CN106420712B - Application of anisomycin in preparing medicine for preventing/treating ovarian cancer - Google Patents

Abstract

The invention provides an application of anisomycin in preparing a medicament for preventing/treating ovarian cancer, which is characterized in that the anisomycin has the following structural formula:

Description

Application of anisomycin in preparing medicine for preventing/treating ovarian cancer

Technical Field

The invention relates to a medicament, in particular to application of anisomycin in preparing a medicament for preventing/treating ovarian cancer.

Background

At present, human ovarian cancer stem cells have been found to be the main cause of ovarian cancer cell resistance to chemotherapeutic drugs and the development of ovarian cancer that is prone to metastasis, relapse and poor prognosis. However, the deep mechanism of the proliferative invasion of OCSCs (ovarian cancer stem cells) is not clear, and there is no effective medical means for inhibition. Fan et al reported that overexpression of microRNA-20a can promote proliferative invASion of ovarian cancer cells by inhibiting expression of amyloid precursor protein (Fan X, Liu Y, Jiang J, Ma Z, WuH, Liu T, Liu M, Li X and Tang H. miR-20a proteins promotion and invASion bytargeting APP in human ovarian cells. acta Biochim Biophys Sin (Shanghai) 2010; 42: 318-. Therefore, amyloid precursor protein is considered to be a direct target gene of microRNA-20 a. Beta amyloid plaques (Α β plaques) are the major histopathological hallmarks of alzheimer's disease. Beta-secretase, amyloid precursor protein cleaving enzyme 1 at the beta site (BACE1), is an enzyme that initiates a β production by cleaving the extracellular domain of amyloid precursor protein. Inhibition of BACE1 (amyloid precursor protein cleaving enzyme 1 at the β site) expression can significantly increase the intracerebral A β concentration. Liu and Faghih et al demonstrate that AS-related BACE1 long non-coding RNA can regulate BACE1mRNA and promote A β 1-42 production by increasing BACE1mRNA stability (Guo X, Wu X, Ren L, Liu G and Li L. emissive mechanisms of amino-beta production in amino-branched SH-SY5Y cells. neuroscience 2011; 194: 272. 281; Faghhi MA, Modaresi F, Khalil AM, Wood DE, Sahagan BG, MorganTE, Finch CE, St Laurent G,3rd, Kenny PJ and Wahletest media C. expression of coding RNA expression in amino acids of beta 14. 2008. A. the present application of FIGS. Nevertheless, the functional role of APP (amyloid precursor protein) in alzheimer's disease needs to be further studied, and its role and mechanism of modulating human OCSCs has not been reported. Previous studies have focused on the effects of a β on neuronal toxicity and side effects, while few have discussed the effects of a β on tumor cell proliferation, invasion and tumor formation.

In a word, anisomycin is a novel potential target for inhibiting proliferation and invasion of human OCSCs by promoting expression of IncRNA BACE1-AS, increasing BACE1mRNA stability, enhancing APP processing activity, increasing A beta 1-42 concentration and toxicity to tumor cells.

Anisomycin, structural formula as follows:

