Cefotaxime sodium (COS), cefmetazole sodium (CMS), cisplatin (DDP) and 5-fluorouracil (5-Fu) were purchased from hospitals and freshly dissolved into PBS for use (stored in shadow at 4℃). The final concentrations of COS and CMS in complete medium were 25, 50, 100, 200 and 400 µg/ml; cisplatin were 0.5, 1.0, 2.0, 3.0 and 4.0 µg/ml; 5-Fu were 1, 2, 4, 8 and 16 µg/ml in the in vitro study. The doses of cefotaxime sodium used in the in vivo xenograft mouse study were 200 mg/kg; DDP was 2 mg/kg.
Cell lines and cell culture
Human non-small cell lung carcinoma (A-549), Xuanwei lung carcinoma (XWLC-05), hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT-116), stomach gastric carcinoma (SGC-7901), nasopharyngeal carcinoma (CNE2), neuroglioma (U-251), breast carcinoma (MCF-7) and human normal epithelia cell of vein (ECV-304) cell lines were friendly provided by Institute of Yunnan Tumor stocks, which were purchased from Cell Bank of Kunming Animal Institute, China Academy of Science. All cell lines were cultured in DMEM/F12 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS) (complete medium) (free of antibiotics) at 37℃ in a 5% CO2, humidified incubator.
6-8 weeks age male balb/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were fed in the temperature- and humidity- controlled Specific Pathogen Free Animal Facility of Kunming Medical University, with a 12-hr light-dark cycle. Mice were fed autoclaved distilled water and autoclaved rodent chow. Animal work described in this manuscript has been approved by the Ethical Committee of Kunming Medical University, China. All methods were carried out in accordance with relevant guidelines and regulations.
Cell viability assay
Cellular viability was determined by MTT assay. Cells were collected when they were cultured in the incubator for 72h and covered about 90% of the flask bottom. 200 µl complete DMEM/F12 medium with applicable cell number were seeded into wells of 96-well plates and incubated overnight. The following day, the original medium was gently removed by injector, 200 µl new complete medium containing different concentrations of samples was added into each well according to experiment design. 8 replicate wells were done for each concentration. Then, cells were incubated for 72 hrs. After removing the medium, 200 µl new complete medium containing 10% MTT（5 mg/ml）was added into each well. Plates were incubated for another 4 hours. After carefully removing the MTT medium, 150 µl DMSO was added into each well. Plates were then shaken in the shadow for 10 minutes. OD (optical density) values were determined by microplate reader at 490 nm. Deleting the highest and the lowest OD value in each group, 6 OD values in each group were remained for statistical analysis. Inhibition rates of cell viability were calculated using the following formula:
Inhibition rate = (Ac - Ad) / Ac × 100%.
Ac: the corrected absorbance (OD value) of the vehicle control group.
Ad: the corrected absorbance (OD value) of the drug group.
Corrected absorbance = OD value of sample group – OD value of blank control group
Plate clone formation assay
1,000 cells were seeded into each well of the 6-well culture plate. Cells were adhered overnight in the incubator. On the next day, after the medium in the wells was removed, 2 ml new medium containing different concentrations of drugs was added into each well, and 3 replicate wells were done in each group. Plates were further incubated for 14 days, during the period the medium was renewed at an interval of 5, 4, 3 days and cell state was observed. On the 14th day, each well was washed with 2 ml 0.01M PBS for one time, and then cells were fixed with 5% paraformaldehyde for 15 minutes. Discarding the paraformaldehyde, each well was washed with 2 ml 0.01M PBS for one time, and stained with 0.5 ml of 0.5% crystalviolet for 10 minutes, and then washed once with 2 ml 0.01M PBS. At last, the pictures were acquired with a camera, and the number of clones was counted.
Inhibition rate = (Nc - Nd) / Nc × 100%.
Nc: the clone number of the vehicle control group.
Nd: the clone number of the drug group.
Detection of apoptosis by flow cytometer
Experimental groups: solvent control group (PBS), cisplatin group (DDP, 2 𝜇g/ml), COS group (200 𝜇g/ml), COS+DDP group. Annex-V FITC/PI kit was used for apoptosis detection. Cells were seeded into 25 cm2 flasks and treated with samples as described in the cell viability assay. At the end, all cells including the cells died and floating in the medium were collected. Cells then were treated according to the protocol of the kit: each sample was added 0.4 ml binding buffer, resuspended, then added 5 𝜇l Annex-V/FITC and mixed to be stained in ice for 15 minutes. Then 10 𝜇l PI was added into each sample to incubate for 30 minutes at room temperature in dark. And then samples were detected by a flow cytometer according to standard protocol, in which flow cytometer would collect more than 10,000 events in each sample. During the process, the live and normal cells were not stained with Annexin-V/FITC and PI (Q3), the early apoptotic cells were only stained with Annexin-V/FITC (Q4), and the late apoptotic cells were stained with both Annexin-V/FITC and PI (Q2), and the necrotic cells and mechanically damaged cells were only stained with PI (Q1).
