<em>Origanum majorana</em> Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway

Cell Culture, Chemicals, and Antibodies

Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany). Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin at 5% CO₂, 37°C and 95% humidity. 3-methyladenine (3-MA) and Z-VAD-FMK were obtained from sigma-Aldrich. Antibodies against target proteins used in this study are: caspase 8, caspase 7, LC3 and Beclin-1 (Cell Signaling, USA); cleaved caspase 3, Cyclin B1, H3 phospho-Ser10, γH2AX (Millipore), TNFα, p62/SQSTMI and cleaved PARP (Abcam), survivin and β-actin (Santa Cruz Biotechnology).

Preparation of Origanum majorana Ethanolic Extract (OME)

The plant was collected from a private commercial farm located at 33° 16′ 54′′ N and 35° 14′ 51′′ E. The farm is located in Tire region, Lebanon and the approval of the owner was obtained before collecting the fruit or commencing any experiments. This plant is neither endangered nor protected by any laws and it is readily and commercially available in the market. Origanum majorana plant, at the time of collection, was identified by Dr. Ali Al-Khatib, a plant biologist at the Lebanese International University (Lebanon). The dried leaves, used for the extraction, were further identified and confirmed by Dr. Mohamed Tahar Moussa, plant taxonomist at the United Arab Emirates University where a voucher specimen of the plant (No. 14670) was deposited at the National Herbarium, College of Science, Department of Biology, United Arab Emirates University.

Origanum marjorana ethanolic extract (OME) was prepared as previously described (10). Briefly, dried leaves powder (5.0 g) was extracted in 100 mL of 70% absolute ethanol and the mixture was kept in the dark for 72 h in a refrigerator without stirring. Afterward, the mixture was filtered, and the filtrate was evaporated to dryness using a rotary evaporator at room temperature. The green residue was kept under vacuum for 2–3 h and its mass was recorded. The residue was stored at −20°C until further use.

HPLC-MS Identification of Constituents in Origanum majorana Ethanolic Extract

The identity of O. majorana was analyzed by LC-MS (6420 Triple Quadrupole, Agilent Technologies). Sample of O. majorana ethanolic extract was filtered using 0.45 μm syringe filter preceding the analyses. The instrument was fitted with a Agilent EclipsePlus-C18 column (1.8 μm particle size, 2.1 × 50 mm length, Agilent Technologies, USA) maintained at 35°C, coupled to a tunable UV-Vis detector (Agilent Technologies, USA) and 6420 Triple Quadrupole LC/MS System (Agilent Technologies, USA). The mobile phases used were A = 0.1% formic acid and B = acetonitrile, and the gradient was: 0–2.5 min: 0% B, 2.5–15 min: 20–100% B, 15–18 min: 100% B, and 18–25 min: 5% B with 0.2 ml/min. Electrospray ionization (ESI) source was used in LC-MS system in positive polarity. LC-MS operating conditions were as follows: capillary voltage: 4 kV; the nebulizer pressure was 45 psi; drying gas flow was 11 L/min and drying temperature was 325°C. The mass range monitored was from 100 to 1,000 Da. Compounds were identified based on their Molecular weight (MW) and retention time (RT).

As shown in Figure 1, HPLC/MS analysis revealed the presence of several peaks which indicated the presence of 12 compounds [Limonene (MW: 136 g/mol, RT: 12.2 min), Terpinen-4-ol (MW: 153 g/mol, RT: 10.2 min), Linalyl acetate (MW: 196 g/mol, RT: 12.2 min), β-Caryophyllene (MW: 204 g/mol, RT: 10.6 min), Apigenin (MW: 270 g/mol, RT: 11.1 min), Hesperetin (MW: 302 g/mol, RT: 15.2 min), Rosmarinic acid (MW: 360 g/mol, RT: 11.3 min), Luteolin (MW: 286 g/mol, RT: 12.9 min), Arbutin (MW: 272 g/mol, RT: 15.9), Quercetin (MW: 302 g/mol, RT: 12.6 min), Ferulic acid (MW: 194 g/mol, RT: 12.7 min), Catechin (MW: 290 g/mol, RT: 12.4 min)].

Figure 1

Chromatograms of compounds identified in ethanolic extract of Origanum majorana L. by HPLC-MS. Name of the compounds, their respective chemical structures, molecular masses, and retention times are shown.

Measurement of Cellular Viability

Cells (7 × 10 ^{cells/well) were seeded in triplicates in 96-well culture plates. After overnight incubation, cells were treated with various concentrations of OME or vehicle for another 24 and 48 h. At the end of incubation, cell viability was measured with the Cell cytotoxicity assay kit (Abcam) following the manufacturer's instructions.}

Cell viability was also measured by counting live cells using the Muse Count and Viability Kit (Millipore) as previously described (12).

