Nanoparticle-induced photocatalytic head and neck ...

PreliminaryRCesearch ommunication Article Nanoparticle-induced photocatalytic head and neck squamous cell carcinoma cell death is associated with autophagy

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Aim: To characterize molecular mechanisms underlying photocatalytic cell death of head and neck squamous cell carcinoma (HNSCC) by zinc oxide nanoparticles (ZnO-NPs). Method: Human HNSCC-derived FaDu cells were incubated with ZnO‑NPs followed by UVA‑1 irradiation. Cytotoxicity was assessed by MTT assay and annexin‑V propidium iodide test. Autophagy was detected by autophagosome accumulation, conversion of light chain 3 (LC3) I to LC3 II, and lysosomal activity. The generation of reactive oxygen species was measured using the 2´,7´‑dichlorofluorescein-diacetate test. Results: Apoptosis-independent cytotoxic effects were induced by 0.2 and 2 µg/ml ZnO‑NPs and UVA‑1. FaDu cells promoted autophagosome formation. Significantly elevated LC3 II and reactive oxygen species were seen after the combined application of both ZnO-NPs and UVA‑1 as photocatalytic treatment. Autophagy probably mediates cell survival under UVA‑1 or ZnO-NP exposure alone but induces self-digestive cell death after combined treatment. Conclusion: The effect of autophagy on HNSCC viability after nanoparticle-induced photocatalytic treatment seems to depend on the impact of the physicochemical trigger. Original submitted 14 June 2012; Revised submitted 19 November 2012

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Keywords: autophagy n HNSCC n nanoparticles n oxidative stress n photocatalytic n zinc oxide

in vitro [6] . ZnO-NPs mediated cell death in two HNSCC cell lines at concentrations of 0.2 and 2 µg/ml in combination with 15 min of UVA‑1 irradiation. These effects were predominant in HNSCC in comparison to non-malignant human oral mucosa cells. Flow cytometry revealed an apoptosis-independent cell death pathway. Other groups observed photocatalytic cell death in colon carcinoma and melanoma cell lines after treatment with TiO2-NPs and UV irradiation [7,8] . The production of reactive oxygen species (ROS) seems to play a crucial role in NP-induced cell death. However, the molecular mechanisms responsible for cell death by photoactivated metal oxide NPs in malignant cells need further evaluation. Furthermore, it is still unclear why these phenomena predominate in malignant cells and are less severe in nontransformed benign cells. Autophagy is a catabolic cellular process that induces the degradation of damaged cellular proteins or organelles [9] . The formation of double membrane vesicles, so-called autophagosomes, is an initial step in the process of autophagy, proceeding with the development of autolysosomes by fusion with lysosomes [10] . Certain stressors may induce autophagy such as starvation, loss of energy, or decreased growth factor input [11] . Autophagy has been reported

doi:10.2217/NNM.12.137 © 2013 Future Medicine Ltd

Nanomedicine (Epub ahead of print)

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Despite advances in treatment strategies for patients suffering from head and neck squamous cell carcinoma (HNSCC), survival rates have not improved over the last three decades [1] . Surgery, radiation, and chemotherapy are treatment modalities in HNSCC patients that may be applied as combination or single therapies. Novel therapeutic regimens need to be evaluated in order to complement present treatment algorithms. Nanotechnology has rapidly increased in importance over time, especially in biomedical applications. For example, in cancer treatment, nanomaterials have been proven to exhibit promising properties that allow them to improve tumor response to chemo- and radiotherapy [2,3] . Due to their photocatalytic properties, zinc oxide nanoparticles (ZnO-NPs) and titanium dioxide nanoparticles (TiO2-NPs) are used as components in cosmetics. Sunscreen products usually contain 5–10% ZnO-NPs or TiO2-NPs, depending on the sun protection factor [4,5] . There is no duty of declaration for the fraction of nanoscaled ZnO or TiO2 in suncreens, so the absolute amount of NPs in cosmetic products remains unknown to the consumer. In a previous study by our group, ZnO-NP-induced photocatalytic elimination of human HNSCC cell lines was demonstrated

