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acquisition of metastatic potential. During the process Withaferin A (WA), a cell-permeable steroidal lactone
[1]
of tumor metastasis, a cellular event termed epithelial-to- extracted from the Indian winter cherry, Withania somnifera,
mesenchymal transition (EMT), is initiated and is believed has been cited for its anti-cancer effects via multiple
to be a prerequisite for tumor dissemination. Accumulating mechanisms. [12-16] For instance, WA has been shown to
[2]
evidence from neoplastic tissues indicates the presence of elicit oxidative stress reactive oxygen species (ROS) and
self-renewing, stem-like cells within tumors called cancer mitochondrial dysfunction in leukemia cells leading to
stem cells (CSCs) or tumor-initiating cells. CSCs, which apoptosis. In breast cancer, WA-induced apoptosis via
[14]
constitute a small subpopulation of neoplastic cells within a the induction of Bim-s and Bim-L in estrogen-responsive
tumor, are defined operationally by their ability to seed new MCF-7 cells and in triple-negative MDA-MB-231
[17]
tumors. Recently, a seminal study has demonstrated that cells. In another study, WA has been shown to exhibit
[3]
disseminating cancer cells require self-renewal capability, anti-tumor and anti-angiogenesis activity by binding to
[18]
similar to that exhibited by stem cells and has indicated the intermediate filaments vimentin and F-actin. More
that the EMT process enriched the CSC population. importantly, WA at low dosages appeared to eliminate cells
[4]
Collectively, these studies provided important evidence expressing breast CSC markers including CD44, CD24,
and insights into CSC biology. The existence of CSCs has CD34, CD117 and Oct 4, and to down-regulate Notch1,
[19]
been shown to contribute to many aspects of tumorigenesis, Hes1 and Hey1 expression, suggesting the potential
especially therapy resistance [5,6] and metastasis. However, of WA as a CSC-targeting compound. Together, these
[7]
studying CSCs has been a great challenge due to their rarity findings provide the rationale to further explore the anti-
and the accuracy of identification methods. Therefore, a cancer effects of WA on the UP-LN1 cell line in terms of
reliable tumor cell model which replicates the physiological the mechanisms involved in blocking the formation of
properties of CSCs and metastatic CSCs (mCSCs) becomes CSCs and/or mCSCs.
a valuable tool for the understanding of CSC biology.
In this study, we also used side-population (SP) method [20,21]
The UP-LN1 lymph node metastatic cell line we have to enrich the CSC subpopulation from UP-LN1 cells, and
established previously exhibits CSC characteristics. [8,9] UP- then showed that F cells harbored the highest percentage of
LN1 is a CEA-producing gastrointestinal (GI) carcinoma CSC-like cells with an elevated expression of few selected
cell line which harbors a unique co-existence of 2 major stemness-related genes. Subsequently, we demonstrated
naturally occurring cell populations, adherent (A) and that WA treatment could inhibit the formation of tumor
floating (F) cells. Between the 2 subpopulations, the F cells aggregates/spheres and induce apoptosis in F cells. More
were characterized to possess several CSC-like properties, importantly, WA treatment could lead to the down-regulation
including CD44high/CD24low phenotype, high expression of CXCR4/CXCL12 and STAT3/IL-6 axes, both being
of multiple drug resistance genes, and tumor-initiating ability key members of a metastatic signaling pathway. Finally,
in NOD/SCID mice with low cell numbers, depressed HLA using non-invasive bioluminescence imaging technique,
class I expression, [9,10] and resistance to natural killer (NK)/ we demonstrated that after treatment with WA, both the
tumor burden and dissemination ability were significantly
lymphokine activated killer (LAK)-mediated cytolysis,
relative to CD44low/CD24high A cells. In addition, F and suppressed in NOD/SCID mice implanted with F cells.
[9]
A cells were found mutually convertible with F to A cells METHODS
at a faster rate. It is also conceivable that F cells may be
more easily separated from the primary lesion than A cells Chemicals and reagents
to enter the bloodstream as circulating tumor cells and then
deposit and proliferate at the new site through extravasation WA was purchased from Sigma-Aldrich (St. Louis, MO,
and intravasation, as the initial step toward metastasis. This USA), and its purity was > 95%. Primary monoclonal
phenomenon appears not to be restricted to GI malignancies antibodies (mAbs) to Oct4, Sox2, c-Myc, Nanog, vimentin,
since a similar result was recently reported with other cancer Fas receptor, caspase-3, caspase-8, caspase-9, poly ADP-
types such as breast cancer recently. Within the CSC cell ribose polymerase (PARP), Bcl-2, survivin, Akt, ERK,
[11]
population, there is an even smaller subset which could be GRK3/2, STAT3, and β-actin were purchased from Cell
become induced to CXCR4-positive mCSCs responsible Signaling Technology (Boston, MA, USA). Additional
for initiating metastatic activity in or migrate toward/invade mAbs used were as follows: Mouse anti-human CXCL12
a new microenvironment where a greater CXCL12 gradient (clone 79018, R and D Systems, Minneapolis, MN, USA),
is present. Moreover, in response to interferon-γ (IFN-γ) and CXCR4 (clone 15G5, R and D Systems, Minneapolis,
[9]
or activated NK or LAK cells, the CXCR4-positive mCSCs MN, USA), Fluorescein isothiocyanate (FITC) or
could only be induced from CSCs, which were harbored in phycoerythrin-conjugated goat-antimouse IgG (Biolegend,
the highly tumorigenic CD44high/CD24low F subset. Thus, San Diego, USA) were used as the secondary Ab for tracing
the UP-LN1 cell line represents an ideal in vitro model for the primary mAb. For multiple color-phenotyping, 5 × 10 5
studying CSCs and screening for effective anti-CSC and cells were directly incubated with FITC-conjugated mouse
anti-mCSCs agents. anti-human CD44 (clone G44-26, BD-Pharmingen, Franklin

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Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ Issue 1 ¦ January 15, 2016 ¦