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

Original Article

Abstract

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.

Keywords

Wheatgrass Cytoxicity Anti-proliferation Migration Apoptosis and cell cycle 

Abbreviations

HWE

Hexane wheatgrass extract

CWE

Chloroform wheatgrass extract

MWE

Methanol wheatgrass extract

AWE

Aqueous wheatgrass extract

5-FU

5 Fluorouracil

Pen-strep

Penicillin-streptomycin

IC50

Concentration which leads to 50 % cell death

CKI

Cyclin dependent kinases inhibitor

Notes

Acknowledgment

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

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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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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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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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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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