Toxicology Letters 225 (2014) 294–304
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Additive effect of zinc oxide nanoparticles and isoorientin on apoptosis in human hepatoma cell line Li Yuan, Yutang Wang, Jing Wang, Haifang Xiao, Xuebo Liu ∗ Laboratory of Functional Chemistry and Nutrition of Food, College of Food Science and Engineering, Northwest A&F University, Yangling, China
h i g h l i g h t s • • • • •
ZnO Nps could exert dose- and time-dependent cytotoxicity in HepG2 cells. The combining treatment resulted in a greater cytotoxicity than single treatment. ZnO Nps could additively potentiate ISO to induce apoptosis. ZnO Nps were uptaked by endocytic and it enhanced the cellular uptake of ISO. No significant injury was found in normal liver cell after the synergistic treatment.
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Article history: Received 8 September 2013 Received in revised form 18 November 2013 Accepted 19 December 2013 Available online 26 December 2013 Keywords: Zinc oxide nanoparticles Isoorientin Apoptosis Mitochondrial stress PI3K/Akt MAPKs
a b s t r a c t Metal nanomaterial could effectively decrease tumour resistance to anti-cancer drugs. In this paper, we have explored the synergistic effect and mechanisms of zinc oxide nanoparticles (ZnO Nps) and isoorientin (ISO) on cytotoxicity in human hepatoma (HepG2) cells. The results showed that ZnO Nps could exert dose- and time-dependent cytotoxicity in HepG2 cells, and the combining treatment resulted in a greater cytotoxicity than single treatment. ZnO Nps could synergistically potentiate ISO to induce apoptosis through resulting in mitochondrial dysfunction, inhibiting the phosphorylation of Akt and ERK1/2, and enhancing the phosphorylation of JNK and P38. Additionally, ZnO Nps were uptaked by cells through endocytic pathway and it enhanced the cellular uptake of ISO, while no significant injury was found in normal liver cells after the combined treatment. These results suggest that the combination of metal nanoparticle with anti-cancer drugs may provide a promising alternative for novel cancer treatments. Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Liver cancer is one of the most common malignancies, and the incidence rates are increasing in many parts of the world, including the United States, Central Europe, Asia and Africa (Jemal et al., 2011). A large number of therapeutic methods, such as radiation therapy and chemotherapy, have been performed to control tumour growth. However, these medical treatments are mainly based on the use of cytotoxic drugs that act on intracellular targets that are generally common to both cancer and normal cells (Tortora et al., 1997). Moreover, cancer cells have different degrees of resistance to cytotoxic anticancer agents that directly affect their treatment (Safrit and Bonavida, 1992). To increase susceptibility of cancer cells to anticancer agents, improve the efficacy of treatment
∗ Corresponding author at: Yangling, Shaanxi 712100, China. Tel.: +86 029 87092325; fax: +86 029 87092817. E-mail address:
[email protected] (X. Liu).
by a reduction in their dosage and reverse drug resistance, many efforts have been made using co-therapy, chemical modification, gene therapy, vector delivery and other means (Dowdy et al., 2006; Wang and Lippard, 2005; Egger et al., 2004). Therefore, it is worthwhile to combine two or more drugs to improve the efficacy of treatment and reduce drug dosage. Nanoparticles of metal oxides are new generation materials that are being widely investigated for biomedical and therapeutic applications (such as drug delivery systems and cancer therapy) because of their unique physical and chemical characteristics (Venkatpurwar et al., 2011; Lanone et al., 2009;Jeng and Swanson, 2006). Some nanomaterials, such as gold nanoparticles and iron oxide nanoparticles, have been specifically utilized in cancer therapy (Liu et al., 2010; Jain et al., 2005). Zinc oxide nanoparticles (ZnO Nps) are amongst the most commonly utilized nanomaterials in biomedical and therapeutic applications, most notably in antibacterial behaviours because they damage the membrane wall and induce oxidative stress and DNA damage in bacteria (Kumar et al., 2011; Zhang et al., 2007; Brayner
0378-4274/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.12.015
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Fig. 1. Chemical structure of ISO (A) and TEM images of ZnO Nps in RPMI-1640 (scale bar = 100 nm) (B).
