Zinc-Doped Copper Oxide Nanocomposites Inhibit the Growth of

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Zinc-doped copper oxide nanocomposites inhibit the growth of human cancer cells through reactive oxygen species-mediated NF-#B activations Ru Yuan, Huanli Xu, Xiaohui Liu, Ye Tian, Cong Li, Xiaoliang Chen, Shuonan Su, Ilana Perelshtein, Aharon Gedanken, and Xiukun Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09542 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 29, 2016

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ACS Applied Materials & Interfaces

Zinc-doped Copper Oxide Nanocomposites Inhibit the Growth of Human Cancer Cells through Reactive Oxygen Species-Mediated NF-κB Activations

Ru Yuan1＃, Huanli Xu1＃, Xiaohui Liu1, Ye Tian1, Cong Li1, Xiaoliang Chen1, Shuonan Su1, Ilana Perelshtein2, Aharon Gedanken2, Xiukun Lin1＊

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Department of Pharmacology, School of Basic Medical Sciences, Capital Medical

University, Beijing 100069, China. 2

Department of Chemistry and Nanomaterials, Bar-Ilan University Center for Advanced

Materialsand Nanotechnology, Ramat-Gan 52900, Israel.

Corresponding author Dr. Xiukun Lin Department of Pharmacology, School of Basic Medical Sciences Capital Medical University No.10, Youanmenwaixitoutiao, Fengtai District, Beijing 100069, China. Tel and Fax: 86+010-83911835. E-Mail: [email protected]

＃

Ru Yuan and Huanli Xu have contributed equally to this work.

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Abstract Purpose: Zinc-doped copper oxide nanocomposites (nZn-CuO NPs) are novel nanparticles synthesized by our group. In the present study, the antitumor effects and the underlying molecular mechanisms of the nZn-CuO NPs were investigated. Methods and results: The cytotoxicity of nZn-CuO NPs against several types of cancer cell lines was studied using MTT assay. Results showed that nZn-CuO NPs exerted obvious anti-proliferation effects on cancer cells and relatively weak anti-proliferation effects on normal cells. The antitumor mechanisms of nZn-CuO NPs were further investigated using human liver cancer HepG2 cells and human pancreatic cancer Panc28 cells. Hoechst 33342 staining and FITC-Annexin V/PI staining showed that nZn-CuO NPs could induce cell apoptosis in a dose dependent manner. Cell-cycle analysis using flow cytometry revealed that nZn-CuO NPs were able to arrest the cell cycle in G2/M phase. Also, nZn-CuO NPs were found to induce reactive oxygen species generation. Further studies confirmed that nZn-CuO NPs could increase p-IKKα/β and nucleus p-NF-κB p65 expressions and decrease IKKα, IKKβ, IκBα, and nucleus NF-κB p65 expressions in both cell lines. Conclusion: Overall, our data demonstrated that nZn-CuO NPs could selectively inhibit the growth of cancer cells via reactive oxygen species-mediated NF-κB activation. The current study provides primary evidence that nZn-CuO NPs possess the potential to be developed as a novel anticancer agent. Keywords: Zinc-doped copper oxide nanocomposite, Cancer cell, Antitumor mechanisms, Apoptosis, Reactive oxygen species, NF-κB activation

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1. Introduction

Nanoparticles (NPs) represent an innovative tool in cancer prevention, diagnosis and treatment. The physical, chemical, and biological properties of NPs differ fundamentally from those of the corresponding bulk material because the quantum mechanical properties of atomic interactions which are influenced by their sizes1. The use of nanotechnology in cancer treatment offers some exciting possibilities, including the possibility of destroying cancer tumors with minimal damage to normal cells, as well as the detection and elimination of cancer cells before forming tumors2. Nano metal oxides (nMeOs) are one of the most promising NPs due to their potential physico-chemical properties such as higher affinity, low molecular weight, and larger surface area3. The most common nMeOs mainly include CuO NPs, ZnO NPs, Fe3O4 NPs, cerium oxide NPs, and more. Previous studies have shown that nMeOs can inhibit cell viability and induce apoptosis of many human cancer cell lines, including lung adenocarcinoma A549 cells, hepatocellular carcinoma HepG2 cells, breast cancer MCF-7 cells, squamous tumor SCL-1 cells, epithelial colorectal adenocarcinoma Caco-2 cells, and glioblastoma U251 cells4-8. It was extensively reported that the effect of nMeOs on apoptosis induction was closely related to reactive oxygen species (ROS) production depending on the presence of defect sites in the structure of nMeOs9. In recent years, ZnO NPs were extensively studied for their implications in cancer

