Mitochondrial pathway mediated apoptosis and cell cycle arrest triggered by aqueous extract of wheatgrass in colon cancer colo-205 cells

  • Shagun Arora
  • Simran TandonEmail author
Original Article


Four different extracts of wheatgrass i.e. hexane, chloroform, methanol and aqueous were used to evaluate the anti-cancer potential on COLO-205 cells. Aqueous extract demonstrated maximum anti-cancer activities with significant inhibition on growth of COLO-205 cells. Anti-proliferative capabilities were also enhanced, as observed from clonogenic survival and in vitro wound scratch assay. Increased apoptotic hallmarks were evidenced in aqueous extract treated cells such as, DNA fragmentation, nuclear condensation and membrane blebbing. Increased expression of caspase-9, caspase-3 and Bax genes, along with down-regulation of Bcl-2 gene was also seen. Arrest of cells at G0/G1 phase by flow cytometric analysis could be attributed to up-regulated expression of inhibitors of cell cycle progression i.e. p16, p21 and p27 in aqueous extract treated cells. GC analysis revealed the presence of various chemicals possessing medicinal properties which could be responsible for the effects seen on the cancer cells. Our findings, for the first time suggest that the aqueous extract of wheatgrass represents a potential plant based anti-cancer agent.


Wheatgrass Cytoxicity Anti-proliferation Migration Apoptosis and cell cycle 



Hexane wheatgrass extract


Chloroform wheatgrass extract


Methanol wheatgrass extract


Aqueous wheatgrass extract


5 Fluorouracil




Concentration which leads to 50 % cell death


Cyclin dependent kinases inhibitor



We are thankful to the Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Solan for providing us the infrastructure to carry out this research.

Conflict of interest statement

We declare that we have no conflict of interest.

Supplementary material

13562_2015_309_Fig4_ESM.gif (10.8 mb)
Fig. 1S

Effect on viability of COLO-205 cells treated with different concentrations of (a) 5-FU, (b) HWE, (c) CWE and (d) MWE and (e) AWE for 48 and 72 hours and trypan blue exclusion assay was performed to calculate percent cell viability. Data presented as mean ± S.D (n=3) and compared as percent viability of untreated cells vs treated cells. *p<0.05, **p<0.01, ***p<0.001 (GIF 11084 kb)

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Fig. 2S

Effect on growth of COLO-205 cells treated with IC50 of each extract for a period of 72 hours. (a) Control, (b) 5-FU (3 μg/ml), (c) HWE (393.94 μg/ml), (d) CWE (445.58 μg/ml), (e) MWE (227.77 μg/ml) and (f) AWE (163.6 μg/ml) (Magnification at 100X). (GIF 455 kb)

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Fig. 3S

Inhibitory effects of wheatgrass extracts on the migration of COLO-205 cells. The images demonstrate cell migration over time, as the width of the wound (open scratch area) narrowed after 24 hours incubation. (a) Initial view of scratch at time 0, prior to any treatment (b) Control, after 24 hours, (c) 5-FU (3 μg/ml), (d) HWE (393.94 μg/ml), (e) CWE (445.58 μg/ml), (f) MWE (227.77 μg/ml) and (g) AWE (163.6 μg/ml) treated cells after 24 hours (GIF 234 kb)

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Fig. 7S

Evaluation of apoptotic changes in COLO-205 cells by AO staining. The effect of wheatgrass extracts on COLO-205 cells was observed by staining cells with AO dye. Cells were observed under fluorescence microscope at 200 X. (a) Control, cell treated with (b) 5-FU (3 μg/ml), (c) HWE (393.94 μg/ml), (d) CWE (445.58 μg/ml), (e) MWE (227.77 μg/ml) and (f) AWE (163.6 μg/ml). (JPEG 309 kb)

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Fig. 4S

Colony formation inhibition of COLO-205 cells on treatment with IC50 of 5-FU (3 μg/ml), HWE (393.94 μg/ml), CWE (445.58 μg/ml), MWE (227.77 μg/ml) and AWE (163.6 μg/ml) for a period of 72 hours followed by 10 days incubation (no treatment) the cells were fixed with 4 % paraformaldehyde and stained with crystal violet . Data presented as percent colony formation of wheatgrass extract treated COLO-205 cells and compared with control (untreated cells) (n=3). *p<0.05, **p<0.01, ***p<0.001 (GIF 4846 kb)

