(M0930-05-29) Piperlongumine Reverses EGFR Resistance as an Adjuvant Treatment in Lung Cancer Cells

Purpose: Lung cancer is the second-most frequently diagnosed cancer and the leading cause of cancer-related mortality worldwide. Overexpression of the epidermal growth factor receptor (EGFR) is associated with the development of lung cancer. EGFR-targeted therapies (EGFR-TKI) such as erlotinib and gefitinib are approved for the treatment of non-small cell lung cancer (NSCLC). However, the high incidence of acquired resistance (c-MET amplification, L858R, and T790M secondary mutation) to these EGFR-TKIs may preclude their effectiveness. Piperlongumine (PPL), an extract from the long pepper fruit (Piper longum) has been shown to possess anticancer properties. We, therefore, hypothesized that PPL will induce a potent anticancer response in NSCLC cells and overcome resistance.

Methods: Anticancer efficacy of PPL, erlotinib (ERL), and gefitinib (GEF) were investigated using a resazurin-dye assay in H1299 (EGFR wild-type) and H1975 (EGFR-T790M-mutation) cell lines, with cisplatin (CIS) as a positive control. Treatment of cells with PPL, ERL, GEF, and CIS along, and in combination was performed at various concentrations, and cell viability was determined after 72 h. The potency (IC50) of the treatment was computed using linear regression analysis. To determine the mechanism of Piperlongumine-induced toxicity, we performed a cellular reactive oxygen assay using DCFDA/H2DCFDA Cellular ROS Assay Kit (Abcam, USA). Apoptosis induction was determined by fluorescence microscopy using acridine orange/ethidium bromide (AO/EB) staining, and by flow cytometry using Muse™ Annexin V and Dead Cell Staining Kit (Luminex, USA). The effect of treatment on EGFR-mediated oncogenic signaling was studied by immunoblotting for markers of MAPK (EGFR mutant (L858R), p-EGFR, Akt, p38 MAPK, p44/p42 MAPK) and apoptotic signaling (Bcl-2, Cleaved caspase 3, Cleaved caspase 7, p53).

Results: PPL exhibited potent cytotoxic effect in H1299 (5.84 ± 0.04 µM) and H1975 (6.05 ± 0.31 µM) cells compared to ERL (138.83 ± 9.62 µM, 51.92 ± 2.34 µM), GEF (219.43 ± 22.69 µM, 47.22 ± 3.97 µM), and CIS (57.64 ± 3.06 µM, 100 ± 3.06 µM) respectively. The combination treatment of PPL with GEF and ERL showed a significant reduction in cancer cells compared to control in both the cell lines. ROS induction was observed with the combination treatment; however, there was no significant difference in the ROS levels compared to control. AO/EB staining indicated cell death via apoptosis. The combination of PPL and GEF had a significant increase in apoptotic cell death in the mutant cell line as compared to control which was confirmed with flow cytometry analysis. Treatment with PPL alone and in combination induced anti-mitogenic and apoptotic responses at a molecular level

Conclusion: EGFR-TKI is the second line of treatment for metastatic NSCLC. Most of these therapies are ineffective due to the development of acquired resistance. PPL sensitized lung cancer cells to EGFR-TKI and induced potent cytotoxic effects in lung cancer cells at low concentrations

Acknowledgements: The project was funded by the Massachusetts College of Pharmacy and Health Sciences. There is no conflict of interest. No disclosures