CN106420712A - Application of anisomycin to preparation of medicine for preventing/treating ovarian cancer - Google Patents

Abstract

The invention provides application of anisomycin to preparation of a medicine for preventing/treating ovarian cancer. The application is characterized in that the structural formula of the anisomycin is as follow: (the formula is as shown in the description); and the medicine is a medicinal composition consisting of the anisomycin serving as the active component, and medicinal auxiliary materials. The medicinal composition is an oral preparation or injection. The result of an experiment about the influence of the anisomycin on OCSCs (ovarian cancer stem cells) indicates that the anisomycin can obviously inhibit OCSCs proliferation and invasion. An experiment of stimulating OCSCs apoptosis related protein lncRNA BACE1-AS expression by the anisomycin is conducted, and data indicates that the anisomycin can induce high expression of starch-like-beta related protein of lncRNA BACE1-AS. Therefore, the anisomycin can be used for preparing the medicine for preventing/treating ovarian cancer. According to the application, a new medicine for preventing/treating ovarian cancer is provided, a good clinical application prospect is achieved and huge social benefits are achieved.

Description

Anisomycin is in preparation prevention / Application in treatment ovarian cancer

Technical field

The present invention relates to medicine and in particular toAnisomycin is in preparation prevention / Application in treatment ovarian cancer.

Background technology

At present it has been found that human ovarian cancer stem cell is to lead to ovarian cancer cell to be easy to the not good main cause of transfer, recurrence and prognosis to chemotherapeutics tolerance and generation ovarian cancer.But the deeper mechanisms of OCSCs (ovary cancer stem cell) increment erosion are unclear, also do not have effective medical procedure to be suppressed.The overexpression that Fan et al. reports microRNA-20a can promote the propagation of ovarian cancer cell to attack (Fan X by suppressing the expression of amyloid precursor protein, Liu Y, Jiang J, Ma Z, Wu H, Liu T, Liu M, Li X and Tang H.miR-20a promotes proliferation and invASion by targeting APP in human ovarian cancer cells.Acta Biochim Biophys Sin (Shanghai) 2010；42:318-324.).It is therefore contemplated that amyloid precursor protein be microRNA-20a be directly targeted gene.Amyloid beta speckle (A β plaque block) is the Main Tissues neuropathological hallmarks of Alzheimer.Beta-secretase, i.e. the amyloid precursor protein lyases 1 (BACE1) in β site, are a kind of enzymes starting A β generation by cracking amyloid precursor protein extracellular domain.Suppression BACE1 (the amyloid precursor protein lyases 1 in β site) expression is remarkably improved big intracerebral A β concentration.Liu with Faghih etc. proves AS related BACE1 long-chain non-coding RNA controllable BACE1 mRNA and promotes generation (the Guo X of A β 1-42 by increasing BACE1 mRNA stability, Wu X, Ren L, Liu G and Li L.Epigenetic mechanisms of amyloid-beta production in anisomycin-treated SH-SY5Y cells.Neuroscience 2011；194:272-281；Faghihi MA,Modarresi F,Khalil AM,Wood DE,Sahagan BG,Morgan TE,Finch CE,St Laurent G,3rd,Kenny PJ and Wahlestedt C.Expression of a noncoding RNA is elevated in Alzheimer's diseASe and drives rapid feed-forward regulation of beta-secretASe.Nat Med 2008；14:723-730.).Even so, function in Alzheimer for the APP (amyloid protein precursor) still needs to study further, it adjusts the effect of people OCSCs and mechanism is not yet reported.Previous studies concern is primarily with the impact of the Side effect to neuron for the A β, and seldom has research to discuss A β on tumor cell proliferation, invasion and attack and neoplastic impact of swelling.

In a word, anisomycin passes through to promote the expression of lncRNA BACE1-AS, increases BACE1mRNA and stablizes, and strengthens it to APP processing activity, improves the concentration of A β 1-42 and its toxicity to tumor cell, is one kind that suppression people OCSCs breeds and attacksNewPotential target.

