br colorectal cancer cells in vitro
colorectal cancer 1449-05-4 in vitro and in vivo. We first conducted MTT and clonal formation experiments to study the anti-proliferation effect of BA in vitro. The results showed that BA had cytotoxic and inhibitory effects on colorectal cancer cells. To further investigate the anti-proliferation mechanism of BA in colorectal cancer cells, FCM analysis was con-ducted to evaluate the effect of BA on apoptosis of HCT116 cells. In addition, expressions of Bax, cleaved caspase-3, and Bcl-2, three key
Fig. 6. Toxicity evaluation of BA in murinetumor xenograft models. (A) Body weight of the mice. (B) BA did not cause obvious pathologic abnormalities in normal tissues. H&E staining of paraffin-embedded sections of the lung, heart, liver, spleen and kidney of the mice.
apoptosis-related proteins were analyzed. The results showed that the expression of Bax and cleaved caspase-3 protein was significantly en-hanced, while the expression of Bcl-2 protein was inhibited. The exo-genous pathway mediated by cell death receptor and the endogenous pathway mediated by mitochondria are two main apoptosis pathways, which have been widely recognized by most scholars.18,19 In this study,
we found that BA-treated HCT116 cells had significantly reduced mi-tochondrial membrane potential and significantly increased ROS levels, suggesting that BA may trigger apoptosis through mitochondrial mediated apoptosis pathways inherent in colorectal cancer cells. It is known that damage to the structural integrity of mitochondrial mem-brane can promote the accumulation of ROS in cells.20 In turn, in-creased ROS levels induce changes in mitochondrial membrane per-meability. In order to determine the effect of BA on the metastasis ability of colorectal cancer cells, we conducted transwell migration and invasion experiments. We found that BA treatment significantly in-hibited the migration and invasion ability of HCT116 cells. It is well known that the up-regulation of MMP-2 and MMP-9 and down-reg-ulation of TIMP-2 are related to the acquisition of invasive and ag-gressive phenotypes in cancer cells. In addition, in vivo experiments have shown that BA can delay tumor growth, which is consistent with the results of in vitro experiments. In addition, we evaluated the toxi-city of BA in the mouse tumor xenotrans plantation model, and the collected visceral organs showed normal performance without obvious histopathological changes. To sum up, we evaluated the anti-cancer activity of BA in human colorectal cancer cells by in vivo and in vitro experiments. Mechanism studies have shown that BA induces apoptosis through mitochondrial mediated apoptosis pathways. In addition, our results showed that BA significantly inhibited the migration and
invasion of colorectal cancer cells. It is worth noting that BA has anti-tumor effect in mice xenotrans plantation model. Overall, our results provide some new evidence that BA may be a potential anticancer drug for colorectal cancer treatment.
4. Materials and methods
4.1. Reagents and antibodies
BA was purchased from (Mansite Chemical Co., Cheng Du, Sichuan, China). Purity (98.9%) and measured by high-performance liquid chromatograph (HPLC) analysis. In vitro assays, BA was initially pre-pared as 80 mM stock solution in dimethyl sulfoxide (DMSO) and keep in dark place at −20 °C. In vivo studies, BA was prepared in 6% DMSO in corn oil, and 6% DMSO in corn oil served as a vehicle control.
4.2. Cell lines and cell culture
HCT116 (ATCC: CCL-247), SW480 (ATCC: CCL-228) and DLD-1 (ATCC: CCL-221) cells were purchased from Shanghai Cell Bank, the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI1640 medium (Gibco, Grand Island, NY) containing 10% defined FBS (Shanghai Sangon Biological Engineering Technology and Services,
Shanghai, China). All the cells were maintained in a humidified in-cubator with 5% CO2 at 37 °C.21
4.3. Cell viability assays
The cell viability of BA-treated thyroid cancer cells was assessed by MTT assay. Briefly, the exponentially growing cells (4 × 103 cells per well) were plated in 96-well plates (100 Ml per well) and incubated for a night. Subsequently, cells were treated with designated concentra-tions of BA (0, 5, 10, 20, 40, 80 μg/mL) for 48 and 72 h. After treatment for 48 h and 72 h, respectively, 20 μL of MTT (5 mg/ml) was added to each well, and the plates were incubated at 37 °C for an additional 2–4 h. Then MTT solution was removed and replaced with 150 μL DMSO. Absorbance was measured at 492 nm by a microplate reader).21 The experiments were performed in triplicates.
4.4. Colony-formation assays
The cells growing to the logarithmic phase were digested and cen-trifuged with trypsin, the supernatant was removed, and the count was resuspended and 6-well plate was laid. Each well of cells was covered with 500–800/2 ml cells. At 37 °C and 5% CO2 incubator in the night. The next day, with designated concentrations of BA (0, 5, 10, 20 μg/ mL) put in plate and in 37 °C and 5% CO2 incubator in continuous culture. Change the solution every two days. Incubate continuously for 10–14 days. When larger clones were formed in the control group fin-ished the administration. The culture medium in the orifice plate was absorbed and discarded, and the cells were fixed with 800 L methanol per well for 10–20 min. Discard methanol and add 1 ml crystal violet dye to each well for 30–40 min to avoid light treatment. The crystal violet dye was recovered and washed with 1–2 ml PBS for 2–3 times per hole. With air dry in a ventilated kitchen and then taking pictures.21 The experiments were performed in triplicates.