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

Abstract

Despite advances in the understanding of tumor biology in recent years, lung cancer remains the leading cause of cancer death worlwide. Non small cell lung cancer (NSCLC; adenocarcinoma, squamous carcinoma, and large cell carcinoma) is the most common type, representing more than 80% of lung cancer diagnoses. Clinical improvements following treatment with traditional chemotherapy have reached a plateau in patients with NSCLC and 5 year survival still remains at 15%. Moreover, the molecularly targeted therapies having already progressed to clinical trials have not proved successful [1,2]. Although NSCLC is very often extremely resistant to chemotherapeutic agents, chemotherapy remains an established treatment for advanced NSCLC. Platinum-based chemotherapy frequently combined with other agents and/or approaches remain the standard in the first-line treatment of patients with advanced NSCLC [3,4]. Methylating agents such as temozolomide, an oral SN1 methylating agent which crosses the blood brain barrier achieving effective concentrations in the Central Nervous System (CNS), have already been used combined with platinum-based regimen and whole brain radiotherapy as prophylaxis against brain metastasis in NSCLC and/or as second-line treatment in NSCLC patients with brain metastases [5–8]. Methylating agents of SN1 type, including temozolomide (TMZ), procarbazine, dacarbazine and streptozotocine, constitute a widely used class of anticancer drugs. These DNA damaging agents are highly cytotoxic, mutagenic, recombinogenic and clastogenic inducing about a dozen DNA methylation adducts [9]. Among them, O6-methylguanine (O6-meG), induced in small amounts (maximally 8% of total methylation products), is the most biologically significant lesion; it mispairs with thymine during DNA replication finally generating G to A transitions in the second round of replication. The DNA mismatches are recognized by the Methyl-directed Mismatch Repair (MMR) system that removes the mis-incorporated base opposite the O6-meG lesion. Repair resynthesis of DNA leads to reinsertion of thymine opposite O6-meG and re-initiation of futile mismatch repair. The DNA double-strand breaks (DSBs) resulting from this process are thought to initiate a series of events including prolonged G2 arrest leading to cell death [9]. A critical factor influencing the cellular response to methylating agents is O6-methylguanine- DNA methyltransferase (MGMT), the DNA repair protein that stoichiometrically and selectively removes methyl lesions from the O6 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 SN1 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 SN1 methylating agents on human NSCLC has not been studied thoroughly. We thus investigated the mechanism of the cell death induced by a model SN1 methylating agent, N-methyl-N-nitrosourea (MNU) in two human NSCLC cell lines, A549 (p53wt) and H157 (p53null) [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. MNU induced cell death through distinct responses at the gene expression level in the above cell lines. Our results overall support the use of SN1 methylating agents in platinum-based combination regimen against advanced NSCLC.