Stephan Hackenberg1, Agmal Scherzed1, Antje Gohla2,3, Antje Technau1, Katrin Froelich1, Christian Ginzkey1, Christian Koehler1, Marc Burghartz1, Rudolf Hagen1 & Norbert Kleinsasser*1 Department of Oto-RhinoLaryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery, University Hospital of Wuerzburg, Germany 2 Rudolf Virchow Center for Experimental Biomedicine, University of Wuerzburg, Germany 3 Institute for Pharmacology and Toxicology, University of Wuerzburg, Germany *Author for correspondence: Tel.: +49 931 201 21323 Fax: +49 931 201 21321 kleinsasser_n@ klinik.uni-wuerzburg.de 1

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ISSN 1743-5889

Preliminary Communication

Hackenberg, Scherzed & Gohla et al.

a high grade of dispersion. 30 µl of bovine serum albumin (1.5 mg/ml) was added to stabilize the suspension. Finally, 100 µl of 10× concentrated phosphate buffered saline (PBS) was added to achieve a physiological salt concentration and pH of 7.4 [18] . The stock suspension was subsequently diluted to the indicated final concentrations with RPMI 1640 medium. Characterization of NPs The particle characterization included shape, size, size distribution, and tendency of aggregation. The morphology and size of ZnO-NPs in the sonicated and protein-supplemented stock dispersion were determined by transmission electron microscopy (TEM). After sonication and stabilization, the TEM samples were prepared by drop coating of the stock suspension on carboncoated copper grids. The films on the grids were dried using a tissue paper prior to measurement. The evaluation was performed with the aid of a Zeiss transmission electron microscope EM 900 (Carl Zeiss, Oberkochen, Germany). Additionally, size distribution of the ZnO-NP aggregates in RPMI 1640 was evaluated by dynamic light scattering (Malvern Instruments Ltd., Herrenberg, Germany). The ZnO-NP stock dispersion was prepared as described above and stored for 1 h before characterization. Next, the stock dispersion was diluted with RPMI 1640 until the working concentration of 20 µg/ml was reached. The surface zeta potential of the dispersion in the aforementioned cell culture medium (pH 7.4) was assessed by a ZetaSizer 3000HSA (Malvern Instruments Ltd.).

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to play a protective role in cancer cells exposed to stressors. However, extended autophagic activity may induce an apoptosis-independent programmed cell death [12] . Thus, autophagy is not only a pro-survival process, but may also induce lethal mechanisms in tumor cells. Several studies describe autophagy-associated, self-digestive tumor cell death in vitro following radiation and/or chemotherapy in human breast cancer cells, human prostate cancer cells, human sarcoma cells, and in human papillary thyroid cancer cells [13–15] . Mechanisms of cell death mediated by photoactivated ZnO-NPs are still largely unknown. The generation of ROS has been demonstrated to be associated with NP-induced cytotoxicity [16] . However, there is a lack of information about the potential role of autophagy in HNSCC elimination by ZnO-NPs. As we previously demonstrated photocatalytic HNSCC cell death by ZnO-NPs [6] , the aim of the current investigation was to further investigate molecular mechanisms of these reactions. In particular, the influence of autophagy and the interaction between autophagy and ROS generation were evaluated.

Materials & methods

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Cell line FaDu cells served as a human head and neck squamous carcinoma cell line [17] . This cell line was established from the lymph node metastasis of a hypopharyngeal squamous cell carcinoma. Tumor cells were cultured in RPMI 1640 Medium (Biochrom AG, Berlin, Germany) supplemented with 10% fetal calf serum (Linaris, Wertheim, Germany), 100 U/ ml penicillin, 100 µg/ml streptomycin, 1% 100 mM sodium pyruvate (all Biochrom AG) and 1% of a 100‑fold concentration of nonessential amino acids (Biochrom AG). The cells were incubated at 37°C/5% CO2 in 150 cm2 flasks, replacing the media every second day and passaging before reaching 80% of cell confluence by trypsinization (0.25% trypsin; Gibco, Eggenstein, Germany), washing, and seeding in new flasks or treatment wells. Reagent preparation ZnO-NPs (99% 404 nm

Surface area Shape Purity Size distribution in media after sonication Zeta potential

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SE: Standard error.

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the control group was demonstrated after photocatalytic treatment with both ZnO-NP concentrations (Figure 6) .

