Figure 3. Glutamine is required for growth of lung cancer cells in vitro.

To test whether SLC1A5 expression is correlated with Gln-dependent growth of lung cancer cells in vitro, 5 lung cancer cell lines that vary in their expression level of SLC1A5 protein (A) were grown in culture media that are supplemented with EGF, Insulin, selenium and transferein and 2mM of L-glutamine (+Gln+Sup), or L-glutamine but no supplements (+Gln-Sup), or no L-glutamine but with supplements (−Gln+Supp) or not supplemented with neither L-glutamine not supplements. (B) The fold change in cell growth was analyzed at day 3 by Cell-Titer 96-Aqueous Colorimetric Assay as a change of OD at 490_nm signal from day 0. Sensitivity of growth inhibition to Gln deprivation was significant in cell lines that overexpress SLC1A5, A549, H520 and HCC15 but not in cell lines that have low or undetectable level of the protein H1819 and H727. (C) The effect of Gln deprivation on cell viability in SLC1A5 expressing cell lines A549 and H520 were measured by Trypan blue Exclusion Dye method after 48 h of culturing in media that either supplemented or deprived of Gln. (D) The anti-growth effect of Gln deprivation was measure by calculating the net increase or decrease of cellular growth after 48 hour of culturing under media that either supplemented or deprived of Gln or fully supplemented media + 1mM of GPNA using the following equation (Ti−Tz)/(C−Tz)×100. Where Ti is the cell number after 48 hours of treatment of growth stimuli or inhibitors (i=inhibition), Tz is the cell number at time zero and C is the cell number of the control cells that were cultured in optimum growth conditions (+Gln +Sup). (E) Dose response decline in cell growth after treatment of A549 and H1819 with increasing concentrations of DON for 48 hours. Statistical significance was assessed by Students t-test and was denoted as *= p≤ 0.05, **= p≤ 0.005 from 3 independent assays.