Introduction

Current therapeutic approaches for patients with non-Hodgkin lymphoma (NHL) include

chemotherapy, signal transduction inhibitors, radiation and immunotherapy; bone marrow

transplantation has become more frequent for patients who fail initial therapies. Although

these treatments are often initially successful, most patients eventually become refractory and die of the disease. NHL is the sixth most common cause of cancer-related death in the United

States [1–3]. The median age of lymphoma patients is 66 years old. The fastest growing segment of the population acquiring NHL is elderly males. Many of these patients cannot tolerate

standard chemotherapy, hence efficacy is severely limited by toxicity. Therefore, less toxic,

more effective therapeutics are needed.

According to a U.S. government survey, approximately 38% of adults and 12% of children

use some form of complementary and alternative medicine (CAM) [4]. The use of many forms

of CAM has significantly increased in prevalence over the last few decades. Although not specifically studied, the use of CAM in oncology patients is likely even higher due to a desperate

attempt of cancer patients to find therapies that are perceived to be more effective and less

toxic than conventional medicines. While the use of CAM has increased, scientific research

has provided little evidence of their efficacy. There needs to be better, more rigorous scientific

studies to back up these claims before they can be recommended for clinical use.

Direct cytotoxicity

Direct cytotoxic activity of FWGE was assayed by incubating 5 x 104 cells/well (96-well plates)

in 100 μl culture medium with the indicated concentrations of FWGE for up to 72 hours at

37˚C, 5% CO2. Cell viability was assessed using an MTS-based assay (Promega) according to

the manufacturer’s instructions and compared to untreated controls. IC50 values were calculated by fitting the dose-response data to a dose–inhibition curve using GraphPad Prism software. Cytotoxicity of heat-inactivated FWGE (80˚C, 90 minutes), proteinase K-treated FWGE

(100 μg/ml, 37˚C, 1 hour) and the protein fraction FWGP were assayed in the same way.

Three replicate wells per condition were used in 3 independent experiments.

Apoptosis and cell cycle

Raji or Ramos cells (1 x 106

/ml) were incubated with 200 μg/ml FWGP for 1, 3, 6, 12, 24 and

48 hours, washed with PBS and resuspended in 100 μl Annexin-V binding buffer (10 mM

HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH = 7.4) with 5 μl Annexin-V-Cy5 (BD Pharmingen)

and 1 μg/ml Sytox Green (Thermo Fisher) according to the manufacturer’s instructions. After

staining for 15 minutes, cells were analyzed by flow cytometry using a FACSCanto instrument

(BD); 30,000 events per sample were acquired. Untreated cells were stained as above and used

as controls. Untreated, unstained or single-stained controls were used for compensation. To

assess caspase activity, cells were incubated with FWGP or PBS control as above and stained

with a Vybrant FAM Poly Caspases Assay Kit (Molecular Probes) according to the manufacturer’s instructions. Briefly, 300 μl of cell suspension (1 x 106 cells/ml) were incubated with

VAD-FMK FLICA reagent and Hoechst 33342 for the detection of activated caspases 1, 2, 4, 5,

6, 8 and 9, washed and analyzed by flow cytometry as above. Data were analyzed using FlowJo

software. For cell cycle analysis, cells were fixed in ethanol, washed, and stained with 20 μg/ml

propidium iodide (PI) as previously described [33]; data (50,000 events/sample) were acquired

as noted above.

Cell staining and flow cytometry

Lymphocyte surface and activation markers were stained by resuspending 106 cells in 10 μl

Fc receptor block (TruStain fcX mouse or human, BioLegend) for 10 minutes at 25˚C followed

by 30 μl antibody cocktail for 30 minutes at 4˚C in 96-well round-bottom plates. Cells were

washed twice with PBS and stained with the fixable viability dye (FVD) Zombie near infra-red

(ZNIR, BioLegend, 100 μl/well of a 1:1,000 dilution in PBS) for 15 minutes at room temperature. After washing with 2% FBS/PBS, samples were either acquired immediately or fixed in

4% paraformaldehyde/PBS for 10 minutes at 25˚C. For intracellular staining, fixed cells were

washed 3 times with permeabilization buffer (BioLegend) and incubated with the appropriate

antibodies for 30 minutes at 25˚C followed by 2 final washes and resuspension in 2% FBS/PBS.

