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vitro model. Therapeutic approaches represent translational number of CXCR4-positive F cells, also known as mCSCs,
strategies, which could improve the clinical outcome for played a pivotal role in lymph node metastasis of some types
patients with malignancies that are currently refractory of GI cancer, including UP-LN1. [8,9,30] This observation
to conventional treatments. [4,5] However, developing such was further supported by a recent study where the positive
an agent or strategy has been hindered by the lack of an CXCR4 expression was shown to be significantly associated
experimental CSC cell model, which could be maintained with lymph node metastases (P = 0.028) and higher stages
with relative ease and replicate most of the clinical CSC III/ IV (P = 0.047) in gastric cancer. In addition, several
[27]
characteristics. other major metastasis-associated pathways such as Akt/
ERK, GRK3/2, and STAT3 could also be attenuated by
Previously, we identified the UP-LN1 cell line which the addition of WA [Figure 4c]. Interestingly, GRK3/2
was characterized by a co-existence of 2 unique cell expression, in particular, has been shown to form complexes
populations. [8,9] The CD44 high /CD24 F cells attracted our with CXCR4 and/or FAK in human primary monocyte-
low
attention particularly due to their phenotypic and cellular macrophages and to play a part in their trafficking, upon
resemblance of CSCs in the form of tumor spheres. Using the stimulus by inflammatory cytokines such as IL-4 and
SP methods, we demonstrated that F cell population was IL-13. Equally important, it has been demonstrated in
[30]
enriched with CSCs with clearly elevated levels of stemness small cell lung cancer where the activation of CXCR4/
markers including Nanog, c-Myc, Oct4 and Sox2 noted, CXCL12 axis leads to the activation of the JAK/STAT3
as compared with the CD44 /CD24 high A-cell population pathway. [31-34] STAT3 has been found and deposit and
low
[Figure 1a]. proliferate at the new site constitutively activated as a result
of carcinogenesis in different cancer types, and aberrant
Having established this CSC cell model, we intended STAT3 signaling has been implicated as an important
[9]
to examine WA as a potential anti-CSC and anti-mCSC [35-37]
agent. WA has been indicated for its anti-cancer effects by process in malignant transformation and induction of
[37]
modulating multiple molecular pathways, predominantly angiogenesis. Activation of CXCR4 and STAT3 has been
through the induction of intracellular oxidative stress. [12,13,17,29] linked to tumor progression in different cell types, such
[38]
WA dose-dependently reduced the percentage of SP cells in as hematopoietic progenitor cells and small cell lung
[39]
A, F, and parental UP-LN1 cells [Figure 2a]. Interestingly, F carcinoma lines with high mobility. Interestingly, STAT3
cells appeared to be more sensitive to WA treatment than A activation was shown to allow a crosstalk between tumor
and parental cells [Figure 2b]. In addition, we demonstrated cells and dendritic cells which forms an immunosuppressive
that WA promoted apoptosis in F cells in a dose-dependent microenvironment favorable for tumor survival and
[40]
manner as evidenced by both cytofluorometric [Figure perpetuation. Thus, the observation that WA treatment
3a and b] and Western blot [Figure 3c] analyses via up- negatively regulated the CXCR4/CXCL12 chemotactic
regulating caspases-3, -8 and -9, as well as PARP, which axis and molecules involved in IFN-γ-stimulated F cells,
are collectively a family of proteins involved in a number leading to making more CXCR4-positive mCSCs in cell
of cellular processes, such as DNA repair and programmed numbers but not in increasing the number of CXCR4 sites
cell death. It has been suggested that CSCs were more on a per-cell basis, provides support for using WA as a
sensitive to ROS-induced apoptosis. [26,27] F cells were found potent anti-metastasis agent. The extent of the inhibitory
to contain a lower intracellular glutathione (GSH) level effect of WA on invasiveness of IFN-γ-treated F cells was
(data not shown), which could partially explain why WA as great as that achieved by IFN-γ-mediated induction of
eliminated F cells more efficiently. On the other hand, A cells CXCR4 in F cells attenuated by neutralizing anti-CXCR4
are in general more sensitive to killing by chemotherapeutic antibody [Figure 5b], or by knocking down the CXCR4
drugs and NK/LAK cells. [8,9] expression using siRNA [Figure 5c]. It should be pointed out
that IFN-γ is not the only agent or means known to induce
[9]
In addition to promotion of apoptosis in F cells, WA mCSCs, since other agents/methods such as HGF and
[41]
[42]
appeared to be potent in suppressing the metastatic potential hypoxia conditions have also been reported to be able to
of F cells. As we observed in our previous study, we have do the same. Interestingly, the fluctuations of vimentin and
[9]
also demonstrated here that the migratory/invasive ability CXCR4 expression in the experiments stated above were
of UP-LN1 cells could be stimulated by low levels of IFN-γ very similar, suggesting the importance of these 2 molecules
via an increase in only the percentage of F cells expressing in the appearance of EMT phenotype during the process of
CXCR4, while the number of CXCR4 density on a per-cell cancer migration and invasion. [26,27] Interestingly, a recent
basis remained relatively constant [Figure 4b]. This study study by Bargagna-Mohan et al. indicated that WA acted as a
showed that WA suppressed IFN-γ-induced invasiveness tumor inhibitor, as well as the antiangiogenic agent through
in the UP-LN1 parental, A and F cells in a dose-dependent targeting the intermediate filament protein vimentin. [18]
manner [Figure 4a], with the greatest effect on F cells, the
intermediate effect on parental cells, and least effect on A Based on our own current findings and those by others, [13,15,17,19]
cells. The WA-mediated effect was found to be through the apoptotic process triggered by WA works through
down-regulation of CXCR4 and vimentin expression multiple mechanisms. They involved the mitochondrial
[Figure 4b]. This finding suggests that the increase in the pathway and associated Bcl-2 down-regulation, caspase-8,

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