Thomas Gehrke

Human mesenchymal stem cells enhance cancer cell proliferation via IL-6 secretion and activation of ERK1/2

Human mesenchymal stem cells (hMSC) are frequently used in tissue engineering. Due to their strong tumor tropism, hMSC seem to be a promising vehicle for anticancer drugs. However, interactions between hMSC and cancer are ambiguous. Particularly the cytokines and growth factors seem to play an important role in cancer progression and metastasis. The present study evaluated the effects of hMSC on head and neck squamous cell carcinoma (HNSCC) cell lines (FaDu and HLaC78) in vitro. hMSC released several cytokines and growth factors. FaDu and HLaC78 showed a significant enhancement of cell proliferation after cultivation with hMSC-conditioned medium as compared to control. This proliferation improvement was inhibited by the addition of anti-IL-6. The western blot showed an activation of Erk1/2 in FaDu and HLaC78 by hMSC-conditioned medium. HNSCC cell lines expressed EGFR. The current study confirms the importance of cytokines secreted by hMSC in cancer biology. Especially IL-6 seems to play a key role in cancer progression. Thus, the use of hMSC as a carrier for cancer therapy must be discussed critically. Future studies should evaluate the possibility of generating genetically engineered hMSC with, for example, the absence of IL-6 secretion.

Time-Dependent Toxic and Genotoxic Effects of Zinc Oxide Nanoparticles after Long-Term and Repetitive Exposure to Human Mesenchymal Stem Cells

Zinc oxide nanoparticles (ZnO-NP) are widely spread in consumer products. Data about the toxicological characteristics of ZnO-NP is still under controversial discussion. The human skin is the most important organ concerning ZnO-NP exposure. Intact skin was demonstrated to be a sufficient barrier against NPs; however, defect skin may allow NP contact to proliferating cells. Within these cells, stem cells are the most important toxicological target for NPs. The aim of this study was to evaluate the genotoxic and cytotoxic effects of ZnO-NP at low-dose concentrations after long-term and repetitive exposure to human mesenchymal stem cells (hMSC). Cytotoxic effects of ZnO-NP were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Furthermore, genotoxicity was evaluated by the comet assay. For long-term observation over 6 weeks, transmission electron microscopy (TEM) was applied. The results of the study indicated cytotoxic effects of ZnO-NP beginning at high concentrations of 50 μg/mL and genotoxic effects in hMSC exposed to 1 and 10 μg/mL ZnO-NP. Repetitive exposure enhanced cyto- but not genotoxicity. Intracellular NP accumulation was observed up to 6 weeks. The results suggest cytotoxic and genotoxic potential of ZnO-NP. Even low doses of ZnO-NP may induce toxic effects as a result of repetitive exposure and long-term cellular accumulation. This data should be considered before using ZnO-NP on damaged skin.

The differentiation of hMSCs counteracts their migration capability and pro‑angiogenic effects in vitro

Human mesenchymal stem cells (hMSCs) have been applied therapeutically in numerous clinical trials. The pro-angiogenic effects of hMSCs, as well as their strong tumor tropism, have been shown both in vitro and in vivo. Some studies suggest using hMSCs as potential drug-carriers for tumor therapy. In previous investigations by our group, the pro-tumorigenic effects of hMSCs on head and neck squamous cell carcinoma (HNSCC) were shown. However, the influence of hMSCs on tumor vascularization as well as the possibility of its inhibition are yet to be elucidated. The cytokine patterns of the HNSCC cell line FaDu, native hMSCs (hMSCs-nat), hMSCs differentiated into adipocytes (hMSCs-adip) and osteocytes (hMSCs-ost) were evaluated. Human umbilical vein endothelial cells (HUVECs) were co-cultured with FaDu cells, hMSCs-nat, hMSCs-adip and hMSCs-ost. The capillary tube formation assay was applied. Furthermore, the migration capability of hMSCs-nat, hMSCs-adip and hMSCs-ost towards FaDu cells was measured in a Transwell system. Spheroids were cultured from hMSCs-nat, FaDu cells and DiI-labeled HUVECs. FaDu cells, hMSCs-nat, hMSCs-adip and hMSCs-ost released a wide range of cytokines and growth factors, e.g., IL-6, IL-8, IL-10, GRO and MCP. In the capillary tube formation assay, HUVECs generated significantly longer tubes after co-cultivation with hMSCs-nat as compared to HUVECs alone and FaDu. Differentiation into adipocytes and osteocytes counteracted the tube formation. The adipogenic differentiation did not alter hMSC motility, whereas osteogenic differentiation significantly inhibited hMSC migration. Generation of multi-cellular spheroids from hMSCs-nat, FaDu cells and DiI-labeled HUVECs was possible. Florescence microscopy revealed that all HUVECs were present in the spheroid core. Taken together, hMSCs-nat have a pro-angiogenic effect. The effects are counteracted by the differentiation of hMSCs towards osteogenic and adipogenic lineages. The differentiation of stem cells into different lineages may be a promising solution to generating carriers for cancer therapy without pro-tumorigenic properties.

