TA was collected from Orbata Mountain, in the city of Gafsa, Tunisia (coordinates: N 34${}^{\circ }$ 22${}^{\prime }$ 49.8 and E 9${}^{\circ }$ 3${}^{\prime }$ 23.4). This plant species (1122) was identified and confirmed by Dr. Hamdi Lazhar, Engineer and Director of Bouhedma Natural Park (Tunisia). Only flowering plants’ leaves were collected. A voucher specimen was deposited in the National Institute of Agronomic of Tunisia (INAT) Herbarium. To profile bioactive compounds and polysaccharids of Teucrium essential oil, Gas Chromatography, Mass Spectrometry analysis (GC/MS) and FTIR were used.

Stock solutions of Oily fraction of Teucrium alopecurus leaves (100 $\mu$g/mL) were prepared in dimethyl sulfoxide (DMSO), (Sigma, St. Louis, MO, USA), stored at -20 ${}^{\circ }$C and then diluted as needed in experiments. Soluble recombinant human TRAIL/Apo2L was purchased from PeproTech (Rocky Hill, NJ, USA). Penicillin, streptomycin, medium (DMEM, RPMI 1640, IMDM) and fetal bovine serum (FBS) were obtained from Mediatech, Inc. (Herndon, VA, USA). Tris, SDS, LPS (Escherichia coli 055 : B5), NaCl, glycine, and antibody against $\beta$-actin were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Bovine serum albumin (BSA) was obtained from Atlanta Biologicals (Norcross, GA, USA). Antibody against VEGF was purchased from NeoMarkers (Fremont, CA, USA). Anti-XIAP was obtained from BD Biosciences (San Diego, CA, USA). Antibodies against survivin were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against cFLIP, Bcl-2, Mcl-1, Bcl-xL, p53, p38, phospho- ERK1/2, ERK2, cIAP1/2, PARP, CHOP, SP1, phospho-JNK1, JNK1, Akt, caspase-3, caspase-8, caspase-9, DR4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-DcR1, anti-DcR2 were obtained by Imgenex. Anti-DR5 was purchased from ProSci, Inc. (Poway, CA).

The human carcinoma cell lines HCT116 (human colon cancer cells), SCC4 (human squamous cell carcinoma), MCF-7 (breast adenocarcinoma), KBM-5 (human chronic leukemic cells), HL-60 (human promyelocytic leukemia), U266 (human multiple myeloma), MIA PaCa-2 (human pancreatic carcinoma cell line), Panc28 (human pancreatic carcinoma) were obtained from the American Type Culture Collection (Manassas, VA, USA). U266 were cultured in RPMI1640; HCT116, Panc-28, MCF-7, MIA PaCa-2 and SCC4 were cultured in DMEM with 10% fetal bovine serum, 100 U penicillin, and 100 U streptomycin; and KBM-5 cells were cultured in IMDM with 15% FBS and penicillin-streptomycin.

Cytotoxicity was investigated by MTT assay. Briefly, colon cancer cells (5 $\times$ 10${}^3$ cells per well) were pretreated with various doses of oily fraction of Teucrium (0, 50, or 100 pg/mL), dissolved in DMSO, in triplicate in 96-well plates for 12 h and removed, then we exposed cells to TRAIL (25 ng/mL) for 24 h. After this step, 20 $\mu$L of MTT solution was added to each well. After incubation for 2 h, an extraction buffer was added. Vehicle was considered as control. OD was measured at 570 nm using an MRX Revelation 96-well multiscanner (Dynex Technologies; MRX Revelation).

Tumor cells (2 $\times$ 10${}^5$) were pretreated with 50 pg/mL of TA, TRAIL/Apo2L, and combination of TA and TRAIL/Apo2L. The treated and untreated cells and incubated in 96-well plates, the medium was removed. After this step, 20 $\mu$L of PBS + Calcein AM + Ethidium homodimer solution was added to each well, mixed properly and cells were incubated at 37 ${}^{\circ }$C for 30 min in the dark to protect it from light. Cells were analyzed under a fluorescence microscope (Labophot-2; Nikon, Melville, NY, USA). The cancer cells in apoptosis can be estimated in terms of percentages.