et al., 2006). Many in vitro studies also suggested that ZnO Nps can be developed as an alternative anticancer therapeutic agent and potentially for multimodal cancer treatment (Zhang et al., 2011). For example, ZnO Nps induced cell toxicity and apoptosis in murine macrophages (RAW264.7 cells) (Ng et al., 2011) and human myeloblastic leukaemia (HL60) cells (Premanathan et al., 2011), leading to the generation of reactive oxygen species (ROS), DNA damage and p53 signalling pathway activation. However, the effect of ZnO Nps on cell toxicity in the human hepatoblastoma cancer cell line (HepG2) and their anticancer properties remain poorly understood. Isoorientin (3 ,4 ,5,7-tetrahydroxy-6-C-glucopyranosyl flavone, ISO), a common C-glycosyl flavone (Fig. 1), could be extracted from many plant species, such as Phyllostachys pubescens (Yong-Chun et al., 2012), Patrinia (Peng et al., 2005), Fagopyrum esculentum (Mitsuru and Miyuki, 2002) and Drosophyllum lusitanicum (Budzianowski et al., 2000). It has been suggested that ISO significantly reduced the proliferation of HepG2 cells (Pacifico et al., 2010), and in our previous study, we demonstrated that ISO induced apoptosis in HepG2 cells through an increase in the generation of ROS. This resulted in mitochondrial dysfunction, which inhibited the PI3K/Akt signalling pathway and increased the phosphorylation of JNK and p38 kinases (Yuan et al., 2012, 2013). Combination of traditional chemotherapy with nanotechnology may provide a promising alternative for novel cancer treatments. The use of nanoparticles to sensitize tumour cells to anticancer agents in vitro and in vivo has been described recently. Apoptosis is a normal physiological process, and numerous studies have showed that activation of the apoptotic pathway in tumour cells is considered to be a major protective mechanism against the development and progression of cancer (Ghobrial et al., 2005). In the present study, to more deeply understand the toxic effects exerted by ZnO Nps and the synergistic effect of ZnO Nps and ISO, we demonstrate the viability of HepG2 cells after treatment with ZnO Nps alone and together with ISO and investigate the possible cellular mechanisms of action of ZnO Nps and ISO by measuring the effects of mitochondrial dysfunction, PI3K/Akt and MAPK signalling pathways. 2. Materials and methods 2.1. Reagents and antibodies ZnO Nps (purity > 97%, average particle size < 50 nm, the structure is shown in Fig. 1B) and isoorientin (purity ≥ 98%) were purchased from Sigma and Forever Biotechnology, Ltd. (Shanghai, China), respectively. RPMI-1640 cell cultures, foetal bovine serum (FBS) and BCA protein kit were purchased from Thermofisher (Shanghai, China). MTT (3-(4,5-dimethylthiazol-2-yl)
-2, 5-diphe-nyltetrazoliunbromide) (purity ≥ 93%) was obtained from Wolesn Biotechnology, Ltd. (Xian, China). Hoechst 33258, acridine orange (AO), ethidium bromide (EB), and JC-1 (5,5 ,6,6 tetra-chloro-1,1 ,3,3 -tetraethylbenzimidazolyl-carbocyanine iodide) were provided by Beyotime Institute of Biotechnology (Jiangsu, China). AST and ALT assay kits were purchased from Nanjing Jiancheng Bioengineering Institue (Nanjing, Jiangsu, China). Polyclonal antibody specific to Bcl-2 (SC-492), Bax (SC-493) cytochrome c (SC-7159), FOXO4 (SC-5221), and monoclonal antibodies specific for ␣-tubulin (SC-5286) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Cleaved caspase-3 (p17) (BS7004) was obtained from Bioworld Technology, Inc. (Louis Park, America). PARP (9542), pAkt (Ser473) (9271), Akt (9272), P-P44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101), P44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9102), P-SAPK/JNK (Thr183/Tyr185) (9251), SAPK/JNK (Thr183/Tyr185) (9252), P-P38 MAP kinase (Thr180/Tyr182) (9211) and P38 MAP kinase (Thr180/Tyr182) (9212) were purchased from Cell Signalling Technology Company (Shanghai, China). All other chemicals made in China were of analytical grade. 2.2. Cell culture and ZnO Nps preparation Human hepatoblastoma cells line (HepG2 cell) was obtained from the Fourth Military Medical University (Xian, China) and cultured in RPMI-1640 medium with 10% FBS, and 1% penicillin–streptomycin at 37 ◦ C in a humidified incubator (5% CO2 , 95% air). ZnO nanoparticles were prepared in sterile PBS (0.1 M, pH 7.4), sterilized by UV exposure for 15 min and bath sonicated for 10 min. This was diluted into different final concentrations (0.5, 1, 2, 3, 4 and 5 g/mL) with serum-free culture medium, and further bath sonicated for 10 min and followed incubated with cells. 2.3. Cell viability measurement The cell viability was determined using MTT assay. Cells were seeded at a density of 1 × 104 cells/well in 96-well polystyrene culture plates, and cultured with 100 L cell culture medium per well at 37 ◦ C with 5% (v/v) CO2 for one day. After 24 h of incubation, 100 L of medium were removed from each well, and 100 L of different concentrations of ZnO Nps were added to the cells for 24 or 48 h. Subsequently, 100 L of 0.5% (w/v) MTT dissolved in phosphate buffered saline, were added to each well. After 4 h of incubation, 100 L of DMSO were added to each well to dissolve the formazan crystals. Absorbance values at 490 nm were measured with a microplate reader (Bio-Rad laboratories Ltd., China). Cell viability was expressed as a percentage of the control (untreated cells).
