Thujone-Rich Fraction of Thuja occidentalis Demonstrates Major Anti-Cancer Potentials: Evidences from In Vitro Studies on A375 Cells

Crude ethanolic extract of Thuja occidentalis (Fam: Cupressaceae) is used as homeopathic mother tincture (TOΦ) to treat various ailments, particularly moles and tumors, and also used in various other systems of traditional medicine. Anti-proliferative and apoptosis-inducing properties of TOΦ and the thujone-rich fraction (TRF) separated from it have been evaluated for their possible anti-cancer potentials in the malignant melanoma cell line A375. On initial trial by S-diphenyltetrazolium bromide assay, both TOΦ and TRF showed maximum cytotoxic effect on A375 cell line while the other three principal fractions separated by chromatography had negligible or no such effect, because of which only TRF was further characterized and subjected to certain other assays for determining its precise anti-proliferative and apoptotic potentials. TRF was reported to have a molecular formula of C10H16O with a molecular weight of 152. Exposure of TRF of Thuja occidentalis to A375 cells in vitro showed more cytotoxic, anti-proliferative and apoptotic effects as compared with TOΦ, but had minimal growth inhibitory responses when exposed to normal cells (peripheral blood mononuclear cell). Furthermore, both TOΦ and TRF also caused a significant decrease in cell viability, induced inter-nucleosomal DNA fragmentation, mitochondrial transmembrane potential collapse, increase in ROS generation, and release of cytochrome c and caspase-3 activation, all of which are closely related to the induction of apoptosis in A375 cells. Thus, TRF showed and matched all the anti-cancer responses of TOΦ and could be the main bio-active fraction. The use of TOΦ in traditional medicines against tumors has, therefore, a scientific basis.


Introduction
Thuja occidentalis (Fam: Cupressaceae), commonly known as Arbor vitae or white cedar, is an ornamental tree grown in Europe [1]. It is used as a medicinal plant in various forms of traditional medicines like folk medicine, homeopathy, and so forth. for treatment of bronchial catarrh, enuresis, cystitis, psoriasis, uterine carcinomas, amenorrhea and rheumatism [2][3][4][5]. In homeopathy, the crude ethanolic extract of T. occidentalis is used as mother tincture (TOΦ). Thuja is also occasionally used for treating diseases of skin, blood, gastrointestinal tract, kidney, brain, warty excrescences, spongy tumors, and so forth. and claimed to have pronounced remedial effects. The extract has been reported to enhance the antibody response to sheep blood cells [6]. Protective effect of T. occidentalis has also been reported against radiation-induced toxicity in mice [7]. Ameliorative effect of T. occidentalis has also been suggested in preventing congestive heart disease [8]. Therefore, it gained attention of pharmacologists to study the major constituents of the dried herbal substances of T. occidentalis [9][10][11]. Some 31 compounds were identified in T. occidentalis "globosa" and 27 in T. occidentalis "gracilis" [9]. Although some minor differences exist in the actual ingredients of several varieties of Thuja, generally the major components found in extracts of all varieties of T. occidentalis comprise essential oil (1.4-4%) as the principal constituent. Other constituents include coumarins (p-coumaric acid, Umbelliferone) flavonoids (Catechine, Gallocatechine, etc.), tannic acid, polysaccharides and proteins [12]. The essential oil of the fresh leaves (related to the monoterpene fraction) contains 65% thujone, 8% isothujone and fenchone each, 2 Evidence-Based Complementary and Alternative Medicine 5% sabines and 2% a-pinen as the main monoterpenes. Among the constituents of the dried herbal substances, it is not known precisely as to which fraction(s) of the extract is(are) the most bioactive agent(s), particularly in respect of its (their) anti-tumor or anti-cancer activities. In homeopathic literature [13], TOΦ has been reported to be effective against various forms of skin diseases, particularly in the treatment of moles and papillomas. Homeopathic TOΦ has been reported earlier to have cytotoxic effect in Dalton's lymphoma ascites (DLA), Ehrlich ascites carcinoma (EAC), and lung carcinoma L929 [14]. Dubey and Batra also reported hepato-protective activities [15] and antioxidant activity [16] of T. occidentalis in CCL 4 -treated liver damage in rats. However, to the best of our knowledge, anticancer potentials of TOΦ or any of its major fractions had not been tested earlier in skin cancer cell line A375 by utilizing some widely acceptable parameters of study, like S-diphenyltetrazolium bromide (MTT) assay, Trypan blue exclusion assay and lactate dehydrogenase (LDH) activitybased cytotoxicity assays, fluorescence microscopy, comet assay, DNA fragmentation assay, analysis of changes in morphological features of cells and in mitochondrial membrane potential, ROS production, flow cytometric analysis, immunofluorescence study and western blot analysis.
Thus, the hypotheses to be tested were: (i) whether TOΦ and all chromatographically separated fractions had potential anti-cancer effects in A375 cells; (ii) if any fraction could be demarcated as the most biologically active one; (iii) if, it is possible, to chemically characterize this fraction; (iv) whether the cytotoxic, anti-proliferative and apoptotic effects of the fraction, vis-à-vis TOΦ, could be compared with focus on its possible mechanism of action; and (v) if it is possible to assess the overall anti-cancer potentials of TOΦ in relation to its preferential cytotoxic effect over normal cells, by analyzing results of different relevant assays including the possible signaling pathways. A375 cells were further treated with various concentrations of thujone-rich fraction (TRF) followed by 24 h of incubation. The control received no drug. After the incubation 10 μL of MTT solution (5 mg mL -1 ) was added to each well. The intracellular formazan crystals formed were solubilized with acidic isopropanol and the absorbance of the solution was measured at 595 nm [19] using an ELISA reader (Multiscan EX, Thermo Electron Corporation, USA). The relative percentage of viability was calculated as follows:

