Activation of stress-related signalling pathway in human cells upon SiO2 nanoparticles exposure as an early indicator of cytotoxicity

<p>Abstract</p> <p>Background</p> <p>Nanomaterials such as SiO₂nanoparticles (SiO₂NP) are finding increasing applications in the biomedical and biotechnological fields such as disease diagnostics, imaging, drug delivery, food, cosmetics and biosensors development. Thus, a mechanistic and systematic evaluation of the potential biological and toxic effects of SiO₂NP becomes crucial in order to assess their complete safe applicability limits.</p> <p>Results</p> <p>In this study, human monocytic leukemia cell line THP-1 and human alveolar epithelial cell line A549 were exposed to a range of amorphous SiO₂NP of various sizes and concentrations (0.01, 0.1 and 0.5 mg/ml). Key biological indicators of cellular functions including cell population density, cellular morphology, membrane permeability, lysosomal mass/pH and activation of transcription factor-2 (ATF-2) were evaluated utilizing quantitative high content screening (HCS) approach and biochemical techniques. Despite the use of extremely high nanoparticle concentrations, our findings showed a low degree of cytotoxicity within the panel of SiO₂NP investigated. However, at these concentrations, we observed the onset of stress-related cellular response induced by SiO₂NP. Interestingly, cells exposed to alumina-coated SiO₂NP showed low level, and in some cases complete absence, of stress response and this was consistent up to the highest dose of 0.5 mg/ml.</p> <p>Conclusions</p> <p>The present study demonstrates and highlights the importance of subtle biological changes downstream of primary membrane and endocytosis-associated phenomena resulting from high dose SiO₂NP exposure. Increased activation of transcription factors, such as ATF-2, was quantitatively assessed as a function of i) human cell line specific stress-response, ii) SiO₂NP size and iii) concentration. Despite the low level of cytotoxicity detected for the amorphous SiO₂NP investigated, these findings prompt an in-depth focus for future SiO₂NP-cell/tissue investigations based on the combined analysis of more subtle signalling pathways associated with accumulation mechanisms, which is essential for establishing the bio-safety of existing and new nanomaterials.</p

Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines

Background Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. Methods HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12 h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. Results HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12 h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6 ± 4.7% (T12) by LDH assay and 19 ± 3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. Conclusions In the presence of effector cells with high cytotoxic capacity, TKIs have the ability to augment the passive immunotherapeutic potential of trastuzumab in SKBR3, a model of HER2+ breast cancer. ADCC levels detected by LDH release assays are extremely low in MCF-7; the flow cytometry-based CFSE/7-AAD method is more sensitive and consistent for the determination of ADCC in HER2-low models

DNA mismatch repair protein MSH2 dictates cellular survival in response to low dose radiation in endometrial carcinoma cells

► MSH2+ cells express hyperradiosensitivity. ► HRS in MSH2+ cells is associated with activation of the early G2-phase cell cycle check point. ► HRS is associated with persistent MRE11 and RAD51 foci after low dose irradiation. ► Low dose radiotherapy regimen could improve the treatment of MSH2 proficient tumors. ► MSH2 proficiency may reduce cancer susceptibility following exposure to low radiation doses. DNA repair and G2-phase cell cycle checkpoint responses are involved in the manifestation of hyper-radiosensitivity (HRS). The low-dose radioresponse of MSH2 isogenic endometrial carcinoma cell lines was examined. Defects in cell cycle checkpoint activation and the DNA damage response in irradiated cells (0.2Gy) were evaluated. HRS was expressed solely in MSH2+ cells and was associated with efficient activation of the early G2-phase cell cycle checkpoint. Maintenance of the arrest was associated with persistent MRE11, γH2AX, RAD51 foci at 2h after irradiation. Persistent MRE11 and RAD51 foci were also evident 24h after 0.2Gy. MSH2 significantly enhances cell radiosensitivity to low dose IR