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2.4. Hoechst 33258 and AO–EB staining assay To investigate whether the cell viability decrease in HepG2 cells mainly by apoptosis or necrosis after treatment, the nuclear morphology and cells time periods were analyzed with Hoechst 33258 and AO–EB staining, respectively. Cells were seeded into 35 mm polystyrene culture dishes at a seeding density of 1 × 104 cells/mL and cultured with 2 mL cell culture medium overnight. After treatment, cells were fixed in 4% paraformaldehyde (in PBS) for 10 min and permeabilized with 0.1% Triton X-100 for 15 min at room temperature of 22 ◦ C, washed with PBS for twice, then cells were stained with 1 mg/mL Hoechst 33258 for 30 min at 37 ◦ C, and observed using a fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan). Cells were incubated with 5 L of 100 g/mL AO–EB at room temperature in the dark, followed by observation under a fluorescence microscope. 2.5. Analysis of DNA fragmentation The fragmented DNA was extracted with the DNA Extraction Kit according to the described method with some slight modifications. In brief, the cell layer was rinsed twice with 2 mL of PBS (pH 7.4) and pooled in 1.5 mL tubes, then lysed overnight at 50 ◦ C in a lysis buffer with proteinase K solution. The DNA fragments were purified by phenol/chloroform extraction and absolute ethyl alcohol precipitation, last resuspended in TE solution. DNA samples were analyzed by electrophoresis (1 h at 80 V/30 mA) on a 1.2% agarose gel containing 0.5 mg/mL ethidium bromide. 2.6. Measurement of mitochondrial membrane potential (MMP) The mitochondrial membrane potential (MMP) was measured by the mitochondria-specific lipophilic cationic fluorescence dye JC-1. JC-1 is capable of selectively entering mitochondria where it forms monomers and emits green fluorescence when MMP collapsed during apoptosis. In normal conditions, JC-1 aggregates and gives red fluorescence. The ratio of red and green fluorescence of JC-1 reflects the change of MMP. Cells were seeded at a density of 1 × 104 cells/well in 96-well polystyrene culture plates, and cultured with 100 L cell culture medium per well overnight. Then, cells were treated with ZnO Nps alone or together with ISO. After 48 h, 100 L of medium was removed from each well and 100 L of 5 g/mL JC-1 was added to the cells for 1 h. Finally, cells were washed twice with PBS, then qualitatively and quantitatively analyzed by fluorescence microscope and microplate reader (Molecular Devices, American), respectively.
expressed as relative DCF fluorescence per microgram of protein (Syama et al., 2013). 2.8. SDS-PAGE and western blot analysis For preparation of samples from the in vitro studies, HepG2 cells were washed twice with PBS (pH7.4), and lysed in cell lysates (P0013, Beyotime Institute of Biotechnology, Jiangsu, China) with 1% of PMSF and 20 mM NaF, then incubated for10 min on ice. The extracted proteins were removed from the culture dish by gentle scraping with a rubber policeman and transferred to a microcentrifuge tube. The samples were centrifuged at 15,000 × g for 10 min at 4 ◦ C, and then the supernatant was transferred to a new tube. Total protein concentration was determined using the BCA Protein Kit (Thermofisher, Shanghai, China). The homogenates prepared from the cells were treated with the SDS sample buffer and then immediately heated at 95 ◦ C for 10 min. The proteins were separated by SDS-PAGE and electro-transferred onto a polyvinylidene fluoride membrane (0.45 m, Millipore) using a semidry transfer apparatus (Bio-Rad, Shanghai, China). The blocking was carried out for 2 h in 5% nonfat dry milk in TBST (20 mM Tris, 166 mM NaCl, 0.05% Tween 20, pH 7.5). Then the membrane was washed for 15 min at room temperature in table concentrator (70 r/min), and repeated three times. The primary antibodies were added as per the manufacturer’s recommended dilution in TBST buffer overnight at 4 ◦ C. Next day, after three washes in TBST, secondary antibodies were added and incubated at 25 ◦ C for 2 h. Followed by three washes with TBST, the blots were detected with chemiluminescent substrate (Thermofisher, China) and developed using Molecular Imager Chemidoc XRS System (Bio-Rad, Shanghai, China). 2.9. Observation of cell morphology and nanoparticle biodistribution by TEM After treatment with ZnO Nps and ISO, cells were washed with PBS and then centrifuged at 1000 r/min for 10 min. The supernatants were removed. The cell pellets were fixed in a 0.1 M PBS solution containing 2.5% glutaraldehyde for 12 h. They were then washed with 0.1 M PBS, embedded in 2% agarose gel, postfixed in 4% osmium tetroxide solution for 1 h, washed with PBS, dehydrated in a graded series of ethanol (30%, 50%, 70%, 80%, 90%, and100%), and embedded in epoxy resin. The resin was polymerized at 30 for 24 h and 60 ◦ C for 48 h, respectively. Ultrathin sections obtained with a ultramicrotome were stained with uranyl acetate and lead citrate, and imaged under the transmission electron microscopy (TEM) (HT7700, Hitachi, Japan). 2.10. HPLC analysis of intracellular ISO concentration
