Expressions of c-MYC, c-FOS and c-JUN were evaluated in the current study in MCF-7, A549, and HepG2 cells with Lanatoside C treatment. significant evidence on the binding sites of Lanatoside C with various key signaling proteins ranging from cell survival to cell death. Our studies provide a novel molecular insight of anti-cancer activities of Lanatoside C in human cancer cells. and studies. 2. Materials and Methods 2.1. Cell Lines and Chemicals Human breast cancer (MCF-7), lung cancer (A549), and hepatocellular carcinoma (HepG2) cell Rabbit Polyclonal to HTR5A lines were purchased from CSIR-Central Drug Research Institute (Lucknow, India) and normal lung (L132) and liver (WRL68) cell lines were purchased from the National Center for Cancer Cell lines (NCCS, Pune, India). All the cells were cultured in DMEM supplemented with 10% FBS (fetal bovine serum), L-glutamine (2 M) and antibiotic-antimycotic solution, and incubated at 37 C in a humidified atmosphere of 5% CO2. Lanatoside C was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) by maintaining the overall DMSO concentration not exceeding 0.001% in all the experiments. MTT, Propidium iodide, and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA). In every experiment, the control contained the highest DMSO percentage (0.001%). Peripheral blood mononuclear cells (PBMC) were used for checking the toxicity of Lanatoside C with a wide range of concentrations (0.01C500 M). PBMCs were purchased from Himedia, Cat#CL003-25 (Mumbai, India). The cells were then revived in the RPMI medium supplemented with 10% FBS and antibiotics. Approximately 1 105 cells were seeded in 96 well Rupatadine Fumarate plates; after 2C4 h incubation, the cells were Rupatadine Fumarate treated with a wide range of Lanatoside C concentrations to check the toxicity. The experiment was done thrice and results were interpreted in Rupatadine Fumarate Origin 9.5. 2.2. Cytotoxicity Assay Approximately 3500 cells were seeded in each well of 96 well plates and allowed to attach overnight (16 h). The cells were treated with Lanatoside C with different doses for 24 h. Then, 0.5 mg/mL of MTT solution was added to the cells and allowed to incubate in the dark for 2C4 h, and the dye was dissolved in DMSO. The absorbance was measured at 570 nm and the baseline correction was set to 630 nm. 2.3. DNA Damage Assay DNA damage has been evaluated by comet assay with minor modifications from . Briefly, around 1000 cells were seeded in a 6 well plate and allowed to incubate for at least 16 h. The cells were then treated with inhibitory concentrations for 24 h. After 24 h, cells were harvested and mixed in 0.6 mL of PBS. 1% low melting agarose was prepared and mixed with cells and layered on scored glass slide without forming air bubbles. The slides were then allowed to dry in the air and incubated in lysis buffer overnight. Next, the slides were washed with 1 TAE three times at 20 min intervals and subjected to electrophoresis at 0.6 V/cm for 25 min. The slides were then stained with 2. 5 g/mL of propidium iodide and washed and distilled for destaining. The cells were visualized Rupatadine Fumarate for DNA damage using a fluorescent microscope under 20 magnification (Leica DMI-3000I microscope- Wetzlar, Germany). 2.4. Cell Cycle Analysis By Flow Cytometry DNA content based cell cycle regulation analysis was performed as follows: Briefly, 1 105 cells were seeded in a 6 well plate and incubated overnight. After 24 h, the media was removed and the cells were treated with inhibitory concentrations for 24 h. Cells were then trypsinized and centrifuged at 3000g for 5 min and the pellet was dissolved in ice-cold ethanol and stored at ?20 C for a minimum of 24 h. The cells were then washed thrice with PBS to remove ethanol content and incubated at 37 C with RNase A. The cells were then stained with 0.5 g/mL of propidium iodide for 30 min and subjected to FACS instrument (BD Biosciences- Allschwil, Switzerland) for cell cycle analysis. 2.5. Real-Time PCR Analysis Total RNA was extracted using TRIzol? (Invitrogen- Carlsbad, CA, USA) reagent by following the.