Human mammary epithelial carcinoma cell lines, MCF-7 and MDA-MB-231, were obtained from NCCS, India. Peripheral blood collected from healthy human volunteers with informed consent (Institutional Review Board 1382) was centrifuged over Ficoll-Hypaque density gradient (Amersham Pharmacia, Uppsala, Sweden) to obtain total peripheral blood mononuclear cells. Cells were routinely maintained in DMEM supplemented with 10% heat inactivated fetal bovine serum (Lonza, Portsmouth, NH, USA), L-glutamine (2 mM), sodium pyruvate (100 mg/ml), non-essential amino acids (100 mM), streptomycin (100 mg/ml), penicillin (50 U/ml; Invitogen, Carlsbad, CA, USA) at 37°C in a humidified 5% CO₂ incubator. Cells were maintained in an exponential growth phase for all experiments. Viable cell numbers were determined by trypan blue exclusion test.

The different strengths (6C, 30C and 200C) of placebo and thuja were procured from Hahnemann Publishing Co. Pvt., Ltd. (Kolkata, India) (authorized manufacturing house sanctioned by both GMP and certified by ISO). The drugs procured were colorless, odorless and endotoxin-free. Drugs were stored at room temperature, away from sunlight and vigorously shaken immediately before each treatment. Cells were treated with thuja or placebo of potencies 6C or 30C or 200C at different concentrations (10, 15, 20 and 30 μl/ml) for different time-points (0, 6, 8, 12, 24, 36 and 48 h) to select the optimum time required for cell killing. To understand the sequence of events leading to apoptosis, cancer cells were treated with mitochondrial pore inhibitor cyclosporine A (CsA) (25 μM; Merck, Darmstadt, Germany) for 1 h prior to incubation with thuja. For transcriptional blockage of p53, cells were treated with pifithrin-α (30 μM; Sigma). NAC (40 mM; Sigma) treatment was carried out for 1 h prior to incubation with thuja for pharmacological inhibition of ROS while H₂O₂ (0.5 mM; Sigma) treatment was applied for ROS enhancement.

For the determination of cell death, cells were stained with 7AAD and Annexin V-PE and analyzed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA). Electronic compensation of the instrument was conducted to exclude overlapping of the emission spectra. A total of 10,000 events were acquired for analysis using CellQuest software (BD Biosciences) (20). For the assessment of mitochondrial transmembrane potential, cells were loaded with potential-sensitive dye 3,3-dihexyloxacarbocyanine iodide (DiOC₆; Merck) during the last 30 min of treatment at 37°C in the dark. Fluorescence of retained DiOC₆ was determined by flow cytometry using logarithmic amplification by CellQuest software (21).

For detection of intracellular ROS, the untreated and thuja-treated cells were incubated during the last 20 min at 37°C in the dark with 10 μM of dichlorofluorescein diacetate (DCF-DA; Sigma). DCF fluorescence was measured by flow cytometry and subjected to analysis using CellQuest 3.2 software. The probe was excited at 488 nm and emission was measured through a 530 nm band-pass filter. For confocal microscopy a Leica fluorescence microscope DM 900 was used to visualize the images of thuja-treated cells incubated with DCF-DA (10 μM). DCF-DA is colorless and non-fluorescent until both of the acetate groups are hydrolyzed and the products are subsequently oxidized to fluorescein derivatives (H₂DCFDA). Digital images were captured with a cool (−25°C) charged coupled device (CCD) camera (Princeton Instruments) controlled with the Andor iQ BioImaging software (BT, London, UK) (19).

Chromatin condensation and nuclear fragmentation were analyzed using a standard protocol. Briefly, cells were grown on coverslips, fixed with 3% p-formaldehyde for 10 min and then permeabilized with 0.1% Triton X-100 for 5 min. Cells were then incubated with 4′6-diamidino-2-phenylindole (DAPI; BD Pharmingen, San Jose, CA, USA). The morphology of the cell nuclei was visualized using a fluorescence microscope (Leitz microscope fitted with epifluorescence illuminator through a ×60 aperture oil immersion lens; Carl Zeiss, Oberkochen, Germany). For fluorescent imaging, cells growing on a coverslip were fixed with 3% p-formaldehyde and were stained with the anti-p53 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), after permeabilization with Triton X-100, followed by addition of the TRITC-conjugated secondary antibody and visualized with a confocal microscope (Carl Zeiss).

