AZD 6244

LDH Cytotoxicity assay was performed using Pierce LDH cytotoxicity assay kit (Thermo scientific 88,954) according to manufacturer’s instructions. Briefly, the optimal number of cells (1 × 10⁵ cells/ml) were seeded in 100 μl of RPMI medium in triplicate wells in a 96-well tissue culture plate. A complete medium control without cells was used to determine LDH background activity present in sera used for media supplementation. A serum-free media control was included to determine the amount of LDH activity in sera. Additional cells were plated in triplicate wells for spontaneous LDH activity controls (water) and maximum LDH activity controls (10X Lysis Buffer). The plate was incubated in an incubator at 37°C, 5% CO₂ for 30 min. To the set of triplicate wells serving as the maximum LDH activity controls, 10 μl of lysis buffer (10X) was added, and mixed by gentle tapping. Different concentrations of glucose (5 mM and 15 mM), and inhibitors AZD6244 (10 μM), GDC0941 (2 μM), GDC0068 (2 μM), were added to cells. The plate was again incubated in an incubator at 37°C, 5% CO₂ for 45 min. Each sample medium (50 μl) (e.g., complete medium, serum-free medium, Spontaneous LDH Activity Controls, glucose-treated, inhibitors-treated and Maximum LDH Activity Controls) was added to a 96-well flat-bottom plate in triplicate wells. Reaction mixture (50 μl) was transferred to each sample well and mixed using a multichannel pipette. The plate was incubated at room temperature for 30 min protected from light. Stop solution (50 μl) was added to each sample well and mixed by gentle tapping. The absorbance was measured at 490 nm and 680 nm. To determine LDH activity, the 680 nm absorbance value (background) was subtracted from the 490 nm absorbance before calculation of % cytotoxicity [(LDH at 490 nm) − (LDH at 680 nm)]. The % cytotoxicity was calculated as follows:
% Cytotoxicity = Glucose/inhibitors-treated LDH activity − Spontaneous LDH activity/Maximum LDH activity − Spontaneous LDH activity × 100.

The HNSCC cell lines Detroit 562 (metastatic pharyngeal carcinoma, CCL-138™), FaDu (hypopharynx squamous cell carcinoma, HTB-43™) and SCC25 (tongue squamous cell carcinoma, CRL-1628™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Detroit 562 cells were cultured in Eagle’s Minimum Essential Medium (EMEM, Lonza, Basel, Switzerland) supplemented with 10% (V/V) fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1 mM sodium pyruvate (Lonza) and 1% (V/V) antibiotic mix (MycoZap Plus-CL, Lonza). The FaDu cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Lonza) supplemented with 10% (V/V) FBS (Gibco), 1 mM sodium pyruvate (Lonza) and 1% (V/V) antibiotic mix (MycoZap Plus-CL, Lonza). The SCC25 cells were cultured in Dulbecco’s Modified Eagle Medium:Nutrient Mixture F-12 (DMEM:F12, Lonza) supplemented with 10% (V/V) FBS (Gibco), 400 ng/mL hydrocortisone (STEMCELL Technologies, Vancouver, BC, Canada) and 1% (V/V) antibiotic mix (MycoZap Plus-CL, Lonza) in humidified atmosphere at 37 °C and 5% CO₂. The authentication of the cell lines was validated by STR DNA analysis (Eurofins Scientific, Luxembourg, Luxembourg). All cell lines were routinely screened for the absence of mycoplasma infection (DAPI staining) [39 (link)]. Afatinib (BIBW2992, cat. no. S1011), erlotinib HCl (OSI-744, cat. no. S1023), selumetinib (AZD6244, cat. no. S1008) and trametinib (GSK1120212, cat. no. S2673) were purchased from Selleckchem (Houston, TX, USA).