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Reagents and antibodies and then fi ltered through a 70-μm fi lter and analyzed
by fl ow cytometry (FACScalibur Becton Dickinson or
®
WS roots were purchased from a local market in the C6 Accuri fl ow cytometer). Data were analyzed with
®
USA and dimethyl sulfoxide (DMSO) from Sigma (St. CellQuest and CFlow software (BD).
Louis, MO, USA). Antibodies (anti-chemokine CCL2,
CXCL1, CXCL2, CXCL3, PARP, and GAPDH) were Immunocytochemistry
from Cell Signaling (Beverly, MA, USA). Human breast Breast cancer MDA-MB-231 cells were seeded in 4-well
cancer MDA-MB-231 cell line and a normal breast cell plates and grown for 16 h. The cells were then treated
line, MCF10A, were obtained from ATCC (Manassas,
VA, USA). The HCA-II human cytokine primer kit was with DMSO (vehicle) or with 25 or 50 μg/mL of WS
obtained from Real Time Primers (Elkins Park, PA, root extract for 18 h. After treatment, the culture medium
USA). was removed, and the cells were fi xed with 10% neutral
buffered formalin. Xenograft tissues were placed in an
Cell culture and treatment automatic tissue processor, embedded in paraffi n, sectioned
at 5-μm thickness, and stained with hematoxylin and
Breast cancer MDA-MB-231 cells were maintained
in Dulbecco’s Modified Eagle’s Medium (ATCC) eosin (HE). For immunohistochemistry, the fi xed cells and
supplemented with 10% fetal bovine serum and tissues from xenografted tumors were stained with CCL2
penicillin/streptomycin. MCF10A cells were maintained antibody because this cytokine is considered to be most
[14]
in complete MEGM (Lonza, Houston, TX, USA). All responsible for metastasis of breast cancer. The sections
cell cultures were incubated at 37 °C with 5% CO in a were de-paraffi nized in xylene and rehydrated through a
2
humidifi ed incubator. series of graded ethanol (100%, 95%, and 70%) and in
water for 5 min each. The sections were then washed three
Assessment of cell viability times for 5 min each in PBS containing 0.05% Tween
To assess the effect of the WS extract on regulation 80 (pH 7.4). Antigen retrieval was achieved by heating
of cell viability, cells were seeded into 96-well, 6-well the sections in a microwave with 0.01 mol/L sodium
3
or 6-cm plates at densities of 10 , 10 or 10 cells per citrate (pH 6.0) solution and subsequently cooling down
5
4
well, respectively. For experiments requiring longer than to room temperature. Endogenous peroxidase activity
48 h, cell numbers were reduced by one half. Viability was blocked by incubating the sections for 30 min in 1%
was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-5- hydrogen peroxide in methanol. Non-specifi c binding was
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo blocked by incubating the sections for 1 h with a normal
lium assay in 96-well plates in triplicate with CellTiter horse serum (Vector Laboratories, Inc., Burlingame, CA,
®
96 AQueous One Solution cell proliferation kits from USA). The sections were then incubated with mouse
Promega (Madison, WI) according to the manufacturer’s anti-CCL2 (MCP-1, eBioscience, San Diego, CA, USA)
instructions. Absorbance was recorded at 490 nm using overnight at 4 °C. On the next day, the sections were
a Synergy HT multimode plate reader or PowerWave rinsed 3 times with PBS at room temperature and then
®
XS2 (BioTek , Winooski, VT, USA) reader. DMSO further incubated with goat anti-mouse IgG-FITC (Santa
was used as a control. To calculate the viability index, Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at
absorbance readings from DMSO-treated control wells room temperature. The fl uorescence was then read using a
were set at 100%, and the relative A490 was calculated wide-fi eld fl uorescent microscope (Olympus, Center Valley,
as a percentage of the control. PA, USA). Stained sections were reviewed and scored
according to the intensity of staining (0, +1, +2 or +3)
Flow cytometry and for the percentage of tumor cells staining positive for
Cells treated with the WS extract were harvested and CCL2 (0%, 0.1-30%, +1; 31-70%, +2; or > 70%, +3). The
prepared for fl ow cytometry as described by Samuel score of the intensity of immunostaining was multiplied by
[13]
et al., with some modifi cations. WS treated and the score of percentage of cell staining to obtain the fi nal
untreated cells were harvested by trypsinization in staining index.
0.25% trypsin/ethylenediaminetetraacetic acid. Prior to RNA isolation and quantitative reverse
trypsinization, fl oating or loose cells were harvested by transcription-polymerase chain reaction
gentle rocking of the culture dishes and transferring the
culture medium containing the cells into centrifuge tubes. Total RNA was isolated from treated and control samples
Trypsinized and detached cells were then combined with RNeasy Mini Kits (Qiagen, Valencia, CA, USA)
and centrifuged. Cell pellets were suspended in 300 μL and reversely transcribed into cDNA using Quantitect
of phosphate-buffered saline (PBS), fi xed with 700 μL Reverse Transcriptase Kits (Qiagen) according to the
of 100% ethanol with vortexing, and stored at -20 °C manufacturer’s instructions. All primers were from
overnight. The fi xed cells were centrifuged and stained SABiosciences (Valencia, CA, USA); and quantitative
in fl uorescence-activated cell sorting staining solution polymerase chain reaction (qPCR) amplifi cation was
(3 mg/mL RNase A, 0.4 mg/mL propidium iodide) in performed using 50 ng of cDNA, 10 μL of Brilliant
PBS without calcium or magnesium for 30 min at 37 °C III Ultra-Fast SYBR Green qPCR Master Mix

Journal of Cancer Metastasis and Treatment ¦ Volume 1 ¦ Issue 2 ¦ July 15, 2015 ¦ 95