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  • br To examine whether BP A

    2019-10-14


    To examine whether BP10A could affect the cytotoxic effect of ox-aliplatin and CPT-11, CRC Elafibranor (GFT505) were exposed to various concentrations of oxaliplatin or CPT-11 with BP10A at minimal cytotoxic dose, which were below 20% cytotoxicity (6.25 μM for HCT-116; 25 μM for KM12SM), and their viabilities were analyzed by Ez-Cytox assay. The cell viabilities of HCT-116 and KM12SM were further reduced by the combination treatment of BP10A with the anti-cancer drugs compared to that of oxaliplatin or CPT-11 alone at each dose (Fig. 2F and G). These results indicate that BP10A acts synergistically with anticancer 
    drugs that are widely used in CRC treatment, enhancing the cytotoxicity of these drugs against CRC.
    3.3. Antitumor efficacy of BP10A in colon PDTX model
    The antitumor activity of the BP10A and anti-cancer drugs against CRC was evaluated using nu/nu mice bearing three F3 xenograft (45 F, 102 F, and 115 F) derived from CRC patients with different genotypic backgrounds. The cell types of the three colon PDTX model were all moderately differentiated adenocarcinoma and the detailed informa-tion of genetic background of each tumor is shown in supplemental Table 1. According to the tumor volume measured every 3–4 days after the treatment, BP10A showed a significant and dose-dependent delay of tumor growth in all three PDTX mice (Fig. 3A). The average tumor volume of control group at the end of study was increased about 2.9–4.2 fold of starting volume, however, daily administration of BP10A attenuated the tumor growth in a dose dependent manner, re-sulted in reducing tumor volume at the end of study about 70% (45 F), 34% (102 F), and 55% (115 F) in the high dose treatment of BP10A group compared to the control group (Fig. 3A). The histological as-sessment by H&E staining showed that the tumor cells of the control group have well-defined cell borders and hyperchromatic nuclei whereas the tumors of the BP10A treated group showed significant morphological differences from the corresponding control groups (Fig. 3B). Because BP10A exhibited in vitro anti-cancer activities through the induction of cancer cell apoptosis (Fig. 2), we next in-vestigated the effects of BP10A on tumor cell proliferation and apop-tosis in the tumor xenografts by immunostaining of the Ki-67 protein as an intrinsic proliferation marker and by TUNEL assay for the apoptosis, respectively. As shown in Fig. 3C, the BP10A treatment remarkably decreased the levels of Ki-67 protein expression in all three PDTX models by more than 80% compared to the control group. In contrast, tumor cell apoptosis in the xenograft assessed by TUNEL assay was increased in a dose dependent manner by the BP10A treatment, com-pared to the control groups which show only basal level of apoptotic cell death (Fig. 3D). The data indicate that the BP10A has a potent effect on inhibiting the proliferation as well as inducing apoptosis in colorectal cancer cells, which was consistent with the attenuation of tumor growth as observed in Fig. 3A.
    Because angiogenesis is known to play an important role in tumor growth, progression and metastasis [24,25], a control of tumor
    Fig. 2. Cytotoxic effect of BP10A in human colorectal cancer cell lines. (A) The effects of various concentrations of BP10A on the viability in HCT-116 and KM12SM cells. The relative viability was calculated as a percentage compared to the viability of the control, incubated with vehicle only. Each experiment was performed at least in duplicate, and the values were reported as the mean ± SD (*, **, ***, and **** are p < 0.05, p < 0.01, p < 0.001, and p < 0.001 vs. vehicle treated control, respectively). (B) Cleavage of the apoptosis-related protein PARP was confirmed with Western blot. Colon cancer cells, HCT-116 and KM12SM, were treated with the indicated concentration of BP10A. The data are presented as one representative experiment done in triplicates. (C) Activity of caspase -3, -8 and -9. Relative activity of caspases in HCT-116 cells in response to various concentrations of BP10A was obtained by comparison with the vehicle treated control. For induction of apoptosis, staurosporin A (STS, 200 nM) was used as a positive control. Data are presented as means ± SD of one representative experiment done in triplicates. **,