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Camptothecin, 20-(N,N-diethyl) glycinate exerts preferential cytotoxicity towards EGFR-transfectant cells. G2/M DNA damage checkpoint regulation, aryl hydrocarbon receptor signaling, xenobiotic metabolism and endoplasmatic reticulum stress are involved.The epidermal growth factor receptor (EGFR) represents a prognostic marker for short survival of patients and therapy resistance of tumors. Despite clinical usefulness of EGFR tyrosine kinase inhibitors, resistance can develop. Therefore, there is an urgent need for novel EGFR inhibitors. Camptothecins have been characterized as inhibitors of DNA topoisomerase I (TOP1), although a correlation between TOP1 expression and activity is not well established in clinical biopsies. Hence, other targets may also be relevant. By molecular docking, we found that camptothecin 20-N,N-glycinate (CPTg) and camptothecin (CPT) bind to the same pharmacophore at EGFR as erlotinib, albeit to partly different amino acids. To validate the in silico results, CPT and CPTg were evaluated for their cytotoxic activity and downstream signaling mechanisms in U87MG glioblastoma cell lines transduced with different expression vectors for EGFR. All transduced cell lines were more susceptible to CPTg or CPT than the non-transduced cells, indicating a preferential activity towards EGFR-expressing tumor cells. Microarray-based mRNA hybridizations were performed in treated a non-treated cell lines. Subsets of genes were commonly regulated between the cell lines. By pathway analyses, we ranked canonical pathways according to differential gene expression after drug treatment. The pathways for G2/M DNA damage checkpoint regulation, aryl hydrocarbon receptor signaling, and xenobiotic metabolism and endoplasmatic reticulum stress were top ranked. In conclusion, binding of CPTg and CPT to the erlotinib pharmacophore and preferential cytotoxicity towards EGFR-overexpressing cells indicate susceptibility towards erlotinib-resistant tumors. Multiple mechanisms may account for cytotoxicity of these camptothecins.