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13562_2015_309_Fig9_ESM.gif (506 kb)
Fig. 5S

Anti-proliferative effect of wheatgrass extracts on COLO-205 cells. Trypan blue viability assay was used to measure the effect of (a) 5-FU, (b) HWE, (c) CWE, (d) MWE and (e) AWE on cell proliferation after a treatment period of 48 hours, followed by performing cell count post 48 hours media replacement and similarly with 72 hours treatment and post 24 hours media replacement. Data presented as percent proliferation of treated cells (n=3) compared to untreated cells. *p<0.05, **p<0.01, ***p<0.001. (GIF 505 kb)

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Fig. 6S

Evaluation of apoptotic changes in COLO-205 cells by Hoechst 33258 staining. A change in nuclear morphology was observed after 72 hours of treatment with wheatgrass extracts. Cells were observed under fluorescence microscope at 100 X. (a) Control, on treatment with (b) 5-FU (3 μg/ml), (c) HWE (393.94 μg/ml), (d) CWE (445.58 μg/ml), (e) MWE (227.77 μg/ml) and (f) AWE (163.6 μg/ml). (GIF 354 kb)

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High resolution image (TIFF 448 kb)
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Fig. 8S

Evaluation of apoptotic changes in COLO-205 cells by Annexin V/PI staining. An exposure of phosphatidylserine on cell surface was observed after 72 hours treatment with IC50 of 5-FU (3 μg/ml), HWE (393.94 μg/ml), CWE (445.58 μg/ml), MWE (227.77 μg/ml) and AWE (163.6 μg/ml). Cells were observed under fluorescence microscope at 100 X. (a) Control, (c) 5-FU, (e) HWE, (g) CWE, (i) MWE and (k) AWE observed for uptake of Annexin V dye and (b) Control, (d) 5-FU, (f) HWE, (h) CWE, (j) MWE and (l) AWE for PI stain. (GIF 453 kb)

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Fig. 9S

Induction of DNA fragmentation in the COLO-205 cells. Fragmentation of genomic DNA was studied in COLO-205 cells exposed to IC50 of wheatgrass extracts for 72 hours. Genomic DNA was isolated and electrophoresed as described earlier. Lane 1- Ladder, Lane 2- untreated cells, lane 3- HWE (393.94 μg/ml) treated cells, Lane 4- CWE (445.58 μg/ml), treated cells, Lane 5- AWE (163.6 μg/ml) treated cells, Lane 6- MWE (227.77 μg/ml) treated cells, Lane7- Ladder, Lane 8- untreated cells and Lane 9- 5-FU (3 μg/ml) treated cells. (GIF 113 kb)

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Fig. 10S

Quantitative assessment of DNA fragmentation by DPA method. The percent DNA fragmentation of 5-FU (3 μg/ml), HWE (393.94 μg/ml), CWE (445.58 μg/ml), MWE (227.77 μg/ml) and AWE (163.6 μg/ml) treated COLO-205 cells was measured by DPA method and compared with control COL0-205 cells. *p<0.05, **p<0.01, ***p<0.001. (GIF 5665 kb)

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Fig. 11S

Enhanced cytotoxic effect of AWE treated COLO-205 cells in presence of β-glucosidase. The percent cytotoxicity was calculated by SRB assay and compared with percent cytotoxicity between AWE + β-glucosidase treated cells and AWE treated cells. Data presented as mean ± S.D (n=3). A comparison was made between the treatments for each dose selected. *p<0.05, **p<0.01. (GIF 386 kb)

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13562_2015_309_MOESM11_ESM.doc (17 kb)
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ESM 2 (DOC 32 kb)


