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Figure 2: Withaferin A reduced side-population and cell aggregates in UP-LN1 cells. (a) Withaferin A reduced the percentage of side-population cells in UP-
LN1 in a dose-dependent manner as indicated by our cytofluorometric analysis. The reduction of side-population cells in different populations of UP-LN1 cells
was plotted against the concentration of withaferin A (right panel); (b) sulforhodamine B viability assay indicated that withaferin A targeted floating (F) cells
most effectively and also in a dose-dependent manner as compared with adherent (A) or parental (P) cells. The difference between F and A cells is signifi cant
(P < 0.01), and so is the difference between F and P cells (P < 0.01) at each of the three withaferin A dose levels (5, 10, 25 μmol/L) tested; (c) microscopic
analysis of F cells under the influence of withaferin A demonstrated a significant reduction in the formation of F-cell aggregates or spheres. At 10 μmol/L,
withaferin A reduced F-cell aggregates by approximately 80%

cancer cells, we wished to determine if it exerted a F-cell population. It was observed that the addition
[16]
similar function in UP-LN1 cells. Using Annexin V as an of IFN-γ increased the invasive ability in all 3 cell
apoptotic indicator, we demonstrated that WA promoted populations of the UP-LN1 cell line, with F cells at the
apoptosis in P, F, and A cells in a dose-dependent manner highest efficiency. In the presence of WA, IFN-γ-induced
[9]
[Figure 3a-c, respectively]. When analyzed quantitatively, invasion was significantly suppressed, particularly in the
WA appeared to preferentially target F cells and triggered F-cell population [Figure 4a]. We subsequently examined
apoptosis to a higher extent in F cells than in P and A cells the 2 major signaling axes involved in cellular trafficking,
[Figure 3b]. This observation corroborates the preference namely CXCR4 and STAT3. [24,25] Using cytofluorometric
[9]
of WA in suppressing the formation of F-cell aggregates analysis, we demonstrated, as shown previously, that the
in the aforementioned section. In addition, Western blot surface expression of CXCR4 was elevated in the presence
analysis of cell lysates obtained from WA-treated F cells of IFN-γ at concentrations as low as 10 U/mL, in terms
indicated an increased expression in some pro-apoptotic of increased percentage of CXCR4-positive cells. Notably,
molecules including caspase-3, -8, -9 and PARP at higher each positive cell bore a relatively constant number of
concentrations [≥ 5 µmol/L, Figure 3c]. The remaining CXCR4 receptor sites, as revealed by a constant value
pro-apoptotic molecule, Fas receptor, and anti-apoptotic of mean fluorescence intensity. The addition of a low
molecules such as Bcl-2 and survivin were clearly down- concentration of WA (2.5 µmol/L) reduced the percentage
regulated in a dose-dependent manner. of CXCR4-positive cells [Figure 4b], and with the addition
of this agent at a higher concentration (5 µmol/L), CXCR4-
WA treatment suppresses two major metastasis positive cells could hardly be detected.
signaling pathways (STAT3 and CXCR4) in F cells
Next, we examined STAT3 and several key signaling
The presence of IFN-γ in tumor microenvironment or pathways involved in cancer metastasis using Western
NK/LAK culture conditioned medium has been reported blot analysis. WA treatment suppressed the expression of
to promote the metastatic ability of cancer cells through Akt/ ERK, CXCR4, STAT3, and GRK3/2 in F cells with or
the modulation of CXCR4 expression in cancer cells. [9,23] without the induction of mCSCs by IFN-γ treatment [Figure 4c].
We wished to determine if WA treatment could overcome A note of explanation is needed regarding the appearance
metastatic potential induced by IFN-γ in the CSC-like of CXCR4 bands in the absence of IFN-γ added, which is

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