Anticancer Properties of Teucrium persicum in PC-3 Prostate Cancer Cells

Plant-derived phytochemicals have been used as therapeutic agents for hundreds of years (Cragg and Newman, 2005). Many such compounds with anti-cancer activities have been identified which some of them (e.g. vinblastine, vincristine, taxol and colchicine), either directly or after chemical modifications, have been used as anticancer drugs (Nirmala et al., 2011). Some of these agents compared to purely synthetic drugs have higher activity and lower toxicity (Fabricant and Fransworth, 2001). Teucrium is a genus of plants belonging to the family of Lamiaceae and includes about 300 species (Moghtader, 2009; Monsef-Esfahani, 2010). The species are mainly distributed in central and southern America, southeast Asia, and Mediterranean region (Kandouz et al., 2010; Miri et al., 2012). The extract isolated from Teucrium species has been used for treatment of diabetes, obesity, inflammation, and hyperlipidemia (Twaij et al., 1987; Tariq et al., 1989; Miri et al., 2012). Also, there are reports on antioxidant, anticancer, and antibacterial activities of some Teucrium species (Kadifkova Panovska et al., 2005; Miri et al., 2012). For example, it has been shown that the organic extracts derived from T polium, T chamaedrys,


Introduction
Plant-derived phytochemicals have been used as therapeutic agents for hundreds of years (Cragg and Newman, 2005).Many such compounds with anti-cancer activities have been identified which some of them (e.g.vinblastine, vincristine, taxol and colchicine), either directly or after chemical modifications, have been used as anticancer drugs (Nirmala et al., 2011).Some of these agents compared to purely synthetic drugs have higher activity and lower toxicity (Fabricant and Fransworth, 2001).
Teucrium is a genus of plants belonging to the family of Lamiaceae and includes about 300 species (Moghtader, 2009;Monsef-Esfahani, 2010).The species are mainly distributed in central and southern America, southeast Asia, and Mediterranean region (Kandouz et al., 2010;Miri et al., 2012).The extract isolated from Teucrium species has been used for treatment of diabetes, obesity, inflammation, and hyperlipidemia (Twaij et al., 1987;Tariq et al., 1989;Miri et al., 2012).Also, there are reports on antioxidant, anticancer, and antibacterial activities of some Teucrium species (Kadifkova Panovska et al., 2005;Miri et al., 2012).For example, it has been shown that the organic extracts derived from T polium, T chamaedrys, and T montanum have remarkable antioxidant activity (Kadifkova Panovska et al., 2005).In addition, it has been reported that the ethanolic extract of T polium has antibacterial effects on both gram positive and gram negative bacteria (Autore et al., 1984).
T persicum is an Iranian endemic plant and grows in Fars province (south of Iran) (Miri et al., 2012).In Iran T persicum has been used for years to relieve headache and abdominal pains (Miri et al., 2012).Several studies have been performed on chemical composition and antioxidant activity of the oils isolated from Teucrium persicum (Monsef-Esfahani et al., 2010;Miri et al., 2012).However, the anti-cancer properties of Teucrium persicum have not yet been investigated.Here, PC-3 prostate cancer cells have been used to study the biological properties of T persicum.PC-3 cells are highly invasive and have a mesenchymal phenotype (Kandouz et al., 2010;Tai et al., 2011).Therefore, this cell line is an appropriate tool to study anti-cancer properties of plant extracts and theirderived compounds (Nyska et al., 2003).The results of this paper show that the methanolic extract of T persicum potently inhibits viability of PC-3 cells.In addition, sublethal concentrations of T persicum extract induce an epithelial-like morphology, leading to an increase in E-Cadherin and β-Catenin membrane localization.
Prostate cancer is a leading cause of cancer death in western countries (Schulz et al., 2003) which is mainly seen as adenocarcinoma of epithelial cell-origin (Mazhar and Waxman, 2002).Epithelial tissue integrity is maintained via E-Cadherin-mediated cell adherens junction (Baum and Georgiou, 2011).Loss of E-Cadherin in epithelial cancers leads to cancer cell invasion and metastasis, a process called EMT (epithelial-mesenchymal transition) (Guarino, 2007).The ability of T persicum extract to restore E-Cadherin to the plasma membrane of PC-3 cells suggests that there are one or more compounds in the extract which can reverse EMT.The findings of this paper suggest that T persicum is a plant with potential anti-cancer properties.