Genotoxic effects of zinc oxide nanoparticles in nasal mucosa cells are antagonized by titanium dioxide nanoparticles

Titanium dioxide nanoparticles (TiO2-NPs) and zinc oxide nanoparticles (ZnO-NPs) are often used in sunscreens and other consumer products due to their photoprotective properties. However, concern exists regarding them possibly causing cyto- and genotoxic effects. The aim of this study was to assess cyto- and genotoxicity of these nanomaterials after single or combined exposure. For this purpose, a battery of cell culture test systems for human nasal mucosa (monolayer, air-liquid interface and mini organ culture) were exposed to 0.1-20μg/ml of TiO2- and ZnO-NPs alone and in combination. Cytotoxicity was measured by the MTT assay, and DNA damage and repair capacity were investigated using the comet assay. TiO2-NPs did not exhibit any cyto- or genotoxic potential within the tested concentrations. However, results of the study indicated cyto- and genotoxicity resulting from ZnO-NPs. The genotoxicity could be antagonized by TiO2-NPs. Furthermore, the DNA repair capacity after ZnO-NP-induced DNA damage was enhanced by TiO2-NPs. The adsorption of dissolved zinc ions onto TiO2-NPs is discussed as the major antagonistic mechanism. The combination of both metal oxide nanoparticles interferes with the genotoxicity of ZnO-NPs and should be discussed as a reasonable and safe alternative to the sole use of ZnO-NPs in consumer products.

A polydopamine peptide coating enables adipose-derived stem cell growth on the silicone surface of cochlear implant electrode arrays

The simultaneous application of neurotrophic factors with cochlear implantation is proposed to enhance the bioelectrical interface between electrodes and auditory neurons, and thus improve speech intelligibility in patients with cochlear implants (CIs). In cell-based approaches, the goal is to colonize CIs with cells producing neurotrophic factors. This study aims to evaluate whether a polydopamine (PD) functionalization of the hydrophobic silicone surface of the electrode carrier enables colonization of adipose-derived stem cells known to deliver neurotrophic factors. Surface characteristics of PD-coated silicone samples and electrode carriers were determined, and the proliferation and viability of adipose-derived stem cells (ASCs) on these surfaces were subsequently analyzed. A homogenous PD coating and cell growth with regular morphology was observed on coated silicone samples and electrode arrays. Hydrophilicity and cell viability was significantly enhanced by PD surface modification. Insertion forces of coated electrode arrays did not increase compared with untreated CIs. Hence, PD coating of the silicone surface of CIs might allow for sufficient colonization with ASCs as a continuous source of neurotrophic factors.