MCF-7-Dn-p53 was derived from MCF-7 cells through an adenoviral vector expression system expressing dominant-negative p53 (Dn-p53) under the control of a cytomegalovirus promoter (Clontech). Similarly, p53 expression was stably knocked down in MCF-7 cells using p53-shRNA (Santa Cruz Biotechnology). These clones (2 μg each/million cells) were introduced into MCF-7 cells using Lipofectamine 2000 (Invitrogen). Stably expressing clones were isolated by limiting dilution and selection with G418 sulphate (Cellgro) at a concentration of 400 μg/ml, and cells surviving this treatment were cloned and screened by western blot analysis with the specific antibody. MCF-7 cells were transfected with 300 pmol of p38-MAPK-/Bax-/control-ds-siRNA (Santa Cruz Biotechnology) and Lipofectamine 2000 separately for 12 h. The protein levels of p53/p38MAPK/Bax were estimated by western blotting.

To obtain whole cell lysates, cells were homogenized in lysis buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM Na-EDTA, 1 mM Na-EGTA and 1 mM DTT) supplemented with protease and phosphatase inhibitor cocktails. Mitochondrial and cytosolic fractions were prepared (21). For direct western blot analysis, a total of 50 μg of protein was resolved using SDS-PAGE and transferred to nitrocellulose membranes for western blotting using the relevant antibodies e.g., anti-caspase-6, anti-caspase-7, anti-caspase-9, anti-p53 (DO-1), anti-p-ser-46-p53, anti-Bax (N-20), anti-p38MAPK, anti-p-p38MAPK (pTGpY; Promega), anti-cytochrome-c and anti-PUMA. The blots were developed with NBT/BCIP (1:1). Equivalent protein loading in cytosolic, nuclear and mitochondrial fractions was verified using anti-α-actin/histone H1/MnSOD antibodies (Santa Cruz Biotechnology), respectively.

Values are shown as standard error of the mean, except when indicated otherwise. Data were analyzed, and appropriate significance (P<0.05) of the differences between mean values was determined by a Student’s t-test.

The effect of thuja on the viability of human breast cancer cell line MCF-7 and normal peripheral blood mononuclear cells (PBMCs) was examined at different potencies and placebos, i.e., 6C, 30C, 200C, where for each potency a differential dose of 0–30 μl/ml was applied (Fig. 1A). Total viable cells were scored by trypan blue dye exclusion assay. It was observed that among all the potencies of thuja, a 30C potency resulted in the most significant decrease (P<0.001) in cell viability as compared to its placebo treatment (Fig. 1A). Moreover, at a 20 μl/ml dose of 30C, the percent MCF-7 cell death reached a plateau (Fig. 1A) while under the same conditions the PBMC viability was found to be >90% (Fig. 1A). These results indicated better efficacy of a 20 μl/ml dose of 30C thuja in MCF-7 cell killing with minimum toxicity leading us to perform all further experiments using this potency and dose of thuja. The time-dependent effect (0–48 h) of thuja at 30C (20 μl/ml) in comparison to the placebo on MCF-7 and PBMCs was examined, and the percentage of cell death was assessed. Thuja at 30C at a concentration of 20 μl/ml exerted significant cell death in MCF-7 cells in a time-dependent manner. However, significant cell death in PBMCs was noted from 24 h onwards following thuja treatment (Fig. 1B).

To confirm the nature of cell death as apoptosis, Annexin V-PE/7AAD binding assay was utilized. Our flow cytometric data demonstrated that, in comparison to placebo-treated MCF-7 cells, thuja-treated unfixed MCF-7 cells showed Annexin V-PE-binding with minimum 7AAD binding (Fig. 1C) indicating that the mode of cell death was apoptosis but not necrosis. These findings were confirmed by the presence of nuclear fragmentation as evidenced by DAPI-stained fluorescent images of the thuja-treated MCF-7 cells (Fig. 1D). Collectivelly, these data revealed that thuja at 30C asserts an apoptogenic effect on mammary epithelial cancer MCF-7 cells.

