Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation
1. Samrat Ghosh (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India )
2. Kausik Bishayee (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India )
3. Avijit Paul (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India )
4. Avinaba Mukherjee (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India )
5. Sourav Sikdar (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India )
6. Debrup Chakraborty (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India )
7. Naoual Boujedaini (Boiron Laboratories, Lyon 69110, France )
8. Anisur Rahman Khuda-Bukhsh (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India E-mail: [email protected].in, [email protected])
OBJECTIVE: Preventive measures against skin melanoma like chemotherapy are useful but suffer from chronic side effects and drug resistance. Ethanolic extract of Phytolacca decandra (PD), used in homeopathy for the treatment of various ailments like chronic rheumatism, regular conjunctivitis, psoriasis, and in some skin diseases was tested for its possible anticancer potential.
METHODS: Cytotoxicity of the drug was tested by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on both normal (peripheral blood mononuclear cells) and A375 cells. Fluorescence microscopic study of 4′,6-diamidino-2-phenylindole dihydrochloride-stained cells was conducted for DNA fragmentation assay, and changes in cellular morphology, if any, were also recorded. Lactate dehydrogenase activity assay was done to evaluate the percentages of apoptosis and necrosis. Reactive oxygen species (ROS) accumulation, if any, and expression study of apoptotic genes also were evaluated to pin-point the actual events of apoptosis.
RESULTS: Results showed that PD administration caused a remarkable reduction in proliferation of A375 cells, without showing much cytotoxicity on peripheral blood mononuclear cells. Generation of ROS and DNA damage, which made the cancer cells prone to apoptosis, were found to be enhanced in PD-treated cells. These results were duly supported by the analytical data on expression of different cellular and nuclear proteins, as for example, by down-regulation of Akt and Bcl-2, up-regulation of p53, Bax and caspase 3, and an increase in number of cell deaths by apoptosis in A375 cells.
CONCLUSION: Overall results demonstrate anticancer potentials of PD on A375 cells through activation of caspase-mediated signaling and ROS generation.
Ghosh S, Bishayee K, Paul A, Mukherjee A, Sikdar S, Chakraborty D, Boujedaini N, Khuda-Bukhsh AR. Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation. J Integr Med. 2013; 11(2): 116-124.
Received June 25, 2012; accepted August 9, 2012.
Open-access article copyright © 2013 Samrat Ghosh et al.
Correspondence: Anisur Rahman Khuda-Bukhsh, PhD, Professor; Tel: +91-33-25828750-315; E-mail: [email protected], [email protected]
A dynamic increase of skin melanoma mortality rate has been observed in the world in recent years. Though this type of skin cancer comprises just 5 % of all malignant skin cancers, it is responsible for a 75%-80% of deaths caused by these tumors. The high rate of malignancy of this neoplasm is associated with rapid proliferation, numerous early metastases and high resistance to conventional treatments involving surgery, chemotherapy and radiation. Although preventive measures like chemotherapy are very useful, these often result in manifestation of chronic side effects[2,3]. Therefore, there is a growing demand for the development of alternative antimelanoma therapies with less side effects. Natural products of various sources, particularly from plants, have been regarded as a precious alternative to modern medicine and investigations on active components with anticancer potential and less side effects have opened up newer avenues.
In cancer treatment, one of the approaches to restrain tumor growth is by activating the apoptotic machinery in the tumor cells. Apoptosis, a highly structured and orchestrated process, performs a significant role in regulating cell number for the growth and homeostasis of tissues by eliminating aged, damaged and unwanted cells[6,7].
Phytolacca decandra (PD), commonly known as poke root, is an ornamental plant of the Phytolaccaceae family. Phytochemical characterisation revealed that terpenoids, saponins, alkaloid, resins, phytolacic acid and tannin are the major bioactive ingredients of Phytolacca species[8-11]. From a long time Phytolacca has been used to combat different ailments in complementary and alternative medicine (CAM). In homeopathy, mother tincture of Phytolacca is used against chronic rheumatism, regular conjunctivitis, psoriasis and some skin diseases. Although to the best of our knowledge, anticancer potential of the ethanolic crude extract of PD is yet to be systematically studied for scientific validation, stray reports exist in literature enlisting some of the ingredients like saponins and alkaloids extracted and separated from other plant sources to have anticancer potentials[13,14]. Since the crude extract of PD is used in homeopathy, and since no earlier study appears to have been conducted on the anticancer effect on the basis of a reliable investigation, we undertook this research. Therefore, in the present set of experiment our main aim was to explore the anticancer activity, if any, of PD against A375 cells in vitro and if positive findings were obtained, to investigate the mode of cell death involved.
2 Materials and methods
2.1 Chemicals and reagents
Plastic wares were obtained from BD Bioscience (USA). Cell culture media Dulbecco’s modified Eagle medium (DMEM) and Roswell Park Memorial Institute (RPMI), fetal bovine serum (FBS) and trypsin-ethylene diamine tetraacetic acid (trypsin-EDTA) were purchased from Invitrogen-Gibco BRL (USA). Primary and secondary antibodies were purchased from Santa Cruz Biotecnology (USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sisco Research Laboratory, India. TRIzol and antibiotics penicillin-streptomycin-neomycin (PSN) were obtained from HiMedia (India). Dichloro-dihydrofluorescein diacetate (H2DCFDA) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma (USA). RNase and p-nitrophenyl phosphate (PNPP) were purchased from Merck (Germany). All the primers used were obtained from Bioserve (India). Ethanolic extract of PD in the form of homeopathic mother tincture was supplied by Boiron Laboratory, Lyon, France. They prepared the drug adopting the guidelines of European Pharmacopoeia and supplied the drug in 65% ethanolic extract. Before the experimental treatment, the drug was diluted in the DMEM media.