Anisomycin, structural formula is as follows：

Chemical name：2 (to methoxy-benzyl) -3,4-pyrrolidinediol-3-acetate, are the first the protozoacide antibiotic through confirming, extract from light gray streptomycete and obtain.It is to eukaryote and the toxic effect of some protozoacidies.Anisomycin can be by suppressing the formation of peptide bond in most cells, thus suppressing the synthesis of protein with reference to 60S ribosomal subunit.Conventional research shows, anisomycin not only can promote A β to produce moreover it is possible to suppress the propagation of some cancerous cell.After Guo et al. reports anisomycin intervention, SH-SY5Y cell transmethylase level declines, three gene promoter demethylations, and then leads to the up-regulated of APP, PS1 BACE1.(Guo X,Wu X,Ren L,Liu G and Li L.Epigenetic mechanisms of amyloid-beta production in anisomycin-treated SH-SY5Y cells.Neuroscience 2011；194:272-281.) simultaneously, anisomycin by raising histone acetyltransferase p300/CREB associated proteins and can lower histon deacetylase (HDAC) expression, improves the total degree of acetylation of albumen H3.In addition, Yu et al. finds, anisomycin passes through to stimulate p53, p21 and p27 signal representation, blocks cell and enters S phase and G2/M phase, suppression Jurkat T cell propagation (Yu C, Xing F, Tang Z, Bronner C, Lu X, Di J, Zeng S and Liu J.Anisomycin suppresses Jurkat T cell growth by the cell cycle-regulating proteins.Pharmacol Rep2013；65:435-444.).SEO et al. research reports anisomycin can be by lowering Bcl-2, c-FLIP (L) and Mcl-1, thus inducing kidney tumor cell apoptosis.(Seo BR,Min KJ,Kim S,Park JW,Park WK,Lee TJ and Kwon TK.Anisomycin treatment enhances TRAIL-mediated apoptosis in renal carcinoma cells through the down-regulation of Bcl-2,c-FLIP(L)and Mcl-1.Biochimie 2012；95:858-865.) simultaneously，Liu et al. also demonstrates anisomycin to be passed through to stimulate p38 mitogen-activated protein kinase phosphorylation and the activation of Jun N terminal kinase to induce acute lymphoblastic leukemia cell apoptosis (the Liu Y to glucocorticoid tolerance,Ge J, Li Q,Gu L,Guo X,Ma ZG and Zhu YP.Anisomycin induces apoptosis of glucocorticoid resistant acute lymphoblAStic leukemia CEM-C1 cells via activation of mitogen-activated protein kinASes p38 and JNK.NeoplASma 2012；60:101-110.).Additionally, Li et al. discloses, anisomycin in vitro can be dead by lowering PP2A catalytic subunit induction gum oncocyte.(Li JY,Huang JY,Li M,Zhang H,Xing B,Chen G,Wei D,Gu PY and Hu WX.Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit in vitro.Acta Pharmacol Sin 2012；33:935-940.) thus, inquire into anisomycin furtherWhether permissibleBy stimulating lncRNA BACE1-AS expression and raising A β concentration thus suppressing people OCSCs propagation and attacking, its new medical usage is found with this.

Content of the invention

The technical problem to be solved be further research design anisomycin to the influencing mechanism of OCSCs it was demonstrated that its effect to cancerous cell, develop new cancer therapy drug.

The invention providesAnisomycin is in preparation prevention / Application in treatment ovarian cancer.

The present inventor, using a kind of micromolecular compound anisomycin with suppression kinds of tumor cells, to treat people OCSCs.Find that anisomycin has a strong impact on OCSCs propagation, invasion and attack and tumor and formed.It is interesting that after processing through anisomycin, the concentration of endogenouss A β 1-42 of OCSCs and BACE1 expression significantly raise, in view of the toxic and side effects to cell for the A β, it is considered herein that after processing through anisomycin, A β 1-42 induces OCSCs apoptosis.However, A β 1-42 is a kind of product, it should not be the direct target of anisomycin.The present inventor employs siRNA targeting lncRNA BACE1-AS.Test result indicate that, after anisomycin processes 72h, the survival rate that siRNA-BACE1-AS transfects OCSCs group significantly raises than siRNA-Mock transfection OCSCs group and untransfected group.Therefore, the expression of siRNA targeted silent lncRNA BACE1-AS can weaken the impact to OCSCs for the anisomycin, and lncRNA BACE1-AS is the new potential target spot of anisomycin.Anisomycin passes through to maintain the stability of BACE1 mRNA, strengthens it and processes activity to APP, improves the concentration of A β 1-42 and its toxicity to tumor cell, is one kind of suppression people OCSCs propagation and invasion and attackNewPotential target.

