Reversal of doxorubicin resistance in lung cancer cells by neferine is explained by nuclear factor erythroid-derived 2-like 2 mediated lung resistance protein down regulation

Aim: Development of multi drug resistance and dose limiting cardiotoxicity are hindering the use of Doxorubicin (Dox) in clinical settings. Augmented dox efflux induced by lung resistance protein (LRP) over expression has been related to multi drug resistance phenotype in various cancers. An alkaloid from lotus, Neferine (Nef) shows both anticancer and cardioprotective effects. Here, we have investigated the interconnection between nuclear factor erythroid-derived 2-like 2 (NRF2) and LRP in Dox resistance and how Nef can overcome Dox resistance in lung cancer cells by altering this signaling. Methods: Anti-proliferative and apoptotic-inducing effects of Nef and Dox combination in Parental and Dox resistant lung cancer cells were determined in monolayers and 3D spheroids. Intracellular Dox was analyzed using flow cytometry, siRNA knockdown and western blot analysis were used to elucidate NRF2-LRP crosstalk mechanism. Results: We observed that the Dox resistant lung cancer cells expressed higher levels of LRP, reduced glutathione (GSH) and NRF2. Combination of Dox and Nef induced apoptosis, leads to reactive oxygen species (ROS) generation, GSH depletion and reduction in LRP levels contributing to higher intracellular and intranuclear Dox accumulation. The use of N-acetylcysteine and knockdown studies confirmed an important role of ROS and NRF2 in LRP down regulation. Presence of NRF2 binding sites in LRP is support of direct interaction between LRP and NRF2. Conclusion: Nef sensitizes lung cancer cells to Dox by increasing intracellular and/or intra nuclear Dox accumulation via LRP down regulation. This is mediated by redox regulating NRF2. This decoded crosstalk mechanism reinforces the role of NRF2 and LRP in Dox resistance and as an important anticancer target.