chemical name: 2 (p-methoxybenzyl) -3, 4-pyrrolidinediol-3-acetate, the first established protozoan antibiotic, was extracted from Streptomyces griseus. It has toxic effects on eukaryotes and certain protozoa. Anisomycin inhibits the formation of peptide bonds in most cells by binding to the 60S ribosomal subunit, thereby inhibiting protein synthesis. Previous studies have shown that anisomycin not only promotes a β production, but also inhibits the proliferation of certain cancer cells. Guo et al reported that SH-SY5Y cell methyltransferase levels decreased and three gene promoters were demethylated after anisomycin intervention, leading to upregulation of APP, PS1 BACE1 expression. (Guo X, Wu X, Ren L, Liu G and Li L. emergent genetic mechanisms of analog-beta production in analytical cell-derived SH-SY5YCels. neuroscience 2011; 194: 272. about. 281.) while anisomycin can increase the total acetylation degree of protein H3 by up-regulating histone acetyltransferase p300/CREB binding protein and down-regulating histone deacetylase expression level. Furthermore, Yu et al found that anisomycin inhibited Jurkat T cell proliferation by stimulating p53, p21, and p27 signal expression (Yu C, Xing F, Tang Z, Bronner C, Lu X, Di J, Zeng S and LiuJ. Anisomycens supressans Jurkat T cell growth by the cell cycle-regulating proteins Pharmacol Rep 2013; 65: 435-. SEO et al reported that anisomycin could induce apoptosis of renal tumor cells by down-regulating Bcl-2, c-FLIP (L) and Mcl-1. (Seo BR, Min KJ, Kim S, Park JW, Park WK, Lee TJ and Kwon TK. Anisomycin treatment of TRAIL-media apoptosis in novel cancer cells through the down-regulation of Bcl-2, C-FLIP (L) and Mcl-1.Biochimie 2012; 95:858 865.) while Liu et al also demonstrated that anisomycin induces glucocorticoid-resistant acute lymphocytic leukemia cell apoptosis by stimulating phosphorylation of mitogen-activated protein kinase p38 and activation of Jun amino-terminal kinase (Liu Y, Ge J, Li, Gu L, Guo X, Ma ZG and ZhuYP. Anisomalt. apoptosis in leukemia cells of leukemia, Ash K1. Aspergillus strain of antibiotic kinase, Aspergillus strain of leukemia cells of leukemia cells of leukemia of cell type II of leukemia of type II of leukemia of cell of strain of leukemia of cell of genus ZU, K7. Furthermore, Li et al disclose that anisomycin induces glioma cell death in vitro by downregulating the PP2A catalytic subunit. (Li JY, Huang JY, Li M, Zhang H, Xing B, Chen G, Wei D, Gu PY and Hu WX. Anisomaltomyces leucoma cell down-regulation of PP2A catalytic subbunit in vitro. acta PharmacolSin 2012; 33: 935) to further investigate whether anisomycin can inhibit proliferation and invasion of human Cs by stimulating lncRNA BACE1-AS expression and up-regulating A beta concentration, thereby finding new medical application of OCSs.

Disclosure of Invention

The technical problem to be solved by the invention is to further research and design the influence mechanism of the anisomycin on the OCSCs, confirm the effect of the anisomycin on cancer cells and develop a new anti-cancer medicament.

The invention provides an application of anisomycin in preparing a medicament for preventing/treating ovarian cancer.

The present inventors used a small molecule compound anisomycin, which inhibits a variety of tumor cells, to treat human OCSCs. Anisomycin was found to severely affect OCSCs proliferation, invasion and tumor formation. Interestingly, the concentration of endogenous a β 1-42 of OCSCs and BACE1 expression were significantly increased after treatment with anisomycin, and in view of the toxic side effects of a β on cells, the present inventors believe that a β 1-42 induces apoptosis of OCSCs after treatment with anisomycin. However, A β 1-42 is only one product and should not be the direct target of anisomycin. The present inventors have used siRNA targeting IncRNA BACE 1-AS. The experimental result shows that after anisomycin is treated for 72 hours, the survival rate of the OCSCs group transfected by siRNA-BACE1-AS is obviously higher than that of the OCSCs group transfected by siRNA-Mock and the OCSCs group not transfected by the siRNA-Mock. Therefore, the effect of anisomycin on OCSCs can be weakened by siRNA targeting silencing expression of IncRNA BACE1-AS, and IncRNA BACE1-AS is a new potential target of anisomycin. Anisomycin is a novel potential target for inhibiting proliferation and invasion of human OCSCs by maintaining the stability of BACE1mRNA, enhancing the APP processing activity of the anisomycin, and improving the concentration of Abeta 1-42 and the toxicity of the anisomycin to tumor cells.

