Thymus hirtus sp. algeriensis Boiss. and Reut. volatile oil enhances TRAIL/Apo2L induced apoptosis and inhibits colon carcinogenesis through upregulation of death receptor pathway

Background: The aim of the study is to determine the anticancer activity of Thymus algeriensis (TS) and its underlying mechanisms using in vitro and in animal models. Methods: HCT116 cells were treated with TS essential oil alone or with TRAIL, and then its anticancer effect was determined by using MTT assay, live dead assay, caspase activation and PARP cleavage. Further mechanisms of its anticancer effects was determined by analyzing expression of death receptor signaling pathway using Western blotting. A mouse model was also used to assess the antitumor potential of thyme essential oil. Results: TS oily fraction showed tumor growth inhibitory effect even at lower concentration. TS induces apoptotic cell death as indicated by cleavage of PARP, and activation of the initiator and effector caspases (caspase-3, -8 and -9). Further, results showed that TS increases the expression of death receptors (DRs) and reduces the expression of TRAIL decoy receptors (DcRs). In addition, upregulation of signaling molecules of MAPK pathway (p38 kinase, ERK, JNK), down-regulation of c-FLIP, and overexpression of SP1 and CHOP were observed by TS. Further in animal model, intragastric administration of TS (12.5 mg/ml and 50 mg/ml) prevented colorectal carcinogenesis by blocking multi-steps in carcinoma. Conclusion: Overall, these results indicate that thymus essential oil promotes apoptosis in HCT116 cells and impedes tumorigenesis in animal model. Moreover, thyme potentiates TRAIL-induced cell death through upregulation of DRs, CHOP and SP1 as well as downregulation of antiapoptotic proteins in HCT116 cells. However, therapeutic potential of TS needs to be further explored.


INTRODUCTION
Cancer is still a major health issue with high care cost and causes physical and emotional difficulties for cancer patients. Besides preventive measures, several therapeutic modalities like surgery, chemotherapy and radiotherapy have been developed [1]. There is an unmet need for novel, affordable and effective anti-neoplastic medications [2]. With the development of new therapeutics, the incidence of colorectal cancer has decreased during the past two decades [3]. However, development of new drugs with higher efficacy and safety are still needed.
Apoptosis generally occurs via two major pathways, intrinsic and extrinsic. The extrinsic pathway of apoptosis AGING in cancer cells takes place by binding of a ligand to the surface death receptor, while intrinsic pathway occurs through mitochondria [4]. TNF-related apoptosis inducing ligands (TRAIL) -bind to the death receptors. It has garnered much attention as a potential anticancer agent and improved effectiveness of therapeutic agents in combination [5]. Moreover, plant-derived secondary metabolites, known as phytochemicals, have also been shown to exert numerous beneficial effects on human health. Many preclinical studies have suggested anticancer functions of phytochemicals at the epigenetic and proteomic levels [6]. Natural compounds are known to target different signaling pathways involved in cancer progression, suggesting their potential to be successful anti-cancer agents [7]. Among various natural compounds, plants of Lamiaceae family have attracted much attention for their health benefits [8]. Many plants of Lamiaceae family have been investigated because of presence of high content of essential oils, which are widely used in pharmaceutical preparations, perfumery and cosmetics [9].
Among various plants of Lamiaceae family Thymus plants are rich in essential oil and contains oxygenated monoterpenes and monoterpene hydrocarbons as its major chemical components. Fifteen species (12 endemic) grow in northwest Africa, north of the Sahara desert (Morocco, Algeria, Tunisia, and Libya), with only three occur in the Iberian Peninsula [10]. In the traditional medicine, Thymus hirtus Willd. Ssp. algeriensis Boiss. and Reut. have been used as antiinflammatory and anticancer agents and also as common tea and infusion in ethnophytotherapy [11]. The essential oil obtained from the aromatic parts of this plant has many phytotherapeutic effects and ontogenic features [12]. Thymus species has demonstrated significant free radical scavenging activity and proapoptotic effects in experimental models [13]. Thyme extract has also been shown to induce leukopoiesis and elevate thrombocyte count in blood [14]. Besides these, essential oil of Thymus species has antibacterial, antifungal and antiviral activities [15,16].
We characterized here one of the aromatic and medicinal plants, Thymus algeriensis, that grows spontaneously in Tunisia. Previously, we and others have demonstrated the anticancer potential of T. algeriensis essential oil using in vitro models [11,17]. However, the detail mechanism of its cancer preventive and therapeutic potential is still lacking. In this study, we report anticancer effects with mechanistic approach of essential oil from the aerial parts of T. algeriensis collected in the mountains of Orbata, Gafsa province of Tunisia. As of now, no reports on in vivo antitumor potential of TS have been described, although few studies have characterized the presence of essential oils in the plants. This plant is a rich composition of approximately 65 bioactive compounds, including viridiflorol, 2-carene, endo-borneol, terpinen-4-ol, camphor, eucalyptol, α-pinene and linalool, which varied from 34.38 to 42.48% of the total essential oil content [12]. To evaluate its possible use as an alternative or complementary cancer treatment, this report sheds light on the potent effect of thyme essential oil on colorectal cancer cells (CRC). This paper focuses on the TS essential oil-induced colon tumor cell apoptosis. We have unveiled the potent antitumor effect of TS, which is related to its ability to enhance TRAIL-induced apoptosis in human colon cancer cells (HCT116) by increasing TRAIL death receptors expression (DRs). Furthermore, in vivo study revealed the antitumor potential of Thymus species.

