Oxidative stress and apoptosis induction in human thyroid carcinoma cells exposed to the essential oil from Pistacia lentiscus aerial parts

Background Essential oils from the aerial parts (leaves, twigs and berries) of Pistacia lentiscus (PLEO) have been well characterized for their antibacterial and anti-inflammatory properties; however, poor information exists on their potential anticancer activity. Methods Increasing concentrations of PLEO (0.01–0.1% v/v, 80–800 μg/ml) were administered to a wide variety of cultured cancer cells from breast, cervix, colon, liver, lung, prostate, and thyroid carcinomas. Fibroblasts were also included as healthy control cells. Cell viability was monitored by WST-8 assay up to 72 hours after PLEO administration. The intracellular formation of reactive oxygen species (ROS), the induction of apoptosis, and the enhancement of chemotherapeutic drug cytotoxicity by PLEO were further investigated in the most responsive cancer cell line. Results A dose-dependent reduction of tumor cell viability was observed upon PLEO exposure; while no cytotoxic effect was revealed in healthy fibroblasts. FTC-133 thyroid cancer cells were found to be the most sensitive cells to PLEO treatment; accordingly, an intracellular accumulation of ROS and an activation of both the extrinsic and intrinsic apoptotic pathways were evidenced in FTC-133 cells after PLEO administration. Furthermore, the cytotoxic effect of the antineoplastic drugs cisplatin, 5-fluorouracil and etoposide was enhanced in PLEO-exposed FTC-133 cells. Conclusion Taking into account its mode of action, PLEO might be considered as a promising source of natural antitumor agents which might have therapeutic potential in integrated oncology.

Among EO-bearing plants, Pistacia lentiscus L. (PL), an evergreen bush of the Anacardiaceae family that extensively thrives in the Mediterranean area, has attracted considerable attention for its wide variety of bioactivities [11,12]. In particular, an increasing number of studies has revealed that PL trunk resin (namely mastic gum) may exert anticancer activity in several types of human neoplasia, including prostate, colon, lung, and pancreatic carcinomas as well as hematological malignancies [13][14][15].
Other than from mastic gum, EOs can be extracted from PL aerial parts such as leaves, twigs, flowers, and berries. However, while antibacterial and anti-inflammatory properties have been widely demonstrated for EOs from PL aerial parts [16,17], poor information exists on their potential anticancer activity. In the present paper, we reported for the first time on the antiproliferative effects of an EO extracted from PL aerial parts on different cultured cancer cells, demonstrating its capability to reduce tumor cell viability through the intracellular accumulation of reactive oxygen species (ROS) and the induction of apoptotic cell death.

Pistacia Lentiscus Essential Oil (PLEO)
PLEO, extracted from leaves, twigs and berries of PL from Sardinia (Italy), was produced by SSA Mediflora (Cagliari, Italy). Its chemical composition is shown in Table 1. The oil was kept in the dark at room temperature; immediately before use, a stock containing 1% PLEO (solubilized in the culture medium containing 1% dimethyl sulfoxide, DMSO) was prepared and sterilized using 0.45 μm filters. The same lot was used for all the experiments on cultured cells.

Apoptosis evaluation
Apoptosis induction by PLEO was investigated in FTC-133 cells by monitoring both caspase-8 and caspase-9 activity (the main effectors of the extrinsic and intrinsic apoptotic pathways, respectively), as well as DNA fragmentation (a common marker of late apoptosis). Briefly, caspase-8 and -9 activation was determined in cell lysates after 6 and 24 hours upon PLEO administration (0.04% v/v, 320 μg/ml) using two colorimetric kits from Biovision (Milpitas, CA, USA) in accordance with the manufacturer's instructions. Assays were based on the spectrophotometric detection at 405 nm of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate ETD-pNA by caspase-8 or LEHD-pNA by caspase-9. Protein concentration in the cytosolic extracts was measured using the Bradford method [19]. Genomic DNA fragmentation was evaluated after 6, 24 and 48 hours upon PLEO administration (0.04% v/v, 320 μg/ml) by agarose gel electrophoresis, as previously described [20]. DNA samples were carefully resuspended in TE buffer; nucleic acid concentration and purity were measured using a NanoDrop1 ND-1000 spectrophotometer (Thermo-Scientific, Wilminton, DE, USA). 2 μg of each sample was loaded onto 1.5% TAE agarose gel; DNA laddering was visualized on a UV transilluminator by ethidium bromide staining. Images were obtained using a Gel Doc 2000 (Bio-Rad Laboratories S.r.l, Segrate, MI, Italy).

