Fig. 2.

Effects of TMZ and doxorubicin on GBM cells. (A–C) Drug-induced cell damage. NSs (open columns) and ACs (hatched columns) were incubated for 72 h with fresh medium (−) or TMZ at 50, 100, and 200 μM (T; panel A), 5 μM doxorubicin for 24 h (dox; panel B), or 50 μM TMZ for 72 h plus 5 μM doxorubicin for the last 24 h (panel C). Human doxorubicin-sensitive colon cancer HT29 cells and the doxorubicin-resistant counterpart HT29-dx cells were used in (B) as positive controls of drug-sensitive and drug-resistant cells, respectively. The culture medium was collected and analyzed in duplicate for the LDH release, as an index of cytotoxicity. Data are presented as means ± SD (n = 4). Significance vs untreated (−) cells: *P < .02; T + dox vs T alone: °P < .05. (D) NSs were seeded at a concentration of 100 cells/well, left untreated (ctrl) or treated at days 4, 11, 18, 25, 32, and 39 with 50 μM TMZ (T) for 72 h, 5 μM doxorubicin for 24 h, or TMZ for 72 h with the addition of doxorubicin in the last 24 h. At the end of each treatment, cells were washed and reseeded in fresh medium. The spheres formed in each well were counted at days 14, 28, and 42. Data are presented as means ± SD (n = 4). Significance vs untreated cells: *P < .02; T + dox vs T alone: °P < .005. (E) NSs incubated as reported in (C) were subjected to cell cycle analysis. Data are presented as means ± SD (n = 3). Significance vs untreated (−) cells: *P < .01; T + dox vs T alone: °P < .02.