The inventor tests the influence of anisomycin on OCSCs, and proves that anisomycin obviously inhibits the proliferation and the invasion of OCSCs. An expression test of anisomycin for stimulating the apoptosis-related protein lncRNA BACE1-AS of OCSCs is carried out, and data show that anisomycin can induce the high expression of lncRNA BACE1-AS and A beta 1-42-related protein. Comparative experiments on the tumorigenicity and tumor growth in mice of anisomycin or DMSO-pretreated human OCSCs showed that the group pretreated with anisomycin formed smaller transplants and had reduced proliferation and invasion than the DMSO-treated group. The targeted regulation of BACE1-AS by siRNA is reflected by the evaluation of the regulation of the anisomycin targeted lncRNA BACE1-AS expression. OCSCs were treated with anisomycin (IC50) 72h after transfection with siRNA-BACE1-AS or siRNA-Mock, respectively. Then MTT detection shows that the survival rate of the anisomycin treatment group of the OCSCs transfected by siRNA-BACE1-AS is obviously higher than that of the anisomycin treatment group of the OCSCs transfected by siRNA-Mock or the OCSCs untransfected. Therefore, the anisomycin can be used for preparing the medicine for preventing/treating ovarian cancer.

The medicine for preventing/treating ovarian cancer is a medicine composition which is prepared from anisomycin serving as an active ingredient and pharmaceutic adjuvants.

The pharmaceutical composition provided by the invention is an oral preparation or an injection.

The invention provides a new medicine for preventing/treating ovarian cancer, has good clinical application prospect and great social benefit.

Drawings

FIG. 1 anisomycin inhibits the proliferation and invasion of human OCSCs. MTT detection indicates that the proliferation inhibition rate of OCSCs is remarkably increased along with the increase of the treatment time (IC50 concentration) of anisomycin; p < 0.01VS untreated group; p <0.05 and untreated group; p # >0.05 and untreated group; n is 3. (b) Flow cytometry examination showed significantly higher early apoptosis in anisomycin treated OCSCs cells than in DMSO treated or untreated groups. (C) Transwell cell migration experiments show that the invasiveness of the OCSCs treated by the anisomycin is obviously reduced compared with that of the OCSCs in a DMSO-treated group or that of the OCSCs in an untreated group; p <0.05vs untreated group; # P >0.05 untreated group; n is 3. (d) The soft agar colony formation experiment shows that after the OCSCs are treated by the anisomycin, the density of colony formation is obviously lower than that of a DMSO treated group or an untreated group; p <0.05vs untreated group; # P >0.05vs untreated group; n is 3.

FIG. 2 Anethycin induced the expression of lncRNA BACE1-AS and apoptosis-related genes in OCSCs. (A) qRT-PCR detection shows that the expression level of lncRNA BACE1-AS in the anisomycin treatment group is obviously higher than that in the dimethyl sulfoxide (DMSO) group or the untreated group after 72 hours of treatment; calibration was performed based on mRNA expression levels of 18S rRNA; p < 0.01VS untreated group; n is 3. (B) The qRT-PCR results show that the expression level of BACE1, APP, BCl-2 and Bax mRNA is significantly higher than that of cells in a dimethyl sulfoxide (DMSO) treated group or an untreated group after the OCSCs are treated by anisomycin for 72 hours; however, Ki67 mRNA expression was significantly reduced; calibrating based on the mRNA expression level of 18 SrRNA; p < 0.01VS untreated group; p <0.05vs untreated group; # P >0.05vs untreated group; n is 3.

FIG. 3 Anethycin induces lncRNA BACE1-AS and apoptotic protein expression in OCSCs. (A) And (B) immunoblot hybridization confirmed that BACE1 and A β 1-42 expression were significantly increased in anisomycin-treated group of OCSCs; however, Ki67 protein expression was significantly reduced; GAPDH is used as an internal reference; p < 0.01vs untreated group; p # >0.05vs untreated group; n is 3. (c) Immunofluorescent staining results show that the expression of BACE1 and A beta 1-42 in the anisomycin-treated group of OCSCs is remarkably increased.

