Elsevier

Clinical Lung Cancer

Volume 16, Issue 5, September 2015, Pages e55-e66
Clinical Lung Cancer

Original Study
Autophagy Inhibition Overcomes the Antagonistic Effect Between Gefitinib and Cisplatin in Epidermal Growth Factor Receptor Mutant Non–Small-Cell Lung Cancer Cells

https://doi.org/10.1016/j.cllc.2015.03.006Get rights and content

Abstract

Background

Four large clinical trials have shown that concurrent administration of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), such as gefitinib or erlotinib, with chemotherapy agents does not improve overall survival (OS) in unselected patients with advanced non–small-cell lung cancer (NSCLC). In the present study, the role of autophagy in the combination of gefitinib and cisplatin on EGFR-TKI–sensitive human lung cancer cell line was investigated. Moreover, whether simultaneous autophagy inhibition treatment increases the antitumor activity was explored.

Materials and Methods

The PC9 cell was exposed to either gefitinib or cisplatin alone or together. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cytotoxic interaction between gefitinib and cisplatin was determined using coefficient of drug interaction (CDI) analysis. The cell cycle distribution and apoptosis were measured using flow cytometry. Alterations in the autophagy and apoptosis signaling pathway were measured using Western blot assays.

Results

Coadministration of gefitinib and cisplatin resulted in antagonistic activity to tumor cell proliferation. However, the addition of chloroquine (CQ), an autophagy inhibitor, overcame the antagonistic effects, as demonstrated by CDI analysis and Annexin V-FITC/propidium iodide (PI) assays. In addition, gefitinib administration led to cell G1 phase arrest, which might have contributed to the antagonistic activity between gefitinib and cisplatin. However, the addition of CQ did not deregulate cell cycle arrest, indicating that other mechanisms might be involved. The Annexin V-FITC/PI assays showed that the addition of CQ significantly the increased the ratio of apoptosis cells. Also, immunoblotting assays exhibited increased Bax and decreased Bcl-2, suggesting that autophagy inhibition by CQ could increase cell apoptosis and thus overcome the antagonistic effects.

Conclusion

The combination of gefitinib with cisplatin generates antagonistic effects on EGFR-TKI–sensitive cells. However, inhibiting autophagy produces a synergistic effect, suggesting that gefitinib and cisplatin combined with an autophagy inhibitor (especially CQ) might be a beneficial strategy to overcome the antagonistic effects between EGFR-TKIs and chemotherapeutic agents.

Introduction

Lung cancer is the leading cause of cancer-related deaths throughout the world. Non–small-cell lung cancer (NSCLC) represents approximately 80% to 85% of all lung cancer cases diagnosed.1, 2 About two thirds of all patients with NSCLC present with an advanced stage at diagnosis.3 Platinum-based, 2-drug combination chemotherapy has been recommended as the standard treatment for such patients. However, the efficacy seems to have reached a plateau in recent decades, although some new agents have been added.

The epidermal growth factor receptor (EGFR) belongs to the receptor tyrosine kinase family, which is overexpressed in a number of solid tumors.4 Thus, blocking the EGFR signaling pathway has been thought to be a promising method for the treatment of cancer with a high expression of EGFR, including NSCLC.5 Gefitinib (ZD1839, Iressa) is an oral, small-molecule EGFR tyrosine kinase inhibitor (EGFR-TKI) that competitively inhibits the EGFR downstream signaling pathways. However, initial studies found that only a few patients with NSCLC responded to gefitinib.6 Investigators soon discovered that the sensitivity to TKIs correlated with somatic mutations of the kinase domain of EGFR, such as either deletions within exon 19 or an L858R mutation in exon 21.7, 8 However, most patients with an EGFR-positive mutation will eventually present with resistance after TKI treatment for 6 to 12 months.9, 10 To improve the efficacy, it would be natural to consider combining TKIs with a traditional chemotherapy drug, such as cisplatin. Several preclinical studies have shown a better treatment effect compared with either gefitinib or a chemotherapy drug alone.11, 12 However, 4 large-scale phase III randomized clinical trials have shown no significant survival benefit from the combination of TKIs and chemotherapy drugs.13, 14, 15, 16 The antagonism between TKIs and chemotherapy drugs was at least in part caused by G1 cell cycle phase arrest induced by TKIs. However, the precise mechanism of antagonism between TKIs and chemotherapy drugs is still not straightforward.

Autophagy is a widely existing and highly conserved biologic phenomenon in eukaryotic cells.17, 18 The specific role of autophagy in cancer development and anticancer therapy is still controversial. In the early stages of tumorigenesis, autophagy inhibit tumor cell growth; however, once a tumor has developed, it can promote tumor cell survival.19 Thus, upregulating autophagy might protect tumor cells against various antitumor therapies, such as chemotherapy or EGFR-TKI treatment. Thus, it has been reported widely that gefitinib and chemotherapy agents, such as cisplatin, can induce prosurvival autophagy and contribute to tumor cell resistance and that inhibition of autophagy by small interfering RNA (siRNA) interference or a pharmacologic inhibitor, such as chloroquine (CQ), could reverse this resistant and enhance antitumor effects through a variety of mechanisms.20, 21, 22

Thus, whether autophagy participates in the antagonistic effects between TKIs and chemotherapy drugs and whether inhibition of autophagy could reverse this antagonistic effect is worthy of study. The present study investigated the role of autophagy in the antagonistic effects between gefitinib and cisplatin and sought to find novel strategies to overcome these antagonistic effects.

Section snippets

Cell Culture and Reagents

The human NSCLC cell line PC9 expressing the EGFR exon 19 deletion mutation (ΔE746-A750) and A549 expressing wild-type EGFR (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were maintained in Roswell Park Memorial Institute 1640 medium (Gibco) and supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL). All the cells were grown as monolayer cultures and maintained in a humidified atmosphere of 5% carbon dioxide in air at 37°C.

Gefitinib Combined With Cisplatin Led to Antagonistic Effects

To address whether gefitinib and cisplatin could, in a dose- and time-dependent manner, inhibit PC9 cell proliferation, we used the MTT assay. As a consequence of increasing the concentration increased or prolonging the exposure time, gefitinib (Figure 1A) or cisplatin (Figure 1B) treatment led to a marked decline in cell proliferation. However, the rates of cell proliferation in the various combinations did not significantly decline compared with cisplatin or gefitinib treatment alone (

Discussion

In the present study, we found that gefitinib combined with cisplatin inhibited cell proliferation and induced apoptosis in EGFR-mutated PC9 cells and simultaneously induced autophagic activity. However, CDI analysis showed an antagonistic effect compared with each agent alone, indicating that autophagy might play an important role in the antagonistic effects. Furthermore, the results from our study have shown that the combination of CQ inhibition autolysosome degradation could reverse this

Conclusion

In the present study, we found that coadministration of gefitinib and cisplatin produced antagonistic effects in vitro and might be caused by autophagy. Autophagy inhibition by CQ improved the therapeutic efficacy of gefitinib combined with cisplatin by increasing apoptosis. Therefore, the combination of EGFR-TKIs and chemotherapy agents with autophagy inhibitors represents a novel and promising strategy for lung cancer treatment. However, the antitumor effects of CQ-mediated autophagy

Disclosure

The authors have stated that they have no conflicts of interest.

Acknowledgments

The present study was supported by grants from the National Natural Science Foundation of China (grants 81402040 and 81272739). We thank Professor Wei Wei for providing the experimental platform to complete the study and Drs. Yu-Jing Wu and Yan Chang for technical support.

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