Cell lines and cell culture
PC-3 and SW480 cell lines were purchased from National Cell Bank of Iran (NCBI, Pasteur institute, Tehran, Iran).T47D and NIH-3T3 cells were kindly provided by Dr. Nasrin Motamed (University of Tehran, Tehran-Iran) and Mr. Mehdi Mohaghegh (Tarbiat Modarres University, Tehran-Iran) respectively.All cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Invitrogen), 100 μg/ml streptomycin, and 100 unit/ml penicillin.

Preparation of methanolic extract of T persicum
50g of the powder obtained from dried aerial parts of Teucrium persicum was soaked in 100 ml of methanol (MeOH) and shaken for 24 hours.The supernatant was collected and evaporated by using a rotary evaporator (Heidolph, Germany) at 37°C.The concentrated extract was dried by a freeze dryer (Christ, Germany) and the dried extract was dissolved in MeOH (20 mg/ml) and stored at -20°C until use.

MTT assay
Cell viability was examined by MTT assay.Briefly, 5.0×10 3 PC-3 cells (in 150 µl growth medium) were cultured in each well of a 96 well plate.24 hours later, the cells were treated with different concentrations of T persicum extract for 48 hours.The medium of cells was removed and 100 µl of PBS containing 5 µg/µl MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) was added to each well.3 hours later, the MTT solution was replaced by 100 µl of DMSO and light absorbency was measured at 590 nm by an Elisa reader (BioTek, USA).

Flow cytometry
Flow cytometry was performed by using an Annexin V/PI kit as described by the supplier (BD Bioscience, USA).Briefly, 7.5×10 4 PC-3 cells were plated in each well of 6-well plates and grown to 70% confluency.The cells were then treated with 50, 100 and 150 μg/ml of T persicum extract for 48 hours.13.96 μg/ml of etoposide (Sigma, Barcelona, Spain) was used as a positive control for induction of apoptosis (Salido et al., 1999;Fukuta et al., 2008).The cells were then harvested and centrifuged at 1500 rpm for 5 min at 4°C.The supernatant was discarded and the cell pellet was resuspended in 100 μl of binding buffer (100 mM HEPES, 140 mM NaCl, 2.5 mM CaCl 2 , pH=7.4). 5 μl annexin V (2.5 μg/ml) and 1 μl propidium iodide (10 μg/ml) was added to each cell suspension followed by incubation at room temperature for 20 minutes.Finally the cells were analyzed by a flow cytometer (Partec, Germany) and the results were quantified by Flow Jo software.

DNA laddering
7.5×10 4 PC-3 cells were plated in each well of 6-well plates and grown to 60-70% confluency.The cells were then treated with 50, 100, 150 and 200 μg/ml of T persicum extract for 48 hours.The cells treated with 13.96 μg/ml of etoposide were used as a positive control (Salido et al., 1999;Fukuta et al., 2008).Genomic DNA was extracted by using a DNA extraction kit as described by the supplier (RCPN, Tabriz, Iran).DNA concentration was measured and then equal amount of DNA from each sample was electrophoresed in a 10% polyacrylamide gel.The gel was fixed twice (3 min, each) in a solution containing 10% v/v EtOH and 0.5% v/v glacial acetic acid (in water).The gel was then washed with water and stained with 0.1% w/v silver nitrate (in water) for 15 minutes.The gel was finally incubated in developing solution (1.5% w/v NaOH and 0.01% v/v formaldehyde) with gentle shaking until the bands appeared.