TS and/or TRAIL induced cytotoxicity in cancer cells
To determine whether TS oil has anticancer activity, we performed cell viability assay. The cell viability of tumor cell lines was investigated by using MTT assay following 24, 48 and 72 hrs of treatment with TS oily fraction at 0.5-50 pg/mL. The results showed a decrease in cell growth with the increasing dose of TS ( Figure  1B). Cancer cell lines responded to the antiproliferative effects of TS oil in a dose-and time-dependent manner. Incubation of cells with 50 pg/mL of essential oil significantly inhibited the growth of MCF7, Panc28 and SCC4 with a percentage of cell viability of 19% ± 0.54, 23% ± 2.08% and 22% ± 1.68% respectively after 24 hrs of treatment, 6% ± 0.22%, 10% ± 1.14% and 17% ± 0.48%, respectively after 48 hrs of treatment and 1% ± 0.08%, 3% ± 0.50% and 6% ± 0.02%, respectively after 72 hrs of treatment. TRAIL is new anticancer drug and it is still under clinical trials. However, some cancer cells display resistance against TRAIL. Therefore, we determined whether TS oil can enhance the anticancer effects of TRAIL. Cancer cells used in this report were sensitive to either TS or TRAIL alone. Moreover, TS AGING essential oil significantly (P < 0.05) enhances TRAILinduced human HCT116 cell growth inhibition in dosedependent fashion ( Figure 1C). As shown in Figure 1D, TS is not cytotoxic to RAW 264.7 macrophage cells.

TS induces apoptosis in HCT116
Next, we confirmed the cytotoxic effects of TS by using live/dead assay. HCT116 cells were treated with different concentration of TS and analyzed under fluorescent microscope after staining with live/dead reagents. As seen in Figure 2Ai, TS volatile fractions-induced a dosedependent cell death in HCT116 cells. In fact, thyme volatile oil dose-dependently increased the number of apoptotic cells from 1.6 to ~ 92.13% (P<0.05).

TS potentiates TRAIL-induced cytotoxic effects in HCT116
As indicated in Figure 2Ai, we found that 0.5 pg/ml TS induce moderate cell death. Therefore, we investigated whether this dose of TS can enhance the apoptotic effect of TRAIL. We observed that 0.5 pg/ml TS and 25 ng/ml TRAIL alone moderately induced cell death. However, the combination of TS and TRAIL produced significantly enhanced cell death (81%) (Figure 2Aii). Thus, it is noteworthy that the combination of TS and TRAIL increased cytotoxicity.

TS oily fractions reduced colony-forming ability of HCT116 cell lines
As growth of cancer cells in colony in vitro mimics the growth of tumor in vivo, we performed colony formation assay. Colony-forming assays measure the ability of cells in culture to grow and divide into groups. The present study was undertaken to investigate the effect of TS oily fraction on the colony-forming ability of colon cancer cell line. HCT116 cell line was seeded in 6-well plates and treated with TS oily fraction. After 9 days of incubation, we observed a large number of cancer cell colonies in control well. However, as seen in Briefly, HCT116 cells (5 × 10 3 cells/well) were seeded in triplicate in 96-well plates. The cells were exposed to different concentrations of thyme volatile oil (0, 0.01 or 0.05 μl/ml) for 12 h, the medium was removed, and tumor cells were then incubated with 25 ng/ml TRAIL for additional 24 h. Cell viability was then determined by the MTT assay. (D) Effect of TS on RAW cell viability. RAW 264.7 cells, seeded in triplicate in 96-well plates, were exposed to different concentrations of TS (0, 0.001, 0.005, 0.01 or 0.05 µl/mL) and incubated for 24 h, 48 h and 72 h. Cell viability was analyzed with the MTT assay. Figure 2Bi, the colony formation ability of cells was declined with increasing doses of TS. For dosedependent investigation, HCT116 colonies were very sensitive to thyme essential oil and displayed dose dependent inhibition in colony formation. TS essential oil even at 0.5 and 1 µg/ml showed ~30% and 100% reduction in colony-forming ability respectively.