Tumor growth study in cancer xenograft model
Nasopharyngeal carcinoma CNE2 cells were collected during logarithmic stage and rinsed with PBS for two times, then resuspended into density of 4×107 cells/ml using fresh DMEM/F-12 medium free of FBS and antibiotics. Each mouse was inoculated 0.1ml cell suspension subcutaneously on the right side flank. Mice were fed autoclaved distilled water and autoclaved rodent chow. 8 days later after cell inoculation, tumor-bearing mice were used for in vivo anticancer study when the average tumor volume was around 200 mm3. 24 tumor-bearing mice were randomly allocated into 4 groups (6 tumor-bearing mice per group): vehicle control group (NS, neutral saline), COS group (200 mg/kg), DDP group (2 mg/kg) and combination group (COS+DDP). Mice were injected intraperitoneally 0.10 ml/10g b.w sample (mice in the vehicle control group were injected the same volume of neutral saline) each time for each mouse, 2 times a day at an interval of 6 hours (9:00 Am and 3:00 Pm). DDP was injected one time a day at one day interval in the morning. Body weight and tumor volume of each mouse were measured every 4 days. Mice were sacrificed at 12th day of drug treatment. Tumors were carefully isolated and weighed. Auto-reading caliper was used to measure the size of tumor. Tumor volume was calculated using the follow formula.
Tumor volume (TV) = (a × b2) / 2, “a” is the longitude range; “b” is the short diameter.
Relative tumor volume (RTV) = VT / V0, V0 is the tumor volume measured when grouping, VT is the tumor volume measured at each experiment time point.
Relative proliferation ratio (RPR) = (TRTV / CRTV) × 100%，TRTV is the relative tumor volume of the drug group, CRTV is the relative tumor volume of the solvent control group.
Microarray gene expression profiling
CNE2 cells were treated with neutral saline (NS), COS (200 µg/ml), DDP group (2 µg/ml) and COS+DDP respectively for 48h. After washed with cooled PBS for 2 times, the total RNA was rapidly extracted from the cells with Trizol and then samples were stored in -80℃ fridge and be sent to Shanghai Qi Ming Biological Information LTD for microarray gene expression profiling analysis using Affymetrix GeneChip® Human Transcriptome Array 2.0 after quality examination of sample RNA and gene chip. Gene chip data was pre-analyzed using RMA (Robust Multiarray Average) method. Gene expression analyses was performed using GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) by experts using Gene Cloud Biotechnology Information (GCBI) software. Genes were filtered using microarray fold change (logFC ≥ ±1.00). The biological process, molecular function, cell component, signal networks, signaling pathway network, and KEGG pathway that were enriched / modulated by samples in CNE2 cell lines were analyzed. The raw microarray data files were submitted to Gene-Cloud of Biotechnology Information (GCBI) repository. The microarray data was validated by profiling the expression of genes through quantitative reverse transcription PCR (RT-qPCR).
Quantitative reverse transcription PCR (RT-qPCR)
The microarray data was validated by profiling the expression of genes through quantitative reverse transcription PCR (RT-qPCR). Each data point presented for the quantitative PCR assay was derived from three biological replicates (BR). Total RNA from each BR was reversed transcribed and the cDNA from each BR was used as a template for the qPCR. Seven genes were selected to validate the microarray data, comprising of four upregulated differential genes (THBS1, SAMD9, PI3 and LAPTM5) and three downregulated differential genes (PPP3CB, STAG1 and NCOA5). Primers were designed according to the relevant target gene sequences published by GenBank. Table 5 lists the primers used for the RT-qPCR. Shanghai Qi Ming Biological Information LTD synthesized the primers.
After CNE2 cells were treated with drugs for 48 h, the total RNA was extracted with Trizol. The extracted total RNA was re-transcribed into cDNA. Finally, the fluorescence quantitative PCR reaction was carried out in a fluorescence quantitative PCR instrument. The thermocycling conditions were 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. GAPDH was selected as the internal control gene to normalize the gene expression data. Relative quantification of the target genes was calculated by comparative 2-△△CT method.
The data from three independent groups or above was analyzed by one-way ANOVA. Statistical comparisons were carried out by independent t-test. Data was represented as mean ± SEM in duplicate assays and analyzed using SPSS statistical software version 21.0. */# meant P<0.05, **/## meant P<0.01, ***/### meant P<0.001. The acceptable level for statistical significance was P < 0.05.