Origanum vulgare is commonly called as Oregano is an aromatic herbaceous perennial shrub grown in dry grassland, road verges, and quarries. It is natively grown in Western and Southwestern Eurasia, and in the Mediterranean regions. The fresh and dried leaves as well as oregano essential oil are used traditionally for treating variety of ailments (Ivanova et al., 2005; Lin et al., 2005; Mahady et al., 2005).
Previous studies were undergone in essential oils and their bioactive compounds for exploring its possible functional application (Gianni et al., 2005; Sawamura, 2000; Ormancy et al., 2001). Therefore, the medicinal and aromatic plants are considered essential because until now the local population primarily depend on treating various diseases using these plants indicating that plant derived components can be used as an alternative therapy for synthesizing new synthetic products (Houghton, 2000).
Increased de novo fatty acid (FA) and cholesterol biosynthesis is a characteristic feature of prostate cancer proliferation and metastasis (Pelton et al., 2012; Zadra et al., 2013). Many solid tumors and cell lines derived from these tumors overexpress FAS for their proliferation and survival (Kuhajda, 2006; Swinnen et al., 2006). Therefore, inhibition of FAS by pharmacological aspects is one of the important strategies to inhibit the cancer cell growth and induce apoptosis (Menendez et al., 2005; Wang et al., 2005).
In case of normal cells, the expression of FAS is low due to the existence of fatty acid in circulation from the diet (Chajes et al., 2006; De Schrijver et al., 2013). Previous study has also mentioned that inhibition of FAS expression is important in the induction of apoptosis (Zhou et al., 2003). Another way of inhibiting fatty acid biosynthesis is by inhibiting SREPB1. SREPB1 is a sterol regulatory element-binding protein 1 which functions is to activate fatty acid biosynthesis genes including FAS. Inhibition of SREPB1 expression could inhibit the cancer cell proliferation (Wu et al., 2005).
In cancer, there is an imbalance between the cell division and cell death due to lack of signals for the cells that need to undergo cell death. This might be due to the difficulty in regulating any of the step in the apoptosis process. For instance, the p53 is a tumor suppressor gene, which function is to keep the cell division and number in check. The downregulation of p53 gene can lead to the uncontrollable cell growth, development and reduced apoptosis.
Therefore, understanding p53 induced apoptosis is crucial and aid in the understanding pathogenesis of conditions. In the intrinsic pathway of apoptosis, the balance between pro-apoptotic protein and anti-apoptotic protein determines the initiation of apoptosis. The proapoptotic proteins such as BAX promote cytochrome-c release and anti-apoptotic protein blocks the release of mitochondrial cytochrome c (Fridman and Lowve, 2003) and induces intrinsic mediated apoptosis. In our current study, we monitored how regulation of fatty acid biosynthesis led to the possible induction of apoptosis pathway after oregano EO treatment. Form our results we conclude that oregano EO might be a potential candidate to induce apoptosis in prostate cancer.
Materials and Methods
Cell viability determination
Anti-proliferative activity of EO in the human prostate cancer (PC3) was evaluated by performing MTT assay (Perumalsamy et al., 2018).
2104 PC3 cells were grown in 96-well culture plates for 24h. The cells were treated with different concentration of oregano EO (0, 6.2, 12.5, 25, 50, and 100 µg/mL) immersed in 0.1% DMSO for 48 h. The untreated cells were used as control whereas cisplatin is used as a reference standard. DMSO treated cells is used as a negative control. After 48h, morphological changes of oregano EO treated and non-treated cells was subjected to light microscopy using Leica DMIL LED equipped with an Integrated 5.0 Mega-Pixel MC 170 HD camera (Wetzlar, Germany).
The lipids in cancer cells were visualized using Oil Red O staining. The cells were rinsed with PBS and fixed in 4% paraformaldehyde for half an hour. Later the cells were stained using 0.5% Oil Red Staining solution for 10 min in 60 ℃. The stained cells were rinsed thrice with 1x PBS and were photographed using light microscopy.
Determination of apoptotic cells by Hoechst method
Hoechst staining 33342 kit was used to differentiate EO induced apoptotic cells from non-apoptotic cells with little alteration. At first, cells were rinsed twice with PBS and fixation was done in 4% paraformaldehyde for 12 min. Later, various concentration of treated and non-treated cells was stained with 11 µg/mL of Hoechst staining solution at 37℃ for 15 min. The cells were rinsed thrice with PBS and photographed under fluorescence microscope using a Leica DMLB fluorescence microscope (Wetzlar, Germany).
Detection of cell death by propidium Iodide method
5104/ well of PC3 cells were grown in 6 well plates and cell death were measured as described previously (Passarinho et al., 2014).