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therapy. Studies have shown that ZnO NPs exert distinct effects on mammalian cell viability, with rapidly proliferative cancer cells being more susceptible and quiescent cells or normal cells being less sensitive6,10,11. Recently, it has been found that doping ZnO with Mg or Sb slightly increased the activity of nanosized ZnO12,13; the enhanced activity of doped metal oxides was due to their increased structural defects and higher ROS production14. Our research group has reported the synthesis of a novel zinc-doped copper oxide nanocomposites (nZn-CuO NPs), Cu0.89Zn0.11O, by the sonochemical method and characterized it by chemo-physical methods including inductive coupled plasma (ICP) analysis, scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), high resolution scanning electron microscopy (HRSEM), and electron spin resonance (ESR)

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. Our previous study also showed that the small

particles of nZn-CuO NPs exhibited enhanced killing of S. aureusand E. coli in both regular and multidrug resistant bacteria when compared to ZnO NPs and CuO NPs15. The improved antimicrobial activity might be associated with structural defects in the solid crystal induced by the sonochemical process that led to an increased ROS production15. Also, we found that compared with nZnO16, the highly effective antibacterial agent nZn-CuO NPs could induce only mild acute toxicity in X. laevis embryos. However, the effects of nZn-CuO NPs on cancer cells have not been studied. In this study, we examined the antitumor effects of nZn-CuO NPs on several cancer cell lines and the underlying molecular mechanisms of the nZn-CuO NPs were also investigated.

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2. Materials and Methods

2.1 Materials and reagents

DMEM medium, RPMI1640 medium, fetal bovine serum and Dulbecco phosphatebuffered saline were purchased from Invitrogen Co. (Carlsbad, CA). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo lium (MTS)/phenazinemethosulfate (PMS) assay kit was purchased from Promega (Madison, WI, USA). The primary antibodies and secondary antibodies were purchased from Cell Signaling Technology, Inc. (Boston, USA). Mammalian whole cell protein extraction kit, Nuclear Protein Extraction Kit, BCA Protein Assay Kit, and sodium dodecylsulfate (SDS) were bought from Nanjing KeyGEN BioTECH. Co. Ltd. in China. All other chemicals used were of the highest purity available from commercial sources.

2.2 Synthesis and characterization of nZn-CuO NPs The detailed description of the synthesis and the characterization of the Zn doped CuO was described elsewhere15,16. Briefly, nZn-CuO NPs were prepared from a mixed solution of copper acetate and zinc acetate at a molar ratio of 3:1, by the sonochemical method. The nZn-CuO NPs were characterized by Ion Coupled Plasma (ICP) analysis, , X-ray diffraction (XRD, Bruker D8 diffractometer with Cu Kα), , High Resolution

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Transmussion Electron Microscope (HRTEM, JEOL 2100, with accelerating voltage of 200 kV), and Electron Spin Resonance (ESR). Ion’s concentration was probed by ICP, by dissolving the nanoparticles in 0.5M HNO3. The molecular formula of nZn-CuO NPs was proved to be Cu0.89Zn0.11O. The crystalline structure of the nanoparticle’s powder was examined by XRD. The morphology and particle size was observed by HRTEM,

2.3 Cell culture and treatment with nZn-CuO NPs Human hepatocellular carcinoma cell line HepG2 and human umbilical vascular endothelium cell line HUVEC were purchased from ATCC, USA. Human hepatoma cell line Bel7402, human lung adenocarcinoma cell line A549, human pancreatic cancer cell line Panc 28, human fibrosarcoma cell line HT1080, human cervical carcinoma cell line Hela, and human hepatocyte L02 cell line were purchased from Cell Resource Center of Peking Union Medical College Beijing, China. HepG2, HUVEC, Panc 28, and L02 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 in air at 37°C. Bel7402, A549, HT1080, and Hela cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 in air at 37°C. Then the cells in logarithmic growth phase were collected for the anti-proliferation assay, ROS detection, cell-cycle analysis, apoptosis analysis and western blot. nZn-CuO NPs were suspended in cell culture medium and diluted to appropriate concentrations. Just before the experiment, different concentrations of nZn-CuO NPs

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were then sonicated at room temperature using a sonicator bath for 10 min at 40 W to avoid NPs agglomeration. Following exposure to nZn-CuO NPs for 48 h, cells were harvested to determine the proliferation, ROS production, cell-cycle perturbations, and apoptosis.