FIG. 4 OCSCs anisomycin inhibits the growth of transplanted tumors. (A) Tumor bearing mouse phenotype, tumors formed by OCSCs within the blue circle. (b) Tumor size; p < 0.01vs DMSO group; n is 7. (C) Tumor weight; p < 0.01vs DMSO group; n is 7. (D) The pathological detection result of hematoxylin-eosin staining on the tumor section indicates that all subcutaneous transplanted tumors are moderate or low-differentiation (II and III grade) mixed epithelial ovarian cancer; immunohistochemical staining shows that the expression of BACE1 and Abeta 1-42 protein in the OCSCs tumor body pretreated by anisomycin is obviously lower than that in the DMSO pretreatment group; however, the expression of Ki67 protein was significantly reduced; original amplification: x 200.

FIG. 5 siRNA targeting silencing IncRNA expression of BACE1-AS can impair anisomycin inhibitory activity on OCSCs. (A) MTT method detection shows that after siRNA-BACE1-AS transfects OCSCs group anisomycin treatment, the cell proliferation inhibition rate is obviously reduced compared with that after siRNA-Mock transfects OCSCs group or untransfected group for 72 h; p <0.05vs untransfected group; # P >0.05 vsvs untransfected group; n is 3. (B) Western blot results show that after siRNA-BACE1-AS transfects OCSCs group anisomycin, the expression of BACE1 protein of cells is obviously reduced after 72 hours compared with siRNA-Mock transfects OCSCs group or untransfected group; p <0.05vs untransfected group; # P >0.05vs untransfected group; n is 3. (C) Northern blot showed that the hybridization signal for lncRNA BACE1-AS was significantly stronger after 72h treatment of OCSCs with anisomycin than in the dimethyl sulfoxide (DMSO) -treated or untreated group; however, after the siRNA-BACE1-AS transfects the OCSCs group and is treated by anisomycin, the hybridization signal of lncRNA BACE1-AS is obviously reduced compared with that of siRNA-Mock transfects the OCSCs group or the untransfected group for 72 h; p <0.05vs untransfected group; # P >0.05vs untransfected group; n is 3.

Detailed Description

The starting materials and reagents used in the following examples were obtained commercially.

Anisomycin (Anisomaltin, purity 98% or more, HPLC grade), powder, available from Sigma reagent company, USA (A9789, Sigma-Aldrich, St. Louis, USA).

The following examples relate to tissue samples and cases of ovarian cancer patients from the public hospital of the Pudong New district, Shanghai. (2014 2 months-2015 4 months)

Example 1 isolation of tumor Stem cells from ovarian cancer patients

Undifferentiated mixed epithelial ovarian cancer 7 ovarian cancer patients. The age, stage, degree of differentiation and histological distribution of all ovarian cancer patient populations are shown in Table 1.

Isolation of tumor Stem cells from patients with ovarian cancer (7 patients with ovarian cancer were surgically excised from their tissues, this experiment collected surgically excised tissues in sterile centrifuge tubes, digested into single cell suspensions with 0.25% trypsin, and 4. mu.L of CD44 and CD117 monoclonal antibody (mouse anti-human CD 117-FITC; rabbit anti-human CD44-PE, eBioscience) were added per 100. mu.L of ovarian cancer cell suspension, the cells were incubated at 4 ℃ for 30 minutes, after completion of the reaction, washed 1 time with Phosphate Buffered Saline (PBS), and then FACS was isolated using a flow cytometer (BD Asia, BD Bioscience, CA, USA) to isolate CD44+/CD117+ OCSCs cell subsets)

TABLE 1 ovarian cancer patient characteristics

Example 2 anisomycin significantly inhibits the proliferation and invasion of OCSCs

MTT method (Thiazolum method) for detecting cell proliferation

Cells were seeded on (2 × 10)³/ml) 96-well plates and 5% CO at 37 ℃₂The cells were cultured in DMEM (modified high glucose cell culture medium) containing 10% FBS (fetal bovine serum) under conditions until 85% of the cells were fused. MTT (3(V/V) -diphenylbromide; from Sigma) reagent (5mg/ml) was added to the cell culture medium at various time points and incubated for 4 hours at 37 ℃. The reaction was stopped by adding 150. mu.L of dimethyl sulfoxide (DMSO, Sigma Chemicals) per well. Cells were lysed for 15min and 96-well plates were gently shaken for 5 min. The absorbance values were measured by enzyme-linked immunosorbent assay (ELISA) at 490nm (model680, Bio-Rad Bole Bio Inc.).