Methods
Relative percentage viability = OD of drug-treated sample OD of control sample × 100. (1)

Trypan Blue Exclusion Assay (Cytotoxicity Assay).
For cytotoxicity assessment, trypan blue exclusion assay was performed [20]. After treatment with TRF (0-200 μg mL -1 ) for 24 h, A375 cells were stained with 0.4% trypan blue and 100 cells were counted at various fields in haemocytometer for each experiment. The level of cytotoxicity was calculated as per the following formula: Relative percentage of cytotoxicity = Total cells − Viable cells Total cells × 100.
Evidence-Based Complementary and Alternative Medicine 3 2.7. LDH Activity-Based Cytotoxicity Assay. LDH activity was assessed using a standardized kinetic determination kit (Enzopak, Recon, India). LDH activity was measured in both floating dead cells and viable adherent cells. The floating cells were collected from culture medium by centrifugation (1000 g) at 4 • C for 5 min, and the LDH content from the pellets was used as an index of apoptotic cell death (LDHp) [21]. The LDH released in the culture supernatant [designated as extracellular LDH (LDHe)] was used as an index of necrotic death, and the LDH present in the adherent viable cells was designated as intracellular LDH (LDHi). The percentages of apoptotic and necrotic cell deaths were calculated as follows:

Observation of Morphological
Changes. Cells plated in six-well culture plates (2 × 10 5 cells per well) in DMEM supplemented with 10% FBS for 24 h were treated with or without TRF at a specified concentration. After 24 h, the cells were observed under inverted phase contrast microscope (Axiscope plus 2, Zeiss, Germany) and photographs were taken.

Fluorescence Microscopy.
To determine the live apoptotic cells, cells treated for 24 h were stained separately with 10 μg mL -1 of DAPI and with acridine orange:ethidium bromide (AO/EB) [22]. After 24 h, the control and treated cells were stained with acridine orange (50 μg mL -1 ) and ethidium bromide (50 μg mL -1 ) mixture. Then the cells were analyzed under fluorescence microscope (Axiscope plus 2, Zeiss, Germany) and representative photographs were taken for further quantitative analysis.