Toxicological characterization of ZnO nanoparticles in malignant and non‐malignant cells

The increasing usage of zinc oxide nanoparticles (ZnO‐NPs) in industrial applications as well as in consumer products raises concern regarding their potential adverse effects to a greater extend. Numerous studies have demonstrated toxic properties of NPs, however there is still a lack of knowledge concerning the underlying mechanisms. This study was designed to systematically investigate cytotoxicity, apoptosis, cell cycle alterations, and genotoxicity induced by ZnO‐NP. Moreover, it was an aim of the investigations to specify the diverse effects of nanoparticle exposure in malignant in comparison with non‐malignant cells. Therefore, human head and neck squamous cell carcinoma‐derived FaDu cells were incubated with 4–20 µg/ml of ZnO‐NPs for 1–48 hr and tested for cell viability, cell cycle alterations, apoptosis and caspase‐3 gene expression as a sensitive marker of molecular apoptotic processes with regard to time‐ and dose‐dependent effects. Human mesenchymal bone marrow stem cells were used as non‐malignant representatives to examine oxidative stress‐related genotoxicity. Results showed a significant reduction in cell viability as well as dose‐ and time‐dependent increase of apoptotic cells following nanoparticle treatment. Likewise, caspase‐3 gene expression enhanced already before first apoptotic cells were detectable. It could be observed that doses that were cytotoxic in tumor cells did not reduce viability in stem cells. However, the same concentrations already induced significant DNA damage. The findings of the study suggest to keep a more critical eye on the use of nanoparticles as anti‐cancer agents. Yet, additional in vivo studies are needed to assess safety concerns for consumers and patients. Environ. Mol. Mutagen. 59:247–259, 2018.

Long‑term changes in the properties of skin-derived fibroblasts following irradiation of the head and neck

The tumor stroma performs an important role in carcinogenesis. It predominantly consists of fibroblasts and the connective tissue produced by them, and undergoes a multitude of interactions with the surrounding cancer cells. Since irradiation is part of the majority of therapeutic strategies for head and neck squamous cell carcinoma, more information regarding the effects of a previous irradiation on the tumor stroma is desirable. In the present study, fibroblasts were cultivated from human non-irradiated and pre-irradiated skin of the neck for 48 h. Subsequently, analyses of cell viability, apoptosis, necrosis and motility were conducted via MTT assay, Annexin V/propidium iodide staining, electronic cell counting for 4 consecutive days, and scratch assay. Pre-irradiated fibroblasts exhibited a significantly slower growth rate as well as increased rates of apoptosis and necrosis. They also exhibited significantly decreased motility compared with non-irradiated fibroblasts. These results indicated the long-term effects of irradiation on fibroblasts, which may affect cancer recurrence in the irradiated region via the tumor stroma. More information, such as that regarding the secretory capacities of pre-irradiated fibroblasts, is required to evaluate the possible therapeutic implications of these findings.

Zinc oxide nanoparticles antagonize the effect of Cetuximab on head and neck squamous cell carcinoma in vitro

ABSTRACT Zinc oxide nanoparticles (ZnO-NPs) are being used in many cosmetic products and have been shown to induce tumor-selective cell death in human head and neck squamous cell carcinoma (HNSCC) in vitro. Cetuximab is a monoclonal antibody directed against the epidermal growth factor receptor (EGFR), whose effectiveness for HNSCC, alone or in combination with cytostatic drugs, has been demonstrated intensively in the last decades. Nanoparticles are known to interact with protein structures and thus may influence their functionality. The aim of the current study was to evaluate the effect of ZnO-NPs on the antitumor properties of Cetuximab in HNSCC in vitro. Two HNSCC cell lines (FaDu and HLaC-78) were treated with 0.1, 1 or 10 μM Cetuximab as well as 0, 0.1 or 1 μg/ml ZnO-NP. Qualitative assessment of ZnO-NP was conducted via transmission electron microscopy (TEM) and immunofluorescence staining. Evaluation was done via the MTT-assay after 24, 48 and 72 hours of incubation with Cetuximab and ZnO-NPs. ZnO-NPs were shown to antagonize the anti-tumor effects of Cetuximab in a time-dependent as well as dose-dependent way. These findings suggest an inhibitory interaction of ZnO-NPs with Cetuximab, which warrants further investigation.