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  • br Immunohistochemistry br At the end of

    2019-10-14


    2.9. Immunohistochemistry
    At the end of each animal study, the tumor tissues were collected from the mice and fixed with 10% (v/v) neutral buffered formalin, and then embedded in paraffin. The paraffin sections were deparaffinized in xylene, rehydrated in a series of graded alcohols and treated with 0.3% H2O2 for 10 min. For the immunohistochemistry, the tissue sections were incubated with 1% (w/v) bovine serum albumin in PBS for 1 h, incubated overnight with each primary antibody at 4 °C, and then sections were incubated with a secondary antibody using the ABC kit (Vector Laboratories. Burlingame, CA, USA) for 1 h at room tempera-ture. Then, the samples were developed with the 3, 3′diaminobenzidine (DAB, Vector Laboratories) reagent and counterstained with hematox-ylin.
    For the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay, the samples were fixed in 4% paraf-ormaldehyde for 30 min and permeabilized with 20 μg /ml proteinase K for 10 min at room temperature. The tissue sections were then in-cubated with the TUNEL reaction buffer in a 37 ℃ humidified chamber for 1 h, rinsed twice with PBS, and then incubated with DAPI for 1 min at room temperature. The nuclear DNA fragmentation in the apoptotic Seladelpar was measured using the DeadEnd™ Fluorometric TUNEL System (FisherScientific) and visualized by fluorescence microscopy. To quan-tify the staining intensity, TUNEL-positive cells, Ki-67-positive cells, and CD31 or vWF-positive cells were analyzed with the ImmunoRatio, a free web application for automated image analysis, and compared be-tween the control and treatment groups. The percentage of positively stained nuclear area was calculated by using a colour deconvolution for separating the staining components (diaminobenzidine or hematoxylin) in at least 3 fields for each slide.
    Statistical analyses were done with GraphPad Prism software (La Jolla, CA, USA) using one-way ANOVA followed by Dunnet post-hoc test. The differences with p < 0.05 were considered significant.
    3. Results
    Constituents Mean (mg/g) SD RSD (%) Source
    Fig. 1. 3-D UPLC profile of BP10A. The ethanol extract at a concentration of 5 mg/ml in 95% methanol was injected, Seladelpar and the spectrum of each constituent was analyzed at 200–400 nm.
    and its quality was verified using an UPLC-diode array detector system. The typical 3-D UPLC chromatogram of BP10A is shown in Fig. 1. Based on quantitative UPLC analysis results, the amounts of 5 constituents in BP10A extract were determined with a concentration range from 0.91 ± 0.01 mg/g dry extract (isorhamnetin-3-o-β-glucopyranoside) to 10.88 ± 0.01 mg/g dry extract ((+)-praeruptoirn A) as presented in Table 1. These data provide the phytochemical information, including the constituents of the BP10A and their quantity, supporting the quality of the BP10A extract.
    3.2. In vitro cytotoxic activity of BP10A and anticancer drugs
    To investigate anti-cancer activity of BP10A, the cell viability in two different human colorectal carcinoma cells, HCT-116 and KM12SM, was measured by the Ez-Cytox assay. When the cells were exposed to serial concentrations of BP10A (0–200 μg/ml) for 48 h, BP10A caused cytotoxic effects in the HCT-116 and KM12SM cells in a dose dependent manner and the half maximal effective concentrations (EC50) were 16.78 and 42.39 μg/ml, respectively (Fig. 2A). In addition, BP10A in-duced a significant degradation of PARP in both cell lines (Fig. 2B). Activities of caspase-3, -8, and -9, key mediators of the apoptotic sig-naling pathway in the HCT-116 cells, were also clearly increased to similar levels induced by a well-known apoptotic inducer staurosporin A (Fig. 2C). To know whether BP10A could influence the cell cycle progression, cell cycle fractions after BP10A treatment were measured in HCT-116 and KM12SM cell lines. The BP10A induced the G2/M ar-rest in both cell lines. As shown in Fig. 2D, the percentages of cells in G2/M phase were increased from 21.95% to 49.73% in HCT-116 and 20.95% to 27.41% in KM12SM. Consistently, BP10A remarkably de-creased the expression of the major mitotic promoting factor cdc2/cdk1 and cyclin B1, and increased the expression of p21 protein, the cyclin dependent kinase inhibitor (Fig. 2E).