Light microscopy
4.0×10 4 PC-3 cells grown on 10% poly L lysine-coated coverslips were treated with 10 or 25 μg/ml of T persicum extract for 48 hours (the medium containing the extract was renewed after 24 hours).The cells were then rinsed with PBS (phosphate buffered saline) and fixed with a cold acetone-methanol solution (50:50 v/v) for 20 min at -20°C.The cells were then rinsed twice with PBS (5 min, each) and each coverslip was pressed down on top of a small drop of mounting medium placed on a clean slide and visualized with a light microscope (Zeiss, Germany).

Indirect immunofluorescence
PC-3 cells grown on coverslips were treated with the indicated concentrations of T persicum extract for 48 hours (the medium containing the extract was renewed after 24 hours).The cells were then rinsed with PBS and fixed with a cold acetone-methanol solution (50:50 v/v) for 20 min at -20°C.The cells were rinsed with PBS twice (5 min, each) and permeabilized in 0.2% Triton X-100 (in PBS) for 4 minutes.The cells were rinsed again with PBS, blocked with 3% BSA (in PBS), and were incubated with primary antibodies against E-Cadherin (DAKO, Denmark) or β-Catenin for 1 hour.The cells were washed three times with PBS, and then incubated with appropriate FITClabeled secondary antibodies for 1 hour.The coverslips were finally washed with PBS three times (10 min, each) and then each one of them was pressed down on top of a small drop of mounting medium placed on a clean slide.The edges of coverslips were sealed with nail polish and the slides were visualized with a fluorescence microscope (Zeiss, Germany).Cell scratch assay 1.0×10 5 cells were plated in each well of a 6-well plate and allowed to grow to confluence.Then a scratch was generated in the monolayer with a sterile plastic yellow tip.The cells were washed with PBS three times to remove any damaged or floating cells and then were treated with the indicated concentrations of T persicum extract for 48 hours.The plates were photographed with an ordinary digital camera and the wound surface area was measured by ImageJ software.

Gelatin Zymography assay
7.5×10 4 PC-3 cells were plated in each well of 6-well plates and grown to 60-70% confluency.The cells were then treated with the indicated concentrations of T persicum extract in a serum free medium for 48 hours.The medium from each well was collected and freeze dried.The dried medium was dissolved in 100 μl H 2 O and protein concentration was measured (Sun et al., 2012).Equal amounts of protein from each sample were then electrophoresed in a 10% SDS-PAGE containing 0.1% (w/v) gelatin.The gel was first incubated in 2.5% Triton X-100 (in H 2 O) at room temperature for 1 hour and then in activating buffer (50 mM Tris pH=7.4,150 mM NaCl, 5 mM CaCl 2 , 1 μM ZnCl 2 ) for 18 hours at 37°C.The gel was then stained with 0.5% commassie blue for 1 hour and destained with a solution containing 10% v/v glacial acetic acid and 30% v/v EtOH (in water) until the bands appeared.The gel was scanned and quantified by Image J software.