The present inventor passes through the impact test to OCSCs for the anisomycin, illustrates that anisomycin substantially suppresses OCSCs propagation and attacks.Having carried out anisomycin stimulates the expression test of OCSCs apoptosis-related protein lncRNA BACE1-AS, as shown by data, and anisomycin can induce the high expression of lncRNA BACE1-AS and A β 1-42 associated protein.Oncogenicity and tumour growth situation comparative test in mice body to anisomycin or DMSO pretreatment people OCSCs, show that the transplanted tumor being formed with anisomycin pretreated group is less than DMSO treatment group, and breed and attack minimizing.By the regulation evaluation that anisomycin targeting lncRNA BACE1-AS is expressed, siRNA adjusts to the targeting of BACE1-AS and is embodied.After OCSCs has transfected siRNA-BACE1-AS or siRNA-Mock respectively, process 72h with anisomycin (IC50).Then mtt assay detection shows, siRNA-BACE1-AS transfects the anisomycin treatment group of the anisomycin treatment group survival rate of OCSCs apparently higher than siRNA-Mock group or untransfected OCSCs.Thus, anisomycin can be used for preparing preventing/treating ovarian cancer.

The medicine preparing preventing/treating ovarian cancer of the present invention is the pharmaceutical composition being made up of as active component and pharmaceutic adjuvant anisomycin.

Pharmaceutical composition of the present invention is oral formulations or injection.

The present invention provides new medicine for preventing/treating ovarian cancer, has good potential applicability in clinical practice, has larger social benefit.

Brief description

Figure 1Anisomycin suppression people OCSCs propagation and invasion and attack.MTT detection prompting processes the increase of (IC50 concentration) time with anisomycin, and OCSCs proliferation inhibition rate dramatically increases；* P ＜ 0.01VS untreated fish group；*P<0.05 and untreated fish group；P#>0.05 and untreated fish group；N=3.There is significantly higher getting up early apoptosis than DMSO treatment group or untreated fish group in (b) flow cytomery display anisomycin treatment group OCSCs cell.(C) Transwell Cell migration assay shows that the OCSCs after anisomycin process is remarkably decreased than the aggressive of DMSO treatment group OCSCs cell or untreated OCSCs group cell；*P<0.05vs untreated fish group；#P ＞ 0.05 untreated fish group；N=3.D () soft-agar cloning forms experiment and shows, after anisomycin processes OCSCs, density ratio DMSO of its Clone formation processes or untreated fish group is significantly low；* P ＜ 0.05vs untreated fish group；#P>0.05vs untreated fish group；N=3.

Figure 2Anisomycin induces the expression of lncRNA BACE1-AS and apoptosis-related genes in OCSCs.(A) qRT-PCR detection display, is processed after 72h, the expression of anisomycin treatment group lncRNA BACE1-AS is apparently higher than dimethyl sulfoxide (DMSO) group or untreated fish group；Calibrated based on the mRNA expression of 18S rRNA；* P ＜ 0.01VS untreated fish group；N=3.(B) qRT-PCR result shows, anisomycin processed OCSCs after 72 hours, and the expression of BACE1, APP, BCl-2 and Bax mRNA is significantly higher than dimethyl sulfoxide (DMSO) treatment group or untreated fish group cell；But, the expression of Ki67 mRNA is but significantly reduced；Calibrated based on the mRNA expression of 18SrRNA；* P ＜ 0.01 VS untreated fish group；*P<0.05vs untreated fish group；#P>0.05vs untreated fish group；N=3.

Figure 3Anisomycin induces lncRNA BACE1-AS and the expression of apoptosis protein of OCSCs.(A) confirm with (B) western blot hybridization method, the expression of BACE1 and A β 1-42 in anisomycin treatment group OCSCs dramatically increases；But, Ki67 protein expression significantly reduces；GAPDH is internal reference；The non-treatment group of * P ＜ 0.01vs；P#>0.05vs untreated fish group；N=3.C () immunofluorescence dyeing result shows, the expression of BACE1 and A β 1-42 in anisomycin treatment group OCSCs dramatically increases.

