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Journal of Integrative Medicine ›› 2014, Vol. 12 ›› Issue (2): 102-114.doi: 10.1016/S2095-4964(14)60015-7

• Research Article • Previous Articles     Next Articles

Oleanolic acid isolated from ethanolic extract of Phytolacca decandra induces apoptosis in A375 skin melanoma cells: Drug-DNA interaction and signaling cascade

Samrat Ghosh, Kausik Bishayee, Anisur Rahman Khuda-Bukhsh   

  1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India
  • Received:2013-11-26 Accepted:2014-01-03 Online:2014-03-10 Published:2018-10-22

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Objective

Oleanolic acid (OA) has been reported to have anticancer effects, but the extent of its cytotoxicity, its ability to interact with nuclear DNA, its action against skin melanoma, as well as the molecular mechanism of its action against cell proliferation and in support of cell death are still unexplored. This led us to examine the efficacy of OA, a bioactive compound isolated from Phytolacca decandra, on these issues in the present investigation. 

Methods

Studies related to analyses of cell viability, drug-DNA interaction, cell proliferation, cell cycle and epidermal growth factor receptor (EGFR) activity were performed. To investigate whether cells undergo apoptosis, studies like fluorescence microscopy, poly (ADP-ribose) polymerase (PARP) degradation, annexin V-fluorescein isothiocyanate/propidium iodide assay, alteration in mitochondrial membrane potential and activity of some relevant signaling proteins were performed. 

Results

OA displayed a minimal and negligible cytotoxic effect on normal HaCaT cells (skin keratinocytes) and peripheral blood mononuclear cells but by contrast it reduced A375 cell viability significantly. OA interacted with nuclear DNA quickly after exposure. It acted as an anti-proliferative agent. It suppressed EGFR activity. OA administration led the cells to mitochondria-dependent caspase 3-mediated apoptosis. 

Conclusion

OA interacts with cellular DNA, inhibits proliferation possibly through modulating EGFR activity and induces mitochondria-dependent caspase 3-mediated apoptosis in A375 cells which would qualify it as a potent anticancer agent.

Key words: Oleanolic acid, Skin melanoma, Drug-DNA interaction, Proliferation, Epidermal growth factor receptor, Apoptosis

Figure 1

Mass spectra and 1H NMR (A) Mass spectra of oleanolic acid isolated from ethanolic extract of Phytolacca decandra. Mass spectroscopy data revealed that the molecular weight of isolated compound from ethanolic extract of P. decandra was m/z: 479.73 [M+23]+, which implied that the actual molecular weight of the compound was approximately (479.73-23.00) or 456.73. (B) 1H NMR of oleanolic acid isolated from ethanolic extract of P. decandra. 1H NHR: 1H nuclear magnetic resonance."

Figure 2

FTIR spectra, molecular structure of OA and cell viability assay (A) FTIR spectra of OA isolated from ethanolic extract of Phytolacca decandra. (B) Molecular structure of OA. (C) Cell viability assay. Cell viabilities were assessed by using MTT assay. A375, HaCaT and PBMCs were exposed to different concentrations of OA (0-48.17 μmol/L) for 24 h. The values of three independent experiments were represented as mean ± standard error of mean. Statistical significance was considered as P<0.05. *P<0.05, vs relevant control. FTIR: Fourier transform infrared; OA: oleanolic acid; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBMCs: peripheral blood mononuclear cells."

Figure 3

Drug-DNA interaction study, monolayer growth assay and cell cycle assayDNA binding capacity of OA was determined by CD spectroscopy. (A) Interaction with CT-DNA. The CT-DNA (100 μmol/L) was exposed to 40.7 μmol/L OA. Uppermost curve indicated in black represents vehicle-treated CT-DNA and the ash coloured curve represents OA-treated CT-DNA. (B) Interaction with cellular DNA. A375 cells were treated with 40.7 μmol/L of OA for 3 h. Thereafter, cellular DNA was isolated, and purified and interaction was checked. (C) Thermal denaturation study of CT-DNA: thermal denaturation of CT-DNA (30 μg/mL) in absence (triangles) and presence (circles) of OA (40.7 μmol/L). (D) Monolayer growth assay. Treatment with 40.70 μmol/L of OA reduced the proliferation of the A375 cells in a time-dependent manner at late hours of treatment. The bars represented the fractional value of cell count, considering the cell count of control as unity. Statistical significance was considered as P<0.05. *P<0.05, vs relevant control. (E) Cell cycle analysis. Propidium iodide-based FACS (FL2 filter) assay was performed to analyze the cell cycle progression. The percentages of cells of various phases have been mentioned. OA: oleanolic acid; CT-DNA: calf thymus-DNA."

