2.1 Cell culture and antibodies
The human Laryngeal cancer cell line HEp-2 was gifted by Dr. Chinmay K. Panda, Department of Oncogene Regulation (ORU), Chittaranjan National Cancer Institute (CNCI), Kolkata. Oral squamous cell carcinoma cell line UPCI: SCC131 was kindly provided by Dr. Susanne M. Gollin, University of Pittsburgh, PA, USA. Both the cells were cultured in Minimum Essential Media with Earle′s salts, EMEM (Invitrogen), supplemented with 10% fetal bovine serum (Gibco, USA) at 37ºC. Mouse monoclonal antibodies against AKT (sc-5298), NF-kB (sc-8008) and COX-2 (sc-376861) were purchased from Santa Cruz Biotechnologies. FITC tagged secondary antibodies (F-2761) was purchased from Invitrogen.
2.2 Clonogenic assay
Cells were seeded at a very low number (500 cells/ well) in six well plates. After 24h, they were treated with the optimum concentration of Lupeol (50 µM) alone and in combination with IR (2Gy) and incubated for 72 hours. The colonies were stained with Harry’s hematoxylin and number of clones was counted under Bright Field Microscope (Leica DM1000, Germany).
2.3 TUNEL assay
HEp-2 and UPCI: SCC 131 cells were seeded in six well plates and treated with Lupeol (50 µM) alone and in combination with IR (2Gy). After 24 hours of incubation TUNEL assay was performed using TUNEL assay kit- HRP- DAB (ab206386). The cells were fixed and permeablilized using Proteinase K solution. Next the cells were quenched using 3% H2O2 solution for endogenous peroxidise blocking followed by equilibration by the TdT equilibration buffer. The cells were then labelled using TdT labelling reaction mixture, blocked using 100 µl of blocking buffer and developed using DAB working solution. The cells were finally counterstained by Methyl green and observed under 10X magnification (Leica DM1000, Germany).
2.4 Detection of apoptosis
To check the induction of apoptosis, cells were seeded in six well plates and treated with Lupeol (50μM) and IR (2Gy) alone and in combination. After 24 hours incubation, the cells were centrifuged (1200 rpm for 5 minutes). The pellet was resuspended in 1x assay buffer (final concentration of 1x106 cells/ ml). Five micro litres (i.e.1μg) of Annexin V FITC and 10 μl of PI were added to the cells and incubated at room temperature in the dark for 15 minutes. The cells were washed twice with PBS, resuspended in sheath fluid (BD FACSFlowTM) containing 2% paraformaldehyde and analyzed using FACSVerseTM.
2.5 Transwell migration assay
HEp-2 and UPCI: SCC 131 cells were seeded in transwell plates (Corning) in serum free media. The bottom layer was filled with complete media as chemoattractant. The cells were further treated with the said treatment regime of Lupeol and IR and after 24 hours Transwell migration assay was performed. The wells were thoroughly washed using 1X PBS, stained with Giemsa stain. After thorough wash the membrane was carefully taken out, mounted on glass slides and photographed under 40X magnification (Leica DM1000, Germany).
2.6 Sphere formation assay
Cells were seeded in 96 well plates with ultra low attachment surface (Corning). The hydrogel of the polystyrene vessel surface forces cells into a suspended state, enabling the formation of 3D spheroid. The cells were treated with Lupeol (50 µM) and 2Gy of IR and incubated for seven days. On the eighth day the plates were observed under inverted phase contrast microscope (Leica DM1000, Germany), the number and diameter of each sphere was calculated and the effect of combination dose of Lupeol and IR was obtained.
2.7 Immunofluorescence staining
In order to analyse the expression of oncoproteins, HEp-2 cells were seeded in cover slips and grown for 24 hours in 37ºC. Next day the cells were treated with Lupeol (50 µM) and in combination with Lupeol (50 µM) and IR (2 Gy). Treated cells were then fixed in Methanol, treated with primary monoclonal (AKT, NF-kB, COX-2) and fluorescence tagged secondary antibodies respectively. The cells were finally incubated with DAPI and observed under fluorescence microscope (Leica DM4000 B, Germany).
2.8 Flow cytometric analysis
0.3x106 cells (HEp-2) were seeded in each well of six well plates and treated with Lupeol (50 µM) and in combination with Lupeol (50 µM) and IR (2 Gy). After respective period of incubation, the cells were centrifuged and the pellet was resuspended in 0.5 ml PBS. For fixation 4.5 ml of 70% ethanol was added to the suspension. The cells were centrifuged for 5 minutes at 1000 rpm and the cell pellet was suspended in anti AKT, NF-kB and COX-2 antibody and incubated for 45 minutes in dark. The cells were next incubated with FITC tagged secondary antibody and Fluorescence was measured using the long-pass filter in BD FACSCaliburTM (USA).
2.9 Western blot
Treated (Lupeol and Lupeol+IR) HEp-2 cells were homogenized and lysed in western blot lysis buffer (15mM Tris, 2mM EDTA, 50mM 2-Mercaptoethanol, 20% Glycerol, 0.1% Triton X100, 1mM PMSF, 1mM Sodium Fluoride, 1mM Sodium Orthovanadate, 1µg/ml Aprotinin, 1µg/ml Leupeptin, 1µg/ml Pepstatin) at room temperature. Fifty microgram of total cell lysates (TLC) were separated on a 10% sodium dodecyl sulphate- polyacrylamide gel SDS-PAGE, and blotted onto nitrocellulose membranes, blocked in TBS-T (0.1 % Triton in 1xTBS) and probed with anti- AKT, NF-kB and COX-2 primary antibody overnight at 4ºC. The membranes were then incubated with the appropriate horseradish peroxidase- conjugated secondary antibodies. The immunoreactive protein bands were developed by enhanced chemiluminescence kit (BioVision ECL Western Blot Substrate, USA) and analyzed by a densitometer (Bio Rad, GS 800, USA) for quantification.