Figure 4OCSCs anisomycin suppresses the growth of transplanted tumor.(A) mice with tumor phenotype, the tumor being formed for OCSCs in Blue circles.(b) tumor size；* P ＜ 0.01vs DMSO group；N=7.(C) tumor weight；* P ＜ 0.01vs DMSO group；N=7.(D) tumor biopsy row hematoxylin-eosin staining pathology testing result prompting, all subcutaneous transplantation tumors are moderate or the mixed epithelium ovarian cancer of low differentiation (II, III grade)；Immunohistochemical staining shows, in anisomycin pretreatment OCSCs tumor body, BACE1 and A β 1-42 protein expression is all significantly lower than DMSO pretreated group；But, the expression of Ki67 albumen significantly reduces；Original amplification：×200.

Figure 5The expression of siRNA targeted silent lncRNA BACE1-AS can weaken anisomycin to OCSCs rejection ability.(A) mtt assay detection shows, after siRNA-BACE1-AS transfection OCSCs group anisomycin is processed, its cell proliferation inhibition rate transfects than siRNA-Mock and significantly reduces after OCSCs group or untransfected group 72h；*P<0.05vs untransfected group；#P ＞ 0.05vs vs untransfected group；N=3.(B) Western blot result shows, after siRNA-BACE1-AS transfection OCSCs group anisomycin is processed, significantly reduces after the expression ratio siRNA-Mock transfection OCSCs group of its cell BACE1 albumen or untransfected group 72h；*P<0.05vs untransfected group；#P ＞ 0.05vs untransfected group；N=3.(C) Northern blot shows, lncRNA BACE1-AS hybridization signal is significantly stronger than dimethyl sulfoxide (DMSO) treatment group or untreated fish group after anisomycin processes OCSCs 72h；But, after siRNA-BACE1-AS transfection OCSCs group anisomycin is processed, its lncRNA BACE1-AS hybridization signal transfects than siRNA-Mock and significantly reduces after OCSCs group or untransfected group 72h；*P<0.05vs untransfected group；#P ＞ 0.05vs untransfected group；N=3.

Specific embodiment

The raw material that following examples use and reagent pass through to be commercially available.

Anisomycin (Anisomycin, purity >=98%, HPLC rank), powder, purchased from Sigma Reagent Company of the U.S. (A9789, Sigma-Aldrich, St.Louis, USA).

Human ovarian cancer patients' tissue samples that following examples are related to and case come from Shanghai City PuEast is newGong Li hospital of area.(2 months in April, -2015 in 2014)

The separation of embodiment 1 ovarian cancer patients tumor stem cell

Undifferentiated 7 ovarian cancer patients of Combination ovarian epithelial carcinoma.The age of all ovarian cancer patients crowds, stage, differentiation degree and Histological distribution situation are shown inTable 1.

Separation (7 human ovarian cancer patients' row excision ovarian cancer tissues of ovarian cancer patients tumor stem cell, this experiment will perform the operation the tissue collecting cut in sterile centrifugation tube, it is digested to single cell suspension with 0.25% pancreatin, every 100 μ L ovarian cancer cell suspensions add CD44 and CD117 monoclonal antibody (mouse anti human CD117-FITC of 4 μ L；Rabbit antihuman CD 44-PE, eBioscience company), cell is incubated 30 minutes under the conditions of 4 DEG C, after reaction terminates, cleaned with phosphate buffered saline (PBS) (PBS) 1 time, then use flow cytometer (BD FACS Aria, BD Bioscience, CA, USA) separate CD44+/CD117+ OCSCs cell subsets)

Table 1Human ovarian cancer patients' feature

Embodiment 2 anisomycin substantially suppresses OCSCs propagation and attacks

Mtt assay (Thiazolyl blue method) detects cell proliferation

Cell is inoculated in (2 × 10³/ ml) 96 orifice plates in 37 DEG C, 5%CO₂Under the conditions of with the culture of the DMEM containing 10%FBS (hyclone) (modified form high sugar cell culture medium) culture fluid, until 85% cell fusion.MTT (3 (V/V)-diphenyl bromides；Sigma produces) reagent (5mg/ml) is added in cell culture medium in different time points, it is incubated 4 hours under the conditions of 37 DEG C.Every hole adds dimethyl sulfoxide (DMSO, the Sigma Chemicals) terminating reaction of 150 μ L.Cell cracking 15min, 96 orifice plates are shaken gently for 5 minutes.Using enzyme linked immunosorbent assay (ELISA) mensuration absorbance value, measure (model680, Bio-Rad Bole biotech firm) in 490nm.