AGING
Further, we determined whether TS can increase the colony forming inhibitory effect of TRAIL. As shown in Figure 2Bii, treatment with TRAIL/Apo2L (25 ng/ml) alone reduced number of colonies whereas exposing colon carcinoma cells to TS and TRAIL in combination synergistically decreased the number of cancer cell colonies.

TS potentiates the effect of TRAIL/Apo2L-mediated apoptosis through PARP cleavage and caspase cascades activation
We investigated whether TS enhances TRAIL/Apo2Linduced apoptosis in HCT116 cells. This was assessed by caspase activation and PARP cleavage. As shown in Figure 2Ci, pretreatment of colon cancer cells with either TS or TRAIL alone mediates significant cleavage of PARP and moderate caspase-3, -8 and -9 activation. However, when tumor cells were cotreated with 0.5 experiments (i); colon cancer cells were exposed to 0.5 pg/ml thyme volatile oil for 12 h and rinsed with PBS. Cells were then treated with 25 ng/ml TRAIL for an additional 24 h. Cell death was analyzed by the LIVE/DEAD assay (ii). Orange arrows indicates necrotic cells; pink arrows indicates live cells and white arrows indicate apoptotic cell. (B) HCT116 (5 × 10 2 ) cells seeded in 6 well plates were treated with different concentrations of TS (0-1 µg/ml), TRAIL (25 ng/ml) and TS + TRAIL for 9 days to form colonies and stained with clonogenic acid reagent to fix cells, and then incubated with crystal violet dye. Colony-forming ability was assessed by counting blue colonies. (C) TS sensitizes TRAIL-induced PARP cleavage and caspase activation in dose-dependent manner (i). Briefly, HCT116 cells (1 × 10 6 per well) were pretreated with vehicle control (DMSO) or indicated doses of thyme essential oil for 12 h and then rinsed, TRAIL was then added for an additional 24 h. Whole-cell lysates were subjected to Western blotting analysis using relevant antibodies. TS sensitizes TRAILinduced PARP cleavage and caspase activation in time-dependent manner. Colon cancer cells were exposed to thyme volatile oil and TRAIL at the indicated time points (ii). Whole-cell lysates were subjected to Western blotting analysis using relevant antibodies. Used blots were stripped and reprobed with β-actin antibodies to verify equal protein loading. These are representative results of three independent experiments. AGING µg/ml or 1 μg/ml TS and 25 ng/ml TRAIL together, cleaved PARP was faintly enhanced and upstream and downstream caspases (casp. 3, 8, and 9) appeared to be activated ( Figure 2Cii). Thus these findings suggest that TS enhances the anticancer effect of TRAIL.

TS upregulates TRAIL receptors DR4 and DR5
This study is to underly the mechanisms of antitumor effects of oily fractions from T. algeriensis, in which HCT116 cells were treated with oily fraction of TS. The lysates were analyzed by Western blotting to examine the effect of TS on expression of death and decoy receptors. The results reveal that TS treatment induced the expression of death receptors in dose-( Figure 3Ai) and time-dependent (Figure 3Aii) manner. The effect of TS was more prominent on expression of DR5. Thus, treatment of human colon cancer cells with TS for 24 h appears to induce the expression of DR5 in MCF-7 and Panc28 cancer cells, indicating its non-specificity to cancer cells. In addition, TS suppressed the expression of decoy receptor DcR1. However, no effect was observed on the expression of DR4 and DcR2 in either MCF-7 or Panc28 cells (Figure 3Aiii).
TS is found to potentiate TRAIL-induced cell death through death receptors and inhibits growth and proliferation of colon cancer cells. Incubation of HCT116 with both TS and TRAIL increased the level of DR4/DR5 even more in dose- (Figure 3Bi, 3Bii) and time- (Figure 3Biii) dependent manner, compared with that in untreated cells.

