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  • br Fig Buforin IIb inhibits the


    Fig. 1. Buforin IIb inhibits the proliferation, growth of PC-3 (left) and Du-145 (right) cells. (A) The growth curve of PC-3 and Du-145 CCK8 treated with different doses of buforin IIb for 6 days. (B) PC-3 and Du-145 Cells were treated with indicated concentrations of buforin IIb and cells viability was determined by MTT assay. (C) The effect of buforin IIb on the survivability of PC-3 and Du-145 cells was evaluated by the quantity of cloning formation. (D) Peptide was added to RWPE and 293T, and cell viability was measured by MTT assay after a 24 h incubation with the peptides. Error bars represent the mean ± SD. *p < 0.05; **p < 0.01.
    Fig. 2. Buforin IIb induced apoptosis in PCa in vitro. (A) Apoptotic analysis of PCa cells by Flow cytometry. PC-3 (left) and Du-145 (right) cells were treated with various doses of buforin IIb and incubated with AV-FITCand PI. Stained cells were analyzed by Flow cytometry. Percentage of intact cells (AV-/PI-) and different stages apoptotic cells (AV+/PI-, AV+/PI+ and AV-/PI+) are presented. (B) Western blot analysis of apoptosis-related proteins in buforin IIb –treated PCa cells. PCa cells were treated with indicated doses of buforin IIb for 24 h, the procaspase-8/9/3, PARP, Bax and Bcl-2 were indicated. β-actin was used as a loading control.
    2.4. Cell viability assay
    Cells (5000/well) were plated at 96-well plate over night and then treated with different concentrations of buforin IIb. 10 μl MTT solution per well was added in plate and incubated for 4 h at 37 °C. Absorbance at 490 nm was examined using a M200pro Multimode Plate Reader (Tecan, Switzerland). Each treatment was performed in triplicate and experiments were repeated at least 3 times.
    2.5. Clonogenic assay
    Cells (2000/dish) were plated in 60 mm dishes and cultured in medium with ddH2O or 8 μM buforin IIb for 2–3 weeks at 37 °C in-cubator. Colonies were fixed with methanol and stained with 0.5% crystal violet (Sigma). Colonies with 50 cells or greater were counted in each well.
    1 × 105 cells grown in 6-well plates were treated with various doses of buforin IIb for 24 h, and stained with Annexin V (AV) conjugated 
    with FITC and propidium iodide (PI) using the Annexin V-FITC Apoptosis Assay Kit following the manufacturer's instructions. Stained cells were analyzed with Flow Cytometer (BD company, US). Data were analyzed using Flow JO 7.6.5 software.
    2.7. RNA isolation and real-time PCR
    Cells were treated with buforin IIb as indicated. Total RNAs were extracted using TRIzol reagent (Invitrogen) according to the manufac-turer's instructions. 1 μg of RNA was converted to cDNA using a 1st Strand cDNA Synthesis kit (TaKaRa). Real-time PCR was performed using a SYBR Premix Ex TaqTM II kit (TaKaRa) and measured by ABI Stepone plus. The mRNA level of each gene was normalized to β-actin with the CT method. The primer sequences for qRT-PCR are listed in Table 1.
    Cells were treated with various doses of buforin IIb for 24 h. Cells were lysed with RIPA lysis buffer, and then Protein samples were subjected to SDS–PAGE. Protein blots were probed with the primary
    antibodies and a secondary antibody and then protein bands were vi-sualized using the ECL system. β-actin was used for the loading control.
    2.9. Statistical analysis
    All the results were expressed as mean ± SD. All the statistics analysis were performed by GraphPad Prism (version 6.01; GraphPad software). A p value < 0.05 was considered as significant.
    3. Results
    3.1. Buforin IIb inhibits the proliferation of PC-3 and Du-145 cells
    To investigate the effect of buforin IIb on the proliferation of an-drogen-independent prostate cells, we performed growth curve assay, cell viability assay and colonic assay using PC-3 and Du-145 cell lines. Cells were treated with buforin IIb at the doses of 4 and 8 μM for the indicated time, H2O was used as a vehicle. As shown in Fig. 1, buforin IIb significantly inhibited the proliferation of PC-3 and Du-145 cells in a dose-dependent manner and the growth of cells was inhibited even at 4 μM (Fig. 1A and B). Meanwhile, in the clonogenic assay, buforin IIb had a stronger inhibitory effect on the colony-forming ability of PC-3  Toxicon 168 (2019) 16–21
    and Du-145 cells in a dose-dependent manner (Fig. 1C). Furthermore, at a concentration of 60 μM, buforin IIb only displayed 20% cytotoxicity to normal cells, 293T and RWPE (Fig. 1D). All the data above revealed that buforin IIb could selectively inhibit the proliferation of PC-3 and Du-145 cells.
    Apoptosis is a normal process in the growth of various cells (Roca et al., 2018; Malumbres et al., 2001). To explore the effect of buforin IIb on the apoptosis of AIPC cell lines, PC-3 and Du-145 cells were stained with PI and Annexin V (AV)-FITC after the treatment with various doses of buforin IIb for 24 h, then analyzed by flow cytometry. As shown in Fig. 2A, the results suggested that buforin IIb could induce the in-creasing percentage of both early (AV+/PI-) and late (AV+/PI+) apoptotic PCa cells in a dose-dependent manner, which implied buforin IIb could result in apoptosis of PC-3 and Du-145. To further determine whether caspase is involved in buforin IIb -induced apoptosis in PCa cells, we detected the caspase-related protein by western blotting. As shown in Fig. 2B, buforin IIb induced significant activation of the PARP and caspase 9/8/3 after the treatment with buforin IIb for 48 h in a dose-dependent manner. We also detected the expression of Bax and Bcl-2 by Western blotting, which plays an important role in cell apop-tosis. Bax induces apoptosis while Bcl-2 inhibits apoptosis. The results indicated that the protein level of Bax was increased while the protein expression of Bcl-2 was decreased by buforin IIb (Fig. 2B). Taken to-gether, these results indicated that buforin IIb induced PC-3 and Du-145 cell apoptosis by caspases and Bcl-2/Bax.