Mitochondrial Membrane Potential (MMP) evaluation
MMP was determined in FTC-133 cells after 2, 4, and 6 hours upon PLEO administration (0.04-0.08% v/v, 320-640 μg/ml) using a fluorometric kit from Biovision (Milpitas, CA, USA). The kit uses TMRE (tetramethylrhodamine, ethyl ester) to label active mitochondria. TMRE is a cell permeant, positively-charged, red-orange dye that readily accumulates in active mitochondria due to their relative negative charge. Depolarized or inactive mitochondria have decreased membrane potential and fail to sequester TMRE. Fluorescence was monitored at Ex/Em 549/575 nm in the multiwell plate reader FluoStar Optima (BMG Labtech, Germany).

Hypoxia-Inducible Factor-1 alpha (HIF-1α) measurement
HIF-1α quantification was performed in FTC-133 cells upon PLEO administration (0.04% v/v, 320 μg/ml) using an enzyme-linked immunosorbent assay kit from Abcam (Cambridge, UK), in accordance with the manufacturer's instructions. Color development was evaluated at 450 nm in a multiwell plate reader (Thermo Fisher Scientific, Milan, Italy). Protein concentration in cell extracts was measured using the Bradford method [19].

Clonogenic assay
The clonogenic cell survival assay determines the ability of a cell to proliferate indefinitely, thereby retaining its reproductive ability to form a large colony or a clone. To test PLEO effects on FTC-133 colony formation capacity, cells (40000/cm 2 ) were pretreated for 24 hours with increasing concentrations of PLEO (0.04-0.08% v/v, 320-640 μg/ml). Cells were harvested, 1000 viable cells were plated in 6-well plates and allowed to grow for 14 days. Colonies were then stained for 90 min at room temperature with 0.25% methylene blue in 50% ethanol; pictures were captured digitally and analyzed using a software for densitometric analysis (Quantity One 4.0.1, Bio-Rad Laboratories, Milan, Italy) to evaluate the colony volumes. Data were expressed as percentage of inhibition of colony formation compared to the control.

Statistical analysis
Data are presented as mean ± standard deviation (SD) of at least three independent experiments and analyzed by Student's t-test to compare treated vs. untreated cells. Significance level was set at p<0.05 for all analysis.

Cell viability inhibition and intracellular ROS formation by PLEO
PLEO was administered to cancer and healthy cell lines up to 72 hours at concentrations ranging from 0.01 to 0.1% v/v (80-800 μg/ml). Overall, a dose-dependent inhibition of cancer cell viability by PL was observed as compared to untreated cells receiving the vehicle (0.1% DMSO) (Fig 1). In most cases, the maximum concentration tested (0.1% v/v, 800 μg/ml) was cytotoxic, leading to the detection of necrotic cells. As reported in Table 2, the most sensitive cancer cell line was FTC-133 (follicular thyroid carcinoma), showing the lowest IC 50 value (376±20 μg/ml) after 24 hours of incubation with PL; on the contrary, AG-09429 healthy fibroblasts were the most resistant cells to PL treatment (IC 50 >800 μg/ml).
The administration of PLEO (0.01-0.08% v/v, 80-640 μg/ml) to cancer cell lines also led to a significant increment of intracellular ROS levels as compared to untreated cells receiving the vehicle (0.1% DMSO) (Fig 2A). FTC-133 cells showed the highest ROS content, while AG-09429 healthy cells the lowest. A significant negative correlation (R = -0.820, p = 0.013) was observed between ROS increment at 6 hours upon PL administration and IC 50 values at 24 hours of treatment ( Fig 2B). In FTC-133 cells, experiments were repeated in the presence of GSH (5 mM). No intracellular ROS accumulation was evidenced after 6 hours of cell incubation with increasing concentrations of PLEO ( Fig 3A); at the same time, no inhibition of cell viability was found up to 24 hours after PL administration (Fig 3B). The protective effect of GSH decreased over time, leading to a significantly reduced cancer cell viability after 48 hours of treatment with PLEO.

