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Original Article

Withaferin A targeting both cancer stem cells and metastatic cancer stem
cells in the UP-LN1 carcinoma cell model

Lai-Lei Ting , Andy Shau-Bin Chou , Chin-Hsuan Hsieh , Shih-Chieh Hsiung , See-Tong Pang , Shuen-Kuei Liao 4,5
1
3
2
3
3
1 Department of Radiation Oncology, Cancer Center, Taipei Medical University Hospital, Taipei 110, Taiwan, China.
2 Department of Radiology, Tzu-Chi General Hospital, Hualien 970, Taiwan, China.
3 Department of Surgery, Division of Uro-Oncology, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Taoyuan 333,
Taiwan, China.
4 The PhD Program of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei 110, Taiwan, China.
5 Department of Research and Development, Vectorite Biomedica Inc., New Taipei City 221, Taiwan, China.
Correspondence to: Dr. Shuen-Kuei Liao, The PhD Program of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei 110, Taiwan,
China. E-mail: liaosk@h.tmu.edu.tw

Dr. Shuen-Kuei Liao earned his PhD degree from McMaster University, Canada, and subsequently received his
postdoctoral training at University of Toronto. He has been working in the areas of cancer biology and immunotherapy
in Canada, USA and Taiwan over the past three decades. Currently he is Professor at Taipei Medical University with his
interests in cancer stem cells and immunotherapy, and the roles of mesenchymal stem cells in transplantation.

A B S T R AC T
Aim: As our understanding of cancer stem cell (CSC) biology improves, search for inhibitory agents of CSCs and metastatic CSCs
(mCSCs) positive for CXCR4 is warranted. Withaferin A (WA), a withanolide extracted from the medicinal plant Withania somnifera,
has been shown to exhibit anti-cancer effects through multiple mechanisms. Whether WA could selectively target CSCs, mCSCs, or non-
CSCs of a gastrointestinal (GI) carcinoma tumor remains unclear. Methods: Side-population (SP) analysis, flow cytometric phenotyping
and sorting, non-invasive imaging in conjunction with xenotransplantation, and immunohistology were used in this investigation. Results:
Using the lymph node metastatic GI cancer cell line UP-LN1, consisting of CD44 high /CD24 floating (F) and CD44 /CD24 high
low
low
adherent (A) cell subsets, this study demonstrated that as compared with parental UP-LN1 cells or A cells, WA preferentially
reduced F-cell proliferation, tumor sphere formation, and SP cells in vitro in greater effi ciencies by apoptosis. This action was
mechanistically mediated via the down-regulation of CXCR4/CXCL12 and STAT3/interleukin-6 axes, both of which are instrumental
in the acquisition of metastatic ability. Attenuation of interferon-γ-induced CXCR4 expression in F cells by knockdown with siRNA
or blocking with an anti-CXCR4 antibody, followed by Western blot analysis, showed signifi cantly reduced metastatic potential in vitro. The
extent of in vitro anti-invasive effect of WA on the IFN-γ-treated F cells was signifi cantly greater than on the F cells without WA treatment,
or F cells treated with control siRNA or with control IgG antibody. The observed in vitro effects of WA on the CSC and mCSC targeting
were validated by data obtained with non-invasive imaging in NOD/SCID mouse xenotransplantation. Conclusion: WA could effi
ciently block the formation of both CSCs and mCSCs in the UP-LN1 cell line, suggesting that WA may be considered an effective
therapeutic agent for this type of GI malignancies.

Key words: Cancer stem cells; CXCR4; gastrointestinal cancer; metastasis; metastatic cancer stem cells; STAT3; withaferin A

INTRODUCTION distant organs. It has been demonstrated both experimentally
and clinically that the tumor microenvironment plays
Distant metastasis represents one of the few most challenging a pivotal role in tumor progression, particularly in the
aspects in cancer management. Cancer cells progress from
the primary lesion site and gain the ability to spread to This is an open access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows
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How to cite this article: Ting LL, Chou AS, Hsieh CH, Hsiung SC,
Pang ST, Liao SK. Withaferin A targeting both cancer stem cells and
DOI: metastatic cancer stem cells in the UP-LN1 carcinoma cell model. J
10.4103/2394-4722.172008 Cancer Metastasis Treat 2016;2:29-40.
Received: 07-06-2015; Accepted: 16-11-2015.

© 2016 Journal of Cancer Metastasis and Treatment ¦ Published by OAE Publishing Inc. 29