We developed and characterized a new tyrosine kinase inhibitor resistant non-small cell lung cancer (NSCLC) line, HCC827GR, which (1) was significantly resistant to both gefitinib and AZD9291; (2) had more in vitro tumorigenic potential as evident by a larger colony size from the anchorage-independent growth assay; and (3) exhibited a total of 26 differentially expressed genes (≥two fold-change) as compared to HCC827. These genes were involved in regulating cell growth, transcription, phase 1 metabolism, cell cycle, and apoptosis. Additionally, AZD9291 in combination with CAPE partially reverted the AZD9291 resistance in HCC827GR cells. This was exhibited by (1) synergistically suppressed cell viability; (2) cell cycle arrest and apoptosis induced through suppressing EGFR activation and modulating p53, p21, cyclin D1, and survivin protein expressions; and (3) differentially regulating genes involved in cancer drug resistance pathways.

Abstract

Epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the first-line therapy for EGFR mutated non-small cell lung cancer (NSCLC); however, resistance rapidly develops. The objective of this study was therefore to establish and characterize a gefitinib resistant NSCLC line (HCC827GR) and evaluate the therapeutic effects of natural products in combination with third-generation EGFR-TKI, AZD9291. The IC₅₀ of gefitinib and AZD9291 in HCC827GR were significantly higher than those of HCC827 (p < 0.05). Furthermore, anchorage-independent colony assay indicated that HCC827GR cells were more aggressive than their predecessors. This was reflected by the gene/protein expression changes observed in HCC827GR versus HCC827 profiled by cancer drug resistance real-time polymerase chain reaction (RT-PCR) array and Western blot. Three natural products were screened and caffeic acid phenethyl ester (CAPE) exhibited the most significant combinative cytotoxic effect with AZD9291. Specifically, flow cytometry revealed that AZD9291 + CAPE considerably increased the fraction of cell in pre-G1 of the cell cycle and caspase-Glo3/7 assay showed a dramatic increase in apoptosis when compared to AZD9291 alone. Furthermore, Western blot showed significant downregulation of p-EGFR/p-AKT in HCC827GR cells treated with AZD9291 + CAPE as compared to AZD9291. Moreover, it is evident that AZD9291 + CAPE specifically resulted in a marked reduction in the protein expressions of the cell-proliferation-related genes p21, cyclin D1, and survivin. Finally, refined RT-PCR/Western blot data indicated that AZD9291 + CAPE may at least partially exert its synergistic effects via the PLK2 pathway. Together, these results suggest that CAPE is a clinically relevant compound to aid AZD9291 in treating EGFR-TKI resistant cells through modulating critical genes/proteins involved in cancer resistance/therapy.