Since tumor-suppressor protein p53 plays an important role in the canonical apoptotic pathway, we explored the changes in the protein profile of p53 after thuja administration in wild-type p53-expressing MCF-7 cells. Our results depicted a significant increase in the levels of p53 in MCF-7 cells after thuja treatment (Fig. 2A). To confirm whether or not thuja-induced cancer cell apoptosis is p53-dependent, the percentage of apoptosis as detected by Annexin binding was assessed in wild-type p53-expressing MCF-7 cells, p53-mutated MDA-MB-231 cells and p53-shRNA-transfected MCF-7 cells following thuja treatment (Fig. 2B). Notably, thuja at 30C at a 20 μl/ml dose significantly (P<0.001) induced apoptosis in the wild-type p53-expressing MCF-7 cells. The apoptogenic insult asserted by thuja was ~38% as compared to the placebo effect which was only 10%, while p53-mutated MDA-MB-231 and p53-knockdown MCF-7 cells were resistant to the thuja-induced insult (Fig. 2B). To understand the mechanism of thuja-induced apoptosis, the p53 cellular localization of p53 in MCF-7 cells following thuja administration was examined by confocal microscopy. Translocation of the p53 protein from the cytosol to the nucleus was noted in the thuja-exposed MCF-7 cells (Fig. 2C). It is known that p53, being a transcription factor, is activated when localized in the nucleus and transactivates its downstream target genes. Therefore, the time-dependent accumulation of p53 both in the nuclear and cytosolic fractions and the status of its transactivated gene products, i.e., Bax and PUMA, in thuja-treated and untreated MCF-7 cells were examined by western blot analysis. Our results revealed that, in comparison to the untreated and placebo-treated tumor cells, thuja-exposed cells showed increased nuclear p53 expression with concomitant increase in the levels of p53 transactivated gene products, Bax and PUMA, in a time-dependent manner from 4 h onwards following thuja administration as detected by western blot analysis (Fig. 2D and E). These results indicated that p53 is imperative for thuja-induced functional p53-expressing MCF-7 cell apoptosis.

Since reactive oxygen species (ROS) have been implicated as potential modulators of p53-dependent apoptosis, we explored the role of ROS in thuja-induced p53-dependent MCF-7 cell apoptosis. Our results revealed that, in comparison to the untreated and placebo-treated MCF-7 cells, exposure to thuja caused a significant increase in the levels of intracellular ROS in these functional p53-expressing cells (Fig. 3A and B). Notably, thuja-treated p53-shRNA-transfected MCF-7 cells showed a much significant reduction in the ROS level as compared to the functional p53-expressing MCF-7 cells although complete abrogation of ROS was not observed (Fig. 3A). Efficiency of p53-shRNA transfection was confirmed by western blot analysis (Fig. 3A, western blot). These findings indicated that thuja-induced ROS production was significantly and predominantly p53-dependent. These data suggest that p53 might regulate the intracellular redox status in these cancer cells and induce apoptosis by an ROS-dependent pathway. In addition, these results also indicated the possibility of p53-independent ROS production in the thuja-treated MCF-7 cells.

To further validate the role of ROS in thuja-induced p53-dependent apoptosis, two approaches were employed. Firstly, MCF-7 cells were pretreated with a pharmacological inhibitor of ROS, i.e., N-acetyl-L-cysteine (NAC), before thuja administration and secondly, H₂O₂, a pharmacological ROS inducer, was administered in the presence and absence of thuja. Thereafter, both the experimental sets were scored for the levels of intracellular ROS, levels of p53 in nuclear and cytosolic fractions and percent apoptosis. Notably, while NAC pretreatment resulted in loss of DCF-DA fluorescence (Fig. 3C), reduction in nuclear p53 levels (Fig. 3D) and significant decrease in percent apoptosis (Fig. 3E) in contrast to thuja administration alone, H₂O₂ treatment significantly upregulated intracellular ROS (Fig. 3C), increased nuclear p53 (Fig. 3D) and increased thuja-induced apoptosis (Fig. 3E). Since the silencing of p53 abrogates ROS production and inhibition of ROS decreases p53 expression, there is the inter-dependency and crosstalk between p53 and ROS in executing thuja-induced apoptosis.

It is known that p53 is a redox-regulated transcription factor. Thus, to delineate the functional impact of thuja-induced ROS on p53 activation, several experiments were designed. Our earlier findings implicated the role of thuja-induced ROS in nuclear translocation of p53, a prerequisite for its transcriptional activation (Fig. 3D). It is known that to circumvent diverse activities in response to varied stress stimuli, p53 protein is under tight regulatory control of post-translational modifications where phosphorylation is one of the key events modulating p53 functions (22). Our search for the factor(s) responsible for functional activation of p53 revealed that, in response to thuja, p53 manifested phosphorylation at serine 46 residues (Fig. 4A). Based on previous research which found that p38MAPK regulates p53 function in response to ROS-induced DNA damage (23), we evaluated the role, if any, of p38MAPK in thuja-induced p53 activation. Our results indicated upregulation of the phosphorylated form of p38MAPK without intervention of the total p38MAPK status in MCF-7 cells following thuja treatment with simultaneous phosphorylation of p53 at Ser46 and an increase in p53 transactivated gene product, Bax, while the placebo failed to display any such effect (Fig. 4A).

