Lately, there is an increasing interest for exploring the tumor preventive actions of mastic gum and its essential oil, a natural blend of bioactive terpenes. Results obtained to date support that mastic gum/oil can inhibit the growth of several cancer types in vitro [13–16]. In addition, the anti-tumor potential of mastic oil has been recently confirmed in vivo on the experimental growth of Lewis lung adenocarcinoma (LLC) tumors, an aggressive type of mouse lung cancer and some of the underlying mechanisms have been investigated . In the present study, through a genome-wide transcriptomic approach we identified for the first time the dynamic alterations occurring in gene expression of LLC cells in response to mastic oil treatment at different incubation periods (3, 6, 12, 24 and 48 h) and undertook a detailed analysis of the involved mechanisms using bioinformatic methodologies. Furthermore, we followed by RT-PCR the expression of four selected genes, namely Pten, Hmox1, E2f7 and Nod1 in LLC as well as in other cancer cell lines of human origin, in order to reveal potential common patterns of response to mastic oil among different tumor types.
Microarray data normalization by two different algorithms provided a list of 925 genes with a significantly modified expression in mastic oil treated cells compared to untreated controls. RT-PCR selectively applied on the above mentioned genes due to their particular interest, further confirmed the expression profiles found by the microarray approach. Meta-analysis performed on the complete set of differentially expressed genes using the GO database (Table 1), comprised a statistically strong cohesive net of functions as derived from the GO tree, thus further supporting the reliability of the adopted fold change expression thresholds. This analysis demonstrated that mastic oil influences significantly a number of fundamental cellular processes such as those related to cell cycle, apoptosis, signal transduction and regulation of transcription. Also some more specific functions (e.g. glutathione transferase activity and metabolism), linked to detoxification of endogenous or exogenous metabolites, a well-known mechanism of cancer chemoprevention by several phytochemicals, including terpenoids [36–38], are affected.
It is worth mentioning that alterations in GO categories related to "negative regulation of cell proliferation", "cell cycle", "positive regulation of I-κB kinase/NF-κB cascade" and "induction of apoptosis" correlated very well with existing experimental evidence supporting that mastic oil causes regression of tumor growth by inhibiting cancer cell proliferation and survival [13, 16, 17], reducing tumor-associated expression of inflammatory mediators  and down-regulating NF-κB transcriptional activity [14, 17].
An interesting result of our analysis was that among the targeted by mastic oil genes corresponding to the GO categories presented in Figure 2, Pten appeared to be relevant to three of them. Its expression was further assessed by RT-PCR and found to be late up-regulated in LLC, HCT116 and K562 cells. This gene which encodes for PTEN, a dual-specificity phosphatase, has lately gained much attention as a tumor-suppressor due to its prominent inhibitory role in cancer expansion and metastasis [33, 34]. Although little is known about the upstream regulatory pathways for PTEN, one of its most recognized down-stream actions is the blockade of PI3K/AKT signaling through de-phosphorylation of phosphatidylinositol (3,4,5) tri-phosphate (PIP3) which is generated by PI3K to mediate AKT activation [31, 36]. Active AKT then phosphorylates a plethora of targets to activate cell cycle, prevent apoptosis and trigger NF-κB signaling . Furthermore PTEN has been shown to antagonize tumor necrosis factor (TNF)-stimulated NF-κB-dependent gene expression, thus sensitizing cells to TNF-induced apoptosis . Thereby the induction of Pten expression by mastic oil may be considered as an essential upstream effect accounting for the anti-proliferative, pro-apoptotic and anti-inflammatory actions of mastic oil through blockade of AKT and NF-κB transcriptional activity. In line with this, we found that pharmacological blockade of PTEN by bpV(phen) partially reversed the anti-tumor growth effects of mastic oil in K562 cells. These results indicate the active involvement of PTEN but also of other target pathways in tumor growth suppression by mastic oil. Compatible to this hypothesis, perillyl alcohol (one of the best studied mastic oil components) and mastic gum have been shown able to reduce the levels of phosphorylated (active) AKT [14, 38] and in addition mastic oil to down-regulate the constitutive and TNF-α-induced NF-κB-dependent gene transcription .