Data were acquired in an LRSFortessa (BD Biosciences, San Jose, CA) instrument equipped

with an automated sampling module. All flow cytometry data were analyzed with FlowJo

(Tree Star, Ashland, OR).

Mouse-specific antibodies were as follows (clones indicated in

between parenthesis): AF-700 anti-CD19 (6D5), PE/Cy5 anti-Ly6G/6C (RB6-8C5), PerCP/

Cy5.5 anti-CD4 (RM4-5), BV570 anti-CD11b (M1/70), BV650 anti-CD25 (PC61), Pacific Blue

anti-CD45 (30-F11), APC anti CD69 (H1.2F3), BV785 anti-CD3 (17A2), BV711 anti-CD8a

(53–6.7), PE anti-CD49b (DX5), FITC anti-CD8a (53–6.7), PE/Cy7 anti-CD49b (DX5), AF647

anti-CD49b (DX5) and BV510 anti-CD3 (17A2) from BioLegend, PE anti-Granzyme B

(NGZB) from eBioscience, and V450 anti-CD107a (1D4B) and AF-488 anti-IFNγ (XMG1.2)

from BD Biosciences. Human-specific antibodies were PerCP/Cy5.5 anti-CD3 (OKT3),

BV785 anti-CD56 (5.1H11), PE anti-CD69 (FN50), APC anti-CD25 (M-A251) from BioLegend, and AF-488 anti-INFγ (B27) from BD Biosciences.

qPCR arrays

Quantitative real-time PCR (qPCR) was performed using the Apoptosis and Survival Tier 1–4

H384 panel (Bio-Rad PrimePCR) to examine over 350 genes associated with cell survival and

apoptosis. Total RNA was extracted from control and treated (200 ng/μl) Raji cells at the indicated time points using an RNeasy kit (Qiagen) and reverse-transcribed with the iScript™

Advanced cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instruction. Reactions were run in a 7900HT instrument (Applied Biosystems) using SsoAdvanced™ Universal SYBR1 Green Supermix (Bio-Rad). Data were normalized and analyzed with the PrimePCR

analysis software (Bio-Rad). Selected genes were validated by immunoblotting.

Immunoblotting

Five million Raji cells were incubated with 200 μg/ml FWGP or PBS control in 5 ml culture

medium at 37˚C, 5% CO2. At 2, 6, 12, 24 and 48 hours, 1-ml aliquots were collected, centrifuged and cells washed with PBS. Cell pellets were lysed in 100 μL of RIPA buffer (150 mM

NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 50 mM Tris-HCl, pH = 7.2) supplemented with protease inhibitors on ice for 30 minutes with occasional vortexing. Immunoblotting was done as previously described [34, 35]. Briefly, cell lysates (50 μg protein/lane in

reducing Laemmli buffer) were run on a 10% SDS-PAGE gel and transferred to nitrocellulose.

Membranes were blocked with 5% BSA or 5% non-fat dry milk in PBS and incubated with primary antibodies (4˚C, overnight) diluted as indicated in 5% BSA in PBS with 0.01% Tween-20

(PBS-T). Membranes were washed with PBS-T and incubated for 1 hour at room temperature

with HRP-labeled secondary antibodies, washed and developed with Luminata Crescendo

(Millipore) detection reagent. Signal intensity was quantified using ImageJ software and normalized to load controls (GAPDH).

