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Objective:The study was to determine the anti-tumor effect of norcantharidin incholangiocarcinoma (CCA) cell line RBE and to investigate the mechanismsby study the changes in apoptosis, apoptosis-related proteins, cell cycle,cytoskeleton, among ultrastructure.Methods:1MTT experiment: the logarithmic growth phase cells were seeded in96-well plates, intervented in200μl RPMI1640medium with0,5,10,20,40μg/ml norcantharidin after adherence follow by incubated for24,48,72hours. MTT working solution was added to each well, incubated in theincubator for4hours. Then with150μl DMSO dissolving crystals, absorbancevalues were read on a microplate reader. The experiment was repeated threetimes.2Detection of the rate of apoptosis by flow cytometry: Collect24hour’sconcentration as above in the experimental group and control group cells,washed the cells in PBS3times and resuspended them with Binding Buffer.After joined the Annexin V-FITC and PI, the samples tested in the flowcytometry.3Flow cytometric wes used to analysis the cell cycle distribution: thelogarithmic growth phase RBE cells intervention in different concentrations ofnorcantharidin for24hours, collected and fixed in70%ice-cold ethanol,4℃overnight, centrifuged to remove ethanol, then washed with PBS and joined PIfor staining. Tested the samples in flow cytometry.4Wright-Giemsa staining for cell morphology: RBE cells were seeded insix-well plates, with different concentrations of norcantharidin treated for24hours. The samples were fixed with Wright-Giemsa, and dyed after added distilled water, then immediately observed under microscope.5AO/EB staining for morphology of apoptosis: the cells treated in thesame manner as described above, covered in AO/EB dye, and observed underthe fluorescence microscope.6Western blot detection the expression of bax, Actived-caspase-3andcytochrome c: cells treated by norcantharidin for24hours then collection for cytoplasmic protein or total protein, followed by separation, transfer film,block, first antibody, rinsed, fluorescent secondary antibody, scanning afterrinsed.7Immunohistochemical staining for study the α-Tubulin distributionchanges: RBE cell after norcantharidin treatment, fixed, first antibody,incubation, second antibody, enhanced color, DAB coloration, gradient alcoholdehydration, neutral gum sealing.8Real-time PCR detection the transcription level of mRNA: Collecttreated by norcantharidin for24hours, then the cells were extracted totalRNA, synthesis the cDNA. bax, bcl-2, Caspase-3and internal referenceβ-actin according to their reaction system, fluorescence PCR detected.9Observed cell surface ultrastructure changes induced by norcantharidinby SEM: The RBE cells were treated with norcantharidin for12hours. Thenfixed with glutaraldehyde, dehydrated, and dried in vacuo, plating gold andscanned with electron microscope.10Observed cell ultrastructure changes induced by norcantharidin byTEM: The RBE cells were treated with norcantharidin for12hours. The cellswere collected, then fixed with glutaraldehyde followed by osmium tetroxide,dehydrated in acetone, propylene oxide replacement, embedded in epoxy,polymerization, sliced by ultra-microtome, uranyl acetate-lead citratestaining finally observed by transmission electron microscopy.Results:1MTT experiments showed that norcantharidin inhibited CCA cellgrowth. With different time and concentrations the norcantharidin threat theinhibition rate of RBE followed the time-dose-dependent manner (P<0.05). The24-hour IC50calculated by nonlinear fitting was36.9μg/ml.2Annexin V-FITC/PI double staining showed after treated bynorcantharidin for24hours, the RBE cells were shown significant earlyapoptosis, and after dose increased, the proportion of late apoptosis raised.The total rate of apoptosis in a dose-dependent manner, greater than10μg/mlexperimental group compared with the control group, the difference wasstatistically significant (P<0.05).3The RBE cells were arrested in G2/M phase after24hours treated bynorcantharidin, compared with the control group, the difference wasstatistically significant (P=0.000, P=0.000, P=0.000, P<0.01).4Wright-Giemsa staining and AO/EB staining showed that cells showedvarying degrees of damage and typical apoptotic changes after differentconcentrations of norcantharidin cantharidin-treated cells.5AO/EB staining showed that the effect of norcantharidin treated RBEcells for24hours: with the dose increases the orange fluorescent cells appearin clusters and the proportion elevated. In40μg/ml NCTD, orange cellsaccounted for83.1%±15.6, significantly higher than the control group(P<0.01).6Real-time PCR and Western blot showed that the concentration ofcytochrome c in the cytoplasm increased. And so did the bax orActived-caspase-3content in cells gradually. The bcl-2mRNA expression wasdecreased.7α-Tubulin immunohistochemical staining showed that the α-Tubulinskeleton structure was destroyed, showing the structure of a cyclic structurewithout uniform. No metaphase appeared in the spindle cells.8Real-time PCR detection of α-Tubulin mRNA expression levels weresignificantly lower (P=0.002, P=0.000, P<0.01), the difference wasstatistically significant.9Observed cell surface ultrastructure changes induced by norcantharidinby SEM.20μg/ml norcantharidin made cell protrusions thinner and fracture，microvilli on surface disappeared, the surface curled, deformed. When 40μg/ml NCTD dose, cell volume was significantly reduced, and theprojection surface microvilli disappeared, membrane structure appeared smallpotholes, some cells formed a complete body’s coated vesicle like structuresprotruding on the cell surface.10Observed cell ultrastructure changes induced by norcantharidin byTEM.20μg/ml norcantharidin made the cell surface microvilli disappeared,reducing the cytoplasm, nuclear membrane outward protruded as acute angle.We can see part of the spherical envelope bulging outward, cytoplasmicvacuoled. When40μg/ml did, cell shrinkaged, surface microvilli disappeared,chromatin condensated nucleus, the volume was reduced, the electron densityincreased and gathered on the side formed like crescent-shaped bodies,endoplasmic reticulum, degranulation, cytoplasm appeared empty bubble,mitochondria disappeared, enveloped spherical bulge outward, some of whichfilled by cytoplasm.Conclusion:1Norcantharidin inhibited the proliferation of RBE-TCHU179humancholangiocarcinoma cell and induced apoptosis followed thetime-does-dependent. Cells showed typical apoptotic changes in lightmicroscopy and electron microscopy.2Norcantharidin induced RBE apoptosis by increasing bax, inhibittranscription and expression of bcl-2, and activated Caspase-3by releasecytochrome c.3. Norcantharidin can prevent the formation of spindle toarrest the cellcycle in G2/M phase, by disrupting Tubulin cytoskeleton, inhibiting α-TubulinmRNA expression, which is one of the mechanisms of Norcantharidin’santi-tumor effect.
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