T persicum extract reduces viability of cancer cells
To find out whether the extract obtained from T persicum had any effect on cell viability, PC-3 prostate cancer cells were exposed to different concentrations of the extract for 48 hours and then the viability of cells was measured by MTT assay.As shown in Figure 1, although treatment of cells with 10 and 25 μg/ml of T persicum extract resulted in a slight increase in the number of viable cells (up to 15%), 50 μg/ml or higher concentrations of the extract significantly reduced cell viability (Figure 1) in a concentration dependent manner.As shown in this Figure, the extract at concentration of 200 μg/ml led to more than 60% decrease in the number of viable cells.The IC 50 value of the extract for PC-3 cells was calculated to be 142 μg/ ml.Staining of PC-3 cells with propidium iodide followed by flow cytometry analysis, further demonstrated that T persicum extract induces cell death (not shown).
Interestingly, the extract of T persicum also inhibited the viability of SW480 colon and T47D breast cancer cells, suggesting that T persicum probably inhibits viability of many cancer cells (Figure 1).The IC 50 values of T persicum extract for SW480 and T47D cells were estimated to be 79 and 50 respectively.These results indicate that the extract obtained from T persicum carries one or more compounds which have a strong negative effect on viability of cancer cells.T persicum extract also reduced the viability of mouse fibroblast NIH-3T3 cells with an IC 50 value of 143 μg/ml (Figure 1).Unfortunately, we did not have access to a normal human tissue cell line and therefore NIH-3T3 was used as a replacement.
We then used flow cytometry analysis to find out whether the reduction of PC-3 cell viability by T persicum extract was due to induction of apoptosis.Since it has been shown that etoposide induces apoptosis in PC-3 cells (Salido et al., 1999;Fukuta et al., 2008), we used this drug  T persicum inhibits invasion of PC-3 cells First, we used wound healing assay to find out whether T persicum extract had any effect on invasion of PC-3 cells.The assay was performed as described in the Method section.As shown in Figure 3A, compared to that of nontreated cells (100%), the wound closure values for the cell sheets (monolayers) treated with 10 and 25 μg/ml of T persicum extract were approximately 50% (Figure 3A).As expected, the wound repair values of the cell sheets treated with higher concentrations of the extract were much less compared to that of non-treated cells (20% and 7.5% for the cell treated with 50 and 75 μg/ml of T persicum extract respectively) (Figure 3A).
The wound closure of a cell monolayer is dependent on both cell proliferation and cell invasion (Zahm et al., 1997).Since we observed a slight increase in viability of cells treated with 10 and 25 μg/ml of T persicum extract (about 15%) (Figure 1), we conclude that the reduction observed in wound healing of the cell monolayers treated with these two concentrations of T persicum extract was probably due to a decrease in cell invasion.
We also tested the effect of T persicum extract on PC-3 cell invasion by using gelatin zymography assay.Matrix metalloproteinases are expressed by many invasive cancer cells and are the key proteins involved in cell invasion and metastasis (Stetler-Stevenson, 2001;Deryugina and Quigley, 2006).PC-3 cells are among the cells that express high levels of matrix metalloproteinases including MMP-2 and MMP-9 (Vijayababu et al., 2006) and therefore, gelatin zymography is an appropriate assay to measure the invasion ability of these cells (see the Methods).PC-3 cells were treated with several concentrations of T persicum extract for 48 hours and then the media of these cells were examined for the presence of matrix metalloproteinases as described in the Methods.As shown in Figure 3B, treatment of cells with T persicum extract significantly as a positive control.We chose three lethal concentrations of the extract including 50, 100, and 150 μg/ml (Figure 1).The extract concentrations greater than 150 μg/ml were excluded because of a dramatic reduction in the number of treated cells.As shown in Figure 2A, while treatment of PC-3 cells with 50 μg/ml of T persicum extract induced a mixture of necrosis and apoptosis, the cells exposed to 100 or 150 μg/ml of the extract underwent late apoptosis (Figure 2A).
We also used DNA fragmentation experiments to test apoptosis.PC-3 cells were treated with 50, 100, 150 and 200 μg/ml of T persicum extract and then genomic DNA was extracted from the cells (see the Methods).Equal amount of DNA from each sample was separated in a 10% polyacrylamide gel followed by silver staining.Despite modification of the assay, we never observed a typical DNA ladder even when etoposide, a known inducer of PC-3 cell apoptosis, was used for treatment of cells (Figure 2B).DNA degradation always appeared as a smear in the gel and the smear intensity increased with concentration of the extract, in expense of the main genomic DNA band on top of the gel (the arrow in Figure 2B).Collectively, the results of flow cytometery and DNA degradation assays suggest that T persicum induces apoptosis in PC-3 cells.lowered (50-60%) the protein levels of MMP-2 and MMP-9 in the culture medium.These results indicate that production and (or) secretion of metalloproteinases decrease significantly in the presence of T persicum extract, supporting the idea that T persicum extract has a negative effect on invasion of PC-3 cells.