Killing assays

Killing assays were performed by incubating effector and target cells at the specified ratios for

4 or 24 hours, followed by flow cytometric quantification of double-labeled target cells. For

mouse samples, 0.5 μl CFSE (stock = 10 mM in DMSO, eBioscience) were added to 5 x 105 target YAC-1 cells in 1 ml 5% HI-FBS/PBS in a 15-ml conical tube, mixed immediately and incubated for 5 minutes at room temperature. Labeling was stopped by adding 2–3 ml HI-FBS and

culture medium to fill the tube. Cells were centrifuged (5 minutes, 300 x g, 24˚C), resuspended

at 1 x 106 cells/ml in culture medium and allowed to recover overnight. Mouse splenocytes

were T-cell depleted by incubating with 1.5 μg/106 cells anti-Thy1.2 (BioLegend, clone

30-H12) for 30 minutes at 4˚C, washing and incubating with rabbit serum complement

(Cedarlane, Burlington, NC) at the lot-specific recommended dilution for 45 minutes at 37˚C.

T-cell depleted (TCD) splenocytes were then washed twice and resuspended in culture

medium. Twenty thousand CFSE-YAC-1 cells were incubated with TCD splenocytes in

96-well round-bottom plates, in a final volume of 200 μl containing recombinant human IL-2

(rhIL-2, 1,000 IU/ml, Biological Resources Branch, NCI, Frederick, MD). After 4 h, cells were

centrifuged, washed with PBS and resuspended in 100 μl FVD eFluor 455UV (1:1000 dilution

in PBS, Thermo Fisher) for 30 minutes at 4˚C. Cells were washed with 2% FBS/PBS and resuspended in the same buffer for acquisition in a Fortessa (BD) flow cytometer. Dead target cells

were defined as the CFSE+

FVD+ population.

Degranulation assays

TCD splenocytes (105 cells/200 μl/well) were plated with YAC-1 cells at the indicated ratios in

RPMI-1640, 10% HI-FBS, 1,000 IU/ml rhIL-2. V450 anti-CD107a (BD, clone 1D4B, 0.25 μg/

well) was added and incubated at 37˚C for 1h. Protein transport inhibitor cocktail (ThermoFisher) was then added to each well and further incubated for 4 h. Cells were then

washed twice with PBS/2% FBS and stained for CD3 and CD49b. Dead cells were stained with

ZNIR (BioLegend). Controls included effector-only cells (non-stimulated) and cells stimulated

with PMA+ionomycin (Cell Stimulation Cocktail, Thermo Fisher).

Animals

For xenograft experiments, female 6-8-week-old nu/nu mice (Harlan, Indianapolis, IN) were

maintained in micro-isolation cages under pathogen-free conditions at the UC Davis animal

facility. All procedures were conducted under an approved protocol according to national and

institutional guidelines. Three days after whole body irradiation (400 rads), Raji human lymphoma cells (1 x 106 in 100 μl PBS) were implanted subcutaneously on the left flank. Either on

the day of tumor implantation (preemptive), or once approximately 300 mm3 tumors had

been established (~20 days), mice were randomly divided into treatment groups (n = 8–10).

Treatment (FWGE, FWGP or PBS) was administered by gavage once daily 5 days per week for

the duration of the study. Tumors were measured twice per week using a digital caliper; tumor

volumes were calculated using the equation: (length x width x depth) x 0.52. Tumor responses

were categorized as follows: cure (C, tumor disappeared and did not re-grow by the end of the

84-day study); complete regression (CR, tumor disappeared for at least 7 days but later regrew); partial regression (PR, tumor volume decreased by 50% or more for at least 7 days then

re-grew).

Ethics

All animal work has been conducted according to relevant national and international guidelines under approved protocols from the University of California Davis Institutional Animal

Care and Use Committee (AAALAC accreditation #000029; PHS Animal Assurance #A3433-

01; USDA Registration #93-R-0433). Human cells were collected from discarded leukapheresis

bags under protocols approved by The University of California Davis Institutional Review

Board Administration. Informed written consent was obtained at the time of collection. The

need of consent for the of discarded, anonymized leukapheresis bags was waived by the ethics

committee. No patient was recruited or sample collected for the sole purpose of this study.