The Role Of ROS In Bcl-2Inhibitors-treated Lymphoma And Hepatocellular Carcinoma Cells And Associated Mechanisms

Background:Inducing apoptosis is the most common strategy for tumor therapy. Bcl-2anti-apoptoticfamily proteins have been studied as potent anti-tumor targets for many years and severalBcl-2inhibitors have been developed. Bcl-2inhibitors showed effective anti-tumor activity indiverse tumor cells in vitro and in vivo. Human lymphoma and hepatocellular carcinoma(HCC) are both common malignancies in our country and have a considerably high mortalityrate. However，current treatments for patients with lymphoma or HCC mainly consists oftraditional therapy which often yields unsatisfactory outcomes.Within this context,examination of novel molecular-targeted compounds is a priority. It’s clear that theoverexpression of Bcl-2anti-apoptotic proteins is tightly associated with the development,progression and therapeutic resistance of human lymphoma and HCC. Bcl-2inhibitors mayrepresent an effective therapeutic strategy and bring hope for patients. Reactive OxygenSpecies(ROS) have been confirmed to be involved in regulation of multiple cellular signalingpathways and important for cancer therapy. Recently,the dual roles of ROS in regulatingapoptosis and autophagy have become attractive.However, the role and mechanisms of ROSinduced by Bcl-2inhibitors’ work in cancer cells are still not clear and need to be figure outmore deeply.Objective:to study the role of ROS in Bcl-2inhibitors-treated lymphoma and hepatocellularcarcinoma cells and associated mechanismsMethods:Human lymphoma cell line Namalwa and HCC cell lines HepG2, Huh7, Hep3B andPLC/PRF/5were used as research model. Cells were treated with Bcl-2inhibitors, then cellviability was quantified by CCK-8assay and total cell death was counted by trypan blue staining. The TUNEL-positive cells were deteced by fluorescent microscopy observationand the protein levels of apoptotic molecules and autophagic marker LC3and P62weredetected by western blot. In addition, caspase-3activity was also measured. DCFH-DAfluorescent probe was to measure the level of ROS. Cells were pretreated with JNKinhibitor or NAC and subsequently treated with Bcl-2inhibitors for different time. AnnexinV-FITC/PI Apoptosis Detection Kit or TUNEL-FITC Apoptosis Detection Kit was utilizedto detect the apoptotic cells and western blot was used to test the apoptotic and autophagicproteins.GFP-LC3punta was observed under fluorescent microscopy.Results:1. The role of ROS in (-)-gossypol-treated lymphoma and associated mechanisms.(1)(-)-Gossypol dose-dependently inhibits proliferation and promotes death oflymphoma Namalwa cells;(2)(-)-Gossypol triggered the generation of ROS in Namalwa cells;(3) Pretreatment with antioxidant NAC attenuated (-)-gossypol-induced autophagy inNamalwa cells;(4)(-)-Gossypol-induced cytosolic translocation of HMGB1is ROS-dependent in partand contributes to augmentation of autophagy;(5) NAC pretreatment enhanced (-)-gossypol-induced apoptosis.2. The role of ROS in ApoG2-treated HCC cells and associated mechanisms..(1) ApoG2induces autophagy in HCC cells;(2) ApoG2interrupts the interaction of Beclin-1and Bcl-2;(3) ApoG2promotes ROS generation in HCC cells;(4) ApoG2induces ROS-mediated autophagy in HCC cells;(5) Antioxidant NAC enhances ApoG2-induced apoptotic cell death.3. The role of ROS in ABT-737-treated HCC cells and associated mechanisms(1) Different HCC cell lines presents diverse sensibilities to BH3mimetic ABT-737.HepG2and Hep3B cell lines which have high level of Bcl-2and Bcl-xl were relativelyresistant to ABT-737, while Huh7and PLC/PRF/5cell lines which have high level of Bcl-xlbut low level of Bcl-2were sensitive to ABT-737.(2) Cytoprotective autophagy is induced more dramatically in ABT-737-resistant HepG2cells than that in ABT-737-sensitive Huh7cells. (3) ABT-737triggers mild ROS in HepG2cells and relatively fierce ROS in Huh7cells.(4) ABT-737induces transient activation of JNK in HepG2cells at the early stage andsustained activation of JNK in Huh7cells.(5) ROS-JNK pathway triggered by ABT-737mainly contributes to autophagy in HepG2cells and primarily mediates apoptosis in Huh7cells.Conclusions:(1)(-)-Gossypol induces cytoprotective autophagy in lymphoma cells in ROS-dependentmanner. Antioxidant NAC can promote cell apoptosis via inhibiting autophagy. Theautophagy triggered by (-)-gossypol may partly due to ROS-mediated increasion of HMGB1in cytoplasm.(2) ApoG2induces ROS-dependent and cytoprotective autophagy in HCC cells which isassociated with increasion of cytosol HMGB1and activation of ERK and JNK pathway. NACenhances the cytotoxicity of ApoG2by attenuating ApoG2-induced autophagy in HCC cells.(3)The activation of ROS-JNK plays critical roles in different sensibilities of HCC cellsto ABT-737. ABT-737differentially activates JNK via ROS in ABT-737-resistant HepG2cellsand ABT-737-sensitive Huh7cells. ROS-JNK pathway induced by ABT-737mainlycontributes to autophagy which leads to drug resistance in HepG2cells and is more prone tomediating apoptosis in Huh7cells.Our study opens a new sight to potential resistance mechanism of Bcl-2inhibitors andmay be beneficial for further clinical application.