Sublethal concentrations of T persicum extract induce an epithelial-like phenotype in PC-3 cells
It was interesting to know whether T persicum extract had any biological activities at sublethal concentrations (10 and 25 μg/ml).First, we simply used a light microscope to compare the morphology of cells treated with either of these two concentrations of T persicum extract with that of non-treated cells.As shown in Figure 4, non-treated PC-3 cells have a mesenchymal phenotype and they look round and separated.Interestingly, treatment of cells with 10 or 25 μg/ml of the extract partially led to a mesenchymalepithelial transition (MET).The population of treated cells had fewer round cells and we could find patches of closely attached epithelial-like cells (Figure 4).This preliminary observation raised the possibility that T persicum extract has a negative effect on EMT (Epithelial-Mesenchymal Transition).
EMT is a feature of many advanced human carcinomas and also, is one of the causes of cancer cell invasion and metastasis (Behrens, 1993;Thiery, 2002;Guarino, 2007).E-Cadherin is a critical protein involved in cell adherens junction and maintenance of epithelial tissue integrity (Leckband and Prakasam, 2006;Van Roy and Berx, 2008).Lack of E-Cadherin expression has been reported in various invasive cancers (Stemmler, 2008).
Although it has been reported that PC-3 cells are negative for E-Cadherin (Nakamura et al., 2003), the results of our immunofluorescence microscopy and western blotting experiments showed that in fact, these cells express E-Cadherin, however, the protein does not appear to be localized at the cell membranes and it has a diffuse staining pattern inside the cells (Figure 5B and not shown).A similar observation has been reported by another research group (Kandouz et al., 2010).Therefore, we tested whether the extract of T persicum had any effect on E-Cadherin cellular localization.Treatment of PC-3 cells with 10 or 25 μg/ml of T persicum extract led to staining of E-Cadherin protein at the cell membrane in a subpopulation of cells, appearing as patches of epithelial cells (Figure 5B).This observation is consistent with the results obtained from our cell invasion assays (Figures 3A and 3B), further suggesting that T persicum inhibits PC-3 cell invasion.It is important to emphasize that only a subpopulation of treated cells had epithelial-like phenotype and the rest of the cells appeared to be remained intact.This might be due to the low degree of homogeneousity among cells in the monolayer.
β-Catenin is a major binding partner of E-Cadherin in cell-cell adherens junctions (Cavallaro and Christofori, 2004) and therefore, we hypothesized that the pattern of cell membrane localization of β-Catenin in the presence and absence of T persicum extract to be similar to that of E-Cadherin.The results of immunofluorescence microscopy supported this hypothesis and we found β-Catenin at cell membrane of the patches of epithelial cells formed in the presence of T persicum extract (Figure 6).In non-treated PC-3 cells, β-Catenin protein was mainly cytoplasmic and had a diffuse staining pattern (Figure 6).