Colony Formation Assay

For anchorage-dependent colony formation assay, HT-29 cells were seeded (2 × 10 ^{cells/well) in 6-well culture plates, and maintained for 10 days at 37°C to form colonies. At day 10, formed colonies were treated with various concentrations of OME and were maintained at 37°C for 5 additional days. At day 15, plates were washed 3 times with PBS, fixed for 15 min with 4% formalin and stained with 0.01% crystal violet for 30 min. The colony number was counted and their surface area in each well was measured using the imageJ software.}

Cell Cycle Analysis

Briefly, HT-29 cells were exposed to different concentrations of OME or vehicle. After 24 h incubation, cells were harvested, washed with 1X PBS and then fixed with ice-cold 70% ethanol and stored at −20°C overnight. Cell cycle analysis was performed using the Muse™ Cell Analyzer (Millipore) as previously described (13). Analysis of DNA content of cells at different phases of cell cycle was determined using the FlowJo software.

Whole Cell Extract and Western Blotting Analysis

HT-29 cells were seeded in 100 mm culture dishes at a density of 3 × 10 ^{and cultured for 24 h. After OME treatment, protein extraction and Western blotting were performed as previously described (}12). All Western blots shown are representative of three independent experiments.

Statistical Analysis

Statistical analyses were carried out using SPSS version 21 software. Data were presented as group mean ± SD. The data were analyzed via one-way ANOVA followed by LSD's post-hoc multiple comparison test. A p < 0.05 was considered statistically significant.

Origanum majorana Extract Inhibits Cellular Viability of Human Colorectal Cancer Cells

We have examined the anti-proliferation effect of OME on two human colorectal cancer cell lines (HT-29 and Caco-2 cells). Cells were treated with increased concentrations of OME for 24 and 48 h. In both cell lines, OME caused significant growth inhibition, in a concentration and time-dependent manner as shown in Figures 2A,B. IC50 values are 498 and 342 μg/mL at 24 and 48 h for HT-29 and 506 and 296 μg/mL at 24 and 48 h for Caco-2 cells. It is worth mentioning that there is no color interference between the extract and the reagent used to measure cellular viability.

Figure 2

Origanum majorana ethanolic extract inhibits cellular viability of colorectal cancer cells. (A) Exponentially growing HT-29 and (B) Caco-2 colon cancer cells were treated with and without of various concentration (0, 150, 300, 450, and 600 μg/mL) OME for 24 and 48 h. Viability was measured using a colorimetric assay as described in section Materials and Methods. Values are represented as mean ± SD of n = 4 (*p < 0.05 and ***p < 0.001). (C) HT-29 cells were exposed to OME for 24 and 48 h and number of viable cells, using a fluorescent dye, was monitored as described in section Materials and Methods using the Muse Cell Analyzer (Millipore). Data represent the mean ± SD of n = 3 carried out in triplicate.

Cell viability was also measured by counting live cells using the Muse cell counting kit, which differentially stains viable and dead cells. Results showed a concentration of 150 μg/mL of OME, reduced cellular proliferation of HT-29 when compared to control cells. However, concentrations of 300, 450, and 600 μg/mL of OME caused time-dependent decline in the number of viable cells when compared to the number of cells counted at the day of treatment (day 0) (Figure 2C) hence, indicating cell death.

Origanum majorana Extract Inhibits HT-29 Colony Growth

The anti-colon cancer effect of OME was further examined by testing the ability of OME to modulate the proliferative capacity of HT-29 to form colonies. After HT-29 cells were allowed to form colonies for 10 days, wells were replaced with fresh media with or without OME and colonies were let to grow for 5 additional days. Results showed that OME caused a significant decrease in concentration-dependent manner, not only, in size (Figure 3C) but also in number (Figure 3B) of the already formed colonies and thus, clearly indicating massive cell death of HT-29 cells (Figures 3A,B).

Figure 3

Origanum majorana inhibits HT-29 colony growth. (A–C) Inhibition of formed HT-29 colony growth by various concentrations of OME (0, 150, 300, 450, and 600 μg/mL) was assessed by measuring the number and average size (surface area) of the colonies obtained in control and OME-treated plate as described in section Materials and methods. Values are represented as mean ± SD of n = 3 (*p < 0.05 and **p < 0.005).

Origanum majorana Extract Induces a Mitotic Arrest

In order to investigate the mechanism through which OME exerts its cytotoxic and colony growth inhibition effect on HT-29 cells, cell cycle analysis was performed. We found that a concentration of 300, 450, and 600 μg/mL of OME caused a significant inhibition of cell cycle progression at G2/M. Indeed, the population arrested cells at G2/M cells arose from 30% in control to 51% in OME-treated cells (Figures 4A,B). To determine at which stage (M or G2) cell cycle arrest occurred, we examined the phosphorylation status of histone H3, a marker of mitosis. OME at concentrations of 300, 450, and 600 μg/mL significantly increased the level of H3p(Ser10) (Figure 4C). The level of cyclin B1, whose upregulation invokes mitotic arrest, was also examined and was found to be upregulated as well (Figure 4C). We also found that OME induces a concentration dependent increase of the cell cycle regulator p21 (Figure 4C), hence suggesting that p21 upregulation may contribute to the mitotic arrest induced by OME.

