Establishment and Characterization of Pemetrexed-resistant NCI-H460/PMT Cells

Author(s):

Yu-Lian Xu, Xiao-Ming Jiang, Le-Le Zhang, Xiuping Chen, Zhang-Jian Huang and Jin-Jian Lu*   Pages 731 - 739 ( 9 )

Abstract:

<P>Background: Pemetrexed (PMT) is a multitargeted antifolate agent that is used for treating patients with Non-Small Cell Lung Cancer (NSCLC). However, patients have presented clinical responses of drug resistance to PMT. </P><P> Objective: This study aimed to explore the underlying mechanisms of PMT resistance in NSCLC cells. </P><P> Methods: PMT-resistant NCI-H460/PMT cells were established by treating with PMT in a concentrationescalation manner. MTT assay and colony formation were performed to detect cell proliferation. Immunofluorescence was used to detect the expression of Ki-67. Transwell assay was performed to measure cell migration ability. qPCR and Western blot were used to detect the mRNA and protein expression levels of indicated genes. Small interfering RNAs (siRNA) were used to knockdown ATP binding cassette subfamily B member 1 (ABCB1) and Thymidylate Synthase (TYMS). </P><P> Results: This study showed that compared with the parental cells, the NCI-H460/PMT cells displayed weakened proliferation and enhanced cell mobility. In addition, the NCI-H460/PMT cells demonstrated cellular senescence, which might result in PMT resistance. The NCI-H460/PMT cells exhibited cross-resistance to other chemotherapeutics, including fluorouracil, paclitaxel, doxorubicin, etoposide and gemcitabine, possibly because of the upregulated expression of ABCB1. However, the ABCB1 knockdown by siRNA failed to eradicate PMT resistance. Moreover, TYMS, a target of PMT, was obviously upregulated in the resistant cells. The genetic silence of TYMS partially abrogated PMT resistance, suggesting that the overexpression of TYMS was a key resistant mechanism of PMT. </P><P> Conclusion: The overexpression of TYMS was an important resistance mechanism of PMT for KRAS-mutated NCI-H460 cells. Cross-resistance to other chemotherapeutics should be considered in addressing PMT resistance.</P>

Keywords:

NSCLC, pemetrexed, resistance, TYMS, ABCB1, chemotherapy.

Affiliation:

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau

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