Discussion
Human cancers, especially carcinomas are complex and multi-step diseases and therefore their treatment has always been a big challenge.Some plants contain powerful anti-cancer compounds which many of them have been purified and used as drugs for cancer prevention or treatment (Cragg and Newman, 2005).In this work, we studied the anticancer properties of Teucrium persicum, an endemic plant of Iran (Monsef-Esfahani et al., 2010).For the study, we chose PC-3 prostate cancer cells which are very invasive, poorly differentiated and have a mesenchymal phenotype (Kandouz et al., 2010;Tai et al. 2011).Therefore, this cell line is an excellent choice to study several anti-cancer features of a candidate compound.
The results of MTT experiments were very interesting to us.The methanolic extract of Teucrium persicum appeared to be very potent in decreasing the viability of PC-3 and a couple of other cancer cell lines including SW480 (colon) and T47D (breast) (Figure 1).When we compared the cytotoxic activity of Teucrium persicum with that of some known anti-cancer drugs, we found that the IC 50 value of Teucrium persicum extract for PC-3 cells was much lower.The IC 50 values reported for doxorubicin, imatinib and etoposide against PC-3 cells are 500, 5700 and 1730 μg/ml respectively (Kubler et al., 2005;Dudley et al., 2008;Tsakalozou et al., 2012) which are much higher than that of Teucrium persicum extract (142μg/ml).
There are at least two possibilities for this observation.Either there is a compound in Teucrium persicum extract which is very toxic to PC-3 cells, or there are two or more compounds in the extract which work synergistically to kill PC-3 cells.Laboratory results have supported both possibilities.For example, it has been reported that Teucrium species are rich in phenolic compounds like flavonoids (Cakir et al., 2006;Ljubuncic et al., 2006;Stankovic et al., 2011).Flavonoids have several anti-cancer activities including carcinogen inactivation, induction of apoptosis, antioxidation, and reversal of the drug resistance (Chahar et al., 2011).It has also been reported that some phytochemicals work synergistically to block cancer cell growth.For example it has been shown that quercetin (a dietary bioflavonoid) synergizes with epigallocathechin gallate (EGCG) to block prostate cancer stem cell invasion, migration and epithelial-mesenchymal transition (Tang et al., 2010).The synergistic effect is an important issue because it can minimize the toxicity and side effects of a single compound.
The results of flow cytometry and DNA laddering experiments showed that the reduction of viability of PC-3 cells by T persicum extract was due to induction of apoptosis.Reduction of cell viability and (or) induction of apoptosis started at concentration of 50 μg/ml of T persicum extract and continued in a dose dependent manner (Figure 1).Interestingly at two lower concentrations of the extract (10 and 25 μg/ml), not only cell viability was not reduced but also we observed a slight increase (up to 15%) in the number of viable cells.Although this increase was not statistically significant, it was reproducibly observed.
Although, sublethal concentrations of T persicum extract (10 and 25 μg/ml) did not reduce cell viability, treatment of PC-3 cells with these concentrations of the extract resulted in a significant decrease (about 50%) in wound healing of the cell sheets.This observation raised the possibility that T persicum extract reduces PC-3 cell invasion.This possibility was very well supported by the results of Gelatin zymography experiments which showed that treatment of PC-3 cells with T persicum extract significantly reduces the production and (or) secretion of at least two matrix metalloproteinases (MMPs), MMP-2 and MMP-9 (Figure 3).These enzymes are gelatinases which belong to matrix metalloproteinase family and are involved in ECM proteolytic hydrolysis, cell migration and cancer cell invasion (Roomi et al., 2009).It has been shown that blockade of MMP-2 and MMP-9 expression inhibits invasion of metastatic prostate cancer cells (Deryugina and Quigley, 2006).Collectively, our results show that T persicum extract has anti-invasion activity against PC-3 cancer cells.
The anti-cell invasion activity of T persicum extract was also supported by the results of immunofluorescence microscopy experiments with E-Cadherin and β-Catenin antibodies.T persicum extract induced an epithelial-like phenotype in PC-3 cells together with cell membrane localization of E-Cadherin and β-Catenin proteins (Figure 5 and Figure 6).PC-3 prostate cancer cells have a mesenchymal phenotype and are very invasive and produce tumors in laboratory animals (Zhou et al., 2009;Kandouz et al., 2010;Zhang and Waxman, 2010;Tai et al., 2011).Our results showed that in these cells, both E-Cadherin and β-Catenin proteins were cytoplasmic and cells having either protein at the cell membrane were extremely rare (Figures 5 and 6).Treatment of PC-3 cells with 10 or 25 μg/ml of T persicum extract (sublethal concentrations) led to appearance of several patches of closely attached epithelial-like cells having E-Cadherin and β-Catenin at the cell membrane, an observation similar to a very important biological process called MET (mesenchymal-epithelial transition) (Thiery, 2002;Guarino, 2007).Epithelial-mesenchymal transition (EMT) is a feature of many advanced human carcinomas and results in cancer cell invasion and metastasis (Thiery, 2002;Guarino, 2007).Loss of epithelial tissue integrity due to downregulation of E-Cadherin is the main feature of EMT (Thiery, 2002;Guarino, 2007;Baum and Georgiou, 2011).Therefore, any therapeutic strategy to reverse EMT can be beneficial to cancer patients.The ability of T persicum extract to enhance membrane localization of E-Cadherin and β-Catenin represents a valuable anti-cancer activity of this plant.It is worth mentioning that T persicum extract led to membrane localization of E-Cadherin and β-Catenin only in a subpopulation of cells (less than 20%) and the rest of the cells had a phenotype similar to that of non-treated cells.More experiments should be performed to find the reason for this difference in cell response.
β-Catenin is a dual function protein that in addition to its role as a component of cell adherens junction, intracellular and intranuclear localization of this protein are oncogenic and may help tumor initiation and progression (Cavallaro and Christofori, 2004;Salmanian et al., 2010).The oncogenic role of β-Catenin, which is believed to be due to upregulation of the canonical Wnt signaling pathway, has been very well studied in colon cancer and some other human malignancies (Polakis, 2000;Salmanian et al., 2010).
Collectively the results of this paper present a new species of Teucrium genus with valuable anti-cancer activities.Definitely more experiments are needed to find out the molecular mechanisms of the biological properties of T persicum.In addition chemical analysis of T persicum extract is necessary to search for the active component(s).