Clonogenic assay is an in vitro cell survival test based on the ability of a single cell to grow into a colony. Colon cancer cells (500 cells) were seeded in 6-well plates and incubated overnight to allow attachment. The following day, the cells were treated with 200 pg/mL TA, TA + TRAIL, 25 ng/mL TRAIL in triplicate for 24 h. The next day, the medium was changed, and the cells were incubated for 9 days to form colonies. Medium was replaced after 4 days. At the end of the ninth day, medium was removed, cells was washed with PBS, fixed cells and incubated for 20 min. Cells were incubated with 0.5% crystal violet dye for 30 min and washed twice with distilled water, and blue colonies were counted.

Cell extracts were prepared in lysis buffer (20 mM, Tris (pH 7.4), 2 Mm EDTA (pH 8.0), 250 Mm NaCl, 0.01 $\mu$g/mL aprotinin, 0.1% triton X-100, 0.4 M phenylmethylsulfonyl fluoride, 0.005 $\mu$g/mL leupeptin, and 4 Mm Na${}_3$VO${}_4$). Lysates were centrifuged (1400 rpm/10 min) to remove insoluble materials. Thirty microgram of lysates were resolved by 10% SDS-PAGE and proteins were then transferred to nitrocellulose membranes where it bound, forming the blot. After these steps, membranes were probed overnight with relevant primary antibodies at 4 ${}^{\circ }$C. Proteins are exposed to thin surface layer for antibody detection after the blotting process. Dilution factors of antibodies are: c-FLIP (1 : 3000); survivin (1 : 2000); c-Myc (1 : 2000), CXCR4 (1 : 5000), MMP-9 (1 : 1000), cIAP-1/2 (1 : 2000), Mcl-1 (1 : 3000), ICAM-1 (1 : 2000), p53 (1 : 3000), Bcl-xL (1 : 2000), cyclin D1 (1 : 3000), PARP (1 : 5000), caspase-3 (1 : 2000), -8 (1 : 2000) and -9 (1 : 2000), Bcl-2 (1 : 3000), $\beta$-actin (1 : 10,000); XIAP (1 : 3000), VEGF (1 : 1000), TRAF1 (1 : 2000).

Then, blots were incubated with secondary antibodies (1 : 5000) diluted in 5% skimmed milk for 1 h, and signals were detected by enhanced chemiluminescence reagent (GE Healthcare, Piscataway, NJ, USA).

Colon cancer cells (1 $\times$ 10${}^6$ cells) cells were labeled with DCF-DA (20 $\mu$M), exposed for 1 h to 200 pg/mL of Teucrium essential oil. The increase in fluorescence resulting from oxidation of DCF-DA to DCF was measured by fluorescence-activated cell sorting (FACS). The mean fluorescence intensity at 530 nm was calculated. Results were collected from at least 10,000 cells at a flow rate of 250-300 cells/second.

Data presented are the mean $\pm$ standard deviation (SD) of experiments done in triplicate. Differences between groups were compared by a one-way analysis of variance (ANOVA). P-value 0.05 was considered significant.

The FT-IR spectral analyses of the TA essential oil are shown in Fig. 1A. Infrared region starting from 3600 through 3200 cm${}^{-1}$ has been correlated to N-H stretching (aliphatic primary amine) and O-H stretching (carboxylic acids). From 3000 to 700 cm-1 spectrum section, 7 peaks were detected nearby 2800, 1750, 1735, 1650, 1600, 1800, and 700 cm-1. The spectrum region from 3000 to 2800 cm-1 may indicate carbohydrate compounds; wave numbers of 1750 does carbonyl, of 1650 cm-1 does aromatic groups. The absorption band from 1800 to 700 cm-1 is correlated to spectral fingerprint.

