium that contains 10% hyclone is housed that are covered with 22 * 22mm coverslip, are divided into two groups.One group is experimental group (Meva+Sim group), every hole add mevalonic acid (mevalonate) (Sigma) to the concentration of mevalonic acid be 250 * 10
-6m, it is 5 * 10 that 37 ℃ of cultivations add simvastatin solution to the concentration of simvastatin after 4 hours again
-6m; Another group for the every hole of experimental group (Meva) add mevalonic acid (mevalonate) (Sigma) to the concentration of mevalonic acid be 250 * 10
-6m, 37 ℃ cultivate after 4 hours, add again with experimental group (Meva+Sim) in the isopyknic DMSO of simvastatin solution adding.These two groups are all carried out immunofluorescence experiment according to 1.2 method in 37 ℃ of cultivations after 24 hours.What in result demonstration experimental group (Meva+Sim group) cell, DAPI dyed is nucleus, YAP is distributed in nucleus and Cytoplasm, wherein mainly be distributed in nucleus, after two pictures stacks, the dyeing site of the location of YAP in nucleus and DAPI is basically identical; The cell of experimental group (Meva) group and the result of experimental group (Meva+Sim) are basically identical.Illustrate and add mevalonate can overcome the result that simvastatin (simvastatin) causes.
The transfer ability of the inhibition MDA-MB-231 cell of 1.5simvastatin significance.
MDA-MB-231 cell is divided into four groups, and first group of MDA-MB-231 cell cultivated and within 24 hours, obtained matched group (Con) cell at 37 ℃ in containing the L-15 culture medium of 10% hyclone; Second group of MDA-MB-231 cell add mevalonic acid (mevalonate) (Sigma) to the concentration of mevalonic acid be 250 * 10
-6m containing in the L-15 culture medium of 10% hyclone 37 ℃ cultivate after 4 hours, then to add simvastatin solution to the concentration of simvastatin be 5 * 10
-6m, then cultivates 24 hours at 37 ℃, obtains Meva+Sim group cell; The 3rd group of MDA-MB-231 cell is 5 * 10
-6m culture medium (is being 5 * 10 containing adding simvastatin solution to the concentration of simvastatin in the L-15 culture medium of 10% hyclone
-6the culture medium that M obtains) in, at 37 ℃, cultivate and within 24 hours, obtain Sim group cell; The 4th group of MDA-MB-231 cell add mevalonic acid (mevalonate) (Sigma) to the concentration of mevalonic acid be 250 * 10
-6m containing in the L-15 culture medium of 10% hyclone 37 ℃ cultivate after 4 hours, then add with second group in the isopyknic DMSO of simvastatin solution that adds, then at 37 ℃, cultivate 24 hours, obtain Meva group cell.Get 5 * 10
4individual above-mentioned cell (the 100 μ l that respectively organize, serum-free) be inoculated in 24 hole Transwell nested, Transwell lower floor adds 600 μ l containing the L-15 culture medium of 20% hyclone, be placed in 37 ℃ of incubators after 6 hours, fixing, violet staining, under microscope, take pictures, detect cell migration situation, result shows, compare with matched group (Con) cell, the cell number of moving in Sim group cell is 0.3 times of matched group (Con) cell, the cell number of moving in Meva group cell is 1.1 times of matched group (Con) cell, the cell number of moving in Meva+Sim group cell is 0.85 times of matched group (Con) cell.Illustrate that simvastatin can suppress the migration of MDA-MB-231 cell, and mevalonate can there is the inhibition (E and G in Fig. 1) of rescue simvastatin on cell migration.
The wetting capacity of the inhibition MDA-MB-231 cell of 1.6simvastatin significance.
