The human colorectal cancer cell lines, SW480 (Catalog No. TCHu172) and SW620 (Catalog No. TCHu101) were purchased from the Cell Bank of the Chinese Academy of Sciences(Shanghai, China). Cells were maintained in DMEM media (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 IU/mL penicillin, and 100 μg/mL streptomycin. All cells were incubated at 37°C in a humidified incubator with 5% CO2. Media were changed every second or third day.
Cell Proliferation Assays
The cell viability of SW480 and SW620 cells after CKI treatment was characterized after a CCK-8 (Cell Counting Kit-8) assay. Briefly, we adjusted the cell concentration to 1 × 106 cells/mL, then seeded 100 μL of SW480 and SW620 cells (5,000 cells per well) into 96-well flat-bottomed plates, respectively. We then added 10 mL of CKI to each well, with the final drug concentration at 2 mg/mL (based on the total alkaloid concentration in CKI). CKI were purchased from Shanxi Zhendong Pharmaceutical Co., Ltd. (20161113, changzhi, shanxi, China). The plates were incubated for 24 or 48 h. Ten μL of CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) was added to each well of the plates and mixed thoroughly. Subsequently, the plates were incubated for another 2h, and optical densities were obtained by reading the plates at 450 nm with a 96-well micro test spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA). Each concentration of CKI was assayed three times in the same plate. Matched control cells received DMEM cell culture medium and 5-Fu (10 mg/mL) instead of CKI. By comparing untreated control cells with CKI-treated cells, we investigated the effect of CKI on cell proliferation.
Flow Cytometry Assays
For cell cycle analysis, SW480 and SW620 cells (1×106 cells/well) were seeded into six-well plates, cultured in completed culture medium overnight, and then exposed to CKI (2 mg/mL) for 24 h. Nuclear DNA was analyzed using a BD Cycletest™ Plus DNA Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Stained cells were detected and quantified with a FACS Flow Cytometer (BD Biosciences). All FACS results were analyzed with FlowJo software (Version 7.6).
Autophagy activity was analyzed by Cell Meter Autophagy assay. First, SW480 and SW620 cells were adjusted to a concentration of 2 × 104 cells/mL, then 100 μL of this solution (2,000 cells per well) was seeded into 96-well flat-bottomed plates and cells incubated overnight. At the same time, a non-induced negative control cell population at the same density as the induced population was cultured for every labeling condition. The medium was then removed, 100 μL of Autophagy Blue™ working solution (AAT Bioquest Co., CA, USA) was added to each well, and the cells incubated at 37°C in a 5% CO2 incubator for 1h. The cells were washed with wash buffer three times. Finally, the fluorescent intensity was monitored with a fluorescence microscope using a DAPI channel.
Animal Model Experiments
For in vivo tumor growth assays, 5-week-old ICR male mice were purchased from Beijing Vital River Laboratory Animal Technology Co. and housed in a dedicated specific pathogen-free facility at the Laboratory Animal Center of Peking University People's Hospital. Time-course observations during AOM/DSS (azoxymethane/dextran sodium sulfate)-induced mouse colorectal carcinogenesis were conducted to determine when colonic tumors occurred in the inflamed colon of mice that received 2% DSS after AOM initiation. Male ICR mice received a single intraperitoneal injection of AOM (10 mg/kg body weight; Sigma-Aldrich, St Louis, MI, USA) and followed by one week administration of 2% DSS (MP Biomedicals, Irvine, CA, USA) in drinking water, after which the mice were maintained on regular water for 28 days. Then, the mice in the model group were randomly divided into three groups (n = 6/each group). At week seven, the three groups of mice were injected intraperitoneally with 200 mL NaCl (original injection), 200 mL 5-Fu (5-fluorouracil, 4 mg/mL), and 200 mL CKI (original injection), respectively. The weight of mice was measured twice a day with a platform scale. At about 4 weeks after drug intervention, the mice were sacrificed by carbon dioxide euthanasia method. Briefly, after the mice were put into the cage, the power of the pressure reducer was switched on and the valve of the carbon dioxide cylinder was slowly opened. The pressure was kept to 6Mpa and the flow rate to 1.3L/ min till the mice were confirmed death, and the gas cylinder valve was then shut down. Following, the colorectal tissues of the mice were removed and photographed. Tissue samples were fixed overnight in 10% paraformaldehyde, embedded in paraffin, and sectioned for histological analysis. The colorectal tissues of mice, which were fixed by formaldehyde and embedded in paraffin, were sectioned at a 4-mm thickness and stained with hematoxylin and eosin according to standard protocols.