In conformity with a negative regulation of cell proliferation and/or survival by mastic oil was also the observed up-regulation of two other genes, namely E2f7 and Hrasls3. The expression of E2f7 was also determined by RT-PCR and found to be induced in LLC, HCT116 and K562 cells. E2F7 is a transcription factor which, in contrast to the other members of E2F family, has been shown to act as a repressor of several genes known to promote tumor cell proliferation . Concerning Hrasls3, a member of the HREV107 type II tumor suppressors, it has been shown to exert anti-proliferative and pro-apoptotic actions and to be down-regulated by Ras-dependent signaling [40–42]. In correlation, mastic oil has been shown able to reduce Ras levels in LLC cells in vivo and in vitro , probably as an indirect consequence of mevalonate pathway blockade by its isoprenoid components , leading to disruption of Ras signaling. Furthermore, although Nod1 was found to be repressed in LLC, it was elevated in HCT116 and K562 cells indicating an heterogeneity in response to mastic oil.
Moreover, transient up-regulation of Hmox1 in all tested cell lines corroborates with the inhibitory effects of mastic oil on tumor-related inflammatory response . This gene has been reported to be highly inducible by a variety of stress stimuli (hypoxia, inflammation, heavy metals, UV radiations) including several phytochemicals . HMOX1, a heme-degrading enzyme, has been shown to exert anti-oxidative as well as a broad range of anti-inflammatory activities attributed to inhibition of pro-inflammatory mediators and negative regulation of NF-κB signaling [43, 44]. Besides, HMOX1 is known to be involved in the process of xenobiotic metabolism by inactivating chemically reactive metabolites and scavenging reactive oxygen species . This finding along with the proposed by GO analysis modification of glutathione transferase activity (Table 1) support that activation of detoxifying enzymes might also contribute to cancer chemoprevention by mastic oil in agreement with previous studies indicating the anti-oxidative potential of mastic oil [11, 46]. In contrast, Doi et al , using a medium-term carcinogenesis assay in rat liver, have shown that Hmox1 was one of the most repressed genes after treatment with mastic oil. In our system however over-expression of Hmox1 was observed as an early response to mastic oil treatment, while in  gene expression profile was determined after administration of mastic oil for 6 weeks. In addition, Doi et al reported the formation of prenoeoplastic lesions in rat liver after treatment with mastic oil which is not in accordance with our results. It is possible that the unique features of each experimental system, the intrinsic differences of each cell type and/or the presence of some distinct components in mastic gum compared to mastic oil, account for such contradictory effects. For instance Paraschos et al  recently reported that a polymer present in mastic gum could hinder its anti-bacterial activity and reduce the bioavailability of bioactive components. In any case, those controversial results support the need of further studies in order to investigate mastic oil metabolism and bioavailability and specify safe dosage limits.
Finally, by applying k-means clustering the complete set of genes was organized in eight clusters each representing a specific gene expression profile, probably indicating a common regulatory transcriptional mechanism (Figure 5). To test this hypothesis, we performed a representative promoter analysis looking for putative common cis-elements in genes grouped in cluster 2 (Table 2). Putative elements belonging to the EGR family were identified in 85% of the promoters of cluster 2 genes. Functional EGR binding sites have been identified in the promoters of several tumor suppressor genes, including Pten [49, 50]. In particular, EGR1 has been shown to directly transactivate Pten. Furthermore, elements of the AHRR family, containing the Xenobiotic Response Element (XRE) and closely related TF binding sites involved in transcriptional stimulation of several xenobiotic metabolizing enzymes by several phytochemicals [51, 52], were identified in 69% of cluster 2 genes.