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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. GopalakrishnanbSujathan 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

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."

[1] Nu?ez G, Benedict MA, Hu Y, Inohara N . Caspases: the proteases of the apoptotic pathway[J]. Oncogene, 1998,17(25):3237-3245
doi: 10.1038/sj.onc.1202581
[2] Kaufmann SH, Earnshaw WC . Induction of apoptosis by cancer chemotherapy[J]. Exp Cell Res, 2000,256(1):42-49
doi: 10.1006/excr.2000.4838
[3] Hammer ML, Johns EA . Tapping an Amaz?nian plethora: four medicinal plants of Marajó Island, Pará(Brazil)[J]. J Ethanopharmocol, 1993,40(1):53-75
doi: 10.1016/0378-8741(93)90089-N
[4] Sankar V, Kalirajan R , Sweetlin Vivian Sales F, Raghuraman S.Antiinflammatory activity of Elephantopus scaber in albino rats[J]. Indian J Pharm Sci, 2001,63(6):523-525
[5] Daisy P, Priya CE . Hypolipidemic and renal functionality potentials of the hexane extract fractions of Elephantopus scaber Linn[J]. Int J Biomed Sci, 2010,6(3):241-245
[6] Rajesh MG, Latha MS . Hepatoprotection by Elephantopus scaber Linn. in CCl4-induced liver injury[J]. Indian J Physiol Pharmacol, 2001,45(4):481-486
[7] Daisy P, Jasmine R, Ignacimuthu S, Murugan E . A novel steroid from Elephantopus scaber L. an ethanomedicinal plant with antidiabetic activity[J]. Phytomedicine, 2009,16(2-3):252-257
doi: 10.1016/j.phymed.2008.06.001
[8] Geetha BS, Latha PG, Remani P . Evaluation of Elephantopus scaber on the inhibition of chemical carcinogenesis and tumor development in mice[J]. Pharm Biol, 2010,48(3):342-348
doi: 10.3109/13880200903133845
[9] Singh SDJ, Krishna V, Mankani KL, Manjunatha BK, Vidya SM, Manohara YN . Wound healing activity of the leaf extracts and deoxyelephantopin isolated from Elephantopus scaber Linn[J]. Indian J Pharmacol, 2005,37(4):238-242
doi: 10.4103/0253-7613.16570
[10] Geetha BS, Nair MS, Latha PG, Remani P . Sesquiterpene lactones isolated from Elephantopus scaber L. inhibits human lymphocyte proliferation and the growth of tumour cell lines and induces apoptosis in vitro[J]. J Biomed Biotechnol, 2012,2012:721285
[11] Huang CC, Lo CP, Chiu CY, Shayur LF . Deoxyelephantopin, a novel multifunctional agent, suppresses mammary tumour growth and lung metastasis and doubles survival time in mice[J]. Br J Pharmacol, 2010,159(4):856-871
doi: 10.1111/j.1476-5381.2009.00581.x
[12] Su M, Wu X, Chung HY, Li Y, Ye W . Antiproliferative activities of five Chinese medicinal herbs and active compounds in Elephantopus scaber[J]. Nat Prod Commun, 2009,4(8):1025-1230
[13] Than NN, Fotso S, Sevvana M, Sheldrick GM, Fiebig HH, Kelter G, Laatsch H . Sesquiterpene lactones from Elephantopus scaber. Z Naturforsch. 2005; 60b:200-204.
[14] Su M, Chung HY, Li Y . Deoxyelephantopin from Elephantopus scaber L. induces cell-cycle arrest and apoptosis in the human nasopharyngeal cancer CNE cells[J]. Biochem Biophys Res Commun, 2011,411(2):342-347
doi: 10.1016/j.bbrc.2011.06.144
[15] Katz D, Ito E, Lau KS, Mocanu JD, Bastianutto C, Schimmer AD, Liu FF . Increased efficiency for performing colony formation assays in 96-well plates: novel applications to combination therapies and high-throughput screening.Biotechniques. 2008; 44(2): ix-xiv.
[16] Liu RH . Potential synergy of phytochemicals in cancer prevention: mechanism of action.[J] Nutr. 2004; 134(12 Suppl):3479S-3485S.
doi: 10.1093/jn/134.12.3479S
[17] Xu G, Liang Q, Gong Z, Yu W, He S, Xi L . Antitumor activities of the four sesquiterpene lactones from Elephantopus scaber L. Exp Oncol. 2006,28(2):106-109.
[18] Lee WL, Shyur LF . Deoxyelephantopin impedes mammary adenocarcinoma cell motility by inhibiting calpain-mediated adhesion dynamics and inducing reactive oxygen species and aggresome formation[J]. Free Radic Biol Med, 2012,52(8):1423-1436
doi: 10.1016/j.freeradbiomed.2012.01.020
[19] Zhang S, Won YK, Ong CN, Shen HM . Anti-cancer potential of sesquiterpene lactones: bioactivity and molecular mechanisms[J]. Curr Med Chem Anticancer Agents, 2005,5(3):239-249
doi: 10.2174/1568011053765976
[20] Varalakshmi KN, Sangeetha CG, Samee US, Irum G, Lakshmi H, Prachi SP . In vitro safety assessment of effect of five medicinal plants on human peripheral lymphocytes[J]. Trop J Pharm Res, 2011,10(1):33-40
[21] Aithal BK, Kumar MR, Rao BN, Udupa N, Rao BS . Juglone, a naphthoquinone from walnut, exerts cytotoxic and genotoxic effects against cultured melanoma tumor cells[J]. Cell Biol Int, 2009,33(10):1039-1049
doi: 10.1016/j.cellbi.2009.06.018
[22] Budihardjo I, Oliver H, Lutter M, Luo X, Wang X . Biochemical pathways of caspase activation during apoptosis[J]. Annu Rev Cell Dev Biol, 1999,15:269-290
doi: 10.1146/annurev.cellbio.15.1.269
[23] Robertson JD, Enoksson M, Suomela M, Zhivotovsky B, Orrenius S . Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis[J]. J Biol Chem, 2002,277(33):29803-29809
doi: 10.1074/jbc.M204185200
[24] Li H, Bergeron L, Cryns V, Pasternack MS, Zhu H, Shi L, Greenberg A, Yuan J . Activation of caspase-2 in apoptosis[J]. J Biol Chem, 1997,272(34):21010-21017
doi: 10.1074/jbc.272.34.21010
[25] Lavrik IN, Golks A, Baumann S, Krammer PH . Caspase-2 is activated at the CD95 death-inducing signaling complex in the course of CD95-induced apoptosis[J]. Blood, 2006,108(2):559-565
doi: 10.1182/blood-2005-07-007096
[26] Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, Munday NA, Raju SM, Smulson ME, Yamin TT, Yu WL, Miller DK . Identi?cation and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis[J]. Nature, 1995,376(6535):37-43
doi: 10.1038/376037a0
[27] Carnero A . Targeting the cell cycle for cancer therapy[J]. Br J Cancer, 2002,87(2):129-133
doi: 10.1038/sj.bjc.6600458
[28] Hartwell LH, Weinert TA . Checkpoints: controls that ensure the order of cell cycle events[J]. Science, 1989,246(4930):629-634
doi: 10.1126/science.2683079
[29] Lewin B . Driving the cell cycle: M phase kinases, its partners, and substrates[J]. Cell, 1990,61(5):743-752
doi: 10.1016/0092-8674(90)90181-D
[30] Makin G, Hickman JA . Apoptosis and cancer chemotherapy[J]. Cell Tissue Res, 2000,301(1):143-152
doi: 10.1007/s004419900160
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