Detection of fragmented nuclei
DNA fragmentation assay was performed using 2105/well of PC3 prostate cancer cells with or without EO treatment. One day after EO treatment, the cells were trypsinzed and spinned down at 1500 rpm for 5 min and supernatant was eliminated. The lysate was thrice rinsed with 1x PBS solution. The genomic DNA was isolated by Exgene cell SV according to manufacturer instructions (GeneAll Biotech, Korea). Before loading DNA onto agarose gel electrophoresis containing ethidium bromide, the concentration of DNA was assured to be equal in all treated and untreated samples. Finally, the gel was examined under ultraviolet light and photographed.
Quantitative Real-Time PCR
PC3 cells were grown in 25 cm2 cell culture flasks treated with or without EO were used for extraction of RNA. cDNA synthesis and real time PCR experiments were performed as stated previously (Perumalsamy et al., 2018).
Western blot assay
PC3 prostate cancer cell line was treated with various concentrations (30, 60, 120 µg/mL) of oregano EO in 0.1mL DMSO for 48h. Western blot analysis was carried out according to the previous published method (Perumalsamy et al., 2017).
Oregano suppressed cell growth in prostate cancer cells (PC3)
The cancer cell growth inhibition was compared with normal cells by treating different concentration of oregano essential oil and MTT assay were performed. For positive control, we have used cisplatin. The results demonstrated that oregano can significantly inhibit prostate cancer cells in a dose dependent manner (Fig 1A-C). The inhibitory concentration (IC50) of oregano treated prostate cancer cells was found to be 13.82µg/mL, Whereas, the cells treated with cisplatin showed (22.08µg/mL) compared to oregano treated prostate cancer cells. Furthermore, the normal cells (MRC-5) treated with oregano showed the least cytotoxic effects (Fig. 1C).
Morphological characteristics affected by oregano treatment
The morphological characteristics of PC3 cells were observed after treatment with varying concentration of oregano EO (Fig. 2). The oregano treatment induced cell death increased gradually from 6.2µg/mL treatment and attained its maximum at 50µg/mL. Furthermore, there is a significant morphological change happened with or without oregano treatment. The cells without treatment showed distinguished morphology by exhibiting 90% confluency while treated cells exhibited dwindle, form-less, significantly diminshed cell size and number which clearly indicates that oregano induced these changes.
Oregano EO altered lipid biosynthesis
The cancer cells proliferate uncontrollably by activating lipid biosynthesis. To determine the lipid concentration in both treated and untreated cells, oil staining was performed. In the control cells, the accumulation of lipids was significantly higher than that of treatment groups (Fig3A1-3) which indicates that oregano EO treatment could inhibit the cancer cell proliferation by altering lipid biosynthesis. The treatment of oregano EO hindered the cancer cell proliferation in the dose dependent manner (Fig 3A2-3).
Furthermore, we studied the key transcript that play essential role in lipid biosynthesis including fatty acid synthase (FASN), HMG-CoA reductase (HMGCR) and regulator of cholesterol and fatty acid metabolism (SREPB1) respectively. We observed significant downregulation of FASN expression after 30µg/mL of treatment (0.71-fold) and the expression further decreased to 0.18-fold when 120 µg/mL of oregano EO was treated to the cells.
The expression level of HMGCR transcript caused drastic decrease in transcript accumulation at 30µg/mL (0.31-fold) and reached its maximum downregulation (0.18-fold) at 120µg/mL oregano EO treatment. Similarly, the expression of SREBP1 caused significant downregulation of transcript level (0.4-fold) at 30µg/mL and recorded lowest of 0.11-fold after 120 µg/mL of treatment.
Detection of apoptosis by Hoechst staining
We carried out hoechst staining to determine the effect of oregano in inducing apoptosis in PC3 cells. The non-treated cells showed intact cell membrane therefore the dye is unable to diffuse into the nuclei and exhibited no fluorescence (Fig.4A1). However, the cells treated with 25 µg/mL and 50µg/mL showed cell damage occurred which is represented by the bright fluorescence in the nuclei. PC3 cells treated with oregano showed apoptosis characteristics changes including shrunken, fragmented nuclei and reduced cell number (Fig. 4A2-3).
Detection of apoptosis by PI staining
Similarly, to identify the cell death and apoptosis of oregano treated cells we performed PI staining. The characteristics of PI staining is to stain the dead cells that undergoes necrosis and apoptosis rather than viable cells. The control cells showed less or no fluorescence when incubated with PI indicating that cells remained a live (Fig. 4B1). On the other hand, the treated cells with oregano showed red fluorescence in the nuclei indicating that the oregano induced damaged to cell membrane and thus left the dye to stain the nuclei (Fig. 4B2-3).
Oregano caused DNA fragmentation in prostate cancer
Fig 5A& B depicts the threshold of DNA damage in the presence or absence of oregano EO treatment. In untreated well, the band of non-fragmented DNA showed intact DNA. Whereas, the treated cells (12.5, 25, and 50µg/mL) gradually started to show DNA degradation and exhibited highest degradation at 50µg/mL (Fig. 5A). The DNA band intensity was determined using an image analyzer with Quantity One software (Fig 5B).