Analysis of Changes in Mitochondrial Membrane
Potential. The changes in mitochondrial membrane potential of the treated cells were determined using a fluorescent probe, Rhodamine 123 [23]. Briefly, 5 μL of Rhodamine 123 (1 mmol L -1 ) were added to the cells (2 × 10 5 ). Then the cells were incubated for 15 min. Next, the cells were washed with PBS and observed under a fluorescence microscope (Axioscope plus 2, Zeiss) and photographs were taken. The mitochondrial membrane potential changes were also determined by flow cytometer (FACS caliber, BD Bioscience) [24]. Briefly, cells after treatment were washed with ice-cold PBS before incubation with Rhodamine 123 (5 mmol L -1 ) in darkness for 15 min at room temperature. The fluorescence emissions were analysed with a flow cytometer.
2.11. Analysis of ROS Production. Cells after treatment were suspended in growth medium and incubated with DCFDA (2 -7 -dichloro-fluorescin diacetate) (5 μM) for 15 min. Cells were washed with PBS and analyzed under fluorescence microscope and photographs were taken.
To evaluate the intra-cellular ROS level, the cells were incubated with 10 μM DCFDA for 30 min at room temperature. After washing the cells twice with PBS, the intensity of DCFDA fluorescence was determined by flow cytometer with an excitation wavelength of 480 nm and an emission wavelength of 530 nm.
2.12. Flow Cytometric Analysis. A375 cells were harvested and washed once in cold phosphate-buffer saline (PBS). Cell pellets were fixed in 3% paraformaldehyde and suspended in 1 mL of PI solution containing 50 mg mL -1 PI, 0.1% (w/v) sodium citrate and 0.1% (v/v) Triton X-100. Cells were incubated at 4 • C in the dark for 15 min and analyzed by a flow cytometer [25].
In order to detect the translocation of phosphatidylserine from inner cell membrane to outer cell membrane (a characteristic feature of cells undergoing apoptosis), cells were subjected to flow cytometric analysis after staining with Annexin V-FITC and PI [23].

Comet Assay.
After treatment, the cells were trypsinized and washed in ice-cold PBS. The cell suspension was mixed with an equal amount of 1% low melting agarose kept at 37 • C. Immediately after mixing, 100 μL of the suspension was pipetted on to microscope slide pre-coated with normal melting agarose, then covered with cover slip and placed on a glass tray on ice. Then the slides were immersed in cold lysis solution (2.5 M Nacl, 100 mM EDTA, 10 mM Tris, PH-10, with freshly added 1% Triton X-100 and 2% DMSO) followed by incubation at 4 • C for at least 1 h. The electrophoresis in weak alkali (0.03 M NaOH, 1 mM EDTA, PH-12) at 1 V cm -1 and 30 mA for 15 min was preceded by a 20 min immersion of the slides in electrophoresis buffer to promote chromatin unwinding. After electrophoresis the slides were neutralized in 0.05 M Tris buffer. Then DNA was stained with ethidium bromide (50 μg mL -1 ) for 10 min, washed in distilled water and examined in a fluorescence microscope (Lyca, USA) [26].
2.14. DNA Fragmentation Assay. The cells were grown to 70% confluence and treated with various concentrations of TRF for 24 h. Following this treatment, the cells were washed twice with phosphate-buffered saline [10 mM Tris (pH 7.5), 150 mM NaCl, 5 mM MgCl 2 and 0.5% Triton X-100], left on ice for 15 min, and pelleted by centrifugation (1000 rpm) at 4 • C. The pellet was incubated with DNA lysis buffer [10 mM Tris (pH 7.5), 400 mM NaCl, 1 mM EDTA and 1% Triton X-100] for 30 min on ice and then centrifuged at 15 000 g at 4 • C. The supernatant that was obtained was incubated overnight with RNase (0.2 mg mL -1 ) at room temperature and then with proteinase K (0.1 mg mL -1 ) for 2 h at 37 • C. DNA was extracted using a phenol/chloroform mixture (1 : 1) and precipitated with 95% ethanol. The DNA precipitate was centrifuged at 15 000 g and 4 • C for 15 min, and the pellet was air-dried and dissolved in 20 μL of Tris-EDTA buffer [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA]. The total amount of DNA was resolved over a 1.5% agarose gel, containing 0.3 mg mL -1  ethidium bromide in 1× TAE buffer. The bands were visualized under an UV transilluminator followed by digital photography.
2.15. Immunofluorescence Study. Cells were plated in sixwell culture plates and allowed to adhere for 24 h before treatment. At the end of the treatments, cells were washed with PBS and fixed in 3% paraformaldehyde for 1 h. The cells were permeabilized with 0.2% CHAPS in PBS for 2 min and were blocked in 2% BSA with 0.2% Tween-20 for 30 min [27]. Then, the cells were incubated for overnight at 4 • C with a 1 : 400 dilution of specific primary antibodies (anti-Bcl-2, anti-Bax, anti-cytochrome c and anti-caspase-3) and further incubated for 1 h with a 1 : 2000 dilution of goat anti-mouse IgG-FITC as a secondary antibody. Immunofluorescence was photographed.