2.2 Cell culture
A375 and HeLa (National Centre for Cell Science, NCCS, India) cells were cultured in DMEM, whereas PC3 cells (NCCS, India) were cultured in RPMI, supplemented with 10% heat-inactivated FBS and 1% PSN and maintained at 37 oC with 5% CO2 in a humidified incubator (Thermo Scientific, USA). Cells were harvested with 1 × trypsin-EDTA in phosphate buffered saline (PBS) and plated at required cell numbers and allowed to adhere before treatment.
2.3 Cell viability assay
Cells were dispensed in 96-well plates at a density of 1×102 cells per well and treated with various concentrations of PD (0 to 780 μg/mL) for 24 and 48 h respectively and peripheral blood mononuclear cells (PBMCs) were isolated from the human blood sample by conventional ficoll gradient method and treated with PD at different concentrations (0 to 780 μg/mL), and incubated for 24 and 48 h. The percentage of cell death was determined by MTT assay.
2.4 Selection of drug doses
On the basis of the result of MTT assay, we selected three different drug doses, namely, D1 (100 μg/mL), D2 (150 μg/mL) and D3 (200 μg/mL), for experiments and treated for 48 h on A375 cells. To calculate the concentration of the drug, the dry weight of PD was measured and expressed as drug doses in concentration unit (μg/mL). The positive (vehicle) control set received only ethanol (placebo) and the negative control neither received any drug nor ethanol. As the positive control had no significant change with respect to the negative control, we did not include negative control group in our experiments.
2.5 Morphological changes
Cells were treated with PD at specified concentrations. After 48 h, the cells were observed under an inverted light microscope (Leica, Germany) equipped with a digital camera and photographs were taken.
2.6 Nucleosomal fragmentation analysis
Treated cells were stained with DAPI (10 μg/mL) and analyzed under a fluorescence microscope (Axioscope 2 Plus, Carl Zeiss, Germany) and representative photographs were taken.
2.7 Lactate dehydrogenase-based cytotoxicity assay
Lactate dehydrogenase (LDH) activity was assessed using a standardized kinetic determination kit (Enzopak, Recon, India). LDH activity was measured according to the method of Biswas et al. LDH activity was measured in both floating dead cells and adherent viable cells for control and drug-treated cells. The floating cells were collected from culture medium by centrifugation (500 × g) at 4 oC for 2 min, and the LDH content from the pellets was used as an index of apoptotic cell death (LDHp). 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:
Apoptosis percentage = (LDHp × 100%)/(LDHp+LDHi+LDHe)
Necrosis percentage = (LDHe × 100%)/(LDHp+LDHi+LDHe)
2.8 Analysis of intracellular reactive oxygen species generation
Treated fixed cells were incubated with 10 μmol/L H2DCFDA and photographed under a fluorescence microscope (Leica DMLS, Germany) for qualitative analyses. For the quantitative estimation of reactive oxygen species (ROS) generation, the treated cells were fixed in 70% chilled methanol and incubated with 10 μmol/L H2DCFDA for 30 min at room temperature in dark. Fluorescence intensity was measured with an excitation wavelength of 480 nm and an emission wavelength of 530 nm by using a FL-1H filter of flow cytometer (BD FACS calibur, USA). Data were analyzed by using Cyflogic software.
2.9 Assessment of protein activity involved in anticancer signaling pathway
The assay was done according to the manufacturer’s protocol (Santa Cruz Biotechnology Inc, USA) using cell lysate. Drug-treated and control cells were lysed in lysis buffer. The lysed material was centrifuged at 13 000 × g for 15 min at 4 °C and the supernatants were collected. Enzyme-linked immunosorbent assay (ELISA) was carried out with the supernatants to study the expressions of p53, Akt/protein kinase B (PKB), BCL-associated X (Bax), B cell lymphoma 2 (Bcl-2) and caspase 3 quantitatively. The assay was standardized by titration of the different concentrations of antigens and antibodies. Wells were coated with 70 μg of proteins in 100 μL of 0.05 mol/L carbonate buffer (pH 9.6) and kept overnight at 4 °C, in a moist chamber. Flicked the plate to remove the unbound antigen solution and wells were blocked with 1% bovine serum albumin (BSA) in 0.1 mol/L PBS (pH 7.2) for 1 h at 4 °C. Flicked and washed wells thrice with 200 μL of PBS containing 0.05% (volume ratio) Tween-20. Wells were then incubated with primary antibody (1∶500), diluted in PBS (containing 0.05% Tween-20 and 1% BSA) and kept overnight at 4 °C. Plate was again washed and incubated with specific secondary antibodies (1∶1 000) for 2 h at 37 °C. Wells were washed further three times as described above. PNPP was used as a colour developer and colour intensity was measured at 405 nm with respect to blank. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) served as the housekeeping gene.
2.10 Analysis of mRNA expressions by semi-quantitative reverse transcriptase-polymerase chain reaction
Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions and the gene expressions were analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to estimate the expression of p53, Akt, Bcl-2, BAX, and caspase 3, at their mRNA level. G3PDH served as the housekeeping gene. Densitometry was performed using Image J software. Primer sequences of RT-PCR were illustrated in Table 1.
The observers were “blinded” during observations as to whether they were observing the “control” or “drug-treated” materials.
2.12 Statistical analysis
Statistical analysis was performed by one-way analysis of variance with post-hoc least significant difference tests, using SPSS 14.0 software to identify if the differences were significant among the mean values of different groups. Results were expressed as mean ± standard error of mean. P