TS regulates Akt and MAPK pathways
Because essential oils mediated apoptosis involves phosphorylation of MAPK [19], we investigated to determine whether TA also modulates MAPK pathway. We found that the treatment of TS in HCT116 cells induces the phosphorylation of ERK1/2, c-jun, JNK, and p38 MAPK in dose and time dependent manner. As MAPKs are known to responsible for upregulation of DR5 [20], induction of MAPK pathway by TS may contribute to the upregulation of DR5. However, TS decrease the phosphorylation of AKT2 in colon cancer cells ( Figure 4A).

TS up-regulates Sp1 and CHOP bZip transcription factors
Activation of MAPKs and other kinases (JNK) led to Sp1 translocation to the nucleus [21]. This Sp1 transcription factor is known to participate in cell surface expression of DR5 [22]. Therefore, we investigated whether TS can induce the Sp1 proteins. As shown in Figure 4B  AGING TS suppresses STAT3 phosphorylation STAT3 activity is known to regulate sensitivity to TRAIL-induced apoptosis in cancer cells [23]. Therefore, we examined whether TS can suppress the activation of STAT3. We observed that TS inhibited STAT3 (Tyr 705 ) phosphorylation at higher dose but no suppression was observed at Ser 727 of STAT3 ( Figure  4Ci). Moreover, suppression in upstream kinase JAK was observed at higher dose of TS in colon cancer cells (Figure 4Cii).

Both thyme essential oil and TRAIL/Apo2L represses expression of multiple gene products in colon carcinoma cell lines
The increase of death receptor level after TS treatment correlated with the down-regulation of various gene products ( Figure 5A) that mediate inflammation, cell proliferation, cell survival, invasion, and angiogenesis.
We observed that treatment of either TS or TRAIL alone decreased the expression of cIAP-1, cIAP-2, survivin, cFLIP, ICAM-1 and VEGF. The effect of these two agents was more in 24 hours exposed cells. We also observed prominent suppression in these proteins when TS combined with TRAIL ( Figure 5B).

TS inhibits LPS-induced inflammation and carcinogenesis in mice
Next we determine the effect of TS in animal model. We observed no morbidity, no signs of toxicity and no spontaneous behavioral changes in mice after 7 days of treatment with low dose of TS. These findings suggest safety of TS oil. However, as seen in Figure 6A, body weight loss, lesser food and water consumption were detected in LPS alone and with combination-treated mice during experiments. In contrast, a moderate gain in body weight in TS-treated mice was observed. After 7 days, we sacrificed the mice and analyzed AGING macroscopically. We observed that oral administration of LPS significantly induce damage to colon that demonstrate shortened length (Figure 6Avi). In contrast, TS administration significantly inhibited colonic shortening.
Colon appearance in untreated group (Figure 6Ba), TS (Figure 6Bb, 6Bc) had normal architecture, whereas in the colon tissues from the LPS-induced group were significantly marked by inflammation, lesions and carcinogenesis (Figure 6Bd, 6C) with tumor buds. As shown in Figure 6Cd, the black dashed line separates the tumor mass on the top left from the neoplastic cells or tumor budding on the bottom right. Notably, in group treated with increasing doses of TS, we clearly found reduction in injuries. 5-Fu and LPS-treated mice have induced tissues damage compared with thyme volatile oil treated group.

