Nanoquinacrine sensitizes 5-FU-resistant cervical cancer stem-like cells by down-regulating Nectin-4 via ADAM-17 mediated NOTCH deregulation
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Cervical cancer is a major cause of cancer-related death in women world-wide. Although the anti-metabolite 5-FU is widely used for its treatment, its clinical utility is limited due to the frequent occurrence of drug resistance during metastasis. Cancer stem-like cells (CSCs), present in the heterogeneous population of CC cells, are thought to contribute to this resistance. Nectin-4, a CSC marker, is known to play an important role in the cellular aggressiveness associated with metastatic CC. This study was designed to assess the role of Nectin-4 in the acquisition of 5-FU resistance by metastatic CC cells, including its relation to the NOTCH signalling pathway.
5FU-resistant CC cell lines were deduced from ME-180 and SiHA cells by continuous exposure to a single concentration of 5-FU. Thymidylate synthase (TS) positive cells were isolated from the 5-FU resistant cells, after which a metastatic model was developed. The role of Nectin-4 in the sensitization of 5-FU resistant metastatic CC cells upon incubation with Nano-formulated Quinacrine (NQC) was investigated using multiple bioassays including MTT, FACS, ELISA, immunoflurescence, Western blotting, comet and in vivo plasmid-based short patch and long patch base excision repair assays.
We found that the expression level of Nectin-4, as well as that of other CSC markers (Oct-4, β-catenin, SOX2) and representative NOTCH signalling components (NOTCH-1, Jagged-1, γ-secretase, ADAM-17) were elevated in the 5-FU resistant metastatic cells compared to those in control cells. Increased nuclear translocation of Nectin-4 and increased proliferation and invasion rates were observed after culturing the metastatic cells under hypoxic conditions. Treatment with NQC inhibited the nuclear translocation of Nectin-4 and decreased the proliferation and invasion rates of the cells by inhibiting the induction of base excision repair (BER) pathway components and ADAM-17 expression levels. After combination treatment of Nectin-4 overexpressing metastatic CC cells with a specific ADAM-17 inhibitor (GW280264) and NQC, a decreased Nectin-4 expression, without alterations in BER and/or other NOTCH pathway components, was noted.
Our data indicate that Nectin-4 may play a prominent role in 5-FU resistance of metastatic CC cells and that NQC sensitizes these cells by Nectin-4 deregulation through ADAM-17 inhibition, a major component of the NOTCH signalling pathway.
KeywordsCervical cancer Cancer stem cells 5-FU resistance Nectin-4 Metastasis
We are very thankful to the Indian Council of Medical Research (ICMR), Government of India for providing a fellowship to AN. The authors sincerely thank Mark Zakshevsky, Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida and Dr. C. R. Patil, Professor and Head, Department of Pharmacology, R. C. Patel Institute of Pharmaceutical Education and Research, India for carefully proofreading the manuscript.
AN performed most experiments and analysed the data. SD, DN and CS performed the statistical analyses. CNK conceived the hypothesis, analysed the data and wrote the manuscript. SN modified the manuscript and edited the English grammar.
This study was funded by the Indian Council of Medical Research, Govt. of India (ref number- ICMR-35/22/2012-BMS).
Compliance with ethical standards
Conflict of interest
All authors declare no conflict of interest.
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