Preparation of Cell Lysates.
Lysates were prepared to examine the expression of Bax, Bcl2, cytochrome c, caspase-3. A375 cells were washed twice with PBS and then lysed in ice-cold lysis buffer containing 50 mM HEPES (pH 7.4), 1% Triton-X 100, 1 mM EDTA, 2 mM sodium orthovanadate, 100 mM Sodium fluoride, 10 μg mL -1 leupeptin, and 1 mM PMSF, 10 μg mL -1 aprotinin. After 60 min of incubation on ice, the cells were swelled and then centrifuged at 12 000 g Data are means ± SD from three different measurements and were analyzed using one-way ANOVA. P < .05 compared to the control group (zero concentration). Con = control. for 20 min. The protein content of the supernatant was determined by Bradford assay with bovine serum albumin as standard and then was stored at −20 • C until analysis [28].

Western Blot Analysis.
Both adherent and floating cells were collected, and then western blot analysis was carried out. Equal amounts of lysate protein were run on 12.5% SDS-PAGE and electrophoretically transferred to PVDF membrane [29]. After blocking, the blots were incubated with specific primary antibodies (anti-Bcl-2, anti-Bax, anticytochrome c and anti-caspase-3) overnight at 4 • C and further incubated for 2 h with a 1 : 2000 dilution of goat antimouse IgG-ALP as a secondary antibody. Bound antibodies were developed by BCIP-NBT and quantification of proteins was done by densitometry using image analyzer (Gel Doc System; Ultra Lum, USA). The same membranes were also immunoblotted against β-actin (house keeping gene) for data normalization. measured by LDH activity-based assay. The ratio of apoptotic cells increased from 23.59% at 50 μg mL -1 to 68.83% at 200 μg mL -1 for TRF, but that of necrotic cells was still negligible in the presence of 200 μg mL −1 TRF. Data are each the mean ± SD (bars) from three independent experiments. Data are means ± SD from three different measurements and were analyzed using one-way ANOVA. P < .05 compared to the control group (zero concentration). Con = control. * = P < .05, * * = P < .01.

Statistical Analysis. Statistical analysis was performed by the Student's t-test
for the significance of difference between the data of control and drug-treated cells. Data were obtained from representative experiments with triplicate and were expressed as mean ± standard error (SE). Additionally, ANOVA was performed to identify if the differences were also significant among the means of different groups. The P-value < .05 was considered to be significant (Tables 1-4). Differences in cell viability between the Fractions 4 and 1, Fractions 2 or 3 were found to be statistically significant (P < .05) (Figure 1).