Based on earlier observations which confirmed the critical role of ROS in manipulating the levels of nuclear p53, we attempted to clarify the mechanistic link between thuja-induced ROS, p38MAPK and p53 phosphorylation. Thus, we analyzed the level of total p38MAPK, p-p38MAPK and p-Ser46-p53 in thuja-induced MCF-7 cells upon administration of either NAC or H₂O₂. As shown in Fig. 4A, NAC pretreatment abolished phosphorylation of p38MAPK without affecting total p38MAPK and simultaneously significantly decreased the p-Ser46-p53 level. Reduction in intracellular ROS levels in NAC-treated cells further confirmed the inhibitor efficiency (Fig. 3C). In contrast, treatment with H₂O₂ resulted in an increase in p-p38MAPK and phosphorylated-p53 levels. An increase in DCF-DA fluorescence in the H₂O₂-treated cells confirmed an increase in intracellular ROS levels (Fig. 3C). All of these results together underscore the role of ROS in sustaining the activity of p38MAPK and p53.

To ascertain the contribution of p38MAPK in thuja-mediated apoptosis through post-translational modification of p53, transiently silenced p38MAPK MCF-7 cells were used and analyzed for the status of p-Ser46-p53 and Bax. The percentage of apoptosis was also determined in these engineered as well as control MCF-7 cells with and without thuja administration. The silencing of p38MAPK downregulated the p-p38MAPK level in the transfectants thereby confirming the transfection efficiency (Fig. 4B, western blot) and this decrease in p-p38MAPK significantly decreased thuja-induced apoptosis (Fig. 4B). It was observed that in contrast to the thuja-treated wild-type MCF-7 cells, p38MAPK-silenced MCF-7 cells exhibited decreased p-Ser46-p53 (Fig. 4C). Consequently, our results confirmed that p38MAPK plays a crucial role in functionally triggering p53 to execute its transcriptional functions and finally to execute apoptosis. To further validate the sequence of events, p38MAPK-silenced MCF-7 cells with and without thuja administration were analyzed for intracellular ROS levels employing DCF-DA staining. Notably, the silencing of p38MAPK in MCF-7 cells resulted in a significant decrease in DCF-DA fluorescence signifying the role of p38MAPK in ROS production following thuja administration (Fig. 4E). Additionally, similar to p53-knockout cells, p38MAPK-silenced MCF-7 cell also could not neutralize the ROS level completely while inhibiting ROS downregulated p-p38MAPK.

Results of the above experiments indicated the potential role of ROS in p53 nuclear translocation. As shown in Fig. 2E, an increase in Bax and PUMA protein levels in MCF-7 cells following thuja exposure confirmed that nuclear p53 is transcriptionally active. Next, to ensure the involvement of p53-transactivated Bax in thuja-induced apoptosis, MCF-7 cells were pretreated with pifithrin-α (24) before evaluating thuja-induced apoptosis. The results demonstrated that blocking p53-mediated transactivation inhibited thuja-induced cancer cell death, thereby confirming the involvement of the p53-mediated transactivation pathway in thuja-induced apoptosis (Fig. 4D). While examining the p53 transactivated product, it was observed that pifithrin-α-exposed cells, although exhibiting an increased p53 level, however, showed a decrease in the expression levels of Bax and PUMA following thuja administration (Fig. 4F).

In a parallel experiment, the role of ROS on the p53 transactivated gene products, ie., Bax, was analyzed in NAC- or H₂O₂-pretreated thuja-exposed MCF-7 cells. It was observed that while the quenching of ROS by NAC diminished the levels of Bax, H₂O₂ exposure upregulated this p53-transactivated product (Fig. 4A). Additionally, to investigate the contribution of p53 transactivation in thuja-mediated ROS generation and subsequent apoptosis, the transactivation of p53 was blocked by pre-exposing MCF-7 cells to pifithrin-α and then intracellular ROS levels and apoptosis were assessed. Abrogating p53 transcriptional functions in MCF-7 cells resulted in significant downregulation of ROS production even upon thuja administration (Fig. 4E). Remarkably, it was found that these pifithrin-α-treated cells, similar to p53-knockout or p38MAPK-silenced MCF-7 cells, also failed to neutralize the intracellular ROS level completely, thereby pointing towards the possibility of p38MAPK-p53 crosstalk independent of ROS generation. These apparently ‘contradictory’ findings point towards the presence of an ROS-p-p38MAPK-p53 feedback loop that executes apoptosis in thuja-treated MCF-7 cells.