Annexin V-FITC/PI (cell membrane phosphatidyl serine-Fluorescein isothiocyanate/propidium iodide) dyeing and flow cytometry analysis

Cell inoculation (3 × 10³/ ml) in 6 orifice plates, and cultivate to 85% fusion.Cells rinsed with PBS three times, 1000 turns of 5min are collected by centrifugation (Allegra X-22R, Beckman Coulter (Beckman Coulter Inc.)).Cell suspension at 0.1ml PBS (phosphate buffer), and with Annexin V-FITC/PI test kit dyeed.After 4 DEG C are incubated 30 minutes, using flow cytometer (FACS) (BD Biosciences company) analysis.Detect every time 20000 cells, data is with CellQuest software analysis (BD Biosciences company).

Transwell (penetrating cell) migration experiment

Cell (2 × 10³/ ml) it is resuspended in 200 μ L serum-free mediums and be seeded in the top in 8 μm of holes, in the polycarbonate membrane filter (Corning) of 6.5 millimeters thick.Add the DMEM+10% hyclone of 600 μ L amounts in bottom.In 5%CO under the conditions of 37 DEG C₂Middle culture 24 hours.The cell being attached to film lower surface fixes 30 minutes with 4% paraformaldehyde at room temperature, then dye (c1002 with 4,6-diamidino-2-phenylindole (DAPI) (4', 6- diamidino -2-phenylindone), green skies Bioisystech Co., Ltd, China).The cell number of count filtering device lower surface under the microscope.Each cell filter counts five visuals field.

Soft-fractrue rock mass is tested

The basic unit in every hole is made up of 2ml ultimate density 1 × medium (DMEM+10% hyclone) and 0.6% low melting-point agarose.Six 4 DEG C of orifice plate cold preservations, until solidification, are subsequently poured into and include 1 × 10⁴Cell suspension is in the 1ml grown layer agar of 1 × medium and 0.3% low melting-point agarose.4 DEG C of cold preservations condense until grown layer six orifice plates again.In addition take being added on grown layer respectively without agarose 1 × medium of 1ml at the 0th day and the 15th day.Cell grows 1 month and carries out at 37 DEG C clones the total counting number of colony.Detection is in triplicate.Result carries out statistical analysis (GraphPad software Inc, Ge Ruifu software company) using paired t-test with PRISM GraphPad software.

First, MTT detection method is visible, with 8.5umol (IC50, half-inhibition concentration) anisomycin, OCSCs is intervened within (0,12 and 24 hour) in different time, its survival ability does not have significant changes.However, with the prolongation of process time, OCSCs proliferation inhibition rate that anisomycin is processed significantly improve (Figure 1A).On the contrary, the growth inhibition effect of same time DMSO group and matched group all inconspicuous (Figure 1A).After cell is processed 72 hours with IC50 concentration anisomycin, FCM detection display, compare with DMSO matched group and blank group, the notable rising of anisomycin treatment group early apoptosis percentage ratio (Annexin V-FITC+/PI-) (Figure 1B).Additionally, Transwell cell migration Matrigel show, compared with DMSO treatment group (102 ± 16) or untreated OCSCs group (115 ± 15), anisomycin treatment group invasion and attack level significantly reduce (63 ± 13) (Figure 1C).Simultaneously, after processing 72h, soft-fractrue rock mass experiment shows, compared with DMSO treatment group (76.71% ± 6.26%) or untreated fish group (82.26% ± 8.32%), anisomycin process OCSCs group Colony forming be greatly decreased (33.01% ± 9.17%) (Figure 1D).These results indicate that anisomycin substantially suppresses OCSCs propagation and attacks.