MDA-MB-231 cell is divided into four groups, and first group of MDA-MB-231 cell cultivated and within 24 hours, obtained matched group (Con) cell at 37 ℃ in containing the L-15 culture medium of 10% hyclone; Second group of MDA-MB-231 cell add mevalonic acid (mevalonate) (Sigma) to the concentration of mevalonic acid be 250 * 10
-6m containing in the L-15 culture medium of 10% hyclone 37 ℃ cultivate after 4 hours, then to add simvastatin solution to the concentration of simvastatin be 5 * 10
-6m, then cultivates 24 hours at 37 ℃, obtains Meva+Sim group cell; The 3rd group of MDA-MB-231 cell is 5 * 10
-6m culture medium (is being 5 * 10 containing adding simvastatin solution to the concentration of simvastatin in the L-15 culture medium of 10% hyclone
-6the culture medium that M obtains) in, at 37 ℃, cultivate and within 24 hours, obtain Sim group cell; The 4th group of MDA-MB-231 cell add mevalonic acid (mevalonate) (Sigma) to the concentration of mevalonic acid be 250 * 10
-6m containing in the L-15 culture medium of 10% hyclone 37 ℃ cultivate after 4 hours, then add with second group in the isopyknic DMSO of simvastatin solution that adds, then at 37 ℃, cultivate 24 hours, obtain Meva group cell.Get 5 * 10
4individual above-mentioned cell (the 100 μ l that respectively organize, serum-free) be inoculated in 24 hole Transwell nested, Transwell lower floor adds 600 μ l containing the L-15 culture medium of 20% hyclone, be placed in 37 ℃ of incubators after 24 hours, fixing, violet staining, under microscope, take pictures, detect cellular infiltration situation, result shows, compare with matched group (Con) cell, the cell number infiltrating in Sim group cell is 0.25 times of matched group (Con) cell, the cell number infiltrating in Meva group cell is 1.1 times of matched group (Con) cell, the cell number infiltrating in Meva+Sim group cell is 0.8 times of matched group (Con) cell.Illustrate that simvastatin can suppress the infiltration of MDA-MB-231 cell, and mevalonate can there is rescue simvastatin to the inhibition of cellular infiltration (F and H in Fig. 1).
In a word, simvastatin can suppress the activity of YAP, thereby impel the protein level of the RHAMM of YAP mediation to decline, comes migration and the infiltration of inhibition tumor cell.
Embodiment 6, simvastatin (simvastatin) in zoopery level to the active of YAP and the inhibitory action of transcribing with translating to RHAMM.
By the simvastatin DMSO(Sigma that to be dissolved in by volume ratio be 0.8:12:8), polyoxyethylene castor oil (Cremophor) is (Sigma) and in the liquid that forms of ethanol (Shanghai Sheng Gong biological engineering company limited), to be dissolvedly add normal saline after completely and supplement volume, making simvastatin final concentration is 1g/L, the volume content of DMSO is 0.8%, the volume content of Cremophor is 12%, the volume content of ethanol is 8%, obtains simvastatin solution.
10 female Mus of SPF level BALB/c-nu/nu in 5 week age (buying from Chinese Academy of Medical Sciences's medical experiment animal center) are divided into two groups at random, and one group is 5 of matched groups; Another group is 5 for the treatment of groups.In the last week of injection MDA-MB-231 cell, treatment group is injected simvastatin solution in advance according to the dosage of 5mg simvastatin/kg (body weight), it (is that DMSO, polyoxyethylene castor oil and ethanol are all dissolved in to the volume content to DMSO in normal saline is 0.8% that matched group is injected isopyknic carrier, the volume content of Cremophor is 12%, and the volume content of ethanol is 8% solution obtaining).After administration 7 days, by every right veutro subcutaneous injection 5 * 10 of Mus
