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, 2014, 753451

Antitumor Activity of Ethanolic Extract of Dendrobium Formosum in T-cell Lymphoma: An in Vitro and in Vivo Study

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Antitumor Activity of Ethanolic Extract of Dendrobium Formosum in T-cell Lymphoma: An in Vitro and in Vivo Study

Ritika Prasad et al. Biomed Res Int.

Abstract

Dendrobium, a genus of orchid, was found to possess useful therapeutic activities like anticancer, hypoglycaemic, antimicrobial, immunomodulatory, hepatoprotective, antioxidant, and neuroprotective activities. The study was aimed to evaluate the anticancer property of the ethanolic extract of Dendrobium formosum on Dalton's lymphoma. In vitro cytotoxicity was determined by MTT assay, apoptosis was determined by fluorescence microscopy, and cell cycle progression was analysed using flow cytometry; in vivo antitumor activity was performed in Dalton's lymphoma bearing mice. The IC50 value of ethanolic extract was obtained at 350 μg/mL in Dalton's lymphoma cells. Fluorescence microscopy analysis showed significant increase in apoptotic cell death in dose- and time-dependent manner which was further confirmed through the resulting DNA fragmentation. Further, flow cytometry analysis showed that the ethanolic extract arrests the cells in G2/M phase of the cell cycle. The in vivo anticancer activity study illustrates significant increase in the survival time of Dalton's lymphoma bearing mice on treatment with ethanolic extract when compared to control. These results substantiate the antitumor properties of ethanolic extract of Dendrobium formosum and suggest an alternative in treatment of cancer. Further studies are required regarding the isolation and characterization of bioactive components along with the analysis of molecular mechanism involved.

Figures

Figure 1
Figure 1
Cell viability was determined by MTT assay. The graph represents the cytotoxicity profile of D. formosum ethanolic extract against DL cells and mouse BMC at different concentrations (200 μg/mL–600 μg/mL) on 24 h incubation. Results are expressed as a percentage of control ± SEM from at least three independent experiments.
Figure 2
Figure 2
PI staining of nuclei was done to examine morphological changes induced by D. formosum ethanolic extract at 50, 100, 150, 200, and 250 μg/mL concentrations and control under fluorescence microscope after treatment for 3 h, 6 h, 16 h, and 20 h, respectively [blue arrow shows live cells, yellow arrow represents apoptotic cells, and dotted yellow arrow shows presence of apoptotic bodies (late stage apoptosis)].
Figure 3
Figure 3
Acridine orange/ethidium bromide staining was carried out to reveal apoptotic cell morphology of DL treated cells with the ethanolic extract at 50, 100, 150, 200, and 250 μg/mL and a control on 3 h, 6 h, 16 h, and 20 h incubation, respectively [blue arrow shows live cells, red arrows show late apoptotic cells, dotted red arrow shows membrane blebbing in early apoptotic cells, and yellow arrows show necrotic cells].
Figure 4
Figure 4
To determine the percentage of apoptotic cells at different times (3 h, 6 h, 16 h, and 20 h) and at different concentrations (50, 100, 150, 200, and 250 μg/mL) acridine orange/ethidium bromide staining was carried out. Data were plotted as percentage of apoptotic cells and represented as mean ± standard error mean (SEM) of at least three independent experiments. “∗” represents significant difference at P < 0.05 compared to their respective control. “#” represents significant difference from 6 h, 16 h, and 20 h versus 3 h whereas “a” represents significant difference from 6 h to 16 h and “b” represents significant difference from 16 h to 20 h at P < 0.05.
Figure 5
Figure 5
1.8% agarose gel electrophoresis was run to separate DNA fragments after incubation of DL cells with various concentrations (50, 100, 150, 200, and 250 μg/mL) of ethanolic extract for (a) 3 h and (b) 16 h, respectively. DNA fragments separated were visualised under UV light. M: 200 bp DNA ladder marker, C: control. Three independent experiments were performed (n = 3).
Figure 6
Figure 6
(a) Cell cycle analysis in DL cells after D. formosum ethanolic extract treatment was done by flow cytometry. DL cells were incubated with the ethanolic extract (250 and 350 μg/mL) for 24 h and propidium iodide (PI) staining was done to determine the DNA content. (b) The graph shows the percentage of DL cells in different phases of cell cycle on incubation with D. formosum ethanolic extract at 250 and 350 μg/mL for 24 h as analyzed by flow cytometry.
Figure 7
Figure 7
Evaluation of antitumor activity of theethanolic extract against Dalton's lymphoma transplanted mice on 100, 150, and 175 mg/Kg body weight treatment was done by computing median survival time. Data were plotted for median survival days and represented as mean ± standard error mean (SEM) of at least three independent experiments. ∗ represents significant difference at (P < 0.05) compared to control. The median survival time of the treated group of animals (T) divided by control group (C) is given as a percentage and an effective T/C value is considered when the T/C ratio is ≥120% (according to NCI).

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