Annexin V-FITC/PI (Phosphoesterylserine-fluorescein isothiocyanate/propylidine iodide) staining and flow cytometry analysis

Cell seeding (3 × 10)³/ml) in 6-well plates and cultured to 85% confluence. Cells were washed three times with PBS and harvested by centrifugation at 1000 rpm for 5min (Allegra X-22R, Beckman Coulter (Beckman Coulter)). Cells were suspended in 0.1ml PBS (phosphate buffered saline) and stained with Annexin V-FITC/PI kit. After incubation at 4 ℃ for 30 minutes, the cells were analyzed by flow cytometry (FACS) (BD Biosciences). For each detection of 20000 cells, the data were analyzed with CellQuest software (BD Biosciences).

Transwell migration experiment

Cell (2 × 10)³Per ml) was resuspended in 200. mu.L of serum-free medium and seeded on the upper part of an 8 μm well, 6.5 mm thick polycarbonate membrane filter (Corning). A600. mu.L quantity of DMEM + 10% fetal bovine serum was added to the lower part. 5% CO at 37 deg.C₂And culturing for 24 hours. Cells attached to the lower surface of the membrane were fixed with 4% paraformaldehyde for 30 minutes at room temperature, and then stained with 4,6-diamidino-2-phenylindole (dapi) (4', 6-diamidino-2-phenylindole) (c1002, piceatan biotechnology limited, china). The number of cells on the lower surface of the filter was counted under a microscope. Five fields were counted per cell filter.

Soft agar colony formation assay

The substrate of each well consisted of 2ml of final concentration 1 × medium (DMEM + 10% fetal bovine serum) and 0.6% low melting agarose, six well plates were refrigerated at 4 ℃ until solidified, then poured to contain 1 × 10⁴Cells were suspended in 1ml growth layer agar of 1 × media and 0.3% low melting point agarose six well plates were again refrigerated at 4 ℃ until the growth layer coagulated, additional 1ml of agarose free 1 × media was added to the growth layer on days 0 and 15, respectively.

First, the MTT assay showed that the survival of OCSCs was not significantly altered by 8.5umol (IC50, half inhibitory concentration) anisomycin at different times (0, 12 and 24 hours). However, the inhibition of proliferation of anisomycin-treated OCSCs increased significantly with increasing treatment time (fig. 1A). In contrast, neither the DMSO nor the control group had significant growth inhibitory effects (fig. 1A). FCM assays showed a significant increase in the percent of early apoptosis (Annexin V-FITC +/PI-) in the anisomycin treated group compared to the DMSO control and blank groups 72 hours after treatment of cells with anisomycin at IC50 concentration (FIG. 1B). Furthermore, the Transwell cell migration invasion assay showed a significant decrease (63 ± 13) in the anismycin-treated group compared to the DMSO-treated group (102 ± 16) or the untreated OCSCs group (115 ± 15) (fig. 1C). Meanwhile, after 72h of treatment, soft agar colony formation experiments showed a large reduction (33.01% ± 9.17%) in anisomycin-treated OCSCs group colony formation compared to DMSO-treated group (76.71% ± 6.26%) or untreated group (82.26% ± 8.32%) (fig. 1D). These results indicate that anisomycin significantly inhibited the proliferation and invasion of OCSCs.

