Nano-chemotherapeutic efficacy of (−) -epigallocatechin 3-gallate mediating apoptosis in A549 cells: Involvement of reactive oxygen species mediated Nrf2/Keap1signaling

Abstract

Chemotherapeutic drugs exert systemic toxicity in lung cancer cells and therefore novel treatment strategies are warranted. Epigallocatechin 3-gallate (EGCG), though possessing beneficial effects in alleviating cancer, its effect has been limited due to ineffective systemic delivery, toxicity and bioavailability. To attain the maximum therapeutic response of EGCG, we have synthesized bovine serum albumin (BSA) encapsulated magnetite nanoparticle (MNPs) loaded with EGCG (nano EGCG). The synthesized nano EGCG was characterized using HR-TEM, XRD and FT-IR. Cytotoxicity analysis of BSA-MNP and nano EGCG using flow cytometry was evaluated in lung adenocarcinoma A549 cells. The effect of native and nano EGCG modulating apoptosis and Nrf2/Keap1 signaling was analysed. Nano EGCG exhibited increased ROS/RNS levels and decreased mitochondrial membrane potential, as evaluated by DCFH and JC1 staining, respectively. Expression of pro-apoptotic Bcl-2 family proteins (Bcl-2, Bax, Bak, Bim and Puma) was evaluated. This study demonstrates that native and nano EGCG induces apoptosis through the involvement of ROS leading to loss in mitochondrial membrane potential. EGCG exhibited an increased expression of Nrf2 and Keap1 that could regulate apoptosis in A549 cells. This study, for the first time reveals the potential of BSA-MNPs loaded EGCG as drug target and renders better efficacy against lung cancer cells.

Introduction

Lung cancer is the second most cause of cancer-related death among men and women worldwide. Around 1.6 million people are affected yearly with survival rate after diagnosis is less than 18% [1]. Notably, many diagnosed patients are reported to have non-small cell lung cancer (NSCLC) which is highly complex in nature as it is difficult to diagnose at the early stage. Since the only available therapy is surgery followed by chemotherapy or radiation therapy, precise and rigorous treatments option for NSCLC after diagnosis are highly warranted [2]. Though several developments in the treatment for NSCLC were proposed [3], several limitations such as non-specific delivery of chemotherapeutic drugs, systemic toxicity, as well as rapid release have been reported for conventional therapies which ultimately reduce the therapeutic efficacy of drugs against tumor progression [4]. Recently, the use of nanomaterials in drug delivery has opened new avenues in the treatment options against various cancers [5]. The advantages of nano based drug delivery over conventional chemotherapy are numerous such as targeted drug delivery at the tumor site, sustained release of drugs and increased bioavailability at lower dosage [6]. Magnetite nanoparticles (MNPs) have gained a great attention because of their specific features such as superparamagnetism, large surface area, high encapsulation capacity and stability that makes MNPs, an ideal carrier for delivery of chemotherapeutic drug and could be used as contrast agents in magnetic resonance imaging (MRI) for earlier detection of cancer [7]. Among several surface modification agents in improving the biocompatibility of MNPs, bovine serum albumin (BSA) is the widely used surfactant macromolecule because of its non-immunogenic property, similarity to human serum albumin and stability in both acidic as well as basic conditions [8]. BSA-MNPs was applied as a potential candidate for magnetic nanothermotherapy and its in vitro efficacy in different cells demonstrated good biocompatibility with minimal cytotoxicity [9,10]. BSA conjugated MNPs are stable and non-toxic to various tested cell lines and rendered effectiveness in MRI visualization of brain tumor cells [11]. Doxorubicin loaded BSA conjugated magnetic nanoparticle provided controlled and targeted delivery of anticancer drugs in nasopharyngeal carcinoma was also reported [12]. Recently, Sodium alginate-polyvinyl alcohol-BSA coated Fe₃O₄ nanoparticles was used as drug delivery vehicles for controlled and targeted release of anticancer drug, thereby minimizing side effects [13]. While all these studies address the effect of BSA-MNPs as potential candidate for targeted drug release in chemotherapy, the role of the BSA-MNPs loaded drug in lung cancer is yet to be documented.

Epigallocatechin-3-gallate (EGCG) is the key abundant catechin contained in green tea possesses attractive health benefits [14,15]. Research evidences demonstrates that EGCG induced apoptotic cell death in various forms of cancers such as colon, breast, kidney, lung and brain. The major limitations of EGCG are its short half-life, poor bioavailability, instability in alkaline and neutral condition [16]. In this context, improving the pharmacokinetics and pharmacodynamics of EGCG could be of great interest. Previously, we have documented that EGCG is a potent activator of Nuclear factor E2- related factor 2 (Nrf2) in a rat model of pulmonary fibrosis [27]. Herein, we attempt to elucidate the chemotherapeutic efficacy of nano EGCG in lung adenocarcinoma A549 cells through modulation of reactive oxygen species (ROS) triggered Nrf2/Keap1 signaling and apoptosis.