DISCUSSION
In Tunisia, ethnobotanical study and biological activity of Thymus algeriensis are poorly recorded. EO of T. hirtus Ssp. algeriensis had a leishmanicidal activity against both Leishmania species with an IC50 value (calculated by using regression analysis and expressed as mean 50% leishmanicidal activity) equal to 0.43 µg mL -1 for L. major and 0.25 µg mL -1 for L. infantum. Interestingly it was not cytotoxic towards murine macrophagic RAW264.7 (ATCC, TIB-71) cells treated with a concentration of 1 µg/ml after 24 h of incubation [24]. Moreover, lack of cytotoxicity towards macrophagic cells was detected at concentration 8.35 µg/ml for thyme extracts obtained by acetonitrile/water (ACN/W) extraction (60%-40%) and >400 µg/ml for water extract thyme [25]. Thyme essential oil cytotoxicity might be due to its lipophilic compounds that accumulate in cancer cell membranes and increase their permeability, resulting in leakage of enzymes and metabolites [26]. Many EOs and their constituents lead to PARP cleavage [27].
This paper focuses on the cancer preventive and therapeutic potential of thyme essence. We report here that TS inhibit cell viability of various types of tumor cells at different time points. Recently, it has been reported that the antiproliferative effect of oily fractions was correlated with major terpenes constituents, α-Bisabolol as a sesquiterpene alcohol, and (+)-epi-Bicyclosesquiphellandrene [28]. Previous research has demonstrated that terpenic compounds-rich volatile oil from many species prevent tumor progression. Cancer is attributed to uncontrolled proliferation resulting from abnormal activity of different cell cycle proteins. Therefore, cell cycle regulators are becoming attractive targets in cancer therapy [29]. Nutraceuticals have been shown to have potential in cancer prevention for halting cell cycle progression by targeting one or more steps in the cell cycle [30]. The colon cancer regression involve various molecular mechanisms like cell cycle arrest, apoptosis induction as observed by caspase activation and PARP cleavage, antiapoptotic proteins downregulation, p53 induction, angiogenesis, invasion and metastatic gene inhibition. In response to TS treatment, tumor cell proliferation was shown to be suppressed by arresting colon cancer HCT116 cells in the G1 and G2/M phase population through decreasing cyclin D1 expression and upregulating p21 and the tumor suppressor gene p53.
TRAIL receptors have been considered as extraordinary promising antitumor targets, since their cell surface expression preferably kills tumor cells while sparing healthy cells [4]. To gain insight into the antitumor potential of Thyme volatile oil and how it sensitizes TRAIL/Apo2L to induce cell death, western blot analysis was applied. This report shows that TS is a potent chemosensitizer of TRAIL-mediated apoptosis in colon cancer cell lines. Apoptosis was mediated via DR5/DR4 upregulation and downregulation of antiapoptotic gene expression. TRAIL induces apoptosis by binding to its cognate death receptors (i.e., DR4 or DR5), which then recruit caspase 8 via the FADD. Activated caspase 8 then directly activates caspases-3, -6, and -7 or activates the intrinsic mitochondriamediated pathway through caspase 8-mediated Bid cleavage, which indirectly activates caspases-3, -6, and -7 [31]. In vitro experiments showed that the cotreatment of HCT116 cells with either the highest concentration (0.5 or 1 µg/ml) of TS or TRAIL/Apo2L (25 ng/ml) alone significantly enhanced caspase 8 (5fold), caspase 9 (2-fold) activation and PARP proteolysis (1.5-fold) in dose-and time-dependent manner.
Indeed, thyme is an antitumor agent blocking molecular pathways of colon cancer development. Here, we show that TS significantly induces DR5 expression, compared with that in untreated control cells. Cotreatment of HCT116 cell lines with TS at 0.5 or 1 µg/ml and TRAIL at 25 ng/ml potentiated the upregulation of death receptors. It has been shown that thyme essential oil significantly regulates interferon signaling, N-glycan biosynthesis and ERK5 signaling [26]. Therefore, it has been thought that activated AKT and ERK can function as key effectors of the PI3K and MAPK signaling pathways to promote cell survival and proliferation [32]. ERK1/2 has previously been evidenced to be able of inhibiting the apoptosis effector molecule caspase-3 [33]. Its activation may be crucial in the regulation of Sp1 phosphorylation and consequently Sp1 dependent proapoptotic