Figure 1 .
Figure 1.T persicum Extract Decreases Viability of Cancer Cells.The cells indicated in the figure have been treated with increasing concentrations of T persicum extract for 48 hours and then the viability of cells has been measured by MTT experiments.The data shown are mean±SD of three separate experiments in which each treatment was repeated in 10 wells (*p<0.05;**p<0.01;***p<0.001vs control)

Figure 2 .
Figure 2. T persicum Extract Induces Apoptosis in PC-3 Cells.A)The cells were treated with 50, 100 and 150 μg/ml of T persicum extract for 48 hours and then used for flow cytometry analysis as described in the method section.Etoposide (13.96 μg/ml) was used as a positive control for apoptosis induction.In each chart, Q1, Q2, Q3, and Q4 respectively represent necrotic cells which have stained with propidium iodide, late apoptotic cells stained with PI and Annexin V, live cells which have not absorbed PI or Annexin V, and early apoptotic cells which have only absorbed Annexin V. B) T persicum extract induces DNA degradation.PC-3 cells were treated with 50, 100, 150 and 200 μg/ml of T persicum extract for 48 hours.Etoposide (13.96 μg/ ml) was used as a positive control for apoptosis induction.The genomic DNA isolated from each sample was electrophoresed on a 10% polyacrylamide gel followed by silver nitrate staining.The thick arrow on top of the gel represents the main genomic DNA band.The control lane represents non-treated cells.Eto; Etoposide

Figure 5 .
Figure 5. Immunofluorescence Staining of PC-3 Cells with the E-Cadherin Antibody (×400).PC-3 cells seeded on poly L-lysine coated coverslips were treated with T persicum extract (10 and 25 μg/ml) for 48 hours and then used for immunofluorescence microscopy experiments (see the methods).An enlarged section of the right panels is shown on the left.The non-treated cells in the bottom panel have only been stained with the FITC-labelled secondary antibody

Figure 4 .
Figure 4. Morphology of PC-3 Cells Treated with T persicum Extract.The cells were remained untreated or treated with 10 and 25 μg/ml of T persicum extract for 48 hours.The cells were then observed under a light microscope (×400) and photographed with an ordinary digital camera