  1. Allen J, Sotos M, Sylte J et al (2001) Use of hoechst 33342 staining to detect apoptotic changes in bovine mononuclear phagocytes infected with Mycobacterium aviumsubsp paratuberculosis. Clin Diagn Lab Immunol 8:460–464PubMedPubMedCentralGoogle Scholar
  2. Ando K, Urano M, Koike S (1978) Cytocidal and cytostatic ability of Corynebacterium liquefaciens in mouse squamous cell carcinoma in vivo. Cancer Res 38:1769–1773PubMedGoogle Scholar
  3. Arora S, Tandon S (2015) DNA fragmentation and cell cycle arrest:a hallmark of apoptosis induced by Ruta graveolens in human colon cancer cells. Homeopathy 104:36–47PubMedCrossRefGoogle Scholar
  4. Arye B, Goldin E, Wengrower D et al (2002) Wheat grass juice in the treatment of active distal ulcerative colitis: a randomized double-blind placebo-controlled trial. Scand J Gastroenterol 37:444–449CrossRefGoogle Scholar
  5. Aydos O, Avci A, Özkan T et al (2011) Antiproliferative, apoptotic and antioxidant activities of wheatgrass (Triticum aestivum l.) extract on cml (k562) cell line. Turkish J Med Sci 41:657–663Google Scholar
  6. Azizi E, Abdolmohammadi M, Fouladdel S et al (2009) Evaluation of p53 and Bcl-2 genes and proteins expression in human breast cancer T47D cells treated with extracts of Astrodaucus persicus (Boiss.) Drude in comparison to Tamoxifen. Daru 17:181–186Google Scholar
  7. Bharathy V, Sumathy M, Uthayakumari F (2012) Determination of phytocomponents by GC – MS in leaves of Jatropha Gossypifolia. L. Sci Res Reporter 2:286–290Google Scholar
  8. Cravotto G, Boffa L, Genzini L, Garella D (2010) Phytotherapeutics: an evaluation of the potential of 1000 plants. J Clin Pharm Ther 35:11–48PubMedCrossRefGoogle Scholar
  9. Deborah R, Nathaniel G, David A, Shellman Y (2005) A Simple Technique for Quantifying Apoptosis in 96-Well Plate. BMC Biotechnol 5: Article ID 1472–6750Google Scholar
  10. Estelle S, Claudie P, Myriam B, Richard B (2007) DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis. J Zhejiang Univ Sci B 8:377–397CrossRefGoogle Scholar
  11. Franken N, Rodermond H, Stap J et al (2006) Clonogenic assay of cells: in vitro. Nat Protoc 1:2315–2319PubMedCrossRefGoogle Scholar
  12. Globocan Colorectal Cancer Estimated Incidence (2012), Mortality and Prevalence Worldwide in 2012.
  13. Hemmati P, Normand G, Verdoodt B et al (2005) Loss of p21 disrupts p14ARF-induced G1 cell cycle arrest but augments p14 ARF-induced apoptosis in human carcinoma cells. Oncogene 24:4114–4128PubMedCrossRefGoogle Scholar
  14. Hohmann J, Forgo P, Schlosser G et al (2003) Isolation and structure determination of new jatrophane diterpenoids from Euphorbia platyphyllos L. Helvetica Chimica Acta 86:3386–3393CrossRefGoogle Scholar
  15. Houghton P, Fang R, Techatanawat I et al (2007) The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods 42:377–387PubMedCrossRefGoogle Scholar
  16. Hu Y, Liu C, Du C et al (2009) Induction of spoptosis in human hepatocarcinoma Smmc-7721 cells in vitro by flavonoids from Astragalus Complanatus. J Ethnopharmacol 123:293–301PubMedCrossRefGoogle Scholar
  17. Kulkarni S, Tilak J, Acharya R et al (2006) Evaluation of the antioxidant activity of wheatgrass (Triticum aestivum L.) as a function of growth under different conditions. Phytotherapy Res 20:218–227CrossRefGoogle Scholar
  18. Ling Q, Xu X, Wei X et al. (2011) Oxymatrine induces human pancreatic cancer PANC-1 cells apoptosis via regulating expression of Bcl-2 and IAP families, and releasing of cytochrome c. J Exp Clin Cancer Res 30: Article ID PMC3141557,6 pagesGoogle Scholar
