Searching in Mother Nature for Anti-Cancer Activity: Anti-Proliferative and Pro-Apoptotic Effect Elicited by Green Barley on Leukemia/Lymphoma Cells

<div><p>Green barley extract (GB) was investigated for possible anti-cancer activity by examining its anti-proliferative and pro-apoptotic properties on human leukemia/lymphoma cell lines. Our results indicate that GB exhibits selective anti-proliferative activity on a panel of leukemia/lymphoma cells in comparison to non-cancerous cells. Specifically, GB disrupted the cell-cycle progression within BJAB cells, as manifested by G2/M phase arrest and DNA fragmentation, and induced apoptosis, as evidenced by phosphatidylserine (PS) translocation to the outer cytoplasmic membrane in two B-lineage leukemia/lymphoma cell lines. The pro-apoptotic effect of GB was found to be independent of mitochondrial depolarization, thus implicating extrinsic cell death pathways to exert its cytotoxicity. Indeed, GB elicited an increase of TNF-α production, caspase-8 and caspase-3 activation, and PARP-1 cleavage within pre-B acute lymphoblastic leukemia Nalm-6 cells. Moreover, caspase-8 and caspase-3 activation and PARP-1 cleavage were strongly inhibited/blocked by the addition of the specific caspase inhibitors Z-VAD-FMK and Ac-DEVD-CHO. Furthermore, intracellular signaling analyses determined that GB treatment enhanced constitutive activation of Lck and Src tyrosine kinases in Nalm-6 cells. Taken together, these findings indicate that GB induced preferential anti-proliferative and pro-apoptotic signals within B-lineage leukemia/lymphoma cells, as determined by the following biochemical hallmarks of apoptosis: PS externalization, enhanced release of TNF-α, caspase-8 and caspase-3 activation, PARP-1 cleavage and DNA fragmentation Our observations reveal that GB has potential as an anti-leukemia/lymphoma agent alone or in combination with standard cancer therapies and thus warrants further evaluation <i>in vivo</i> to support these findings.</p> </div

Dose-dependent activation of caspase-8 pathway by GB in Nalm-6 cells monitored <i>via</i> flow cytometry.

<p>(A) After 4 h of GB treatment, the percentage of caspase-8-positive cells exhibiting green fluorescence signal is indicated on the <i>y</i>-axis, whereas the different cell treatments are indicated on the <i>x</i>-axis. Each bar represents the average of three independent measurements, and the error bars are their corresponding standard deviations. Representative flow cytometric dot plots (B–G) that were used to determine the distribution of caspase-8-positive cells are depicted. The diverse dot (event) color in each plot, designates just a density gradient; low-density region blue and high-density red. The flow cytometer acquisition settings were as following: FL1 and FL2 detectors were plotted at <i>x</i>-axis versus <i>y</i>-axis, respectively. Cells were exposed to 10 µl (B) and 50 µl (C) of GB for 4h and then, stained with FITC-IETD-FMK, as detailed in Materials and methods; this bar is imperceptible due a low value (<0.1%). (D) A set of GB-treated cells was concurrently exposed to 50 µl of GB and 20 µM of Z-VAD-FMK cell-permeant pan-caspase inhibitor (VAD). (E) PBS solvent control, where an error bar 0.11% is not noticeable and (G) untreated cells were included. (F) As a positive control for induction of caspase-8 activation, 2 mM of H₂O₂ was utilized. Approximately, 1x10⁴ events were acquired and analyzed per sample using CXP software. GB 10 µl = 0.3 ± 0.009 mg/ml, and GB 50 µl = 1.5 ±0.048 mg/ml lyophilized powder. The significance of the differences between 10 µl GB-treated cells as compared to 50 µl PBS-treated cells, and also, with untreated cells, is of <i>P</i> = 0.00039 (*) and <i>P</i> = 0.00203 (‡), respectively; whereas 50 µl GB-treated cells as compared with 50 µl PBS-treated cells was consistently <i>P</i><0.0001(**) in both data sets. GB 10 µl = 0.3 ± 0.009 mg/ml, and GB 50 µl = 1.5 ± 0.048 mg/ml lyophilized powder.</p

GB treatment resulted in caspase-3 activation and PARP-1 cleavage in Nalm-6 cells.