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Measurement of ROS generation Exposure of FaDu cells to ZnO-NPs and UVA‑1 induced ROS generation as measured by flow cytometry. The level of ROS was equal for both solitary and combination treatment (Figure 7) .

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relation to the survival rate of the untreated control, which was defined as 100%. FaDu cells did not show any reduction in cell viability after exposure to ZnO-NPs at concentrations of 0.2 and 2 µg/ml in the absence of UVA‑1 irradiation. Cytotoxic effects could be seen at concentrations higher than 20 µg/ml. Likewise, UVA‑1 irradiation alone without NP exposure did not induce cytotoxicity in the tumor cells. After pretreatment of FaDu with 3‑MA and exposure to 2 µg/ml ZnO-NPs, slightly reduced cell viability was observed, but these effects were not significant in comparison to the untreated control. However, the combination of ZnONPs with 15 min of UVA‑1 irradiation caused a significant reduction in the percentage of viable cells at 0.2 µg/ml. These effects were inhibited by a pretreatment with 3‑MA, resulting in a neutralization of photocatalytic cytotoxic capacity of 0.2 µg/ml ZnO-NPs and a significant inhibition of the reaction with 2 µg/ml ZnONPs. Data are presented in Figure 3. The annexin‑V propidium iodide assay was performed to measure apoptosis and necrosis. Flow cytometry revealed a cell viability of 87.5% in the control group, which was neither treated with NPs nor irradiated by UVA‑1. In this group, 7.3% of the cells were necrotic, 3.0% were apoptotic and 2.1% in the Q1 sector. Solitary exposure to UVA‑1 did not influence cell viability significantly (viability: 83.7%; necrotic: 10.0%; apoptotic: 3.2%; Q1: 3.1%). A slight elevation of necrosis was seen after exposure to 2 µg/ml ZnO-NPs (viability: 80.1%; necrotic: 12.6%; apoptotic: 3.3%; Q1: 4.0%). As a result of photocatalytic treatment with 0.2 µg/ml ZnO-NPs and UVA‑1, the necrosis rate was 45.0% and the apoptosis rate was 10.7%. Therapy with 2 µg/ml ZnO-NPs and UVA‑1 increased the necrosis rate to 55.4%, whereas apoptosis was enhanced to 13.7% as shown in Figure 4.


Western blotting & LysoTracker LC3 I and II expression were detected by immunoblotting and quantif ied by densitometry. Low levels of LC3 I protein were observed in all groups. The levels of LC3 II were also detected in all groups including the control group. However, expression was signif icantly higher after photocatalytic therapy with ZnO-NP concentrations of 0.2 and 2 µg/ml. LC3 II levels after exposure to 2 µg/ml and UVA‑1 were even higher compared to the positive control (F igur e 5) . Enhanced lysosomal activity compared to future science group

Figure 2. Autophagosome formation is induced in FaDu cells upon photocatalytic treatment. Autophagosomes are characterized by a double membrane, and contain cytoplasm and organelles (indicated by white arrow). Scale bar: 500 nm; images were taken at 20,000× magnification.

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doi:10.2217/NNM.12.137


Hackenberg, Scherzed & Gohla et al.

Data of Q4 in the population were as follows: control 1.6%, ZnO-NPs (2 µg/ml) 6.0%, UVA‑1 (15 min) 5.3%, photocatalytic treatment (2 µg/ml ZnO-NPs and 15 min UVA‑1) 26.9%. 10,000 events, n = 3. Photocatalytic cell death was inhibited by pretreatment with 1 mM NAC (see Figure 8 ). The mean survival rate after photocatalytic reaction with 0.2 µg/ml ZnO-NPs was 66%; however, after pretreatment with NAC, viability increased up to 86%. After treatment with 2 µg/ml

ZnO-NPs and UVA‑1, the mean viability rate was 42% without NAC and 69% with NAC.

Discussion The role of autophagy in cancer therapy seems to be contradictory. Since autophagy entails the degradation and recycling of organelles in order to promote cell survival in stress conditions, it is a potential inducer of resistance to cancer therapy. However, autophagy may also support cancer cell elimination in terms of programmed With UVA

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Figure 3. Survival of FaDu cells as a function of zinc oxide nanoparticle concentration (n = 18). Columns show the mean values and the standard deviation. Significance compared to the control with a p