  19. Manoj J, Kumar T, Varghese S et al (2012) The effect of methanol and water extract of leucobryum bowringii Mitt on growth, migration and invasion of MCF-7 human breast cancer cell lines in vitro. Ind J Exp Biol 50:602–611Google Scholar
  20. Meyerowitz S (1999) From Nutrition in Grass- Wheatgrass Natures’s Finest Medicine. In The Complete Guide to Using Grass Foods & Juices to Revitalize Your Health. 6th edition. Edited by Sproutman Publications 53Google Scholar
  21. Moongkarndi P, Kosem N, Kaslungka S et al (2004) Antiproliferation, antioxidation and induction of apoptosis by Garcinia Mangostona (Mangosteen) on Skbr3 human breast cancer cell line. J Ethnopharmacol 90:161–166PubMedCrossRefGoogle Scholar
  22. Patel J, Patel P (2013) Anticancer and cytotoxic of Triticum Aestivum extract on hela cancer cell line. Int Res J Pharm 4:103–105CrossRefGoogle Scholar
  23. Pigni A, Brunelli C, Caraceni A (2011) The role of hydromorphone in cancer pain treatment: a systematic review. Palliative Med 25:471–477CrossRefGoogle Scholar
  24. Pozarowski P, Darzynkiewicz Z (2004) From Checkpoint Controls and Cancer, Activation and Regulation Protocols in Methods in Molecular Biology. Humana Press Inc., 281(2), 303Google Scholar
  25. Pucci B, Kasten M, Giordano A (2000) Cell cycle and apoptosis. Neoplasia 2:291–299PubMedPubMedCentralCrossRefGoogle Scholar
  26. Samarghandian S, Boskabady MH, Davoodi S (2010) Use of in vitro assays to assess the potential antiproliferative and cytotoxic effects of saffron (Crocus sativus L.) in human lung cancer cell line. Pharmacogn Mag 6:309–314PubMedPubMedCentralCrossRefGoogle Scholar
  27. Shahneh FZ, Valiyari S, Azadmehr A et al (2013) Inhibition of growth and induction of apoptosis in fibrosarcoma cell lines by echinophora platyloba dc: in vitro analysis. Adv Pharmacol Sci 2013:512931. doi: 10.1155/2013/512931 PubMedPubMedCentralGoogle Scholar
  28. Shiohara M, Koike K, Komiyama A et al (1997) p21/WAF1 mutations and human malignancies. Leuk Lymphoma 26:35–41PubMedCrossRefGoogle Scholar
  29. Shukla V, Vashistha M, Singh S (2009) Evaluation of antioxidant profile and activity of amalaki (Emblica Officinalis), spirulina and wheat grass. Ind J of Clin Biochem 24:70–75CrossRefGoogle Scholar
  30. Silva S, Filho M, Anazetti M et al (2011) Xylodiol from Xylopia langsdorfiana induces apoptosis in HL60 cells. Brazil J Pharmacognosy 21:1035–1042Google Scholar
  31. Singh N, Verma P, Pandey B (2012) Therapeutic potential of organic Triticum aestivum Linn. (Wheat Grass) in prevention and treatment of chronic diseases: an overview. Int J of Pharm Sci Drug Res 4:10–14Google Scholar
  32. Singha C, Boopathya N, Kathiresana K et al (2013) Effect of bioactive substances from mangroves on anti-oxidant, anti-bacterial activity and molecular docking study against lung and oral cancer. J Free Rad Antioxidants (Photon) 139:226–236Google Scholar
  33. Tandon S, Arora A, Singh S et al (2011) Antioxidant profiling of Triticum Aestivum (Wheatgrass) and Its antiproliferative activity in MCF-7 breast cancer cell line. J Pharm Res 4:4601–4604Google Scholar
  34. Zinadah O, Khalil K, Ashmaoui H et al (2013) Evaluation of the anti- genotoxicity and growth performance impacts of green algae on Mugil cephalus. Life Sci J 10:1543–1554Google Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2015

Authors and Affiliations

  1. 1.Amity Directorate of Science & InnovationAmity UniversityNoidaIndia
  2. 2.Department of Biotechnology & BioinformaticsJaypee University of Information TechnologySolanIndia

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