Apoptosis induction by PLEO
The administration of PLEO (0.04% v/v, 320 μg/ml) to FTC-133 cells led to the activation of both the extrinsic and intrinsic apoptotic pathways. As shown in Fig 4A, a significant caspase-8 activation was observed after 24 hours upon PL administration; while caspase-9 activation was evidenced mostly after 6 hours of treatment. In accord to the early intrinsic pathway induction, a significant reduction of MMP was found at 2, 4, and 6 hours of incubation with PLEO as compared to untreated cells (Fig 4B). The presence of DNA fragmentation after 24 and 48 hours upon PL treatment confirmed apoptosis induction by the EO in FTC-133 cells ( Fig 4C). Interestingly, PLEO-induced apoptosis was associated with a significant decrement of the intracellular levels of HIF-1α (-31.4% vs. untreated cells) (Fig 4D).

Colony formation inhibition by PLEO
As shown in Fig 5, a significant inhibition of FTC-133 colony formation capacity was observed upon PLEO administration (0.04-0.08% v/v, 320-640 μg/ml) to cancer cells in comparison to untreated cancer cells.

Enhancement of chemotherapeutic drug cytotoxicity by PLEO
The antiproliferative effects of PLEO (0.04% v/v, 320 μg/ml) were determined in FTC-133 cancer cells also in presence of CDDP, 5-FU, and VP16. As shown in Fig 6, cell incubation with increasing concentrations of CDDP alone (5-80 μM) led to a significant decrement of cell viability as compared to untreated cells. The co-treatment with PL further decreased cell viability in particular after 24 hours upon administration (IC 50 values equal to 43.5 μM and 4.2 μM in absence and presence of PLEO, respectively). As regards 5-FU, IC 50 values were not reached up to 48 hours of incubation with the drug alone (25-500 μM); on the contrary, the co-treatment with PL allowed a decrease of cell viability so as to achieve the IC 50 value both at 24 (172 μM) and 48 hours (33 μM). In the case of VP16 (5-100 μM), IC 50 values, achieved only after 48 hours of incubation, were the same in absence or presence of PLEO (4.4 μM).