2.7. Detection of intracellular ROS production Cellular ROS was measured with 2 ,7 dichlorodihydrofluorescein diacetate (H2 DCFDA, Sigma). This dye is a stable nonpolar compound, which diffuses readily into the cells and yields DCFH. Intracellular ROS in the presence of peroxidase changes DCFH to the highly fluorescent compound DCF. Thus, the fluorescent intensity is proportional to the amount of ROS, which is produced by the cells. For the assay, the cells were plated in 6-well polystyrene culture plates at a density of 1 × 104 cells/mL and cultured with 2 mL cell culture medium overnight. After treatment, 10 M H2 DCFDA was added to the wells for 30 min at 37 ◦ C. Then, cells were washed twice with PBS, and immediately determined using a fluorescence microscope. For quantitative testing, cells were lysed in cell lysates and centrifuged at 15,000 × g for 10 min at 4 ◦ C. The DCF fluorescence intensity of supernatant was measured via fluorescence microplate reader at 485 nm excitation and 535 nm emission. Cellular ROS levels were
After treatment, cells were washed with PBS and lysed by cell lysate (P0013, Beyotime Institute of Biotechnology, Jiangsu, China), then centrifuged at 15,000 × g for 10 min at 4 ◦ C. The supernatant was transferred to a new tube and added methanol. 5 L of the samples were run on the Agilent HPLC system on a shim-pack upODS (250 L × 4.6) (Shimadzu, Japan). The mobile phases were 100% acetonitrile and 0.1% phosphoric acid. The separation of the product was performed using gradient elution at 25 ◦ C with a flow rate of 1.0 mL/min. The products were monitored using UV at 350 nm. The peak areas were quantified with ISO standard (purity ≥ 98%). 2.11. Assay of intracellular alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities Briefly, after treatment, cells were rinsed with PBS (pH 7.4), and lysed in cell lysates (P0013, Beyotime Institute of Biotechnology, Jiangsu, China). The samples were centrifuged at 15,000 × g for
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Fig. 2. ZnO Nps induces cytotoxicity of HepG2 cells in a dose- and time-dependent manner. HepG2 cells were incubated with different concentration of ZnO Nps for 24 h or 48 h, and then processed for MTT assay (A). The results are shown as the mean ± SD of nine separate experiments. **p < 0.01 versus control. (B) The images of cell morphologic changes in ZnO Nps-exposed cells were also observed by optic microscope. ZnO Nps-treated cells became circular, and smaller than control cells. Scale bar = 100 m.
10 min at 4 ◦ C. An aliquot of the supernatant was used for the assay of ALT and AST by commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols. Total protein concentration was determined using the BCA Protein Kit (Thermofisher, Shanghai, China).
cells with 0.5 g/mL ZnO Nps for 24 h. Therefore, for this study, we selected 1.0 g/mL ZnO Nps as the optimal treatment concentration.
2.12. Statistical analysis
As shown in Fig. 3A, combined treatment more notably changed the cell morphology than any single administration. ZnO Npstreated cells became circular and smaller than control cells. We also found that either ZnO Nps or ISO alone, as well as together, had significant inhibitory effects on HepG2 cellular activity, and combining treatment resulted in a greater inhibition of cell activity than either single treatment. The treatment with a combination of ZnO Nps and ISO markedly (p < 0.01) reduced the cell activity by 60.5%, 13.2% and 97.7% compared to the control, ISO and ZnO Nps treated cells, respectively (Fig. 3B). In addition, to confirm this effect, we examined the effect of this combined treatment on human hepatocarcinoma Hep3B and Huh7 cell lines, the higher cytotoxicity was observed in combined treatment group than ZnO Nps or ISO alone treatment group (Fig. 3C). These results suggest that the combined treatment of ZnO Nps and ISO could significantly inhibit proliferation of liver cancer cells.