Figure 4

OME induces a mitotic arrest in HT-29 cells. (A,B) Cell cycle distribution analysis in HT-29 cells treated with and without OME (0, 150, 300, 450, and 600 μg/mL) for 24 h. Values are represented as mean ± SD of n = 3 (*p < 0.05, **p < 0.005, and ***p < 0.001). (C) Alteration in proteins associated with cell cycle regulation in OME-treated HT-29 cells.

Origanum majorana Extract Activates Caspase 3 and Caspase 7-Dependent Extrinsic Apoptotic Pathway

We have shown that OME induced a concentration-dependent cell death of HT-29 (Figure 2C). Therefore, we examined whether this cell death was associated with the activation of apoptosis. Western blotting analysis showed an accumulation of cleaved PARP, active caspase 8, 3, and 7 in response to OME (Figure 5A). Interestingly, we found that TNF-α, a cytokine involved in the initiation of the extrinsic apoptotic pathway, was also upregulated by OME (Figure 5B) suggesting that OME activates the extrinsic apoptotic pathway through activation of the TNF-α signaling pathway.

Figure 5

Activation of extrinsic apoptotic pathway and upregulation of TNF-α in OME-treated HT-29 cells. (A) Western blot analysis of caspase 3, 7, and 8 activation and PARP cleavage in HT-29 cells. Cells were treated with or without increasing concentration (0, 150, 300, 450, and 600 μg/mL) of OME for 48 h, then whole cell proteins were extracted and subjected to Western blot analysis for the markers of apoptosis (B) Western blot analysis of TNF-α (C) Western blot analysis of cleaved PARP in cells pretreated for 1 h with and without Z-VAD-FMK (50 μM) followed by treatment with OME (450 μg/mL) for 48 h. (D) Inhibition of apoptosis has a minimal effect of OME-induced cell death. HT-29 cells were pretreated with Z-VAD-FMK as described above and then treated for 48 h with 450 μg/mL OME. Cell viability was determined as described in section Material and Methods. Values are represented as mean ± SD of n = 3 (*p < 0.05 and ***p < 0.001).

Next, we examined whether apoptosis is the sole mechanism of cell death activated by OME. Blockade of apoptosis by the pancaspase inhibitor, Z-VAD-FMK (50 μM), revealed by the absence of cleaved PARP and cleaved caspase 8 (Figure 5C), led to a minimal recovery of cell viability. Cell viability increased from 37% in control cell treated with OME only to 43% when cells were pre-treated with Z-VAD-FMK (Figure 5D), suggesting that apoptosis is not the main way through which cell dies and that another mechanism of cell death might be activated as well.

Origanum majorana Extract Induces Abortive Autophagy in Colon Cancer Cells

Having shown that apoptosis is not the sole mechanism of cell death activated by OME, we interrogated whether autophagy is activated by OME in colon cancer cells. We found that that OME caused an increase in the accumulation of lipidized LC3II, a marker of autophagy, in a concentration-dependent manner, suggesting that autophagy is occurring or at least initiated in HT-29 cells (Figure 6). Next, we examined the expression of Beclin-1, an autophagy effector playing a key role in autophagosome. Western blot analysis showed that the level of Beclin-1 slightly decreased in OME-treated cells compared to control cells (Figure 6) and hence, suggesting that autophagy is occurring through Beclin-1-independent mechanism. Further, we scored for p62(SQSTM1), a ubiquitin-binding protein, a marker of autophagic flux degraded in productive autophagy. Strikingly, we found that level of p62(SQSTM1 increased dramatically in concentration-dependent and sustained manner (Figure 6). It is well-documented that p62 is preferentially degraded during autophagy; however, its level remains unchanged or increased during abortive autophagy. Hence our results suggest that autophagy flux was disrupted leading to abortive autophagy in HT-29 cells.

Figure 6

OME induces abortive autophagy in HT-29 cells. Western blotting analysis of LC3II, p62(SQSTM1), and Beclin-1 expression OME-treated HT-29 cells. Cells were treated with or without increasing concentration (0, 150, 300, 450, and 600 μg/mL) of OME for 48 h, then whole cell proteins were extracted and subjected to Western blot analysis, as described in section Materials and Methods, for LC3II, 62(SQSTM1), and Beclin-1.