Discussion
In the present study we investigated for the first time the antiproliferative properties of an essential oil extracted from the aerial parts of Pistacia lentiscus (PLEO) on a wide variety of cultured cancer cells from human breast, cervix, colon, liver, lung, prostate, and thyroid carcinomas. Fibroblasts were also included as healthy control cells.
Overall, a dose-dependent inhibition of cancer cell viability by PLEO was observed as compared to untreated cells. On the basis of IC 50 values, FTC-133 (follicular thyroid carcinoma) was the most sensitive cancer cell line, while AG-09429 healthy fibroblasts were the most resistant cells to PL administration, thus evidencing a selective cytotoxic action of the EO against tumor cells. Other than cell viability, PLEO also inhibited the colony formation capacity of FTC-133 cells, proposing that some EO constituents might affect single tumor cell survival so as to suppress cancer cell colonization, as previously demonstrated [21].
On the basis of the chemical composition, the monoterpenes myrcene and α-pinene were the most abundant compounds of PLEO, suggesting that the antiproliferative effects of PL might be possibly mediated by these two compounds. In accord, previous evidence indicated that myrcene and α-pinene might exert significant cytotoxic effects on different cancer cell lines [22][23][24]. Nevertheless, the involvement of other PLEO minor constituents should not be ruled out; indeed, the terpenes limonene, β-caryophyllene, and β-elemene have also shown significant anticancer activities both in vitro and in vivo models [9], indicating potential synergies among EO components.
The administration of PLEO to cancer cell lines was also associated to a significant increment of intracellular ROS levels as compared to untreated cells. FTC-133 cells showed the highest increase, while AG-09429 healthy cells the lowest. Consequently, a significant negative correlation was observed between ROS increment and cell proliferation upon PL treatment. When experiments were repeated in the presence of GSH as antioxidant, no intracellular ROS accumulation and no inhibition of cancer cell growth were observed in FTC-133 cells after PL administration, thus indicating that PLEO might act as antiproliferative agent in a ROS-dependent manner. In accord, several terpenic EO constituents, such as α-pinene and β-caryophyllene, have demonstrated to specifically induce the production of ROS within cancer cells without increasing oxidative stress in normal cells [22,25].
The specific action of PLEO towards cancer cells might be partially related even to cell mitotic rate. Indeed, FTC-133 cell line, the most sensitive to PLEO administration, also presented the lowest doubling time (approximately 27 hours) among the cell lines tested, possibly indicating that some PLEO components (such as limonene and β-elemene) might interfere with cell cycle and DNA synthesis [9,23].
To clarify the mechanisms underlying PLEO antiproliferative effects, apoptosis was investigated in FTC-133 cells. We found that PLEO led to the activation of both the intrinsic and extrinsic apoptotic pathways, as revealed by caspase-9 activation after 6 hours of treatment and by caspase-8 activation after 24 hours upon PL administration. In accord to the early activation of the intrinsic pathway, a significant loss of mitochondrial membrane potential was found up In vitro anticancer activity of Pistacia lentiscus essential oil to 6 hours of incubation with PLEO. The presence of nuclear DNA fragmentation after 24 and 48 hours upon PL treatment confirmed apoptosis induction by the EO in FTC-133 cells.
Interestingly, FTC-133 is a cancer cell line characterized by the mutation of the tumor suppressor gene PTEN (phosphatase and tensin homologue) [26], which makes the transcription factor HIF-1α functionally expressed in thyroid carcinomas independently of lowered oxygen tension, thus promoting apoptosis resistance and tumor cell survival [27]. In this context, we observed that PLEO-induced apoptosis in FTC-133 cells was associated with a significant decrement of HIF-1α levels, possibly indicating a negative modulation of the hypoxic factor by some PLEO components such as the sesquiterpene β-elemene, which has demonstrated to enhance radiosensitivity in vivo via HIF-1α downregulation [28]. Positively, we evidenced that PLEO enhanced the inhibitory effects of the chemotherapeutic drugs CDDP, 5-FU and VP16 on FTC-133 cell proliferation. This action was particularly effective in the case of 5-FU; indeed, PLEO co-administration favourably allowed the achievement of IC 50 values, thus confirming the role of EO constituents in potentiating the anticancer capabilities of conventional chemotherapeutic agents [9].
In conclusion, this study provided new insights into the antitumor action of the essential oils from Pistacia lentiscus aerial parts, for which poor investigations exist. Being a complex mixture of numerous constituents, PLEO action on cancer cells via intracellular ROS accumulation and apoptosis induction might be the sum of each individual activity, modulated by all the potential interactions. Taking into account its mode of action and its ability to enhance the cytotoxic effect of conventional antineoplastic drugs, PLEO might be considered as a promising source of natural antitumor agents and might have therapeutic potential in integrated oncology as a support to the standard anticancer therapies.