gene transcription [34]. In this report, higher dose of TS can activate MAPKs signalling pathway, including ERK1/2, c-jun, JNK, p38 MAPK in a dose-and time-dependent manner. Western blot analysis showed that TS mediates the induction of MAPKs at as little as 0.25 and 1 µg/ml concentration in 12 and 24 h of treatment. JNK could be correlated with DR5 expression via Sp1 activation [22]. Therefore, Sp1 can directly bind to the DR5 promoter regions and regulate transcriptional expression [34]. The activation of MAPKs and other kinases (JNK) lead to Sp1 translocation to the nucleus [21]. In this study, we have found that dose-dependent up-regulation of CHOP and the transcription factor Sp1 was detected in TS ethereal oil-treated cells and the highest upregulation was seen at 0.25 µg/ml. Thus, Sp1 and CHOP up-regulation by TS oil involve in death receptor expression. In addition, thyme volatile oil showed HCT116 cell death via JAK/STAT pathway suppression.
In this study, we found that exposure of human HCT116 cells to TS sensitized the JAK/STAT and ROS-MAPKs-Sp1/CHOP signal pathway to mediate death receptors expression (Figure 7). TS essence serves as a potent inhibitor of JAK2, which is STAT's upstream protein kinase [35]. The JAK-STAT pathway plays a pivotal role in transducing signals from the cell surface to target genes in response to cytokines such as IFN-α, IFN-β, IFN-γ, interleukins and growth factors such as EGF, PDGF, GMCSF, and G-CSF [36]. Besides TS oil, several other phytoconstituents belonging to the category of natural terpenoids has shown to inhibit the JAK/STAT pathway by inhibiting the phosphorylation of STAT3 in order to impede cancer cell growth [36].
It is also shown that tumor cells cotreated with TS at 1 µg/ml or TRAIL at 25 ng/ml downregulate the expression of STAT3 target genes (cFLIP, Bcl-xL, ICAM1, VEGF, TRAF1 and the chemokine receptor CXCR4). Chang et al. [37] has shown that c-FLIPL, a protease-deficient caspase homolog widely regarded as an apoptosis inhibitor, is enriched in the CD95 deathinducing signalling complex (DISC) and potently promotes procaspase-8 activation through its proteaselike domain. Another decreasing expression protein by TS oil was the anti-apoptotic protein Bcl-2. The Bcl-2 family of proteins are key regulators of apoptosis and AGING have been implicated in colorectal cancer (CRC) initiation, progression and resistance to therapy [38]. Combined treatment of cells with TRAIL and thyme essential oil also downregulate anti-apoptotic proteins like survivin, cIAP1/2, XIAP that bind to caspase-3 and -9 and thereby inhibit caspase activity [39]. Transcription factor Sp1 is known to regulate expression of DR5 [40]. In our study, we observed increased expression of Sp1 by TS oil that might participated in induction of apoptosis through upregulation of DR5.
On the viewpoint of cancer events, pharmacological activation of caspase cascades and cleavage of PARP enhances the upregulation of cancer suppressor genes and downregulation of oncogenes, thereby suppressing LPSinduced colon adenocarcinoma. Here again, volatile oil protected mice from LPS-induced colon carcinogenesis and mediated inhibition of inflammation. The protective effect of TS was investigated by the histological examination. We observed antitumor effect of Thymus phytocompounds, which was evidenced by decrease in numbers of tumor buds that is a marker of colorectal cancer progression. Notably, tumor budding is defined as single cells or small groups of tumor cells (up to 4 cell clusters) within the tumor or at the invasive front [41]. Tumor buds or small tumor cell clusters have also been closely related to the epithelial-mesenchymal transition [42]. Increasing evidence has shown that epithelialmesenchymal transition plays a critical role in tumor cell metastasis [30]. In colon cancer patients, high budding correlated with high tumor grade, infiltrating growth pattern, venous invasion, lymphovascular invasion, and infiltration of a free serosal surface [43].
In light of the above facts, TS potentiates TRAIL-induced cell death through death receptors and inhibits growth and proliferation of colon cancer cells. Thus, plant essential oils have the potential application as protective natural drug towards colon carcinogenesis. However, the molecular mechanisms of the volatile oil of Thymus algeriensis, its phytopharmacological benefits and safety should be investigated by its application as nutraceutical in clinical trials in human body.