Fig. 1.

(A) FTIR and (B) GC/MS spectrum of Teucrium alopecurus essential oil.

Aerial parts of Teucrium alopecurus oils possess different chemical compositions. The compositional analysis of TA by GC/MS has demonstrated that it is composed of various terpenic compounds, including (+)-Epi-Bicyclo sesquiphellandrene, d-limonene, $\alpha$-Bisabolol, $\alpha$-Cadinol, $\alpha$-humulene (Fig. 1B). The presence of terpenic compounds in TA may attribute to its anticancer properties.

We used the MTT assay to examine whether TA has the ability to enhance TRAIL-induced cytotoxicity. TA alone increased cytotoxicity of HCT116 cells dependent on dose. As indicated in Fig. 2A, the viability of human colon cancer cells was decreased by 30% and 37% at 100 pg/mL of TA and 25 ng/mL of TRAIL, respectively, however, combination treatment with both TA (100 pg/mL) and TRAIL induces a much higher level (~80%) of cytotoxicity. Pretreatment with TA increased TRAIL-induced growth inhibition.

Fig. 2.

Teucrium alopecurus enhances colon cancer cells to TRAIL-induced cell death. (A) TA enhances TRAIL-induced cytotoxicity. In 96-well plates, HCT116 (5,000 cells) were pretreated with two doses of TA (0.005 and 0.01 $\mu$L/mL) and then exposed with 25 ng/mL of TRAIL. After indicated period of time, cytotoxicity was examined by the MTT assay. (B) Oily fractions of Teucrium potentiates apoptosis induced by TRAIL. HCT116 cells (1 $\times$ 10${}^6$) were first pretreated with TA. After 4 h cells were incubated with TRAIL for 24 hours. Live/Dead assay was performed as per the instruction and then analyzed under a fluorescence microscope. Images were captured at magnification = 20$\times$. Percentages were calculated based on counted live (green) and dead (red) cells. (C) Effects of TA on the clonogenic potential of tumor cells. (D) TA induces ROS production. HCT116 (1 $\times$ 10${}^6$ cells) were exposed with the indicated concentrations of TA. After 1 h, labeled with dichlorodihydrofluorescein diacetate (DCF-DA) and then flow cytometeric analysis was performed to determine the ROS level. Effect of TA on TRAIL-induced cleavage of PARP and activation of caspases (E) dependent on dose and (F) time.

To assess cell death induced by TRAIL with and without TA, we also used the Live/Dead assay. As shown in Fig. 2B, TA and TRAIL showed modest cell death (20% and 15% respectively). However, when TA and TRAIL were given in combination, a dramatic increase in cell death (87%) was observed. This finding indicates apoptosis potentiating effect of TA.

We also investigated whether TA increases TRAIL-induced suppression of colony-forming ability of HCT116 cells. As shown in Fig. 2C, we found that TA alone represses colony formation. Treatment with TRAIL also moderately suppressed the number of colonies. However, in wells cotreated with TA and TRAIL, we noted almost complete suppression of colony formation.

Since most of the anticancer drugs induce apoptosis through reactive oxygen species (ROS) production, therefore we determined whether TA also generates ROS. We found that TA was able to generate ROS in HCT116 cells. We found a significant production of ROS at 200 pg/mL concentration of TA (MFI 142.4) compared to the control (MFI 114.2) in colorectal cancer cells (Fig. 2D).

To assess whether cotreatment of HCT116 with oily fractions of TA and TRAIL increases apoptosis through cleavage of caspases and PARP dependent on dose- and -time (Fig. 2E,F), we used HCT116 cells. Caspase-8 and -9 activated through extrinsic and intrinsic apoptotic pathway, which further activate effector caspase-3 and then induces PARP cleavage [18]. Administration of TA alone at 100 pg/mL induces little changes in cleaved caspases and PARP levels. Exposure to TRAIL alone induced moderate level of cleaved PARP and caspases (caspase-8, -9 and -3), whereas increased activation of caspases and PARP cleavage were clearly observed in combination of TA (Fig. 2E). When two different doses of TA (100 pg/mL or 200 pg/mL) used alone or in combination with TRAIL (25 ng/mL), both doses increased the apoptotic effect of TRAIL by activating caspases and cleaving PARP (Fig. 2F). Taken together, these results indicate that TA increases TRAIL-induced cell death in colon cancer cells.

