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    2020-08-07

    On the basis of the observed changes in the expression patterns of apoptosis-associated proteins, we speculated that BA-induced apoptosis may be via mitochondria‐mediated apoptotic pathway. To verify the hypothesis, we detected the alterations in the mitochondrial membrane potential by FCM using a mitochondria‐specific and voltage-dependent dye RH123. It can be clearly seen from Fig. 3A, in comparison with the vehicle group, treatment of HCT116 Trizma maleate with BA resulted in a sig-nificant loss of mitochondrial membrane potential. It is proved that mitochondria are the main source of ROS. With a view to assessing the accumulation of ROS in HCT116 cells after BA treatment, we analyzed the cells via FCM in the presence of DCFH-DA staining. As we can see the results from Fig. 3B, the treatment augmented the production of ROS in a concentration-dependent manner in HCT116 cells by BA. These results show that BA induced apoptosis through mitochondria-mediated apoptotic pathway in colorectal cancer cells.
    2.4. BA represses migration and invasion inhuman colorectal cells
    As we all known that migration and invasion are the important steps in the metastatic cascade of cancer cells. To determine whether BA affects the mobility of HCT116 cells, transwell migration and invasion assays were conducted after treatment with different concentrations of BA for 48 h. As exhibited in Fig. 4A, B, BA treatment can slow down the HCT116 cell migration and invasion. To further investigate the in-hibitory effects of BA on colorectal cancer cells migration and invasion, we assessed the expression of cell‐motility-associated proteins by Western blot analysis. As evident from Fig. 4C, BA-treated HCT116 cells showed higher expression levels of MMP-2, MMP-9, and lower ex-pression levels of TIMP-2 compared with the vehicle group. Taken to-gether, the data suggested that BA represses migration and invasion in human colorectal cells.
    2.5. Antitumor efficacy of BA in a tumor xenograft model of HCT116 colorectal cells
    To explore whether the antitumor activity of BA in vivo is consistent with its effects in vitro, HCT116-bearing mice were dosed daily at the designated doses (control, 10 and 20 mg/kg) for 21 days. As a key in-dicator of health, the average body weight of the control (corn oil treated) and BA‐treated mice did not differ significantly throughout the experiment (Fig. 5A). As presented in Fig. 5B, C. BA treatment retarded growth in vivo in comparison with the control group. As evident from the results of immune histochemistry staining, less Ki67-positive and MMP-2-positive cells were observed in the tumors removed from BA-treated mice compared with the control mice (Fig. 5D). These result indicated that BA impedes human colorectal tumor growth in vivo, which is consistent with the findings in vitro.
    2.6. Toxicity evaluation of BA in murinetumor xenograft models
    To evaluate the toxicity of BA in the murine tumor xenograft models, all the HCT116-bearing mice were killed at the end of the ex-periment and then subjected to histopathological examination via he-matoxylin and eosin staining. As shown in Fig. 6, no significant pa-thological alterations were observed in the visceral organs (heart, liver, spleen, lung, and kidney) of the mice following administration of BA. These results indicated that BA may be a safe agent for therapy.
    3. Discussion
    Colorectal cancer is the third most common malignant tumor in the world. It is a deadly malignant tumor with high morbidity and mor-tality, posing a great threat to people's life and health all over the world.
    Fig. 3. BA induces apoptosis through mitochondria-mediated apoptotic pathway inhuman colorectal cells. (A) HCT116 cells were exposed to various concentrations of BA (0–20 μM) for 48 h. The mean fluorescence intensity detected in the mitochondrial membrane potential assay was treated statistically. Data are presented as mean ± SD (**p < 0.01compared to control). (B) HCT116 cells were treated with BA at indicated doses for 48 h followed by analysis of ROS by flow cytometry and the variation of ROS was calculated. Data are expressed as mean ± SD. **p < 0.01 compared to control.
    Fig. 4. BA represses migration and invasion in-human colorectal cells. (A, B) HCT116 cells were seeded in the top chamber of transwell with serum-free medium and treated with vehicle or different concentrations (0,5,10,20 μg/mL) of BA. After about 48 h, migrated and invasion cells were fixed, stained, photographed and quantified. (C) HCT116 cells were treated with BA. After 48 h, cells were harvested, and western blot assay was conducted to
    Therefore, the development of new effective anti-colorectal cancer drug treatment has become an urgent need for us. Although the chemical drugs have a greater destruction of tumor cells but also a strong toxic side effect. Natural extracts from plants and animals have the char-acteristics of high efficiency and low toxicity. Previous studies have shown that BA is cytotoxic to different types of human cancer cells. In this study, we evaluated the anti-cancer activity of BA in human