The obtained results portrayed the potential role of ROS in thuja-induced apoptosis. It was also revealed that the ROS-induced apoptosis was not only p53- and p38MAPK-dependent but could also act independently. In these circumstances the possibility for the production of early ROS was assumed which may be produced before p38MAPK and p53 activation. The above assumption was confirmed when the time frame of ROS production was determined. Our effort to unveil this ‘mystery’ divulged a bi-phasic increase in ROS, with the first peak being at 2 h (Fig. 4G), even before p53 nuclear translocation that had an onset from 4 h following thuja exposure (Fig. 2D). This was followed by a second higher peak of ROS production at 8 h following thuja exposure (Fig. 4G) the timing of which matched with the p53 transactivated Bax expression following thuja exposure (Fig. 2E). Our earlier results revealed that the knockout of p53 (Fig. 3A) or the blocking of p53 transcriptional activity (Fig. 4E) resulted in a significant decrease in ROS levels. Therefore it was hypothesized that the second peak of ROS was induced by activated p53. To further confirm this crosstalk, Bax-deprived MCF-7 cells with or without thuja administration were analyzed for intracellular ROS levels. These findings confirmed the role of thuja in the generation of p53-independent early phase ROS in p53 activation via p38MAPK that finally potentiated p53 nuclear translocation and Bax transactivation which directed the second peak of ROS. Additionally, these findings allowed us to hypothesize that the p53-dependent second phase of ROS is instrumental in maintaining p53 activation through the ROS-p38MAPK-p53 feedback loop while executing thuja-induced apoptosis. Subsequent experiments were designed to identify the source of ROS generation after 8 h of thuja exposure in MCF-7 cells. Notably, blocking of mitochondrial disruption abrogated an increase in ROS levels at 8 h (Fig. 5A) thereby indicating the contribution of mitochondrial membrane disruption in thuja-induced ROS generation at a late time period.

An increase in Bax in apoptogenic cells tempted us to explore the involvement of Bax in the mitochondrial cascade-mediated apoptosis. For this two approaches were employed. Firstly, MCF-7 cells were transfected with Bax-siRNA and secondly, MCF-7 cells were pretreated with cyclosporine A (CsA), the mitochondrial pore formation blocker, before thuja exposure. The silencing of Bax or the inhibition of mitochondrial pore formation resisted the thuja-induced ROS production (Fig. 5A) and significantly decreased the percentage of apoptosis of MCF-7 cells (Fig. 5B). Gene manipulation or pharmacological inhibition of p53-transactivation product Bax failed to neutralize ROS completely reflecting the results of p53-knockout or the p38MAPK-silencing. In the downstream of Bax, the involvement of mitochondria was further confirmed by DiOC₆-florescent dye that demonstrated significant mitochondrial transmembrane potential (MTP) loss in thuja-exposed MCF-7 cells as compared to the control and placebo-treated MCF-7 cells (Fig. 5C). These findings confirmed the role of mitochondria in executing the thuja-induced p53-transactivated Bax-mediated apoptotic cascade. Detail inspection determined that unlike the untreated and placebo-treated cells, the thuja-treated MCF-7 cells showed a significant decrease in cytochrome c level in mitochondria with a simultaneous increase in the cytosol (Fig. 5D).

This ROS-p53 feedback loop activates the mitochondrial death signal through cytochrome c release finally culminating in apoptosis. Further search for the downstream mechanism revealed that thuja influenced the execution phase of apoptosis through activation of the initiator caspase-9 followed by effector caspase-6 and -7 (Fig. 5E) in these MCF-7 cells that lack functional caspase-3. Blocking mitochondrial pore formation downregulated these caspases and thereby implicated mitochondrial membrane disruption in thuja-induced caspase activation and apoptosis (Fig. 5E). Mitochondrial membrane disruption was downstream of Bax transactivation by p53. These results further signify the existence of a feedback loop in thuja-treated MCF-7 cells in which ROS act both as an initiator of p53 activation as well as plays a significant role in maintaining p53 in an activated state via p38MAPK.