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CXC195 Induces Apoptosis and Endoplastic Reticulum Stress in Human Hepatocellular Carcinoma Cells by Inhibiting the PI3K/Akt/Mtor Signaling Pathway

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CXC195 Induces Apoptosis and Endoplastic Reticulum Stress in Human Hepatocellular Carcinoma Cells by Inhibiting the PI3K/Akt/Mtor Signaling Pathway MOLECULAR MEDICINE REPORTS 12: 8229-8236, 2015 CXC195 induces apoptosis and endoplastic reticulum stress in human hepatocellular carcinoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway XIAO-LIANG CHEN1,2*, JIAN-PING FU2*, JUN SHI3, PING WAN2, HONG CAO2 and ZHI-MOU TANG4 1Department of Surgery, School of Medicine, Nanchang University; 2Department of Hepatobiliary Surgery, Jiangxi Provincial People's Hospital; 3Department of Hepatobiliary Surgery, The First Affiliated Hospital of Nanchang University; 4Department of Oncology, Jiangxi Provincial People's Hospital, Nanchang, Jiangxi 330006, P.R. China Received December 18, 2014; Accepted September 16, 2015 DOI: 10.3892/mmr.2015.4479 Abstract. CXC195 exhibits strong protective effects against in the HepG2 cells. In addition, CXC195 inhibited the phos- neuronal apoptosis by exerting antioxidant activity. However, phorylation of phosphoinositide 3-kinase (PI3K), Akt and the pharmacological function of CXC195 in cancer remains to mammalian target of rapamycin (mTOR) in the HepG2 cells. be elucidated. The present study demonstrated that CXC195 These effects were enhanced following treatment with selected exhibited significant cytotoxic effects, and induced cell cycle inhibitors of PI3K (LY294002), Akt (SH‑6) and mTOR arrest and apoptosis in HepG2 human hepatocellular carci- (rapamycin). Furthermore, these inhibitors enhanced the noma (HCC) cell lines. Following treatment of HepG2 cells pro-apoptotic effects of CXC195 in the HepG2 cells. In conclu- with 150 µΜ CXC195 for 24 , cell viability and the apoptotic sion, the results of the present study indicated that CXC195 rate were assessed using an MTT assay and Annexin V/prop- induced apoptosis and ER stress in HepG2 cells through the idium iodide staining followed by flow cytometric analysis. inhibition of the PI3K/Akt/mTOR signaling pathway. Molecular markers of the cell cycle, apoptosis, mitochondrial function and endoplasmic reticulum (ER) stress were analyzed Introduction by western blot or polymerase chain reaction analysis. Caspase activation, cytochrome c and apoptosis-inducing factor release, Hepatocellular carcinoma (HCC), a primary hepatic tumor and analysis of the B cell lymphoma 2 (Bcl-2)-associated X with aggressive malignancy and high prevalence, is the third protein/Bcl-2 ratio demonstrated that the anticancer effects leading cause of cancer-associated mortality worldwide (1,2). of CXC195 in HepG2 cells were mediated by caspase and Although progress in HCC treatment has been achieved during mitochondria-dependent apoptosis. CXC195 also induced the last three decades, patients with advanced and terminal the expression of ER stress-associated proteins, including stages of HCC continue to have a poor prognosis, owing to the CCAAT-enhancer-binding protein homologous protein, and paucity of effective and tolerable systemic chemotherapy strat- glucose-regulated proteins 94 and 78, and led to the activa- egies (3,4). Due to the multi‑fold molecular pathogenesis and tion of multiple branches of ER stress transducers, including high chemoresistance of HCC, as well as the low selectivity of inositol-requiring enzyme 1α‑apoptosis signal-regulating chemotherapeutic drugs, it is essential to develop multi-target kinase-p38/c-Jun N-terminal kinase, and protein kinase R-like compounds, which can target several aberrant signaling path- endoplasmic reticulum kinase-eukaryotic translation initiation ways concurrently, which may not only improve medication factor 2α-activating transcription factor (ATF)4 and ATF6, efficacy, but also minimize drug toxicity (5,6). Multiple pathophysiological processes, including viral replication, protein overloading, oxidative stress and the inflammatory reaction, are involved in the oncogenesis of HCC caused by chronic liver diseases, including hepatitis B Correspondence to: Professor Zhi-Mou Tang, Department of and C viral infection, alcoholic cirrhosis and obesity-associ- Oncology, Jiangxi Provincial People's Hospital, 92 Aiguo Road, ated non‑alcoholic fatty liver disease (7). Notably, these events Nanchang, Jiangxi 330006, P.R. China in hepatocytes induce a state of stress in the endoplasmic E-mail: [email protected] reticulum (ER), termed ER stress (8,9). There has been *Contributed equally increasing awareness with regards to the role of ER stress in the homeostasis of cancer cells (5,9). ER stress occurs when Key words: CXC195, hepatocellular carcinoma, apoptosis, ER homeostasis is lost due to an overload of protein folding endoplasmic reticulum stress, phosphoinositide 3-kinase/protein in the ER. Abnormalities in ER function can cause ER stress, kinase B/mammalian target of rapamycin which results in an unfolded protein response (UPR), including three key signaling proteins: Inositol-requiring enzyme 1α (IRE1α)‑apoptosis signal-regulating kinase (ASK)-p38/c-Jun 8230 CHEN et al: CXC195 INDUCES HepG2 CELL APOPTOSIS AND ER STRESS VIA THE PI3K/Akt/mTOR PATHWAY N-terminal kinase (JNK), protein kinase R-like endoplasmic Double staining for Annexin V‑fluorescein isothiocyanate reticulum kinase (PERK)-eukaryotic translation initiation (FITC) and propidium iodide (PI). Briefly, the HepG2 cells were factor 2α (eIF2α)‑activating transcription factor (ATF)4 and detached with 0.25% trypsin (Invitrogen) and washed twice ATF6, which act as the sensors of ER stresses. Furthermore, with phosphate-buffered saline (PBS), followed by centrifuga- several mechanisms have been suggested to link ER stress tion at 12,000 x g for 5 min. A total of 1x106 cells were then with apoptosis, including the B cell lymphoma 2 (Bcl-2) and suspended in 1.25 mM extracellular calcium (131 mM NaCl, caspase family (10,11). 