Embodiment 3 anisomycin stimulates OCSCs apoptosis-related protein and the expression of lncRNA BACE1-AS (long-chain non-encoding ribonucleic acid β catabolic enzyme translates nucleotide sequence)

QRT-PCR (real time fluorescent quantitative polymerase chain reaction) carries out extraction and the analysis of RNA

Using Trizol (cell/tissue total RNA extraction buffer) reagent from each cell separation total serum IgE.RNA sample is processed with Dnase I (Sigma-Aldrich) (I type DNA (deoxyribonucleic acid) catabolic enzyme (reagent company limited of Sigma)), and quantifies to become cDNA with reverse transcription using the enzyme reagent kit (TOYOBO) of revertra α (α type ribonucleic acid reverse transcription) first chain cDNA synthesis.Real-time fluorescence quantitative RT-PCR adopts realplex4 (fluorescence 4 chrominance channel) the real-time fluorescence quantitative RT-PCR detecting system of Eppendorf company (German), using SYBR green fluorescence quantitative PCR Master Mix (Japan's spinning) as detection dyestuff.Amplification, with 95 DEG C of degeneration 15 seconds, is annealed 35 seconds for 58 DEG C, and 72 DEG C are prolonged raw 42 seconds, carry out 40 circular response.Application relative quantitation method carries out quantitative analyses to target gene.Target gene CT value computing formula is：Δ Ct (initial amplification period)=Ct_genes Ct_18sRNA is normalized.The detection of relative expression levels adopts below equation：Δ Δ Ct=Δ Ct_all_groups Δ Ct_blankcontrol_group is carried out.This value expression formula 2^{- ΔΔ Ct}Calculated, can be used for calculating target gene relative expression's situation.MRNA level in-site can be calibrated based on 18S rRNA (rRNA) gene level.As previously described, the cDNA (complementary DNA (cDNA)) of each gene is expanded using primer.

Western blot analysis (protein immunoblotting hybrid experiment)

Each group total protein of cell extract carries out electrophoresis using 12%SDS-PAGE, and is transfected on PVDF membrane (PVDF, Millipore).After closing, pvdf membrane at room temperature with the Tris containing polysorbas20 (trishydroxymethylaminomethane) buffer (TBST, the trishydroxymethylaminomethane containing polysorbas20) clean 4 times, 15 minutes every time, and carry out one anti-incubation 45 minutes.After washing film, it is incubated 1 hour again with HRP (horseradish peroxidase) labelling goat anti-rabbit igg antibody.At room temperature with cleaning 4 times, 15 minutes every time, immunocompetence, after chemiluminescent enhancement (ECL test kit, Pierre's Si biotechnology) excites, exposed display with Kodak's xar-5 film (Sigma-Aldrich company).

Immunofluorescence dyeing

The cell of culture is to be washed with PBS 3 times, and fixes 30min with 4% paraformaldehyde (Sigma-Aldrich, St.Louis, USA).After closing, cell resists overnight incubation under the conditions of 4 DEG C with one first, then the goat anti-rabbit igg antibody of FITC (guanidinium isothiocyanate) labelling and 5 μ g/ml DAPI (4', 6- diamidino -2-phenylindone) (Sigma-Aldrich company) are used to be incubated 30min at room temperature.The cell thorough wash clean of TBST, and pass through fluorescence microscope (DMI3000；Leica, Allendale, NJ, USA) observed.

QRT-PCR (Fluorescent quantitative PCR) result shows, after processing 72h, lncRNAs BACE1-AS (long-chain non-encoding ribonucleic acid) expression of anisomycin treatment group be significantly higher than DMSO group or blank group (Figure.2A).But, there is no notable difference at 0 hour between three groups.Meanwhile, qRT-PCR result also shows, after processing 72h, anisomycin group BACE1, APP, the mRNA expression of Bcl-2 and Bax be significantly higher than DMSO group or blank group (Figure.2B).But, compare with DMSO group or blank group, process 72h anisomycin group Ki67 mRNA expression significantly reduce (Figure.2B).On the other hand, Western blotting result shows, process 72h after, group anisomycin group compared with DMSO group or blank beta amyloid associated protein (BACE1 and A β 1-42) expression dramatically increase (Figure.3A, 3B).However, after processing 72h, the expression of anisomycin group Ki67 albumen compared with other groups is decreased obviously, (Figure.3A, 3B).Additionally, IF coloration result shows, compare other groups, process 72h after, anisomycin treatment group BACE1 enzyme and A β 1-42 expression dramatically increase (Figure.3C).These as shown by datas, anisomycin can induce the high expression of lncRNA BACE1-AS and beta-amyloyd peptide associated protein.