All experiments were performed three times, and all the data were presented as the mean ± standard errors (SE). Statistical differences between measurements for the control and treated samples were analyzed using one-way factorial analysis of variance (ANOVA), followed by Duncan’s post hoc test (SPSS 16.0). 3. Results 3.1. Induction of cytotoxicity by ZnO Nps in HepG2 Cells ZnO Nps induced a dose- and time-dependent cytotoxic effect on HepG2 cells. Compared with the control cells, treatment of the cells with 1.0, 2.0, 3.0, 4.0 and 5.0 g/mL ZnO Nps for significantly (p < 0.01) decreased cell viability (Fig. 2A) and changed the morphology of the cells (Fig. 2B). Treatment with 0.5, 1.0 and 2.0 g/mL ZnO Nps for 48 h induced a significant (p < 0.01) decrease in cell viability by 8.9%, 33% and 64%, respectively. ZnO Nps decreased cell viability with IC50 of 2.22 g/mL and 1.54 g/mL for 24 h and 48 h, respectively. Almost all of the cells which were treated with 5 g/mL ZnO Nps lost activity, and no significant cytotoxicity or visible morphological changes were observed in the treatment of
3.2. Cytotoxicity by ZnO Nps and ISO in HepG2 Cells
3.3. Induction of apoptosis with the ZnO Nps and ISO in HepG2 cells Treatment with ZnO Nps and ISO resulted in cell shrinkage and high AO signals, indicating that the population of apoptotic cells resulting from the combined treatment was greater than from
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Fig. 3. Synergistic cytotoxicity by the combination of ZnO Nps and ISO in HepG2 cells. HepG2 cells were incubated with ZnO Nps (1 g/mL) and ISO (80 M) alone or the combination for 48 h. (A) The images of cell morphologic changes following ISO and ZnO Nps treatment, alone and incombination were observed by optic microscope. The treated cells became circular, and smaller than control cells (arrowheads). Scale bar = 50 m. (B) Cytotoxicity for HepG2 cells was processed by MTT assay. The results are shown as the mean ± SD of nine separate experiments. **p < 0.01 versus other treatment groups. (C) Cytotoxicity was assayed by MTT in Hep3B and Huh 7 cells. **p < 0.01 versus control group.
individual treatments (Fig. 4A). The protein expression of caspase8 was significantly decreased when cells were treated with ZnO Nps or ISO, and in particular, after treatment with the combination of ZnO Nps and ISO. Additionally, cleaved caspase-3 protein expression, PARP cleavage and DNA fragments also increased when cells were exposed to ZnO Nps or ISO, and a much more significant effect than that observed with either agent alone (Fig. 4B and C). All of these findings indicated that ZnO Nps could synergistically potentiate ISO to induce apoptosis in HepG2 cells.
Fluorescence intensities of DCF signals in the cells remarkably increased after ZnO Nps and ISO combined treatment (Fig. 5C). ROS level in the combined treatment group was 2.29, 1.52 and 2.00 times higher than that in control group, ISO group and ZnO Nps group, respectively.
3.4. Induction of mitochondrial dysfunction by two agents in HepG2 cells
To assess the effects on the PI3K/Akt and MAPK signalling pathways by ZnO Nps- and ISO-induced apoptosis, the expression of relative proteins was examined (Fig. 6). p-Akt expression was reduced by ZnO Nps and ISO treatment. The p-Akt positivity in the synergistically treated cells was significantly lower than that in the ZnO Nps- or ISO-treated cells, whereas the Akt protein remained constant among all treatments. The FOXO4 expression was up-regulated by the treatment with ZnO Nps or ISO, but ZnO Nps plus ISO-treated cells showed the highest value (Fig. 6A). As shown in Fig. 6B, phospho-ERK notably decreased after ZnO Nps or ISO treatment, and the combined treatment showed the strongest effect. The levels of phospho-JNK were markedly increased in all treatment group, and the combined treatment showed the highest values (Fig. 6B). Treatment with ISO effectively increased the expression of phospho-P38 MAPK, whereas the phospho-P38 MAPK expression was not significantly altered by ZnO Nps treatment. However, it was strengthened in ZnO Nps plus ISOtreated cells, and the level was higher than that in ISO-treated cells (Fig. 6B).
