Abstract
Most gefitinib-treated patients with non-small cell lung cancer (NSCLC) would eventually develop resistance. Lysimachia capillipes (LC) capilliposide extracts from LC Hemsl. show both in vitro and in vivo anti-cancer effects. In this study we investigated whether LC capilliposide in combination with gefitinib could overcome the resistance of NSCLC cells to gefitinib and identified the signaling pathways involved. Treatment with LC capilliposide alone inhibited the growth of a panel of NSCLC cell lines (PC-9, H460, H1975, H1299 and PC-9-GR) sensitive or resistant to gefitinib with IC50 values in the range of μg/mL. In the gefitinib-resistant PC-9-GR cells (which have a T790M EGFR mutation), LC capilliposide (at the IC30, i.e.1.2 μg/mL) markedly enhanced the inhibitory effects of gefitinib with its IC50 value being decreased from 6.80±1.00 to 0.77±0.12 μmol/L. By using the median effect analysis we showed that combination treatment of LC capilliposide and gefitinib could restore gefitinib sensitivity in PC-9-GR cells. Furthermore, LC capilliposide (1.2 μg/mL) significantly increased the apoptotic responses to gefitinib (0.77 μmol/L) in PC-9-GR cells, but did not affect gefitinib-induced G0/G1 arrest. Moreover, LC capilliposide (1.2 μg/mL) in combination with gefitinib (0.77, 1.0 μmol/L) markedly decreased the phosphorylation of the EGFR downstream signaling molecule AKT, which neither LC capilliposide nor gefitinib alone affected. In PC-9-GR cells with siRNA knockdown of AKT, addition of LC capilliposide was unable to increase gefitinib sensitivity. In a PC-9-GR xenograft mouse model, combination treatment with LC capilliposide (15 mg·kg−1·d−1, ip) and gefitinib (50 mg·kg−1·d−1, ip) dramatically enhanced tumor growth suppression (with a TGI of 109.3%), compared with TGIs of 22.6% and 56.6%, respectively, in mice were treated with LC capilliposide or gefitinib alone. LC capilliposide can restore the cells' sensitivity to gefitinib through modulation of pAKT levels, suggesting that a combination of LC capilliposide and gefitinib may be a promising therapeutic strategy to overcome gefitinib resistance in NSCLCs with a T790M mutation.
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Acknowledgements
This work was supported by the Natural Science Foundation of Zhejiang Province (LY15H160010), the Medical Science Foundation of Zhejiang Province (2014KYA178), the Hangzhou Key Disease and Discipline Foundation (20140733Q15), and the Science and Technology Project of Zhejiang Provincial Bureau (2015ZA133).
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Supplementary Figure S1
The phospho-kinase array also showed a significant decrease of AKT phosphorylation in PC-9-GR cells treated with the combination of gefitinib and LC capilliposide when compared to that of gefitinib treatment alone. (JPG 156 kb)
Supplementary Figure S2
Cotreatment with gefitinib and LC capilliposide had no obvious effects on the body weight of mice with PC-9-GR xenografts. (JPG 82 kb)
Supplementary Table S1
IHC analysis of tumor sections showing modulation of pEGFR, Ki67, CC3, and pAKT treated with LC capilliposide and gefitinib. (DOC 16 kb)
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Zhang, Sr., Xu, Ys., Jin, E. et al. Capilliposide from Lysimachia capillipes inhibits AKT activation and restores gefitinib sensitivity in human non-small cell lung cancer cells with acquired gefitinib resistance. Acta Pharmacol Sin 38, 100–109 (2017). https://doi.org/10.1038/aps.2016.116
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DOI: https://doi.org/10.1038/aps.2016.116
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