Oregano EO treatment increased p53, BAX level and decreased BCL2 expression
The occurrence of apoptosis through intrinsic pathway could be monitored by studying two key genes such as pro-apoptotic namely BAX and an anti-apoptotic genes namely BCL2 respectively. On the other hand, p53 tumor suppressor protein expression is an essential factor to activate apoptosis process (Han and Parher, 2017). Thus, we determined the transcriptional and translational changes of these proteins to identify the effect of oregano in inducing apoptosis.
The expression of p53 was gradually increased and reached up to 3.08-fold after 120µg/mL post-treatment. Whereas, the transcripts of BCL2 starts declining gradually and showed transcript level of 2.5-fold under 120 µg/mL oregano EO treatment. The expression level of pro-apoptotic protein BAX expression tends to show increased expression pattern of 1.25-fold under 30µg/mL treatment and recorded highest accumulation of 2.5-fold when 120µg/mL of treatment respectively.
Origanum vulgare is widely used as spices in Mediterranean region and exhibited many pharmacological properties including anti-microbial and anti-cancer. Due to its anti-microbial properties, oregano essential oil is used as a food preservative (Savini et al., 2009). So far only few studies reported on oregano essential oil’s anti-cancer effects against colorectal cancer, liver cancer and skin cancer (Elshafie et al., 2015; Mounier et al., 2014; Kuhajda, 2000).
However, these studies didn’t study the molecular signaling pathway involvement in the induction of cancer cell death. For the first time, we attempted to study the anti-proliferative effects of oregano essential oil (EO) toward prostate cancer cells. In this study we identified the oregano EO ability to undergo apoptosis by regulating BAX, BCL2 expression, inhibiting cancer cell proliferation and metastasis in vitro.
Recently, there is a wide range of interest developing toward the development of natural drug for the treatment of cancer. This is because, the drugs used in the treatment of cancer severe cause cytotoxic effects to normal cells. To prevent drug induced side effects, combinational approaches are recommended to the cancer patients which could show better results with very little side effects. In our previous study, we identified the four major active principles in the oregano thymol, ρ-cymene, γ-terpinene and carvacrol, comprised as 65.8, 9.86, 6.73 and 4.96% of the oil, respectively (Perumalsamy et al., 2018).
There is a need of high levels of energy and lipid metabolism for the growth of solid tumors. In the normal cells, energy providing lipids mainly come from circulating lipids whereas in cancer cells, the energy supply is mainly from the de novo synthesis (Horiguchi et al., 2008). The enzymes such as HMGCR, FASN, and SREPB1 genes play an important role in cancer cell progression. For instance, the inhibition of FAS expression decreases tumor growth and induces apoptosis (Kerr et al., 1972; Vogt Sionov and Haupt, 1998).
Similarly, SREPB1c also plays a crucial role in the conversion of cancer cells to normal cells (Guo et al., 2008). It is also speculated that lipogenesis might provides energy supply to cancer cells by activating cell division and cancer cell survival. To study the effect of oregano EO inhibited lipogenesis and induced apoptosis, we studied the transcriptional changes of HMGCR, FASN and SREPB1 expression. From the fig.3 we identified that oregano EO treatment caused the cancer cell damage and decreased the expression of lipogenesis pathway genes.
The ability of p53 to eliminate excess, damaged or infected cells through the process known as apoptosis was previously studied (Bauer and Hefand, 2006). Additionally, the growth inhibitory action of p53 terminates the proliferation of cells containing damaged DNA; contributes to DNA repair and angiogenesis (Fridman and Lowe, 2003). To monitor p53 activity induced apoptosis, we have studied the expression of p53 along with BAX and BCL2 with or without oregano EO treatment. p53 expression was significantly increased under oregano EO treatment in a gradual manner. Whereas BAX (pro-apoptotic protein) and BCL2 (anti-apoptotic protein).
Our data conclude that oregano EO treatment downregulate the BCL2 and upregulates the expression of BAX and induced apoptosis. Apart from the transcript and protein targets, we have also characterized apoptosis by performing DNA fragmentation, Hoechst and PI staining. Oregano EO induced cell membrane damage, apoptotic bodies and reduction in cell number were monitored by Hoechst staining. Similarly, the cells that have undergone apoptosis was also detected by dark red fluorescence by PI staining.
The oregano EO induced fragmentation of DNA in a dose dependent manner. Apoptosis of a cell is described by distinct morphological feature including shrinkage of cell, fragmentation of membrane bound-apoptotic bodies, and rapid phagocytosis by neighboring cells (Kerr et al., 1972). Our morphological data showed that oregano EO caused cell shrinkage, dose dependent reduction of cell number and shape.
In conclusion, our present work concludes that oregano EO can be potent anti-proliferative drug against prostate cancer in vitro. We demonstrated that oregano EO induced apoptosis by suppressing cancer cell growth and proliferation, cell circumference and shape, reduced fatty acid biosynthesis and resulted in the activation of BAX and downregulating BCL2 expression (Fig.7).