Abstract
BackgroundMastic 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.MethodsTo 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.ResultsIn this work we demonstrated that exposure of Lewis lung carcinomas to mastic oil caused a time-dependent 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-κB 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, pro-apoptotic 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.ConclusionsPresent 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.
Highlights
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
For RNA isolation, Lewis lung adenocarcinoma (LLC) cells were plated in 6-well plates at 2.3 × 105 cells/well, K562 in 24-well plates at 3 × 105 cells/well, HCT116 in 12-well plates at 4 × 105 cells/ well and A549 were plated in 12-well plates at 1.5 × 105 cells/well. 24 h later cells were treated with mastic oil (0.01% for LLC and 0.02% v/v for the other cell lines) or 0.1% DMSO vehicle for the indicated periods of time (3 to 48 h)
Significant differentiated genes To obtain the gene expression profile after treatment with mastic oil for five distinct time points (3, 6, 12, 24 and 48 h), LLC cells were cultured with 0.01% v/v mastic oil or 0.1% DMSO, mRNA was isolated from and subjected to high-throughput gene expression profiling using high density oligonucleotide Illumina beadchips to analyze gene expression changes
Summary
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. 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]. 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. 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]
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