Treatment with ZnO Nps and ISO could lead to MMP collapse, as the ZnO Nps and ISO combination resulted in the greatest effect on mitochondrial dysfunction. Cells exposed to ZnO Nps plus ISO for 48 h showed a significant (p < 0.01) loss of MMP that was 49%, 15.3% and 32.4% higher than that in control, ISO and ZnO Nps-treated cells, respectively (Fig. 5A). The combination of ZnO Nps and ISO on MMP loss was supported by observations from fluorescence microscopy, which showed an increase in JC-1 monomers (Fig. 5A). With the MMP collapse, the combination enhanced cytochrome c release to the cytosol, increased Bax protein expression and inhibited Bcl-2 expression compared to ZnO Nps or ISO treatment alone. The Bax:Bcl-2 ratio in ISO-treated cells, ZnO Nps-treated cells and the combined treatment group was 3.22, 2.05 and 14.43 times higher than that in control group, respectively (Fig. 5B). Because accumulating evidence supports that intracellular ROS is derived from mitochondria, in this study, ROS generation in HepG2 cells was investigated by the fluorescent dye, DCFH-DA.
3.5. Effect of ZnO Nps and ISO on PI3K/Akt and MAPK signalling pathways
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Fig. 4. Induction of apoptosis with the combination of ZnO Nps and ISO in HepG2 cells. (A) The nuclear morphology and cells time periods were stained with Hoechst 33258 and AO–EB respectively. Cells were incubated with ISO (80 M) alone, ZnO Nps (1 g/mL), or the combination for 48 h, and then exposed to 10 M Hoechst 33258 and 100 g/mL AO–EB, respectively. Green cell is liver cells, yellow cells indicated early apoptotic cells, orange cell is in the late apoptotic stage, red, dead cells. Scale bar = 50 m. (B) Caspase-8, cleaved caspase-3 and PARP cleavage were assessed by western blot analysis, ␣-tubulin was used as internal control. (C) DNA fragments were purified and separated on an 1.2% agarose gel.
3.6. TEM images of cell morphological changes and ZnO Nps distribution The normally distributed cytoplasm, uniform cell membranes, and oval nuclei were observed in the control cells (Fig. 7A and B). Cell membranes and nuclei significantly changed and shrunk when cells were treated with ISO (Fig. 7C and D) or ZnO Nps (Fig. 7E and F), and vesicles appeared within the cytoplasm. Much smaller nuclei and more vesicles were observed in the combined treatment than that in the treatment of either agent alone (Fig. 7G and H). The results also showed the uptake process of ZnO Nps in HepG2 cells. ZnO Nps could enter the cells (Fig. 7D) and then be enveloped in the different stages of endocytic vesicles, such as endosomes (Fig. 7A) and autophagosomes (Fig. 7C), and ultimately dispersed inside the nucleus (Fig. 7B). These findings suggested that ZnO Nps could enter the cells by endocytosis. In addition, silica nanoparticles were found to deposit inside the cell as clusters or as individual forms.
3.7. Effect of ZnO Nps on the intake of ISO in HepG2 cells As shown in Fig. 8B, with the increasing treatment time, the intracellular concentration of ISO showed an increasing trend in cells treated by ISO both alone and together with ZnO Nps. ZnO Nps could significantly enhance the levels of ISO in HepG2 cells, and the concentrations of ISO in cells treated by ISO plus ZnO Nps were 69.6% and 70.7% higher than that in cells treated by ISO alone for 24 h and 48 h, respectively.
3.8. Effect of ZnO Nps and ISO on normal liver cells To investigate the effect of ZnO Nps and ISO on two normal liver cell lines, buffalo rat liver cells (BRL-3A) and human liver cells (HL7702) were chosen. Liver cells were treated with ZnO Nps and ISO at the same concentration. As shown in Fig. 9A, no significant cytotoxicity was found in the treatment of cells with ZnO Nps and ISO alone, whereas the combined treatment with ZnO Nps and ISO decreased the BRL-3A and HL-7702 cell variability to 94.4% and 88.8%, respectively, and the inhibition ratio of cell variability in two normal liver cell lines was lower than that in HepG2 cells. Then we tested the effect of the combined treatment on normal liver cells morphology through AO–EB staining. The results show that no significant effect of ZnO Nps or ISO individual treatment on liver cells morphology, while small amount of cell shrinkage and AO signals were observed in the combined treatment (Fig. 9B). Next we assessed hepatic injury by measuring the levels of ALT and AST in cells. Our results showed that the levels of ALT and AST were increased in HL-7702 cells by all treatment group; whereas they were decreased in BRL-3A cells treated by ISO or ZnO Nps alone, and the combined treatment obviously reversed the decrease of ALT and AST (Fig. 9C and D). The levels of ALT and AST both in HL-7702 and BRL-3A cells were lower than the upper limit of what is considered normal for these cell lines (40 U/L), suggesting that no significant hepatic injury was found in HL-7702 and BRL-3A cells after treatment. Additionally, we examined the effect of the combined treatment on PI3K/Akt and MAPK signalling pathways (Fig. 9E). In HL-7702 cells, although ISO effectively increased the phosphorylation of Akt,