2.4 Anti-proliferation assay

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-te trazolium (MTS)/phenazinemethosulfate (PMS) assay was performed to analyze the antiproliferative effect of nZn-CuO NPs. Briefly, cells in logarithmic growth phase were plated at a density of 3 × 103 cells/well in 96-well plates. After 24 h incubation, the cells were incubated with certainconcentrations of nZn-CuO NPs for 48 h. Then, MTS and PMS mixed at the ratio of 20:1 (20 µL/well) were immediately added to the culture medium. Formazan production was analyzed at 490 nm in a plate reader after incubation for additional 2 h. The inhibitory rates and half inhibitory concentration (IC50) values were then calculated.

2.5 Cell-cycle analysis using flow cytometry

Cell-cycle perturbations were analyzed using flow cytometry after PI staining. Briefly, HepG2 and Panc28 cells in logarithmic growth phase were collected and cultured

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in 60 mm plates at 3 × 105 cells/well in serum free culture medium for 24 h. Then cells were exposed to certain concentrations (2.5, 5.0, and 10.0 µg/mL) of nZn-CuO NPs for 48 h. Then the cells were harvested, washed twice with cold PBS, and fixed in ice-cold 70% ethanol at 4°C overnight. Then, cells were washed twice with cold PBS, and resuspended in detection buffer containing 50 µg/mL PI and 50 µg/mL RNase A. After incubation at 37°C for 30 min, the cells were analyzed by flow cytometry (BD Biosciences, Le Pont de Claix, France).

2.6 Apoptotic analysis

The apoptotic morphology of the treated cells was detected by Hoechst 33342 staining. Briefly, cells were collected and seeded in 96-well plates at 3000 cells/well. After incubation overnight, the cells were exposed to certain concentrations (2.5, 5.0, and 10.0 µg/mL) of nZn-CuO NPs for 48 h. Then the cells were washed and stained with Hoechst 33342 (10.0 µg/mL) for 20 min at 37°C. Finally, the cells were washed with PBS, and morphologic changes of the cells were observed under a fluorescence microscope and photographed. Apoptosis was quantified by flow cytometry after staining with annexin V-FITC/PI staining kit according to the manufacturer’s instruction. Briefly, cells were collected and seeded in 60 mm culture dishes at 3×105 cells/mL. After incubation overnight, the cells were exposed to certain concentrations (2.5, 5.0, and 10.0 µg/mL) of nZn-CuO NPs for

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48 h. Then cells were collected and 1×105 cells were resuspended in 500 µL detection buffer. Then, 5 µL PI and 5 µL Annexin V-FITC were added to the detection buffer. The cells were incubated for another 15 min and analyzed by a BD FACS Calibur™ system.

2.7 ROS detection assay

Intracellular production of ROS was measured using Reactive Oxygen Species Assay Kit (Jiancheng Bio-engineering, Nanjing, China). Briefly, cells were collected and seeded in 60 mm culture dishes at 3×105 cells/mL and incubated overnight. Then, the cells were exposed to certain concentrations (2.5, 5.0, and 10.0 µg/mL) of nZn-CuO NPs for 48 h. Then, cells were washed twice with HBSS and then incubated in 1 mL DCFH-DA working solution (100 µM) at 37°C for 30 min. Cells were then washed twice with PBS and resuspended in 200 µL PBS. The fluorescence intensities of intracellular dichlorofluorescein (DCF) were detected by flow cytometry with excitation at 525 nm.

2.8 Western blotting analysis

After the cells were exposed to certain concentrations (2.5, 5.0, and 10.0 µg/mL) of nZn-CuO NPs for 48 h, the cells were harvested. Whole cell proteins and nuclear proteins were extracted using mammalian whole cell protein extraction kit and Nuclear

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Protein Extraction Kit according to the manufacturer’s instructions. The protein concentrations of cellular lysates were determined using BCA Protein Assay Kit. Equal amounts of cellular lysates were subjected to 10% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. After being blocked with 5% BSA, the membranes were incubated with the first antibodies overnight at 4°C. After washing, the membranes were incubated with HRP-labeled secondary antibodies (1:4000-dilution) for 1 h. Finally, protein bands were visualized by an enhanced chemiluminescence system. The intensity of bands was quantified using Image J 1.43 software (National Institutes of Health, Bethesda, MD).

2.9 Statical analysis

Statistical analyses for these experiments were done using SPSS Software version 16.0 (Chicago, IL, USA). Data were presented as means ± S.D. A P-value of

Zinc-Doped Copper Oxide Nanocomposites Inhibit the Growth of

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