Figure 4

EGFR activity assay and activity study on its downstream signaling proteins EGFR activity has been assessed by immunoblot analysis following treatment with OA (40.70 μmol/L) for 3 h and 6 h in presence or absence of EGF (10 nmol/L). Cells were cultured in serum-free medium for 12 h before being treated with EGF only or with EGF and OA. (A) Immunoblotting pictures of EGFR and pEGFR. (B) The corresponding densitometric analysis of intensities of immunoblots. Statistical significance was considered as P<0.05. *P<0.05, vs control for EGFR 3 h; △P<0.05, vs control for EGFR 6 h; ▲P<0.05, vs control for pEGFR 3 h; □P<0.05, vs control for pEGFR 6 h, respectively. (C) Immunoblotting pictures of pAkt, Erk1/2, and pSTAT5 after exposing the A375 cells to OA (D1 = 20 μmol/L, D2 = 30 μmol/L, D3 = 40.7 μmol/L) for 24 h. (D) The corresponding densitometric analysis of intensities of immunoblots has been plotted. Statistical significance was considered as P<0.05. *P<0.05, vs control. EGFR: epidermal growth factor receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

Figure 5

Apoptosis analysis Cells were treated with OA of specified doses (D1 = 20 μmol/L, D2 = 30 μmol/L, D3 = 40.7 μmol/L) for 24 h. (A) Nucleosomal fragmentation analysis. OA-treated cells showed DAPI-positive whereas control cells were DAPI-negative. Magnification was 200×. (B) PARP degradation study by immunoblot assay. The densitometric plot of corresponding band intensity shows the increase in intensity in a dose-dependent manner. Statistical significance was considered as P<0.05. *P<0.05, vs control. (C) Annexin V-FITC/PI dual stain assay. Assessment of externalization of phosphatidyl serine by annexin V/PI assay was done by FACS analysis. LL–: lower left (Annexin V–/PI–); UL–: upper left (Annexin V–/PI+); LR–: lower right (Annexin V+/PI–); UR–: upper right (Annexin V+/PI+). The corresponding percentages of cells present on those quadrants were mentioned in upper right corners. DAPI: 4′-6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate; PI: propidium iodide; PARP: poly (ADP-ribose) polymerase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

Figure 6

MMP alteration detection, immunoblotting of some signaling proteins and caspase 3 activity assay (A) Detection of MMP alteration. Change in mitochondrial membrane potential was evaluated by FACS analysis, after treating the cells with 40.70 μmol/L of OA for 12, 18, and 24 h. The shifting of fluorescence peak in the overlay histogram, obtained from flow cytometric data (using FL1 filter), was towards the y-axis and the shifting was most at the 24th hour of OA treatment. ‘M’ represents an arbitrary area along the rhodamine 123 fluorescence axis. The cell populations (within the area of ‘M’) are calculated in percentages and mentioned in the figure. (B) Immunoblotting. Cells were treated for 24 h with specified doses (D1 = 20 μmol/L, D2 = 30 μmol/L, D3 = 40.7 μmol/L). GAPDH was served as an equal loading control. The densitometric plot of relative band intensities revealed that the regulation was dose-dependent. (C) Caspase 3 activity assay. Caspase 3 activity was detected by immunoblot assay. Cells were treated with 40.70 μmol/L of OA only for 24 h or treated with 40.70 μmol/L of OA for 24 h after pre-treated with z-DEVD-fmk (caspase 3 inhibitor, 10 μmol/L) for 2 h. Relative band intensities of cleaved caspase 3 and cleaved PARP fragment revealed up-regulated expression of both upon treatment with OA only whereas substantial inhibition of caspase 3 and cleaved PARP levels was observed following pre-treatment with caspase 3 inhibitor with respect to cells treated with OA only. Statistical significance was considered as P<0.05. *P<0.05, vs control. MMP: mitochondrial membrane potential; OA: oleanolic acid; PARP: poly (ADP-ribose) polymerase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

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