6individual MDA-MB-231 cell.After inoculation breast cancer cell, every Mus for the treatment of group gives above-mentioned simvastatin solution to make dosage is every kg body weight 5mg simvastatin according to measuring in real time body weight abdominal cavity; The isopyknic carrier of every intraperitoneal administration of matched group (ditto).After 3 weeks, nude mice is put to death, get tumor tissues.Detect as follows: (1) is got tumor cell and carried out Western blot immunoblot experiment detection phosphorylation YAP protein expression level according to the method for 1.1.1 in embodiment 1, experimental result shows that phosphorylation YAP protein level in treatment group tumor tissues is 4 times of matched group, in treatment group tumor tissues, phosphorylation YAP protein level is significantly higher than matched group (seeing A in Fig. 2), and the YAP activity in treatment group tumor tissues is significantly lower than matched group; (2) get tumor cell and according to the method for 1.1.2 in embodiment 1, pass through the variation of the transcriptional level of RHAMM in fluorescence quantitative PCR detection tumor tissues.Experimental result shows, in treatment group tumor tissues, the mRNA level of RHAMM is 0.4 times of matched group, illustrates that the transcriptional level of RHAMM in treatment group tumor tissues is significantly lower than matched group (being shown in B in Fig. 1); (3) get tumor cell and detect RHAMM protein expression level according to the Western blot immunoblot experiment of 1.1.1 in embodiment 1, in treatment group tumor tissues, the content of RHAMM is 0.2 times of matched group, illustrates that RHAMM protein level in treatment group tumor tissues is significantly lower than matched group (being shown in C in Fig. 2).What Fig. 2 showed is the experimental result of each three tumor tissues in random choose matched group and treatment group.
In sum, thus simvastatin can suppress by suppressing the transcriptional activity of YAP migration and the infiltration that the protein level of RHAMM suppresses breast carcinoma MDA-MB-231 cell.
Claims (10)
1. the application in the product of simvastatin YAP activity in preparation inhibition cell.
2. application according to claim 1, is characterized in that: in described inhibition cell, YAP is active in improving the phosphorylation level and the core that goes out that promotes YAP of YAP in cell.
3. application according to claim 1 and 2, is characterized in that: the active core that goes out that promotes YAP in cell of YAP in described inhibition cell.
4. application according to claim 1 and 2, is characterized in that: in described inhibition cell, YAP is active in improving the phosphorylation level of YAP in cell.
5. the application in the product of simvastatin RHAMM expression in preparation reduction cell.
6. according to arbitrary described application in claim 1-5, it is characterized in that: described cell is tumor cell.
7. application according to claim 6, is characterized in that: described tumor cell is breast cancer cell.
8. application according to claim 7, is characterized in that: described breast cancer cell is three negative breast cancer cells.
9. simvastatin is being prepared inhibition tumor cell migration and/or is infiltrating the application in product.
10. application according to claim 9, is characterized in that: described tumor cell is breast cancer cell; Further described breast cancer cell is specially three negative breast cancer cells.
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| CN201310703878.7A CN103705505A (en) | 2013-12-19 | 2013-12-19 | Novel application of simvastatin |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016201682A1 (en) * | 2015-06-18 | 2016-12-22 | 高雄医学大学 | Use of pharmaceutical composition in preparation of drug for promoting chondrocyte generation |
| CN109223742A (en) * | 2018-10-09 | 2019-01-18 | 西安交通大学医学院第附属医院 | The purposes of joint Simvastatin and melbine |
| CN109260195A (en) * | 2018-10-09 | 2019-01-25 | 西安交通大学医学院第附属医院 | The purposes of Simvastatin |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010101888A2 (en) * | 2009-03-02 | 2010-09-10 | Biocure Pharma, Llc | Methods and compositions for treatment of tumor metastasis |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010101888A2 (en) * | 2009-03-02 | 2010-09-10 | Biocure Pharma, Llc | Methods and compositions for treatment of tumor metastasis |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016201682A1 (en) * | 2015-06-18 | 2016-12-22 | 高雄医学大学 | Use of pharmaceutical composition in preparation of drug for promoting chondrocyte generation |
| CN109223742A (en) * | 2018-10-09 | 2019-01-18 | 西安交通大学医学院第附属医院 | The purposes of joint Simvastatin and melbine |
| CN109260195A (en) * | 2018-10-09 | 2019-01-25 | 西安交通大学医学院第附属医院 | The purposes of Simvastatin |
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