Origanum majorana Extract Induces DNA Damage in Colon Cancer Cells

It is well-established that DNA damage can trigger cell cycle arrest, autophagy and apoptosis in cancer cells. Because the three events were observed in OME-treated cells, we decided to investigate whether OME exert its anti-colon cancer affect through induction of DNA damage. We found that OME treatment caused an increase in the levels of γH2AX (Figure 7), indicative of double strand breaks.

Figure 7

OME induces DNA damage in response to OME treatment in HT-29 cells. HT-29 cells were treated with increasing concentrations (0, 150, 300, 450, and 600 μg/mL) of OME for 48 h. DNA damage was examined by western blotting by measuring the level of phosphorylated H2AX.

Abortive Autophagy, and DNA Damage Precedes Apoptosis Activation

We next monitored the accumulation of markers of DNA damage (γH2AX), autophagy (LC3II and p62) and apoptosis (cleaved caspase 8 and cleaved PARP) over time. We found that lipidized LC3II, upregulation of p62 and accumulation of DNA damage were detected concomitantly as early as 4 h post-OME treatment (Figure 8A). Again, LC3II and p62 showed sustained accumulation over time, further confirming the occurrence of abortive autophagy. Apoptosis, however, occurred only 24 h post-treatment and was maximal at 48 h. These results suggest that DNA damage and autophagy are the earliest consequence of OME-treatment to HT-29 cells.

Figure 8

DNA damage and autophagy precedes apoptosis in OME-treated HT-29 cells. (A) Time-course analysis, by Western blotting, of PARP and caspase 8 cleavage, LC3-II, p62 (SQSTM1), γH2AX, and H3pser10 accumulation in OME-treated HT-29 cells. Cells were treated with 450 μg/mL OME and proteins were extracted at the indicated time-points (0, 4, 8, 24, and 48 h) as described in section Materials and Methods. (B) Western blot analysis of γH2AX accumulation in HT-29 cells pre-treated with 3MA. Cells were pretreated with or without 3-MA (5 mM) for 1 h and then OME (450 μg/mL) was added, and cells were incubated for 48 h.

To determine which event (DNA damage or autophagy) occurred first, we measured the level of γH2AX in HT-29 cells pre-treated with 3-MA (5 mM), an autophagy inhibitor of autophagosome formation, and then treated with OME. We found that the inhibition of autophagy initiation did not prevent DNA damage induced by OME (Figure 8B), hence, suggesting that DNA damage preceded autophagy activation.

Inhibition of Autophagy Initiation Partially Reduced Cell Death Induced by OME and Apoptosis Activation Depend on the Induction of Autophagy

We have shown that OME induced abortive autophagy as early as 4 h post-treatment while apoptosis was activated only at 24 h. The contribution of autophagy in OME-induced cell death was, therefore, examined in cells pre-treated with 3-MA (5 mM). Blockade of autophagy by 3-MA was confirmed by the observed decrease in the conversion of LC3-I to LC3-II (Figure 9A). Interestingly, cell viability markedly increased when autophagy was blocked compared with control group treated with OME only (Figure 9B). Indeed, cell viability arose from 38% in control group to 67% in group pre-treated with 3-MA before addition of OME (Figure 9B). It is noteworthy to mention that the blockade of autophagy reduced the level of cleaved PARP in HT-29 cells (Figure 9A); hence suggesting that activation of apoptosis is somehow dependent on the initiation of autophagy.

Figure 9

Inhibition of autophagy decreases OME-induced cell death in HT-29 cells. (A) Analysis of LC3-II and cleaved PARP accumulation in HT-29 cells pre-treated with 3-MA. Cells were pretreated with or without 3-MA (5 mM) for 1 h and then OME (450 μg/mL) was added, and cells were incubated for 48 h. (B) Inhibition of autophagy reduces cell death induced by OME. HT-29 cells were pretreated with 3-MA for 1 h and then for 48 h with 450 μg/mL OME. Cell viability was determined as described in Material and Methods. Values are represented as mean ± SD of n = 3 (***p < 0.001).

Origanum majorana Downregulates the Level of Survivin

In addition to its role in mitosis and apoptosis, survivin might also protect cell from death through a mechanism involving autophagy. We found that OME significantly downregulated the level of survivin in HT-29 cells (Figure 10). This result suggests that downregulation of survivin by OME could account, although may be not solely, in sensitization of HT-29 cells to autophagic and apoptotic cell death. It is noteworthy to mention that emerging evidences suggests that survivin can also repress autophagy, it is then tempting to think that downregulation of survivin could contribute, at least partly, in promoting sustained autophagy in response to OME.

Figure 10

Downregulation of survivin by OME in HT-29 cells. HT-29 cells were treated with increasing concentrations (0, 150, 300, 450, and 600 μg/mL) of OME for 48 h and the level of survivin was assessed by Western blotting.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.