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Fig. 5. Induction of mitochondrial dysfunction with the combination of ZnO Nps and ISO in HepG2 cells. (A) The cells were cultured with ISO (80 M) alone, ZnO Nps (1 g/mL), or the combination for 48 h, and stained with JC-1 at 37 ◦ C for 1 h. The images were detected by fluorescence microscope. Red fluorescence represents the mitochondrial aggregate form of JC-1, indicating intact MMP. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of MMP. Scale bar = 50 m. The graph depicts the fuorescence intensity that detected by multifunctional microplate reader. The results are expressed as the mean ± SD of nine separate experiments. **p < 0.01 versus other treatment groups. (B) Representative image of immunoblots for Bcl-2, Bax and cytochrome c (in cytosol) (western blotting analysis). ␣-Tubulin was used as internal control. The graph shows the ratio of values of Bax/Bcl-2. Densitometric analysis is mean ± SD of four independent experiments. **p < 0.01 versus other treatment groups. (C) Stimulated effect of the combination of ZnO Nps and ISO on the ROS generation in HepG2 cells. Cells were stained with 10 M H2 DCFDA for 30 min, and were observed under a fluorescent microscope. Scale bar = 50 m. The graph depicts the DCF fuorescence intensity that detected by fluorescence microplate reader. The results are expressed as the mean ± SD of nine separate experiments. **p < 0.01 versus control group.
the phospho-Akt expression was not significantly altered by ZnO Nps treatment. Conversely, in BRL-3A cells, no significant effect of ISO with or without ZnO Nps on the phosphorylation of Akt was found. No obvious change in the phospho-Akt expression was observed in the ZnO Nps group. The phosphorylation of ERK was apparently increased by the combination of ISO and ZnO Nps in two types of liver cell lines. The phosphorylation of JNK and P38 could be decreased in two liver cell lines exposed to ISO, but ZnO Nps had no significant effect on the expression of the phosphorylation of JNK and P38; ZnO Nps could also enhance the decrease of phosphoJNK. These results demonstrated that ISO treatment promoted the phosphorylation of Akt and reversed the phosphorylation of MAPK. ZnO Nps could enhance the effect of ISO on PI3K/Akt and MAPK signalling pathways, though there was no significant effect of ZnO Nps alone on these two signalling pathways. 4. Discussion In the process of chemotherapy, some agents cooperate with other anticancer drugs to induce apoptosis of cancer cells and may increase drug efficacy, providing more effective strategies for therapeutic intervention (Lin et al., 2005). In the present study, we combine ISO with ZnO Nps to demonstrate the effect on HepG2 cells. The results showed that the combination of ZnO Nps and
ISO dramatically and significantly increased apoptosis of HepG2 cells (Fig. 4), and no significant affects on HL-7702 and BRL-3A cell growth was found (Fig. 9A). It has been reported that ZnO Nps can also induce apoptosis in human myeloblastic leukaemia (HL60) cells (Premanathan et al., 2011), human colon carcinoma (LoVo) cells (Berardis et al., 2010) and leukaemia K562 cells (Guo et al., 2008). Apoptosis is a normal physiological process and is driven by two different death signalling pathways, the cell death receptormediated extrinsic pathway and the mitochondria-mediated intrinsic pathway. The mitochondria-mediated apoptotic pathway involves the mitochondrial membrane potential (MMP) loss, cytochrome c release and the cleavage of caspase-3, which ultimately results in chromatin condensation, DNA fragmentation and cell apoptosis (Circu and Aw, 2010). Additionally, the Bcl-2 family of proteins is the essential regulator of cytochrome c release through changing its conformation and controlling mitochondrial permeability transition pores (mPTPs) (Lei et al., 2006). Our results have shown that a much more severe mitochondrial dysfunction was caused by ISO in the presence of ZnO Nps (Fig. 5). The combined treatment significantly decreased the MMP, and enhanced the Bax/Bcl-2 ratio and the release of cytochrome c to the cytosolic fraction. In the present study, the level of cellular ROS was apparently increased by all treatments, and the cooperative treatment
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Fig. 6. Effect of the combination of ZnO Nps and ISO on the PI3K/Akt and MAPK signalling pathways of HepG2 cells. Cells were treated with ISO (80 M) alone, ZnO Nps (1 g/mL), or the combination for 48 h, and then relative proteins of PI3K/Akt (A) and MAPK (B) signalling pathways were analyzed by western blotting. ␣-Tubulin was used as internal control. The results shown here are representative of three independent experiments. **p < 0.01, compared with control groups. ## p < 0.01 versus ZnO Nps or ISO individual groups.