To investigate whether TA up-regulates the expression of TRAIL receptors, HCT116 cells was used and treated with TA and found that it increased both DR4 and DR5 receptors dependent on dose- (Fig. 3A) and time (Fig. 3B). Increased DRs expression were detected even at 50 pg/mL of TA. Furthermore, it was observed that TA suppressed the expression of decoy receptors DcR1 and DcR2 (Fig. 3A,B). Our findings indicate that TA upregulates the receptors for TRAIL as well as abolishes the expression of decoy receptors for induction of cell death.

Fig. 3.

Upregulation of death receptors and down-regulation of Decoy receptors by TA in colon cancer cells. (A) TA induces DRs expression and down-regulates DcRs in HCT116 cancer cells in dose-dependent manner. (B) TA induces DRs expression and down-regulates DcRs in HCT116 cancer cells in time-dependent manner. (C) TA increases expression of DR5 and DR4 in multiple types of cancer cells including MIA Paca-2, U266, HL60 and SCC4 cells. (D) TA induced expression of DR5 and DR4 in breast cancer cells in time dependent manner. (E) TA upregulates expression of DR5 and DR4 dependent on time.

We also investigated whether TA increases DR5 and DR4 expression in HCT116 cells and other tumor cells. As shown in Fig. 3C, induction of TRAIL receptors was noted in MIA Paca-2, U266 cells, HL60, and SCC4 cells. In MCF-7 (Fig. 3D), KBM5 and U266 cells (Fig. 3E), TA increased expression of DR5 and DR4 dependent on time. Taken together, this data indicates that induction of either DR5 or DR4 expression by TA was not specific to the cell-types.

ERK1/2 phosphorylation and JNK have shown to be involved in the upregulation of DR [19]. Moreover, activation of Sp1 [20] and induction of TRAIL-induced apoptosis has been related to the phosphorylation of ERK1/2 [21]. Therefore, we examined whether the TA is able to activate MAPKs such as ERK1/2, JNK, and p38; and inhibit Akt activation. Our results show that TA increased the phosphorylation of ERK1/2 dependent on dose (Fig. 4A) and time (Fig. 4B), and the optimum induction was detected when HCT116 was exposed with 50 pg/mL of this essential oil. It is depicted in this Figure that the oily fractions of Teucrium trigger apoptosis in HCT116 by activating JNK and p38. Overall, these results suggest that TA activates ERK1/2, p38 and JNK molecules dependent on dose and time.

Fig. 4.

Activation of ERK1/2, p38 and JNK and downregulation of Akt by TA in colon cancer cells. (A) HCT116 cells was treated with indicated dose of TA for 6 hours and subjected to western blotting for ERK1/2 (pERK1/2), p38 (pp38), and JNK (pJNK) expression. Total proteins were used as loading control. (B) HCT116 cells was treated with TA1 (100 pg/mL) for indicated time and subjected to western blotting. Total proteins were used as loading control. (C) HCT116 cells was treated with indicated dose of TA for 6 hours and subjected to western blotting for CHOP and SP1 proteins. $\beta$-actin was used as loading control. (D) HCT116 cells was treated with TA (100 pg/mL) for indicated time and subjected to western blotting. $\beta$-actin was used as loading control.

