A transcriptomic computational analysis of mastic oil-treated Lewis lung carcinomas reveals molecular mechanisms targeting tumor cell growth and survival

BMC Medical Genomics A transcriptomic computational analysis of mastic oil-treated Lewis lung carcinomas reveals molecular mechanisms targeting tumor cell growth and survival Panagiotis Moulos 1 Olga Papadodima 1 Aristotelis Chatziioannou 1 Heleni Loutrari 0 Charis Roussos 0 Fragiskos N Kolisis 1 0 "G.P. Livanos and M. Simou Laboratories", Evangelismos Hospital, Department of Critical Care and Pulmonary Services, School of Medicine, University of Athens , 2 Ploutarchou st., 10676, Athens , Greece 1 Metabolic Engineering and Bioinformatics Group, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation , 48 Vasileos Constantinou ave. 11635, Athens , Greece Background: Mastic oil from Pistacia lentiscus variation chia, a blend of bioactive terpenes with recognized medicinal properties, has been recently shown to exert anti-tumor growth activity through inhibition of cancer cell proliferation, survival, angiogenesis and inflammatory response. However, no studies have addressed its mechanisms of action at genome-wide gene expression level. Methods: To investigate molecular mechanisms triggered by mastic oil, Lewis Lung Carcinoma cells were treated with mastic oil or DMSO and RNA was collected at five distinct time points (3-48 h). Microarray expression profiling was performed using Illumina mouse-6 v1 beadchips, followed by computational analysis. For a number of selected genes, RT-PCR validation was performed in LLC cells as well as in three human cancer cell lines of different origin (A549, HCT116, K562). PTEN specific inhibition by a bisperovanadium compound was applied to validate its contribution to mastic oil-mediated anti-tumor growth effects. Results: In this work we demonstrated that exposure of Lewis lung carcinomas to mastic oil caused a timedependent alteration in the expression of 925 genes. GO analysis associated expression profiles with several biological processes and functions. Among them, modifications on cell cycle/proliferation, survival and NF-B cascade in conjunction with concomitant regulation of genes encoding for PTEN, E2F7, HMOX1 (up-regulation) and NOD1 (down-regulation) indicated some important mechanistic links underlying the anti-proliferative, proapoptotic and anti-inflammatory effects of mastic oil. The expression profiles of Hmox1, Pten and E2f7 genes were similarly altered by mastic oil in the majority of test cancer cell lines. Inhibition of PTEN partially reversed mastic oil effects on tumor cell growth, indicating a multi-target mechanism of action. Finally, k-means clustering, organized the significant gene list in eight clusters demonstrating a similar expression profile. Promoter analysis in a representative cluster revealed shared putative cis-elements suggesting a common regulatory transcription mechanism. Conclusions: Present results provide novel evidence on the molecular basis of tumor growth inhibition mediated by mastic oil and set a rational basis for application of genomics and bioinformatic methodologies in the screening of natural compounds with potential cancer chemopreventive activities. - Background Lung cancer is the leading cause of cancer deaths in the US among both men and women [1]. Nowadays the search for new chemopreventive and chemotherapeutic agents to treat malignancies, especially the most mortal types characterized by rapid metastasis and frequent resistance to current chemotherapy/radiotherapy regimens has recently increased and the interest is mainly focused on natural compounds with low toxicity [2]. A large body of pre-clinical, clinical and epidemiological studies support that many phytochemicals, i.e. bioactive compounds isolated from plants, can delay tumor progression and metastasis [3,4]. While most of the available evidence refers to isolated substances, recent data support that natural combinations of phytochemicals in extracts often possess enhanced reactivity due to their additive and/or synergistic interactions [5]. Plant essential oils containing a wide spectrum of compounds seem to be promising in this respect. Mastic oil, the essential oil of mastic gum, a natural resin obtained from Pistacia lentiscus variation chia has been extensively used in the Mediterranean and Middle Eastern countries as food/beverages flavouring additive and traditional medicine since antiquity without any reported toxicity. Chemical composition analysis of mastic oil revealed that it is a complex mixture of volatile compounds, mainly terpenes, with established beneficial biological properties [6,7]. Although these compounds have been shown to inhibit a variety of tumor-promoting cellular pathways in cancer cells, their precise mechanism(s) of action is still uncertain. It appears that plantderived terpenes act primarily as inhibitors of the mevalonate pathway which regulates the biosynthesis of specific isoprenoids that are indispensable to the post-translational modification of small GTPase [8,9]. Regarding the health beneficial properties of mastic oil, it has been proved to act as antimicrobial [6,7], anti-inflammatory [10] and anti-atherogenic [11] agent without substantial side effects in humans and animals [10,12]. Furthermore, recent studies have revealed that mastic extracts can also exert anti-tumor growth activities against several cancer types (leukemia, prostate, colon, lung and melanoma cancer cells) through mechanisms involving inhibition of tumor cell proliferation and survival, restriction of angiogenesis and modulation of pro-tumor inflammatory response [13-17]. In addition, mastic oil treatment has been shown to target the expression and function of key signaling and transcription regulators implicated in malignant phenotype like Ras/RhoA GTPases and NF-B [14,17]. Despite the great number of reports analyzing the action mechanisms of plant derived compounds, studies focusing on their effects at the gene expression level are very limited. In this work, by combining sensitive highthroughput transcriptomic technology and bioinformatics on Lewis lung adenocarcinoma (LLC) cells, a susceptible to mastic oil cancer cell line [17], we were able to evaluate for the first time the differential expression of tumor genes in a genome-wide scale and identify target pathways modified in response to mastic oil treatment. Furthermore, by analyzing the expression of selected target genes in three additional human cancer cell lines we confirmed that the anti-tumor effects of mastic oil were more general. Our results might help to delineate the molecular basis of mastic oil chemopreventive/chemotherapeutic actions. Methods Cell culture and treatment LLC and K562 cells were cultured in DMEM and RPMI 1640, respectively. Media were supplemented with 10% FCS, L-glutamine and antibiotics. HCT116 cells were maintained in DMEM supplemented with 10% FCS, nonessential amino acids and antibiotics. A549 cells were maintained in F19-K supplemented with 10% FCS and antibiotics. All cell (...truncated)