showed the highest level (Fig. 5C). The same results were found in ZnO Nps-treated human colon carcinoma (LoVo) cells (Berardis et al., 2010), normal human lung fibroblasts (WI-38) (Park et al., 2011), human bronchial epithelial (BEAS-2B) cells, human neonatal foreskin fibroblasts (BJ) and murine macrophages (RAW264.7) (Ng et al., 2011). At physiological levels, ROS is involved in and regulates the intracellular signalling pathways as “redox messengers,” whereas excessive production of ROS can result in mitochondrial oxidative stress, induce oxidative modification of cellular macromolecules, inhibit protein function and promote apoptotic cell death (Xu et al., 2012; Raza et al., 2011). Oxidative stress is a common mechanism for cell apoptosis induced by nanoparticles (Muller et al., 2010; AshaRani et al., 2009). Numerous data show that oxidative stress caused by ZnO Nps could occur through the acidic dissolution of ZnO Nps in an intra-endosomal reaction to release Zn2+ ions within the cytoplasmic space, ultimately triggering ROS generation (Ng et al., 2011; Xia et al., 2008). However, the mechanism of how Zn2+ triggers the ROS generation remains unresolved. Accumulating evidence has indicated that the PI3K/Akt pathway is one of the most important signalling pathways in regulating cell proliferation, growth, apoptosis, survival and metabolism. Akt is the major effector of the PI3K/Akt signalling pathway, and it can inhibit the levels of pro-apoptotic proteins (e.g. Bad, Bax, caspase-9) (Liu et al., 2011; Franke et al., 2003), transcription factors (Babchia et al., 2010) and another protein kinase (Tang and Laurence, 2003). FOXO4 is a member of the forkhead transcription factor family and is a downstream factor in the PI3K/Akt signalling pathway. FOXO4 can inhibit cell cycle progression and induce apoptosis through inactivating the anti-apoptotic proteins (Bim, Bcl-6) (Tang et al., 2002; Urbich et al., 2005; Medema et al., 2000). In accordance
with these previous studies, our results have shown that the phosphorylation of Akt was markedly decreased both in ZnO Nps- or ISO-treated cells either alone or together; the combined treatment showed the highest value, and the protein expression of FOXO4 in the cytosol was also increased by all treatments (Fig. 6A). Furthermore, MAPKs involves the extracellular signal-regulated kinases: ERK1/2 (p44/p42), c-Jun amino-terminal kinase JNK (p46/p54) and P38 kinase. ERK1/2 is activated primarily by mitogenic stimuli such as growth factors leading to cell growth and survival (Chang and Karin, 2001). JNK and P38 are activated by hydrogen peroxide, UV irradiation, heat, osmotic shock and chemotherapeutic drugs, ultimately resulting in apoptotic cell death (Mansouri et al., 2003; Ichijo, 1999). Our results showed that ZnO Nps significantly decreased the phosphorylation of ERK1/2 and increased the phosphorylation of JNK in the presence or absence of ISO. No significant change was found in the protein expression of p-P38 in ZnO Np-treated cells, whereas ZnO Nps could enhance the effect of ISO on the p-P38 protein expression. These results suggest that the synergistic induction of apoptosis by the zinc oxide nanoparticles and isoorientin is related to the inhibition of PI3K/Akt and ERK1/2 and the activation of JNK and P38 in HepG2 cells. It has been reported that nanoparticles overcome tumour resistance to anti-cancer drugs through restoring defective endocytosis, and they increase the intracellular accumulation of anti-cancer drugs (Guo et al., 2008; Liang et al., 2010). Consistent with previous research, our results showed that ZnO Nps could increase the intracellular concentration of ISO in a time dependent manner (Fig. 8). The TEM pictures also demonstrated that ZnO Nps increased the cytotoxicity of ISO in HepG2 cells. Morphological changes of cell injury were also observed clearly from the TEM images, such as shrinkage of the cell nucleus. Numerous reports indicate that in
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Fig. 7. The TME images of cell morphology, and ZnO Nps cellular uptake process and cellular distribution in HepG2 cells. (A) and (B) Control, (B) and (C) ISO treated cells, (E) and (F) ZnO Nps treated cells, (G) and (H) the combining treatment of ZnO Nps and ISO. Scale bar = 2 m. (a) Silica ZnO Nps were enclosed in lysosomes and dispersed in cytoplasm with clusters. (b) The ZnO Nps were dispersed in cell nucleus with clusters. (c) The ZnO Nps were dispersed in cytoplasm with clusters. (d) The ZnO Nps entered the cells and were enclosed in endocytic vesicles. Scale bar = 500 nm.
the uptake process of nanoparticles, nanoparticles (usual diameter