<p>Caspase -3 activation was monitored <i>via</i> (A) flow cytometry; whereas PARP-1 was analyzed <i>via</i> (G) Western blotting analysis. (A) The percentage of NucView caspase-3-positive cells exhibiting green fluorescence signal is indicated on the <i>y</i>-axis, whereas the different cell treatments are indicated on the <i>x</i>-axis. Each bar represents the average of three independent measurements, and the error bars are the corresponding standard deviations. After being exposed to GB for 6 (B) and 8 h (C), cells were incubated with a caspase-3 fluorogenic indicator substrate. (D) A set of GB-treated cells was pre-exposed to an Ac-DEVD-CHO caspase-3 inhibitor (DEVD) to add the NucView substrate. (E) As a positive control for induction of caspase-3 activation, 2 µg/ml camptothecin (CPT) was utilized. (F) Untreated cells (Unt) were used as a negative control. (B–F) Representative single-parameter histograms utilized to obtain the percentage of NucView caspase-3 substrate-positive cells: the number of event counts (cells) is plotted on the <i>y</i>-axis, whereas the measurement parameter, cells with green fluorescence signal NuvView positives, is plotted on the <i>x</i>-axis. Approximately, 1x10⁴ events were acquired and analyzed per sample using CXP software. (<b>G</b>) <b>Full-length PARP-1 (116 kDa) and its large fragment (89 kDa) were analyzed on GB-treated Nalm-6 cells after 24 h</b>. As a positive control for PARP-1 cleavage, 2 µg/ml camptothecin was used. Where indicated, some cells were incubated concurrently with GB and the caspase-3 inhibitor DEVD. β-actin was used as a housekeeping protein loading control (bottom panel). The mobility of the protein molecular weight markers is specified on the left. GB 50 µl = 1.5 ± 0.048 mg/ml, and GB 100 µl = 3.0 ± 0.009 mg/ml lyophilized powder.</p

GB caused perturbation of the cell-cycle profile of BJAB cells.

<p>After 96 h GB induced apoptotic DNA fragmentation and G2/M phase arrest in a dose-dependent modality. Cells were harvested, permeabilized and stained and analyzed <i>via</i> flow cytometry. Each bar represents the average of three independent replicates, and the error bars represent the corresponding standard deviation. (A–D) The percentage of cell frequency is graphed along the <i>y</i>-axis, and the different treatments are plotted along the <i>x</i>-axis. (E–I) Representative single parameter histograms where four gates are annotated exhibiting the percentage of cell frequency in each phase of the cell cycle. Gates from left to right: sub-G0/G1 (hypodiploid; counted as an apoptotic subpopulation), G0/G1 (diploid), S (hyperdiploid) and G2/M (tetraploid). This series of experiments included several controls: two compounds provoking cell-cycle alteration, (G) 80 nM etoposide (ETO) and (H) 1 mg/ml G418; (F) the GB diluent, PBS, as contained in the experimental samples; and (I) untreated (Unt) cells, as a negative control. The significance of the differences between 50 µl GB-treated cells as compared to 50 µl PBS-treated cells, and also, with untreated cells, is of <i>P</i> < 0.03 (*) and <i>P</i> < 0.04 (‡), respectively. GB 10 µl = 0.3 ± 0.009 mg/ml, and GB 50 µl = 1.5 ± 0.048 mg/ml lyophilized powder.</p

GB-mediated cytotoxicity is independent of mitochondrial <i>ΔΨm</i> disruption in Nalm-6 cells.

<p>Cells were exposed for 4 h to GB and detection of changes in the mitochondrial <i>ΔΨm</i> was determined by staining with the aggregate-forming lipophilic cationic fluorophore JC-1. After dissipation of mitochondrial <i>ΔΨm</i>, the JC-1 reagent emitted a green fluorescence signal, whereas cells with polarized mitochondrial membrane emitted a red signal. (A) Percentages of cells emitting green fluorescence signal (<i>y</i>-axis) versus treatment type (<i>x</i>-axis) are depicted. Each bar represents the average of four replicates, and the error bars represent the standard deviation. (B–C) Representative flow cytometric dot plots used to quantify the percentages of green and red signals. (C) Cells exposed to the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP; 50 µM) were used as positive controls. PBS solvent controls and untreated cells were included. The different dot (event) color in each plot, designates just a density gradient; low-density region blue and high-density red. Approximately 1x10⁴ events were acquired and analyzed per sample using CXP software. GB 50 µl = 1.5 ± 0.048 mg/ml, and GB 100 µl = 3.0 ± 0.009 mg/ml lyophilized powder.</p

GB exhibited selective anti-proliferative activity on malignant human leukemia/lymphoma cells.

<p>The total numbers of cells per milliliter (<i>y</i>-axis) were quantified using a hemocytometer after 96 h of treatment: (A) Nalm-6, (B) BJAB, (C) YT NK-like, (D) Jurkat and (E) Hs27 cells. As a positive control of an anti-proliferative effect, 1 mg/ml G418 was included. Untreated cells were used as an indicator of cellular viability during all the incubation time periods. Additional controls of the diluent of the extracts, PBS, were also examined. Each bar represents the average of four replicates and the error bars represent the standard deviation. Cell proliferation, annotated of the top of 50 µl GB treated cells, is shown as a percentage of the cell proliferation of PBS-treated cells, which is considered 100% of growth. Fifty µl GB in PBS is equivalent to 1.5 ± 0.048 mg/ml lyophilized powder.</p