Example 3 Anethomycin stimulation of expression of OCSCs apoptosis-related protein and lncRNA BACE1-AS (Long non-coding ribonucleic acid beta-lyase translated nucleic acid sequence)

Extraction and analysis of RNA by qRT-PCR (real-time fluorescent quantitative polymerase chain reaction)

Using Trizol (cell/tissue total ribonucleic acid extraction buffer) reagentRNA samples were isolated from each cell and treated with Dnase I (Sigma-Aldrich) (type I deoxyribonucleic acid degrading enzyme (Sigma reagent Co., Ltd)), and quantified and reverse-transcribed into cDNA using an enzyme kit for first strand cDNA Synthesis (TOYOBO) reverse α (type α ribonucleic acid reverse transcription). real-time fluorescent quantitative RT-PCR Using realplex4 (fluorescent 4-channel) real-time fluorescent quantitative RT-PCR detection system from Eppendorf corporation (Germany), SYBR Green fluorescent quantitative PCR Master Mix (Toyobo) as a detection dye, amplification was carried out with denaturation at 95 ℃ for 15 seconds, annealing at 58 ℃ for 35 seconds, and extension at 72 ℃ for 42 seconds, 40 cycles of amplification was carried out.A quantitative analysis was carried out on the target gene using a relative quantitative method.A CT value of the target gene was calculated by using the equation of the normalized relative expression level of. delta. Ct (initial amplification) or. delta. Ct _ genes-Ct _18 sRNA. the relative expression level was measured using the equation of the expression of delta. Ct [ delta. Ct-Aldrich (initial amplification) and. delta. the expression of the same type [ delta^-ΔΔCtAnd (4) performing calculation, and calculating the relative expression condition of the target gene. The mRNA level can be calibrated based on the 18S rRNA (ribosomal ribonucleic acid) gene level. As described above, cDNA (complementary deoxyribonucleic acid) of each gene is amplified using primers.

Western blot analysis (Western blot hybridization experiment)

Total protein extracts from each group of cells were electrophoresed using 12% SDS-PAGE and transfected onto polyvinylidene difluoride membranes (PVDF, Millipore). After blocking, the PVDF membrane was washed 4 times with Tris (Tris buffer containing Tween 20) (TBST, Tris buffer containing Tween 20) at room temperature for 15 minutes each, and incubated for one antibody for 45 minutes. After washing the membrane, the goat anti-rabbit IgG antibody was again incubated with HRP (horseradish peroxidase) for 1 hour. After 4 washes at room temperature for 15 minutes each, the immunoreactivity was stimulated by chemiluminescence enhancement (ECL kit, Pierce Biotech) and revealed by exposure to Kodak xar-5 film (Sigma-Aldrich).

Immunofluorescence staining

Cultured cells were washed 3 times with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, st. louis, USA) for 30 min. After blocking, the cells were first incubated overnight with primary antibody at 4 ℃ and then with FITC (guanidinium isothiocyanate) labeled goat anti-rabbit IgG antibody and 5. mu.g/ml DAPI (4', 6-diamidino-2-phenylindole) (Sigma-Aldrich) for 30min at room temperature. Cells were washed thoroughly with TBST and observed by fluorescence microscopy (DMI 3000; Leica, Allendale, NJ, USA).

The qRT-PCR (real-time fluorescent quantitative polymerase chain reaction) results showed that the expression levels of lncRNAs BACE1-AS (long-chain non-coding ribonucleic acid) were significantly higher in the anisomycin-treated group than in the DMSO or blank group after 72h treatment (fig. 2A). However, there was no significant difference between the three groups at 0 hours. Meanwhile, the qRT-PCR results also showed that the mRNA expression levels of BACE1, APP, Bcl-2 and Bax in the anisomycin group were significantly higher than those in the DMSO group or blank group after 72h treatment (FIG. 2B). However, the anismycin group treated for 72h had significantly reduced Ki67 mRNA expression levels compared to the DMSO group or blank group (fig. 2B). On the other hand, Western blotting results showed that the p-amyloid associated protein (BACE1 and a β 1-42) expression was significantly increased in the group of granicolin compared to the DMSO group or blank after 72h of treatment (fig. 3A, 3B). However, after 72h of treatment, the expression of the anismycin group Ki67 protein was significantly reduced compared to the other groups (fig. 3A, 3B). In addition, IF staining results showed a significant increase in BACE1 enzyme and a β 1-42 expression in the anisomycin treated group compared to the other groups after 72h of treatment (fig. 3C). These data indicate that anisomycin can induce high expression of lncRNA BACE1-AS and amyloid-beta-related proteins.