Embodiment 4 anisomycin suppresses the growth of OCSCs transplanted tumor

Internal heterogenous cell is migrated to tumor experiment

OCSCs is randomly divided into two groups：Negative control group (OCSCs DMSO pretreatment), processes 72h with dimethyl sulfoxide (DMSO)；Experimental group (OCSCs anisomycin pretreatment), through anisomycin (in half amount of its maximum suppression concentration, IC₅₀=8.5 μm of ol) process 72h.About 1 × 10⁵The OCSCs of exponential phase is inoculated in BALB/C nude mice (mature B cell and T cell double defect mice).Each experimental group comprises seven mices.Put to death mice after observing seven weeks and take tumor.Tumor is weighed and is calculated volume according to formula：Gross tumor volume (mm3)=(ab²)/2, a represents major axis (mm), and b represents short axle (mm).

Hematoxylin-eosin staining (HE dyeing)

All fresh ovarian carcinomas are washed with PBS 3 times respectively, and fix 30 minutes with 4% paraformaldehyde (Sigma-Aldrich, St.Louis, USA), are dehydrated by ethanol concentration gradient, dimethylbenzene is transparent, paraffin embedding.Microtome continuously cuts the slice, thin piece of 6 μ m thick, and carries out hematoxylin-eosin staining (HE).

Immunohistochemical staining is tested

All flesh tissues PBS washes 3 times, and 4% paraformaldehyde (Sigma-Aldrich, St.Louis, USA) fixes 30 minutes, is dehydrated by ethanol concentration gradient, dimethylbenzene is transparent, paraffin embedding.Next, microtome continuously cuts the slice, thin piece of 6 μ m thick, the phosphate buffer with 3% rinses, and carries out microwave thermal reparation.Sample adds one and resists and be incubated.Then, each sample adds the two of horseradish peroxidase-labeled and resists and be incubated.Finally, BCA colour reagent is added to observe section color change.PBS (pH 7.4) replacement one is anti-to make negative control.Randomly choose five visuals field (200 ×) and observe each tissue slice, result is analyzed using IPP software (Olympus Optical Instrument Ltd.).

All inoculation anisomycin or the pretreatment of mice OCSCs tumor of DMSO about start to grow tumor although two experimental grouies finally all form tumor after injection for 7 weeks, anisomycin pretreated group than DMSO pretreated group tumor formed more slowly (Figure 4A).Additionally, putting to death after injected in mice 7 weeks, the tumor that anisomycin pretreated group obtains may be significantly smaller than DMSO pretreated group, lighter (Figure 4B, 4C).Hematoxylin-eosin staining section display, all subcutaneous transplantation tumors belong to moderate or less differentiated (II, III level) mixed epithelium ovarian cancer (Figure.4D).Additionally, immunohistochemical staining result shows, in tumor tissues the expression of anisomycin pretreated group BACE1 and A β 1-42 albumen apparently higher than DMSO group (Figure.4D) and expression in anisomycin group for the Ki67 albumen be significantly lower than DMSO group (Figure.4D).Therefore, the transplanted tumor being formed with anisomycin pretreatment OCSCs is less than DMSO treatment group, and propagation in nude mice body for the OCSCs cell and aggressive weaken.

The expression of embodiment 5 siRAN (small interference ribonucleic acid) targeted inhibition lncRNA BACE1-AS can weaken the effect that anisomycin suppresses to OCSCs

Mtt assay detects cell proliferation

Cell is inoculated in (2 × 10³) 96 orifice plates in 37 DEG C, 5%CO₂Under the conditions of with containing 10%FBS DMEM culture fluid culture, until 85% cell fusion.MTT (3 (V/V)-diphenyl bromides；Sigma produces) reagent (5mg/ml) is added in cell culture medium in different time points, it is incubated 4 hours under the conditions of 37 DEG C.Every hole adds dimethyl sulfoxide (DMSO, the Sigma Chemicals) terminating reaction of 150 μ L.Cell cracking 15min, 96 orifice plates are shaken gently for 5 minutes.Using enzyme linked immunosorbent assay (ELISA) mensuration absorbance value, measure (model 680, Bio-Rad) in 490nm.

