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Journal of Chinese Integrative Medicine ›› 2013, Vol. 11 ›› Issue (4): 269-277.doi: 10.3736/jintegrmed2013040

• Research Article • Previous Articles     Next Articles

Antineoplastic effects of deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, on lung adenocarcinoma (A549) cells

Farha A. Kabeera, Geetha B. Sreedevib, Mangalam S. Nairc, Dhanya S. Rajalekshmic, Latha P. Gopalakrishnanb, Sujathan Kunjuramana, Remani Prathapana   

  1. a. Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Pin 695011, Kerala, India
    b. Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Pin 695562, Thiruvananthapuram, India
    c. National Institute of Interdisciplinary Science and Technology, Pin 695019, Thiruvananthapuram, India
  • Received:2013-01-23 Accepted:2013-05-29 Online:2013-07-10 Published:2013-07-15

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Objective

Deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, showed inhibition of the growth of various tumor cells in vitro. In the present study, we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma (A549) cells.


Methods

The cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 50% inhibitory concentration (IC50) value was determined. The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay. Cellular morphology of deoxyelephantopin-treated cells was observed using phase-contrast microscopy. The induction of apoptosis was evaluated using acridine orange and ethidium bromide staining, Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) assay, DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry. Activation of caspases was detected using ?uorogenic substrate speci?c to caspases 2, 3, 8 and 9 and flow cytometric analysis. The total cellular DNA content and expression of cleaved poly (ADP-ribose) polymerase was also analyzed.


Results

Deoxyelephantopin exhibited cytotoxicity to A549 cells (IC50 = 12.287 μg/mL), however, there was no toxicity towards normal human lymphocytes. Deoxyelephantopin suppressed the colony-forming ability of A549 cells in a dose-dependent manner. Acridine orange, ethidium bromide and Hoechst 33342 staining showed cell shrinkage, chromosomal condensation and nuclear fragmentation, indicating induction of apoptosis. Deoxyelephantopin increased apoptosis of A549 cells, as evidenced by more TUNEL-positive cells. DNA fragmentation and Annexin V staining revealed late-stage apoptotic cell population. Deoxyelephantopin inhibited A549 cell growth by cell cycle arrest at G2/M phase and induced apoptosis through both extrinsic and intrinsic pathways.


Conclusion

These results suggest that deoxyelephantopin has great potential as a new chemotherapeutic agent to be developed further for the treatment of lung cancer.

Key words: Herbal medicine, Lung neoplasms, Elephantopus scaber, Deoxyelephantopin, Apoptosis, Caspases, Cell line, Tumor, A549 cells

Figure 1

Chemical structure of deoxyelephantopin"

Figure 2

Cytotoxic effect of deoxyelephantopinA: A549 cells were treated with varying concentration of deoxyelephantopin for 24, 48 and 72 h and viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 50% inhibitory concentration value was plotted using Easy plot software. B: Effect of deoxyelephantopin on the viability of lymphocytes after 72 h of incubation. All experiments were performed in triplicate and data are presented as mean ± standard deviation."

Figure 3

Effects of deoxyelephantopin on colony formationA549 cells were treated with deoxyelephantopin for 2 h and colony formation rate was calculated after 14 d. Data are presented as mean ± standard deviation. **P < 0.01, vs control group. C: control."

Figure 4

Apoptosis induction in A549 cells by deoxyelephantopinA: Morphological changes analyzed by inverted microscopy; B: Acridine orange and ethidium bromide staining; C: Hoechst 33342 staining; D: Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling staining by flourescent microscopy (20 × magnification)."

Figure 5

Annexin V-propidium iodide staining for detecting deoxyelephantopin-induced apoptosis in A549 cellsA: Control A549 cells; B: A549 cells treated with deoxyelephantopin at dose of 12.287 μg/mL. Apoptotic percentages of cells were analyzed by flow cytometric analysis of Annexin V- and propidium iodide-stained cells."

Figure 6

DNA fragmentation in A549 cells treated with deoxyelephantopinLane 1: marker; Lane 2: A549 cells control; Lanes 3 and 4: A549 cells treated with 12.287 μg/mL deoxyelephantopin. DNA fragmentation was detected by agarose gel electrophoresis and visualized by ethidium bromide staining under ultraviolet light."

Figure 7

Effects of deoxyelephantopin on expression of caspases in A549 cellsA: Flow cytometric analysis of caspase-3 expression; B: Caspase 2/3/8/9 expression tested by flourimetric analysis; C: Effect of caspase-2, -3, -8 and -9 inhibitors in the viability of DOE-treated A549 cells tested by MTT assay. Data are presented as mean ± standard deviation. **P < 0.01, vs control group."

Figure 8

Cell cycle profile in A549 cells treated with deoxyelephantopinA: A549 control cells; B: A549 cells treated with 12.287 μg/mL deoxyelephantopin; C: Representative graph showing cell cycle phase distribution of A549 cells treated with deoxyelephantopin. A549 cells were subjected to flow cytometric analysis by propidium iodide staining. Data are presented as mean ± standard deviation. *P < 0.05, vs control group; ns: no signicant."

Figure 9

Effects of deoxyelephantopin on PARP expression in A549 cellsA549 cells were treated with deoxyelephantopin and cleaved PARP expression was analyzed by Western blot method. β-actin was used as a loading control for normalization of protein expression. PARP: poly (ADP-ribose) polymerase."

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