Example 4 growth inhibition of OCSCs transplants by Anethomycin

In vivo xenogeneic cell transplantation tumor formation experiment

OCSCs are randomly divided into two groups: negative control group (OCSCs pretreated with DMSO), treated with dimethyl sulfoxide (DMSO) for 72 h; experimental group (OCSCs pretreated with anisomycin) treated with anisomycin (half its maximum inhibitory concentration, IC)₅₀8.5 μmol) for 72h, about 1 × 10⁵OCSCs in log phase were inoculated into BALB/C nude mice (mature B-cell and T-cell double deficient mice). Each experimental group contained seven mice. Seven weeks after observation mice were sacrificed and tumors were removed. Tumors were weighed and volume was calculated according to the formula: tumor volume (mm3) ═ ab²) And/2, a represents a major axis (mm), and b represents a minor axis (mm).

Hematoxylin-eosin staining (HE staining)

All fresh ovarian tumor tissues were washed 3 times with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, st. louis, USA) for 30 minutes, dehydrated through an ethanol concentration gradient, xylene transparent, and paraffin embedded. The microtome continuously slices 6 μm thick and performed hematoxylin-eosin (HE) staining.

Immunohistochemical staining experiment

All fresh tissues were washed 3 times with PBS, fixed in 4% paraformaldehyde (Sigma-Aldrich, st. louis, USA) for 30 minutes, dehydrated through a gradient of ethanol concentration, xylene transparent, and paraffin embedded. Next, the microtome continuously cuts the sheet with a thickness of 6 μm, washes with 3% phosphate buffer, and performs microwave heat-treatment. The samples were added with primary antibody and incubated. Then, a horseradish peroxidase-labeled secondary antibody was added to each sample and incubated. Finally, BCA color reagent was added to observe the color change of the sections. PBS (pH 7.4) was used as a negative control instead of the primary antibody. Five fields (200 ×) were randomly selected for viewing each tissue section and the results were analyzed using IPP software (olympus optics ltd).

All anisomycin or DMSO-inoculated pre-treated mouse OCSCs tumors began to outgrow tumors approximately 7 weeks after injection, although both experimental groups eventually developed tumors, with the anisomycin pre-treated group developing more slowly than the DMSO pre-treated group (fig. 4A). In addition, when mice were sacrificed 7 weeks after injection, tumors obtained in the anisomycin-pretreated group were significantly smaller and lighter than those obtained in the DMSO-pretreated group (fig. 4B, 4C). Hematoxylin-eosin stained sections showed that all subcutaneous transplantable tumors were of moderately or poorly differentiated (grade II, III) mixed epithelial ovarian cancer (fig. 4D). In addition, immunohistochemical staining results showed that expression of BACE1 and A β 1-42 proteins was significantly higher in the anisomycin pretreated group and Ki67 protein was significantly lower in the anisomycin group than in the DMSO group (FIG. 4D) in tumor tissues. Thus, pretreatment of OCSCs with anisomycin resulted in smaller transplants than the DMSO-treated group, and OCSCs cells were less proliferating and invasive in nude mice.

Example 5 SiRAN (Small interfering ribonucleic acid) Targeted inhibition of the expression of IncRNA BACE1-AS attenuates the Effect of anisomycin on the inhibition of OCSCs

MTT method for detecting cell proliferation

Cells were seeded on (2 × 10)³)96 well plates in 37 ℃ 5% CO₂The cells were cultured in DMEM medium containing 10% FBS under the conditions until 85% of the cells were fused. MTT (3(V/V) -diphenylbromide; from Sigma) reagent (5mg/ml) was added to the cell culture medium at various time points and incubated for 4 hours at 37 ℃. The reaction was stopped by adding 150. mu.L of dimethylsulfoxide (DMSO, Sigma Chemicals) per well. Cells were lysed for 15min and 96-well plates were gently shaken for 5 min. Absorbance was measured by enzyme-linked immunosorbent assay (ELISA) at 490nm (model680, Bio-Rad).

