Analysis of ABCG2 expression and side population identifies intrinsic drug efflux in the HCC cell line MHCC-97L and its modulation by Akt signaling
Chen Huy
0
2
Hong Li
0
1
2
y
0
2
Jinjun Li
0
1
2
Zheng Zhu
0
1
2
Shengyong Yin
0
2
Xiangfang Hao
0
1
2
Ming Yao
0
1
2
Shusen Zheng
0
2
Jianren Gu
0
1
2
0
Abbreviations: ABC
,
adenosine triphosphate-binding cassette; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FTC, fumi- tremorgin C; HCC, hepatocellular carcinoma; mTOR, mammalian target of rapamycin; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PI3K, phosphoinositide 3-kinase; siRNA, small-interfering RNA; SP, side population
1
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute and Cancer Institute of Shanghai Jiao Tong University
,
25/Ln 2200 Xietu Road, Shanghai 200032
,
People's Republic of China
2
Department of General Surgery, the First Affiliated Hospital, School of Medicine, Zhejiang University
,
79 Qingchun Road, Hangzhou 310003
,
People's Republic of China
To whom correspondence should be addressed. Tel: 86 21 64432140; Fax: 86 21 64432140; Email: Correspondence may also be addressed to Shusen Zheng. Tel: 86 571 87236466; Fax: 86 571 87236609; Email: Active drug efflux by the adenosine triphosphate-binding cassette (ABC) transporter ABCG2 is one of the common mechanisms causing multiple drug resistance in various human cancers. In the intrinsic drug resistance of hepatocellular carcinoma (HCC), the role of ABCG2 is closely associated with 'side population (SP)', a minor subset of cancer stem-like cells with unique capacity to extrude lipophilic dye Hoechst 33342 and many chemotherapeutic agents. In this study, we showed that ABCG2 was intrinsically expressed in a subgroup of HCC tissues and its expression pattern significantly influenced the levels of drug efflux from HCC cell lines. In MHCC-97L HCC cell line with intrinsic ABCG2 expression, we confirmed the importance of SP cells to the drug effluxrelated chemotherapy resistance and found that the SP analysis provided an efficient method to evaluate the functional activity of ABCG2 transporter. In this cell line, we discovered that the SP proportion was modulated by the treatments of Akt signaling inhibitors and serum supplement, which led to the finding that Akt signaling was able to regulate the SP cells' efflux activity via altering the subcellular localization of ABCG2 transporter. We further demonstrated that the Akt signaling inhibition attenuated the doxorubicin efflux from MHCC-97L cells and increased the drug efficacy. Our results indicate the protective role of intrinsic ABCG2 expression in HCC cells and suggest that suppressing Akt signaling could help overcome the drug efflux by ABCG2 transporter.
Introduction
Hepatocellular carcinoma (HCC) is one of most common cancers
worldwide, and its resistance to conventional drugs is a major impediment to
successful chemotherapy (1,2). Chemotherapy resistance can be
developed by various mechanisms, including altered drug target, enhanced
detoxication capacity, impaired cell death pathway and reduced drug
accumulation in cancer cells (3). In HCC, a leading cause for
chemotherapeutic failure has been attributed to the fact that a large proportion
of cancer cells express relatively high levels of several adenosine
triphosphate-binding cassette (ABC) transporters (2,4), which actively
pump out a broad spectrum of clinically relevant compounds and
decrease the intracellular drug accumulation (5). The ABCG2
transporter, first identified in a doxorubicin-selected MCF-7 breast cancer
yThese authors contributed equally to this work.
cell line, has been shown to mediate multiple drug resistance in various
cancers (6,7); its substrates include a variety of anticancer agents such
as mitoxantrone, camptothecins, anthracyclines, flavopiridol and
antifolates (7). In HCC, ABCG2 expression is known to be preferentially
upregulated in the stem/stem-like cell compartment (8,9), suggesting
a role of ABCG2 in protecting this population against chemotherapy.
Recently, the role of ABCG2 was underlined with the identification
of side population (SP) cells in many cancers (10,11). The SP, a rare
subset of cells distinguished by their low Hoechst dye staining in flow
cytometry, was first described by Goodell et al. (12) in identifying
hematopoietic stem cells in bone marrow. Unlike the common
methods that involve recognition of cell surface markers, SP phenotype is
functionally defined by the cells performance in pumping out the
DNA-binding dye Hoechst 33342 (11). Studies in leukemia and some
solid tumors have shown that the SP phenotype could be used to
enrich cancer stem cells (10,1315), although it appeared
heterogenous in terms of cell surface marker profile (11). Experimental
evidence from the Abcg2 / knockout mice model has demonstrated that
ABCG2 is the primary transporter mediating the SP phenotype, and
several other ABC family members have overlapping function in
Hoechst dye efflux (16,17). The SP cells have been shown to possess
extreme tumorigenicity and intrinsic drug resistance, sugge (...truncated)