All animal studies were performed in accordance with rules and guidelines concerning the use and care of laboratory animals and approved by the Animal Care Committee of Peking University People's Hospital.
Validation of Results by Real Time RT-qPCR Assays
SW480 and SW620 cells were incubated at 37°C in a humidified incubator after treatment with CKI (2 mg/mL) for 24 h. At the same time, phosphate-buffered saline (PBS) was used as a control. Total RNA was extracted using an RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and cDNA was synthesized with a SuperScript Ⅲ Synthesis Reagent Kit (Invitrogen, Carlsbad, CA, USA). Reverse transcription quantitative PCR (RT-qPCR) analyses for mRNA levels of p53, p21, CHEK1, RBL1 in SW480 cells and p53, p21, YWHAZ, BUB3, WEE1 in SW620 cells were performed using KAPA SYBR Green FAST Universal qPCR kit (Roche, Basel, Switzerland) on a Bio-Rad CFX96TM Real-Time PCR System. The program was run as follows: initial denaturation was at 94°C for 2 min. The following steps were reiterated for 44 cycles: denaturation at 94°C for 30 sec; annealing at 60°C for 30 sec; and extension at 72°C for 45 sec. The final extension was set at 72°C for 5 min. β-actin was identified as a suitable internal control, and the results were expressed as the threshold cycle (Ct). The relative quantification of target transcripts was determined by a comparative Ct method (ΔΔCt) according to the manufacturer’s protocol. The 2−ΔΔCt method was used to analyze relative changes in gene expression. Control PCR experiments in the absence of reverse transcription were performed to confirm that total RNA was not contaminated with genomic DNA. The primers used are listed in Table 1.
Western blot analysis
To analyze the relative expression of cell cycle proteins, western blot analysis was performed. The cells of the two colorectal cancer cell lines, SW480 and SW620, were collected in the logarithmic growth phase, and the cell concentration was adjusted to 5 × 105 cells/mL. CKI (2 mg/mL) was added to each cell culture, and cells were incubated for 24h. The cells were then collected, washed twice with cold PBS, and harvested in 500 mL cell lysis buffer (containing incubation buffer, 20% Triton X-100, proteinase inhibitor, sodium fluoride, and sodium orthovanadate solution) for 30 min on ice, briefly mixed, and centrifuged at 14,000×g at 4°C for 30 min. The total protein concentration was determined by a BCA Protein Assay Kit (Thermo Scientific Pierce, Waltham, MA, USA). Equal amounts of cellular proteins were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with Tris-buffered saline (TBS) buffer containing 5% skim milk powder at room temperature for 1h, and then incubated with primary antibodies, including rabbit polyclonal antibody against p53 (1:1000; Proteintech, Rosemont, IL, USA), p21 (1:1000; Cell Signaling, Danvers, MA, USA) and mouse monoclonal anti-β-actin (1:3000; Sigma-Aldrich) overnight at 4°C. The blots were washed three times in TBS with Triton X-100 and incubated with horse radish peroxidase-conjugated secondary antibody (1:1000; ZSGB Biotechnology, Beijing, China) for 1h at room temperature. Blots were then visualized with enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA). All grey values of the protein bands were performed using custom ImageJ (National Institutes of Health; http://rsb.info.nih.gov/ij/) script.
Prediction of Ingredients Acting on p53 in CKI
BATMAN-TCM is the first tool in bioinformatics analysis for studying the molecular mechanism of TCMs by predicting potential targets for ingredients in TCMs (http://bionet.ncpsb.org/BATMAN-TCM). After that, compounds were entered as search queries in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP; http://lsp.nwu.edu.cn/tcmsp.php.) and in the Traditional Chinese Medicine Integrated Database (TCMID; http://www.megabionet.org/tcmid/search/). The following parameter settings were used: Target Prediction’s Score cutoff was 20; Target Analysis’s Adjusted p-value was less than 0.05.
Statistical analysis was performed using GraphPad Prism 5.0. In this study, all experiments were repeated at least three times. Student’s t-test was used to evaluate the differences between two groups. All p values were two-sided and differences between the groups were identified as statistically significant at three levels: p<0.05, p<0.01, and p<0.001.