Viability of A375 Cells.
MTT assay was conducted on the A375 cell treated with different concentrations of TOΦ. The results revealed 50% of cell death at 226.18 μg mL -1 of TOΦ (Figure 2(a)). The cell cytotoxicity on normal peripheral blood mononuclear cell (PBMC) was also measured in the mentioned dose of TRF that showed minimal cytotoxic effects (∼14%) even at the highest dose (Figure 2(b)). Table 2 shows the results of the statistical analysis done by one way ANOVA.
MTT assay showed that TRF had significantly stronger inhibitory effects (P < .05) on proliferation of A375 cells. Treatment of A375 cells with 100 and 200 μg mL -1 for 24 h resulted in ∼42 and 59% cell deaths, respectively   ( Figure 3(a)). The cell cytotoxicity in response to treatment in three concentrations has been shown in Figure 3(b). These results demonstrate that one of the causes of A375 cell death induced by TRF could be attributed to apoptosis. The results were statistically significant at various levels (P < .05 through P < .01). The statistical data of ANOVA have been shown in Table 3.
To further characterize TRF-induced A375 cell death, the ratios of LDH release from viable cells, floating dead cells, and the culture medium were compared (Figure 3(c)). There was a significant (P < .05 to P < .01) increase in the ratio of apoptotic cells from 23.59% at 50 μg mL -1 to 68.83% at 200 μg mL -1 for TRF, but that of the necrotic cells was still negligible in the presence of 200 μg mL -1 TRF. Table 4 summarizes the statistical findings of ANOVA performed to identify if the differences were significant among the means of different groups.

Morphological Changes.
The morphological changes of A375 cells treated with TRF were also observed. Results revealed that morphological changes (specifically for chromatin condensation and cell shrinkage) were typical of apoptosis. These changes were apparent at 24 h, and became common in ∼50% or more of the treated A375 cells after 24 h. Exposure to 100 μg mL -1 for 24 h caused the majority of A375 cells to shrink, round up and detach from the culture dish. The effect was more pronounced in the cells treated with the highest dose of 200 μg mL -1 (Figure 4).

Fluorescence Microscopy.
The untreated A375 cells did not take positive staining with DAPI ( Figure 5) and showed no cells with visible chromatin condensation. However, with different concentrations of treatment, cells with chromatin condensation appeared to increase in number along with the increase in dose ( Figure 6).
The cells stained with AO/EB in untreated culture (Figure 7) showed intact chromatin without fragmentation; while there were nearly 39% cells with fragmented and condensed chromatin visible at 100 μg mL -1 TRF-treated culture. In the 200 μg mL -1 TRF-treated culture; nearly 50% cells were fragmented and condensed (Figure 8).

Mitochondrial Membrane Potential.
As compared to the control (Figure 9(a)), A375 cells showed greenish stain in greater intensity. However, in the TOΦ and TRF-treated cells (Figures 9(b)-9(e)) reddish stain was more prevalent, indicating mitochondrial potential depolarization, and that  was more pronounced in the higher dose (Figure 9(e)). Quantitative data obtained by flow cytometry (Figures 9(f)-9(j), resp.) would also support the mitochondrial membrane potential depolarization convincingly.

Reactive Oxygen Species Generation.
In the TRFuntreated control (Figure 10(a)), the A375 cells did not take up much stain, showing less generation of reactive oxygen species (ROS). TOΦ showed intense staining as compared to the control (Figure 10(b)). In contrast, there were number of cells that showed more intense staining with the increase in dose (Figures 10(c)-10(e)) signifying more amount of ROS generation.
The quantitative data generated by flow cytometry (Figures 10(f)-10(j)) would corroborate the same findings.

Flowcytometric Analysis.
To further confirm whether the cause of A375 cell death induced by TRF was apoptosis, flow cytometric analysis was performed. Incubation of fixed and permeabilized cells with fluorochrome PI results in quantitative PI binding with total cellular DNA, and the fluorescence intensity of PI-labeled cells was proportional to DNA contents ( Figure 11). Apoptotic nuclei with hypodiploid DNA correspond to the sub G 0 /G 1 peak. The maximal increase in the frequency of apoptotic cells was observed upon 24 h treatment with 200 μg mL -1 TRF (Figure 11(d)). These results demonstrate that the treatment with TRF at 200 μg mL -1 induced A375 cell apoptosis maximally.
The percentages of apoptosis in TRF (200 μg mL −1 ) treated cells were found to be 34.69% as compared with a very low percentage of apoptotic cells (8.23%) in the untreated cell ( Figure 12; Table 5).