Western blot analysis (protein immunoblotting hybrid experiment)

Each group total protein of cell extract carries out electrophoresis using 12%SDS-PAGE, and is transfected on PVDF membrane (PVDF, Millipore).After closing, pvdf membrane cleans 4 times at room temperature with the Tris buffer (TBST) containing polysorbas20,15 minutes every time, and carries out an anti-incubation 45 minutes.After washing film, it is incubated 1 hour again with HRP labelling goat anti-rabbit igg antibody.At room temperature with cleaning 4 times, 15 minutes every time, immunocompetence, after chemiluminescent enhancement (ECL test kit, Pierre's Si biotechnology) excites, exposed display with Kodak's xar-5 film (Sigma-Aldrich company).

Northern blot analysis (northern blot hybrid experiment)

For all of sample, 20 μ g high-quality total serum IgE are dissolved in 7.5M carbamide 12% polyacrylamide denaturant gel, and transfer to N⁺Nylon membrane (Amersham, Freiburg, Germany).Film uses 1200mJ/cm²UV irradiate carry out crosslinking within 30 seconds.Hybridization is made with lncRNA Bace1-as (long-chain non-encoding ribonucleic acid) fragment and carries out hybridization check with antisense probe (Shanghai Sheng Gong biological engineering company limited).After cleaning, film is exposed to 20-40 hour to carry out xar-5 Kodak development (Sigma-Aldrich).As positive control, all of film and people's U6 snRNA probe (5 '-GCAGGGGCCATGCTAATCTTCTCTGTATCG-3 ', Shanghai Sheng Gong biological engineering company limited) are hybridized.U6 control probe time of exposure controls in 15-30min.

LncRNA BACE1-AS (long-chain non-encoding ribonucleic acid) can affect propagation and the invasion and attack of OCSCs, by the targeted inhibition to lncRNA BACE1-AS for the siRNA, anisomycin Targeted-control lncRNA BACE1-AS expression can be inquired into the activity of OCSCs is suppressed with this.After OCSCs has transfected siRNA siRNA-BACE1-AS or siRNA-Mock (small molecule disturbance ribonucleic acid that random sequences are constituted) respectively, process 72h with anisomycin (IC50).Then mtt assay detection shows, anisomycin process siRNA-BACE1-AS transfect the siRNA-Mock group that OCSCs group survival rate processes apparently higher than anisomycin or untransfected OCSCs group (Figure.5A).Additionally, Western blot result shows, after processing 72, anisomycin process siRNA-BACE1-AS transfection OCSCs group BACE1 protein expression level and anisomycin process siRNA-Mock transfect OCSCs group or untransfected group compared with significantly reduce (Figure.5B).Additionally, Northern blot testing result shows, after intervening 72h, anisomycin process OCSCs group lncRNA BACE1-AS hybridization signal be significantly stronger than DMSO group or matched group (Figure.5C).But, anisomycin processes in siRNA-BACE1-AS transfection group OCSCs, lncRNA BACE1-AS hybridization signal be significantly lower than anisomycin process siRNA-mock group or untransfected OCSCs group (Figure.5D).These results indicate that siRNA can significantly attenuate the impact to OCSCs for the anisomycin to the targeted silent effect of lncRNA BACE1-AS.

Claims (3)

1. application in preparing preventing/treating ovarian cancer for the anisomycin it is characterised in that Described anisomycin structural formula is as follows：

2. anisomycin according to claim 1 is preparing preventing/treating ovarian cancer In application it is characterised in that described medicine be by anisomycin as active component with medicinal The pharmaceutical composition of adjuvant composition.

3. anisomycin according to claim 2 is preparing preventing/treating ovarian cancer In application it is characterised in that described pharmaceutical composition be oral formulations or injection.