Cell death induced by N-methyl-N-nitrosourea, a model SN1 methylating agent, in two lung cancer cell lines of human origin

Abstract

New therapeutic approaches are needed for lung cancer, the leading cause of cancer death. Methylating agents constitute a widely used class of anticancer drugs, the effect of which on human non small cell lung cancer (NSCLC) has not been adequately studied. N-methyl-N-nitrosourea (MNU), a model S(N)1 methylating agent, induced cell death through a distinct mechanism in two human NSCLC cell lines studied, A549(p53(wt)) and H157(p53(null)). In A549(p53(wt)), MNU induced G2/M arrest, accompanied by cdc25A degradation, hnRNP B1 induction, hnRNP C1/C2 downregulation. Non-apoptotic cell death was confirmed by the lack of increase in the sub-G1 DNA content, Poly (ADP-ribose) polymerase cleavage and caspase-3, -7 activation. In H157(p53(null)), MNU induced apoptotic cell death, confirmed by cytofluorometry of DNA content and immunodetection of apoptotic markers, accompanied by overexpression of hnRNP B1 and C1/C2. Thus, the mechanism of the cell death induced by S(N)1 methylating agents is cell type-dependent and must be assessed prior treatment.

Modulation of Pathways Underlying Distinct Cell Death Mechanisms in Two Human Lung Cancer Cell Lines in Response to SN1 Methylating Agents Treatment

“…The human NSCLC lines NCI-H157 [p53 null ] and NCI-A549 [p53 wt ] were grown and treated as previously described [ 13 ]. Cells were harvested at 24, 48 and 72 h post MNU treatment and cytotoxicity and clonogenic cell survival were assayed as previously described [ 13 ]. DMSO treated cells (0.1% v/v) were used in all cases as controls.…”

“…A critical factor influencing the cellular response to methylating agents is O 6 -methylguanine- DNA methyltransferase (MGMT), the DNA repair protein that stoichiometrically and selectively removes methyl lesions from the O 6 position of guanine and returns the DNA to its pre-lesioned state [ 10 ]. Pre-replicative repair by MGMT as well as post-replicative MMR determine the level of methylating agent-induced genotoxicity and cell death [ 11 – 13 ].…”

“…Chemotherapeutic agents inducing DNA damage, such as S N 1 methylating agents and cisplatin, may activate cell death by apoptosis or necrosis. They could also induce autophagy, senescence or mitotic catastrophe, which may then be followed by apoptosis or necrosis [ 13 – 15 ]. The molecular basis underlying the decision- making process is currently the subject of intense investigation because a deeper understanding of how a given chemotherapy affects all of the signalling pathways involved in cell death is highly relevant in order to develop more effective therapeutics.…”

“…Despite their use in combination therapies, the effect of S N 1 methylating agents on human NSCLC has not been studied thoroughly. We thus investigated the mechanism of the cell death induced by a model S N 1 methylating agent, N-methyl-N-nitrosourea (MNU) in two human NSCLC cell lines, A549 (p53 wt ) and H157 (p53 null ) [ 13 ] through a time course gene expression profiling study 24, 48 and 72 hours after treatment. The list of differentiated genes, biological processes and cellular pathways were identified using appropriate bioinformatics tools and the results were further validated through RT-PCR of selected genes.…”

Proteomic identification of differentially expressed proteins associated with the multiple drug resistance in methotrexate-resistant human breast cancer cells

“…Previously, hnRNP C1/C2 has been considered as an apoptosis-related protein, and the protein level of hnRNP C1/C2 was p53-dependently upregulated, but its gene transcription did not depend on the p53 role in colon cancer cells after mitomycin C treatment (28,29). Furthermore, in A549 lung cancer cells of wild-type p53, the expression of hnRNP C1/C2 was significantly reduced in the process of apoptosis, it regulated the synthesis of p53 isoforms with p53 IRES interaction and affected tumor cell death (30,31). On the contrary, hnRNP C1/C2 was associated with DNA repair function, knockdown of hnRNP C1/C2 by RNA interference could increase the sensitivity of HeLa cells to chemotherapeutic drugs (32).…”