Western blot analysis (Western blot hybridization experiment)

Total protein extracts from each group of cells were electrophoresed using 12% SDS-PAGE and transfected onto polyvinylidene difluoride membranes (PVDF, Millipore). After blocking, the PVDF membrane was washed 4 times with Tris Buffer (TBST) containing Tween 20 at room temperature for 15 minutes each, and incubated for 45 minutes as a primary antibody. After washing the membrane, the goat anti-rabbit IgG antibody was incubated with HRP labeled for another 1 hour. After 4 washes at room temperature for 15 minutes each, the immunoreactivity was stimulated by chemiluminescence enhancement (ECL kit, Pierce Biotech) and revealed by exposure to Kodak xar-5 film (Sigma-Aldrich).

Northern blot analysis (RNA blot hybridization experiment)

For all samples, 20. mu.g of high quality total RNA was dissolved in 7.5M urea 12% polyacrylamide denaturing gel and transferred to N⁺Nylon membranes (Amersham, Freiburg, Germany). The membrane used was 1200mJ/cm²UV irradiation for 30 seconds. An antisense probe (Shanghai Biotechnology engineering Co., Ltd.) for hybridization was prepared from a segment of lncRNA Bace1-as (Long non-coding ribonucleic acid) and hybridization was detected. After washing, the film was exposed to xar-5 Kodak development (Sigma-Aldrich) for 20-40 hours. As a positive control, all membranes were probed with the human U6 snRNA (5' -GCAGGGGCCATGCTAATCTTCTCTGTATCG-3)', Shanghai Bioengineering Co., Ltd.) was used. The exposure time of the U6 control probe is controlled to be 15-30 min.

IncRNA BACE1-AS (long-chain non-coding ribonucleic acid) can influence the proliferation and invasion of OCSCs, and through the targeted inhibition of siRNA to IncRNA BACE1-AS, the activity of inhibiting OCSCs by the expression of IncRNA BACE1-AS under the targeted regulation of anisomycin can be discussed. OCSCs were transfected with siRNA siRNA-BACE1-AS or siRNA-Mock (small interfering ribonucleic acid consisting of random sequences), respectively, and treated with anisomycin (IC50) for 72 h. Then MTT assay showed that the survival rate of anisomycin treated siRNA-BACE1-AS transfected OCSCs group was significantly higher than that of anisomycin treated siRNA-Mock group or untransfected OCSCs group (FIG. 5A). In addition, the Western blot results showed that the expression level of BACE1 protein in the group of anisomycin treated siRNA-BACE1-AS transfected OCSCs was significantly reduced after treatment 72 compared to the group of anisomycin treated siRNA-Mock transfected OCSCs or untransfected group (fig. 5B). In addition, Northern blot assay results showed that the hybridization signal of the lncotrna BACE1-AS in the group of anisomycin-treated OCSCs was significantly stronger than that in the DMSO group or control group 72h after the intervention (fig. 5C). However, in the anisomycin treated siRNA-BACE1-AS transfected group of OCSCs, the lncRNA BACE1-AS hybridization signal was significantly lower than in the anisomycin treated siRNA-mock group or the untransfected group of OCSCs (FIG. 5D). These results indicate that targeted silencing of lncRNA BACE1-AS by siRNA can significantly attenuate the effect of anisomycin on OCSCs.

Claims (3)

1. The application of the anisomycin in preparing the medicine for preventing/treating ovarian cancer is characterized in that the anisomycin has the following structural formula:

2. the use of anisomycin according to claim 1 in the preparation of a medicament for the prevention/treatment of ovarian cancer, wherein said medicament is a pharmaceutical composition consisting of anisomycin as active ingredient together with pharmaceutical excipients.

3. The use of anisomycin according to claim 2 in the preparation of a medicament for the prevention/treatment of ovarian cancer, characterized in that said pharmaceutical composition is an oral formulation or an injection.

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