Comet Assay.
In comet assay, the nuclei were intact and round, without any fragmented DNA (Figure 13   showing signs of more DNA damage with extended comet tails.

DNA Fragmentation Assay.
The DNA fragmentation was also tested by agarose gel electrophoresis. Figure 15 indicates a significant increase in inter-nucleosomal DNA fragmentation of A375 cells. When the DNA isolated from TRF-treated cells was subjected to agarose gel electrophoresis, a DNA ladder characteristic of apoptotic DNA was observed in the cells treated with different concentrations.

Immunofluorescence Study.
With the increase in dose of TRF treatment, there was an increase in fluorescence intensity of Bax, cytochrome c and caspase-3 and decrease in fluorescence of Bcl-2 ( Figure 16) consistent with the immunoblot data ( Figure 17).  3.11. Immunoblot Analysis. Along with the increase in dose of TRF, the expression levels of Bax, cytochrome c in cytosolic fraction and caspase-3 in the total cell lysate were increased while the expression of Bcl-2 was downregulated ( Figure 17).

Discussion
Results of the present study would indicate that the homeopathic drug T. occidentalis Φ had four chromatographically separated fractions of which the thujone-rich fraction was found to be the most bioactive (anti-cancer, pro-apoptotic) component. Unfortunately, despite our best effort, the oily nature of this fraction did not allow us to purify it to 100%, for which we were able to perform only mass spectrometry of TRF. The mass spectrophotometric data obtained by us supported the published data on the chemical structure of the C 10 H 16 O with a molecular weight 152 as reported by Tarcisia [30]. Anti-cancer activity of the alcoholic extract of T. occidentalis had earlier been reported by Sunila and Kuttan [14] from their in vivo studies in rats. In our present in vitro study involving several protocols, we confirm the proapoptotic and anti-cancer potential of T. occidentalis mother tincture. Additionally, it was revealed that the thujonerich component was possibly the key bioactive compound showing its promising anti-cancer potentials in the skin cancer cell line A375, a fact which had not earlier been reported ( Figure 18). Furthermore, the anti-cancer activity was mediated through activation of pro-apoptotic signaling via the activation of Bax, caspase-3 and cytochrome c. We determined the optimum dose through a range finding trial. We also studied the probable cytotoxic effects of both TOΦ and the TRF on a relative basis. The result revealed that the 220.18 μg mL -1 of TOΦ could produce 50% cell death in A375 while that dose was relatively nontoxic to PBMC. Correspondingly, of the three doses of 50, 100, 200 μg mL -1 of TRF used, 200 μg mL -1 TRF showed the maximum effect surpassing the effect of treatment with the TOΦ as revealed from the result of the several protocols. Thus, the TRF appeared to be more potent anti-cancer agent when treated alone, a fact which may be of therapeutic importance in drug design and development of anti-cancer drug from natural sources.
In recent years, the use of complementary and alternative medicine (CAM) and other traditional medicines (TM) is increasingly becoming popular [31][32][33][34][35][36][37]. However, many people are hesitant to use such drugs either because they are not scientifically validated for their action or because the mechanism of their action is not properly known. Therefore, search should be on for finding out safe, affordable and efficient natural plant products that are experimentally proven to be effective and are relatively non-toxic in nature, because most of the orthodox anti-cancer drugs used in cancer therapy are toxic and have adverse side-effects. Thus, studies pinpointing confirmed efficacy of a particular fraction or compound among several found in crude extract are important for therapeutic purposes. Although the efficacy of TRF and TOΦ has presently been tested against an in vitro cancer cell line, it is very likely that the result can be extrapolated in animal or in human. However, to determine this, more experiments should be carried out on in vivo animal models, which will hopefully be taken up in the next phase of our program using mice as a model.