Native musk and synthetic musk ketone
The native musk sample was obtained from the gland capsule of a dead musk deer. This musk deer died of natural causes. The dead musk deer was provided by Li-Jiang City, Yunnan Province, China. The musk deer belongs to Moschus berezovskii. The study was approved by the Ethics Committee for Animal Experimentation, Kunming Institute of Zoology, Chinese Academy of Sciences. The native musk sample (0.076 g) was added to 1 ml of ethanol and the mixture was shaken for 1 hour (h). The supernatant was filtered through a 0.22 μm filter and stored at 4 °C. Gas chromatograph-mass spectrometer-computer (GC-MS) analysis confirmed that main ingredients of native musk were extracted. The sample of synthetic musk ketone (the purity: 98%) was purchased from Chengdu Preferred Biotechnology. Co. Ltd (CAS: 541-91-3, Lot No.13709; Chengdu, China), and this sample was dissolved in ethanol, filtered through a 0.22 mm filter and stored at 4 °C. The chemical structure of synthetic musk ketone was showed in Additional file 1.
Cell lines and cell culture
Up to 22 human cancer cell lines were used in this study. These cell lines included 11 types of cancer such as lung squamous cell carcinoma, lung adenocarcinoma, lung large cell carcinoma, lung small cell carcinoma, mammary carcinoma, esophageal carcinoma, gastric carcinoma, colorectal carcinoma, hepatocellular carcinoma, acute myelogenous leukemia, and B cell lymphoma. These cell lines were cultured with RPMI 1640 or DMEM medium (GIBCO Invitrogen, Grand Island, NY, USA) containing 10% foetal bovine serum and maintained in a humidified incubator with 5% CO2 at 37 °C. XLA-07 and XL-JT were provided by Dr LJ Ma [15]. Detailed information about the cell lines is presented in Additional file 2.
Cell proliferation assay
Cultured cells were seeded in 96-well plates at a density of 1 × 104 cells per well. Native musk and synthetic musk ketone (four replicates in each group) were added at varying concentrations. After 24 h of treatment, 20 μl of MTS solution [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Promega Corporation, Madison, WI, USA] was added to each well, and the plates were incubated for 4 h at 37 °C. Absorbance at 490 nm was measured using a spectrophotometer. The same amount of solvent (ethanol) was used as the control.
Flow cytometry (FCM) analysis
Apoptotic cells were determined by FCM analysis with the Fluorescein Isothiocyanate (FITC)-labeled Annexin V Kit (BD Pharmingen, San Diego, CA, USA) in accordance with the manufacture’ instruction. Treated cells were washed twice with phosphate buffer saline (PBS) and adjusted at a density of 5 × 105 cells/100 μl. Cell suspensions were added to each tube; afterward, the cells were stained with annexin V-FITC and propidium iodide and then analyzed under FCM (BD Biosciences, San Jose, CA, USA). Collected data for 10,000 cells and WinMDL software were used for the analysis of FCM data files.
GC-MS analysis
The native musk sample was extracted with ethanol as described above. The supernatant was filtered and determined by GC-MS (Agilent Technologies, Palo Alto, CA, USA; HP6890GC/5973MS) as previous described [16]. Similarly, 1 μl of the musk ketone sample was dissolved in 100 μl of ethanol and then determined by GC-MS. Qualitative analysis was performed with the Wiley7n.l standard library.
mRNA expression profiling
Harvested cells were washed twice with cold PBS, and total RNA was isolated using Trizol reagent (TaKaRa, Tokyo, Japan). Determination of mRNA profiling was performed in Eplc-32M1 and XL-JT cells with or without native musk treatment by using Agilent Human Gene Expression array (CapitalBio Technology, Beijing, China; http://www.capitalbio.com). Differentially expressed genes were subjected to Gene Ontology (GO) as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using Molecule Annotation System (MAS) 3.0 (http://bioinfo.capitalbio.com/mas3).
Quantitative real–time polymerase chain reaction (qRT-PCR)
For mRNA expression assay, total RNA was isolated from cells by using TRlzol reagent (TaKaRa). Synthesis of cDNA was carried out by M-MLV Reverse Transcriptase (Promega) with random primer and amplified with specific primers on the StepOne Realtime PCR System (Applied Biosystems, Foster City, CA, USA). QRT-PCR analysis was performed using miScript SYBR Green PCR kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer’s instructions. The PCR reaction process was first incubated at 95 °C for 3 minutes (min), followed by 40 cycles of thermal cycling at 95 °C for 15 seconds (s) and 60 °C for 30 s. The mRNA levels of the target genes were demonstrated using ΔCT (ΔCT = CT, Target-CT, actin; CT, Target, the average threshold cycle number of the target gene; CT, actin, the average threshold cycle number of the β-actin). The primer sequences were described as follows. TNFRSF25: 5′-CCCAGAACACACCTACTCTGC-3′(R), 5′-AGAGATACTGACTGT-GGGACC-3′(F); IL-24: 5′-TCCAACTGTTTGAATGCTCTCC-3′(R), 5′-CTTTGTTCT-CATCGTGTCACAAC-3′(F); DDIT3: 5′-CTGCTTGAGCCGTTCATTCTC-3′(R), 5′-GGAAACAGAGTGGTCATTCCC-3′(F).
Statistical analysis
Statistical significance was calculated using the Student’s t-test. SPSS 17.0 software package (Chicago, IL, USA) was used for all statistical analyses. The level of statistical significance was set at 0.05 for all the tests.
