Dose-response curves were drawn to assess the drug concentration reducing survival. The initial cytotoxicity experiments were carried out utilizing toxins concentrations that in other publications were reported to be effective in a7 nAChR-positive NSCLC cell lines but not toxic in normal cells. However, at these concentrations we could not detect appreciable cytotoxic activity and the IC50 could not be reached with any of the two toxins. At higher concentrations the a-cobrotoxin showed a Ifenprodil hemitartrate limited non dose-dependent toxicity that remained essentially constant in a wide range of concentrations in all 5 cell lines. At high concentrations the a-cobratoxin showed a clear dosedependent toxicity. However the IC50 concentration observed in our study for A549 and SK-MES 1 was 2466 and 105 fold higher than that reported in an earlier publication. The difference was much lower for H1650 a result compatible with the utilization of a different toxin preparation. The limited toxic effect of the short chain a-cobrotoxin could be explained by its inability to bind to the a7 receptor. Surprisingly however, the a-cobratoxin was effective also on a7 nAChRnegative cells suggesting that the toxic activity is not mediated by the binding of the toxin to the a7 receptor. To confirm and extend this observation we have conducted a colony formation assay with the a7 nAChR+A549 and with the a7 nAChR2NCI-H1975 cell lines. Since the IC50 was not reached with a-cobrotoxin, we utilized the two highest concentrations tested by MTT. For a-cobratoxin we utilized the concentrations corresponding to the IC50 for A549 and NCI-H1975 and concentrations corresponding to half and twice the IC50. As shown in Figure 3, the clonogenic activity of the two cell lines was not SCH 23390 hydrochloride affected by the treatment and by the presence of the a7 nACh receptor. The activation of the apoptotic cascade was considered the key effect of the binding of the a-cobratoxin to the a7 nAChR. In view of the major differences between our results and those previously published regarding the dosage at which the IC50 could be obtained and the absence of selective action of acobratoxin on a7 nAChR-positive cells, we tried to understand if activation of apoptosis indeed occurs upon treatment with acobratoxin by Annexin V-PI flow cytometry staining. It was reported that the maximum induction of apoptosis in A549 cells could be obtained by treatment with toxin for 24 hours.