Sp1 also regulate the expression of DR5 gene by binding on its promoter region [19]. Previously Yoshida [22] also shown that the promoter region of DR5 gene contains binding sites to Sp1. This overexpression induced by Sp1 helps in enhancing TRAIL-mediated apoptosis. Therefore, we determine whether TA also induces the transcription factor Sp1. Our results showed that TA enhances expression of Sp1, which regulates DR5 expression. We also observed induced expression of CHOP by TA in colon cancer cells dependent on dose (Fig. 4C) and time (Fig. 4D). Similar as Sp1 transcription factor, CHOP is also reported to induce apoptosis in cancer cells by upregulating DR5 [23]. The expression of CHOP was induced at as low as 6h after exposure to TA. These observations suggest that induction of DR4 and DR5 by TA is mediated through upregulation of Sp1 and CHOP that further leads to potentiation of TRAIL-induced cell death in cancer cells.

To understand the mechanism by which TA induced apoptosis in HCT116 cells, western blotting has been done. This method used to determine the level of antiapoptotic proteins because Bcl-2 and IAP family proteins play major role in cancer cell growth and proliferation [24, 25]. We noted that TA reduces the expression of antiapoptotic proteins c-myc, cIAPs, Mcl-1, survivin, Bcl-2, metastatic protein ICAM-1 and induces expression of tumor suppressive protein p53 in dose dependent manner (Fig. 5A).

Fig. 5.

TA suppresses expression of antiapoptotic proteins, induces p53 and increases effect of TRAIL. (A–B) HCT116 cells were treated with indicated dose of TA for 24 hours and then western blot was performed against mentioned proteins. $\beta$-actin was used as loading control. (C) HCT116 cells was treated with TA (100 pg/mL) for 24 hours and expression of cFLIP was determined by western blotting. $\beta$-actin was used as loading control. (D–E) HCT116 cells were treated with indicated doses of TA with or without TRAIL for 24 hours. Western blotting was done to determine the expression of mentioned proteins. $\beta$-actin was used as loading control. (F–H) HCT116 cells were treated with TA (100 pg/mL) with or without TRAIL (25 ng/mL) for 24 hours. Western blotting was done to determine the expression of mentioned proteins. $\beta$-actin was used as loading control.

The overexpression of c-FLIP protein, a caspase-8 inhibitor, is known to regulate apoptosis mediated by TRAIL in colon cancer cells [26] by conferring resistance to death receptor [13]. The data obtained from Western blotting indicated that the administration of TA to colon cancer cells reduced both c-FLIP isoforms in a time course (Fig. 5B). Besides HCT116 cells, this protein was also downregulated in U266 cells dependent on time (Fig. 5C). Overall, our result showed that suppression of c-FLIP${}_L$ and/or c-FLIP${}_S$ expression may involve in apoptosis induced by TRAIL.

Next, we determined whether TA increases the suppression of antiapoptotic proteins when combined with TRAIL. HCT116 cells was exposed with TA (100–200 pg/mL) alone or in combination with TRAIL and then performed western blotting to determine the effect of this combination. As shown in Fig. 5D, we observed that TA alone dose dependently suppressed the expression cIAP2, Bcl-2, survivin (Fig. 5D), and metastatic protein ICAM1 (Fig. 5E), however, in combination with TRAIL this suppression is significantly increased. The antiapoptotic protein XIAP and angiogenic protein VEGF suppressed at even lower dose of both TA and TRAIL either treated alone or in combination.

We further determined whether TA increases modulation of p21, p53 and antiapoptotic proteins TRAF1 and Mcl1 when combined with TRAIL. For this we treated cells for 12 hours and 24 hours with TA alone or in combination with TRAIL. We found that TA suppressed the expression of p21, TRAF1, and Mcl1 alone or in combination with TRAIL in both 12 and 24 hours of exposure. The effect was more prominent at 24 hours of exposure compared to 12 hours. Similarly we observed increased expression of p53 by TA not by TRAIL at both 12 and 24 hours of exposure (Fig. 5F). Besides these, we also found decreased level of c-Myc, Cyclin D1 (Fig. 5G), and CXCR4 (Fig. 5H) by TA alone or in combination. The effect of TA and/or TRAIL was also more prominent at 24 hours of exposure compared to 12 hours.