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Journal of Chinese Integrative Medicine ›› 2013, Vol. 11 ›› Issue (2): 116-124.doi: 10.3736/jintegrmed2013014

• Research Article • Previous Articles     Next Articles

Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation

Samrat Ghosha, Kausik Bishayeea, Avijit Paula, Avinaba Mukherjeea, Sourav Sikdara, Debrup Chakrabortya, Naoual BoujedainibAnisur Rahman Khuda-Bukhshb()   

  1. a. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India
    b. Boiron Laboratories, Lyon 69110, France
  • Received:2012-06-25 Accepted:2012-08-09 Online:2013-03-10 Published:2013-03-15

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.

Key words: Phytolacca decandra, Skin neoplasms, Reactive oxygen species, Apoptosis, Gene expression

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Figure 1

Results of cell viability assay Different concentrations (0 to 780 μg/mL at the interval of 78 μg/mL) of Phytolacca decandra on A375 cells (A), HeLa cells (B), PC3 cells (C) and peripheral blood mononuclear cells (D) incubated for 24 h and 48 h. The cell viability and also the corresponding median lethal dose were then detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at each concentration. The results are expressed as mean ± standard error of mean, n=3; *P<0.05, vs 0 μg/mL."

Figure 2

Changes in cellular morphology (Inverted light microscopy, ×20)D1: 100 μg/mL; D2: 150 μg/mL; D3: 200 μg/mL."

Figure 3

DAPI-stained cells for the detection of DNA damage (Fluorescence microscopy, ×40)D1: 100 μg/mL; D2: 150 μg/mL; D3: 200 μg/mL; DAPI: 4′,6-diamidino-2-phenylindole dihydrochloride."

Figure 4

Determination of apoptosis and necrosis percentages by lactate dehydrogenase activity assay of control and drug-treated A375 cells The drug treatment lasted for 48 h. The results are expressed as mean ± standard error of mean, n=3; △P<0.05, vs control group. D1: 100 μg/mL; D2: 150 μg/mL; D3: 200 μg/mL."

Figure 5

Analysis of accumulation of reactive oxygen species A: Fluorescence microscopic study (×20); B: FACS analysis; C: H2DCFDA-stained A375 cells. In figure B, ‘M’ represents an arbitrary area. The cell populations (within the area of ‘M’s) are calculated in percentages considering the cell populations of the total area as 100 % and represented in a graph in figure C. The results in figure C are expressed as mean ± standard error of mean, n=3; △P<0.05, vs control group. D1: 100 μg/mL; D2: 150 μg/mL; D3: 200 μg/mL; FACS: fluorescence-activated cell sorting; H2DCFDA: dichloro-dihydrofluorescein diacetate."

Figure 6

Expression of mRNA and proteinA: Up-regulated gene expression: the bar diagram represents the expression level of mRNA and protein; B: Down-regulated gene expression: the bar diagram represents the expression level of mRNA and protein; C: Expression of G3PHD at mRNA and protein level. G3PDH serves as the housekeeping gene. The results are expressed as mean ± standard error of mean, n=3; △P<0.05, vs control group. D1: 100 μg/mL; D2: 150 μg/mL; D3: 200 μg/mL; G3PDH: glyceraldehyde 3-phosphate dehydrogenase."

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