GC/MS analysis
To obtain the chemical profile of TS terpenic compounds, gas chromatography coupled with mass spectrometry (GC/MS) analysis was conducted on an Agilent 6890 gas chromatograph with an autosampler coupled with an Agilent 5973 Mass Selective Detector (MSD) (Agilent Technologies, Palo Alto, CA, USA) with an electron impact ionization of 70 eV. A Phenomenex ZB-5MSi capillary column (30 m × 0.25 mm, 0.5 μm film thickness; Agilent Technologies, Hewlett-Packard, CA, USA) was used at a temperature programmed to rise from 40 to 280° C at a rate of 5° C/min using helium (99.999% purity) as a gas carrier, with a flow rate of 0.7 mL/min, a split ratio of 60:1, and a scan time and mass range of 1s and 50-550 m/z, respectively. EO constituents were AGING identified by matching the mass spectra recorded with those stored in the Wiley 09 NIST 2011 mass spectral library of the GC/MS data system.

Live/dead assay
To measure TS-induced cell death, we used Live/Dead assay (Esterase Staining Method). HCT116 (2×10 3 ) were seeded in 96-well plates and treated with increasing dose of TS essential oil (0.1-5 pg/ml) at 37° C. The medium was removed and washed with PBS and then incubated with live/dead reagent (Calcein AM+PBS+ Ethidium homodimer) for 30 min. Calcein AM, a non-fluorescent dye, produce intense green fluorescence when retained within live cells and Ethidium homodimer solution produce red fluorescence when retained by damaged plasma membrane of dead cancer cells. The percentages of cells in apoptosis were calculated by counting live and dead cell numbers. Tumor cells were analyzed under a fluorescence microscope (Labophot-2; Nikon, Melville, NY, USA).

Colony formation assays
Single tumor cell growth into a colony was investigated through clonogenic assay. The clonogenic assay represents the capacity of a cell to grow after a series of mitosis over a 9-day period [45]. For this, HCT116 cells (5 × 10 2 cells per well) were seeded into 6-well plates. Following attachment, cells were exposed to different concentrations of diluted TS essential oil (0.01, 0.05, 0.1, 0.5, 1 µg/ml), TRAIL (25 ng/ml), and TS+TRAIL for 24 h. One day later, the medium was replaced with fresh medium, and allowed to form colonies for an additional 9 days. During 9 days of incubation, medium was replaced once with fresh medium. At the end of experiment, colon cancer cells were washed with PBS, fixed with clonogenic acid reagent and incubated for 20 min. After that, colonies were stained with crystal violet dye (0.5%) for 30 min, washed twice, and blue colonies were counted.

Western blot analysis
Tumor cells were treated with TS and/or TRAIL. After completion of exposure, cells were harvested and incubated with lysis buffer. Lysed cells were centrifuged (1400 rpm g/10 min) at 4° C and then supernatant was collected in fresh Eppendorf tube. Total protein content was measured by BCA method (Uptima,  2000) and β-actin (1:10,000). Membranes were blocked with nonfat dry milk (5% in PBS-T) and then incubated with secondary antibodies, with dilution factor of 1:5000. Blot signals were detected by Enhanced chemiluminescence reagent (GE Healthcare, Piscataway, NJ, USA). The resulting bands were quantitated by NIH imaging software (https://imagej .nih.gov/ij/download.html).

Animals
Forty eight Swiss albino mice (20 ± 2 g weight, 5-weekold) were kept in propylene cages with 6 mice/group under cycle of 12 h light/dark at room temperature (18-25° C) and a relative humidity of 55-65%. Animal experiment design was carried out according to Guide for the Care and Use of Laboratory Animals (8th edition, National Academies Press) and approved by the Animal Ethics Committee, University of Carthage at Tunis, Tunisia (No. LNSP/Pro 152012). Animals received standard diet and free access to tap water.

Study setting
Animals were divided into 8 groups (n=6 mice/group) as follows: (1) saline-treated sham group, (2) LPS (oral 10 µg/ml) treated group, (3) TS essential oil (oral 12.5 mg/ml) treated group, (4) TS essential oil (oral 50 AGING mg/ml) treated group, (5) 5-FU (oral 20 mg/ kg/day) treated group, (6) both TS and LPS (oral 12.5 mg/ml and 10 µg/ml, respectively) treated group (7) both TS and LPS (oral 50 mg/ml and 10 µg/ml, respectively) treated group, (8) both 5-FU and LPS (oral 20 mg/kg and 10 µg/ml, respectively) treated group. Thyme oily fraction was obtained by diluting TS stock solution in the vehicle (2% Tween 80). Intragastric administration of essential oil and comparator drug were given daily 1 h prior to LPS treatment for 1 week. Observations of spontaneous mouse behavior, physical parameters like body weight, water consumption and food intake and clinical responses throughout experiments were recorded daily before the drug dosing. At the end of experiments, mice were sacrificed and organ tissues (colon, liver, heart, spleen, kidney and lung) were immediately collected and processed for macroscopical and microscopical analysis. Colon length of different groups was determined. For histopathological process, organ portions were fixed in buffered formalin solution (10%) and embedded in paraffin wax. Thick sections (5µm) were cut, deparaffinized and stained with hematoxylin and eosin (H&E). Observations were done with Motic SFC-28 SERIES microscopy.

Statistical analysis
Data were analyzed with mean ± SD. One-way ANOVA followed by a Tukey post hoc test was performed to compare difference between groups.