5 mM KCl, 1.3 mM MgSO4, 1.3 mM CaCl2, 0.4 mM KH2PO4, Tetramethylpyrazine (TMP) is one of the major bioactive 6 mM glucose, 20 mM HEPES; pH 7.4) and double-stained with components purified from the Chinese herb, Chuanxiong Annexin V-FITC and PI (1:100; BD Biosciences, San Jose, CA, (Ligusticum wallichi Franchat), and has been widely used in USA) at 4˚C for 30 min at room temperature. The fluorescence the treatment of various types of human cancer, including of each sample was subsequently quantitatively analyzed using lung cancer (12), osteosarcoma (13), gliomas (14,15), ovarian a FACSCalibur flow cytometer (BD Biosciences) and CellQuest carcinoma (16), breast cancer (17), gastric cancer (18) and software (version 5.1; BD Biosciences). HCC (19). CXC195 is a TMP analogue, which can protect human umbilical vein endothelial cells from H2O2-induced Measurement of the cell cycle. The cells were treated with apoptosis through inhibition of the mitochondria- and 20 µg/ml RNase A (Invitrogen), followed by 25 µg/ml PI. The caspase 3-dependent signaling pathways (20,21). In addition, cell population at each stage of the cell cycle was then deter- CXC195 exhibits antioxidant activity and anti-apoptotic mined by examining the intensity of PI fluorescence on a flow effects in transient focal ischemia by inhibiting the expres- cytometer using an argon laser and a 570 nm bandpass filter sion levels of NADPH oxidase and nitric oxide synthase (22), (FACSort; BD Biosciences). and by regulating the PI3K/Akt/glycogen synthase kinase 3β signaling pathway (23). Despite evidence indicating the anti- Caspase 3, 8, and 9 activity assays. HepG2 cells were gently cancer effects of TMP, there is a lack of data describing the lysed in ice-cold lysis buffer [250 mM sucrose, 1 mM EDTA, anticancer activity of its analogue, CXC195. The present study 0.05% digitonin, 25 mM Tris (pH 6.8), 1 mM dithiothreitol, examined whether CXC195 induced apoptosis and ER stress 1 µg/ml leupeptin, 1 μg/ml pepstatin, 1 µg/ml aprotinin, 1 mM in human HCC HepG2 cells, and investigated the possible benzamidine and 0.1 mM phenylmethylsulphonyl fluoride] for mechanisms underlying these effects. 30 min, and centrifuged at 12,000 xg at 4˚C. The cell lysates (30 µg) from the HepG2 cells were determined spectrophoto- Materials and methods metrically at 405 nm using a microtiter plate reader (model M100L; Microfluidics, Newton, MA, USA). The assays to Cell lines and culture. Human HCC HepG2 cells were purchased determine the activities of caspase 3, 8 and 9 were performed from the American Type Culture Collection (Manassas, VA, by incubating the cell lysates with 0.2 mM of one of the USA) and maintained in Dulbecco's modified Eagle's medium following caspase‑specific colorimetric tetrapeptide substrates: (Invitrogen; Thermo Fisher Scientific., Inc., Waltham, MA, Ac-DEVD-pNA for caspase 3, Ac-IETD-pNA for caspase 8 USA) supplemented with 10% heat-inactivated fetal bovine and Ac-LEHD-p-nitroaniline (pNA) for caspase 9, for 1 h at serum (Invitrogen; Thermo Fisher Scientific., Inc.), 100 U/ml 37˚C, as previously described (24). The increase in the absor- penicillin (Invitrogen) and 10 µg/ml streptomycin (Invitrogen) bance at 405 nm, which corresponds to the quantity of pNA at 37˚C in an atmosphere containing 5% CO2. The media were released from the peptide substrates was then converted into replaced every 2-3 days and sub-cultured when the cell popula- units of enzyme activity using a standard curve generated with tion density reached 70‑80% confluence. CXC195 (Fig. 1A) was free pNA. One unit of caspase 3, 8, or 9 activity corresponded synthesized by the direct reaction of 2-chloromethyl-3,5,6-tri- to the quantity of enzyme that releases 1 pmol/min pNA from methylpyrazine hydrochloride (Sigma-Aldrich, St. Louis, 0.2 mM DEVD-pNA, IETD-pNA or Ac-LEHD-pNA, respec- MO, USA) with 4,4'‑difluorobenzhydrylpiperazine (Sigma- tively. Lysates from HepG2 cells treated with DMSO were also Aldrich). The purity of CXC195 (>98%) was determined using used in these assays, which served as a control group. high-performance liquid chromatography (Waters, Milford, MA, USA). The examined compounds and positive control Determining the release of cytochrome c (Cyt‑c) and were dissolved in 0.1% dimethylsulfoxide (DMSO; Sigma- apoptosis‑inducing factor (AIF). The HepG2 cells were Aldrich)). LY294002, SH‑6 and rapamycin were purchased collected by centrifugation at 12,000 xg at 4˚C for 5 min at from Santa Cruz Biotechnology, Inc.
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