TMEM97 and PGRMC1 do not mediate sigma-2 ligand-induced cell death

Abstract Sigma-2 receptors have been implicated in both tumor proliferation and neurodegenerative diseases. Recently the sigma-2 receptor was identified as transmembrane protein 97 (TMEM97). Progesterone receptor membrane component 1 (PGRMC1) was also recently reported to form a complex with TMEM97 and the low density lipoprotein (LDL) receptor, and this trimeric complex is responsible for the rapid internalization of LDL. Sigma-2 receptor ligands with various structures have been shown to induce cell death in cancer cells. In the current study, we examined the role of TMEM97 and PGRMC1 in mediating sigma-2 ligand-induced cell death. Cell viability and caspase-3 assays were performed in control, TMEM97 knockout (KO), PGRMC1 KO, and TMEM97/PGRMC1 double KO cell lines treated with several sigma-2 ligands. The data showed that knockout of TMEM97, PGRMC1, or both did not affect the concentrations of sigma-2 ligands that induced 50% of cell death (EC50), suggesting that cytotoxic effects of these compounds are not mediated by TMEM97 or PGRMC1. Sigma-1 receptor ligands, (+)-pentazocine and NE-100, did not block sigma-2 ligand cytotoxicity, suggesting that sigma-1 receptor was not responsible for sigma-2 ligand cytotoxicity. We also examined whether the alternative, residual binding site (RBS) of 1,3-Di-o-tolylguanidine (DTG) could be responsible for sigma-2 ligand cytotoxicity. Our data showed that the binding affinities (K i) of sigma-2 ligands on the DTG RBS did not correlate with the cytotoxicity potency (EC50) of these ligands, suggesting that the DTG RBS was not fully responsible for sigma-2 ligand cytotoxicity. In addition, we showed that knocking out TMEM97, PGRMC1, or both reduced the initial internalization rate of a sigma-2 fluorescent ligand, SW120. However, concentrations of internalized SW120 became identical later in the control and knockout cells. These data suggest that the initial internalization process of sigma-2 ligands does not appear to mediate the cell-killing effect of sigma-2 ligands. In summary, we have provided evidence that sigma-2 receptor/TMEM97 and PGRMC1 do not mediate sigma-2 ligand cytotoxicity. Our work will facilitate elucidating mechanisms of sigma-2 ligand cytotoxicity

Production of Transgenic Cloned Miniature Pigs with Membrane-bound Human Fas Ligand (FasL) by Somatic Cell Nuclear Transfer

Cell-mediated xenograft rejection, including NK cells and CD8+ CTL, is a major obstacle in successful pig-to-human xenotransplantation. Human CD8+ CTL and NK cells display high cytotoxicity for pig cells, mediated at least in part by the Fas/FasL pathway. To prevent cell-mediated xenocytotoxicity, a membrane-bound form of human FasL (mFasL) was generated as an inhibitor for CTL and NK cell cytotoxicity that could not be cleaved by metalloproteinase to produce putative soluble FasL. We produced two healthy transgenic pigs harboring the mFasL gene via somatic cell nuclear transfer (SCNT). In a cytotoxicity assay using transgenic clonal cell lines and transgenic pig ear cells, the rate of CD8+ CTL-mediated cytotoxicity was significantly reduced in transgenic pig's ear cells compared with that in normal minipig fetal fibroblasts. Our data indicate that grafts of transgenic pigs expressing membrane-bound human FasL control the cellular immune response to xenografts, creating a window of opportunity to facilitate xenograft survival

Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3

Colicin E3 is a cytotoxic ribonuclease that specifically cleaves 16S rRNA at the ribosomal A-site to abolish protein synthesis in sensitive Escherichia coli cells. We have performed extensive mutagenesis of the 96-residue colicin E3 cytotoxic domain (E3 rRNase), assayed mutant colicins for in vivo cytotoxicity, and tested the corresponding E3 rRNase domains for their ability to inactivate ribosome function in vitro. From 21 alanine mutants, we identified five positions where mutation resulted in a colicin with no measurable cytotoxicity (Y52, D55, H58, E62, and Y64) and four positions (R40, R42, E60, and R90) where mutation caused a significant reduction in cytotoxicity. Mutations that were found to have large in vivo and in vitro effects were tested for structural integrity through circular dichroism and fluorescence spectroscopy using purified rRNase domains. Our data indicate that H58 and E62 likely act as the acid–base pair during catalysis with other residues likely involved in transition state stabilization. Both the Y52 and Y64 mutants were found to be highly destabilized and this is the likely origin of the loss of their cytotoxicity. The identification of important active site residues and sequence alignments of known rRNase homologs has allowed us to identify other proteins containing the putative rRNase active site motif. Proteins that contained this active site motif included three hemagglutinin-type adhesins and we speculate that these have evolved to deliver a cytotoxic rRNase into eukaryotic cells during pathogenesis

Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements

BACKGROUND: When evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action. In this regard we evaluated the use of gene expression and common cytotoxicity measurements using as test materials, two selected nanoparticles with known differences in toxicity, 5 nm mercaptoundecanoic acid (MUA)-capped InP and CdSe quantum dots (QDs). We tested the effects of these QDs at concentrations ranging from 0.5 to 160 µg/mL on cultured normal human bronchial epithelial (NHBE) cells using four common cytotoxicity assays: the dichlorofluorescein assay for reactive oxygen species (ROS), the lactate dehydrogenase assay for membrane viability (LDH), the mitochondrial dehydrogenase assay for mitochondrial function, and the Comet assay for DNA strand breaks. RESULTS: The cytotoxicity assays showed similar trends when exposed to nanoparticles for 24 h at 80 µg/mL with a threefold increase in ROS with exposure to CdSe QDs compared to an insignificant change in ROS levels after exposure to InP QDs, a twofold increase in the LDH necrosis assay in NHBE cells with exposure to CdSe QDs compared to a 50% decrease for InP QDs, a 60% decrease in the mitochondrial function assay upon exposure to CdSe QDs compared to a minimal increase in the case of InP and significant DNA strand breaks after exposure to CdSe QDs compared to no significant DNA strand breaks with InP. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) data for cells exposed for 6 h at a concentration of 80 µg/mL were consistent with the cytotoxicity assays showing major differences in DNA damage, DNA repair and mitochondrial function gene regulatory responses to the CdSe and InP QDs. The BRCA2, CYP1A1, CYP1B1, CDK1, SFN and VEGFA genes were observed to be upregulated specifically from increased CdSe exposure and suggests their possible utility as biomarkers for toxicity. CONCLUSIONS: This study can serve as a model for comparing traditional cytotoxicity assays and gene expression measurements and to determine candidate biomarkers for assessing the biocompatibility of ENMs.1R01GM84702-01 - National Institute of General Medical Science

Comparison of cytotoxicity of IQOS aerosols to smoke from Marlboro Red and 3R4F reference cigarettes.

This study compared the cytotoxicity of IQOS aerosols to smoke from Marlboro Red (MR) and 3R4F reference cigarettes. Aerosol/smoke solutions were tested as the gas vapor phase (GVP), particulate phase (total particulate matter or TPM), or whole aerosol/smoke (WA), the latter being what smokers actually inhale. Cytotoxicities were evaluated using the LDH, MTT and neutral red uptake (NRU) assays in conjunction with eight different cell types, mainly from the respiratory system. Most test solutions did not compromise the plasma membranes of cells (LDH). However, mitochondrial activity (MTT) and dye uptake/lysosomal activity (NRU) were equally depressed by IOQS aerosols and cigarette smoke solutions at the high concentrations. Our NRU data with mouse NIH/3T3 transformed fibroblasts were similar to those previously reported by the IQOS manufacturer and showed little cytotoxicity in the NRU assay. In both studies with NIH/3T3 cells, the results were significantly different from 3RF4 cigarette smoke, suggesting reduced toxicity with IQOS. However, by expanding evaluations to a broader spectrum of cells that included respiratory system cells and by including higher concentrations of GVP, as well as WA, cytotoxicity equivalent to that of Marlboro Red and 3R4F cigarettes was frequently observed with IQOS aerosols in the MTT and NRU assays

Clustering of Time Series Cytotoxicity Data

To study the effect of various toxicants on cells’ growth, the Alberta Centre for Toxicology did several in-vitro experiments, and concentration response curves (TCRCs) were generated. Each TCRC represents a time series that gives the temporal evolution of the number of cells, after exposure to a chemical with a certain concentration. Here we use the wavelet transform to extract important features from the original TCRC data, and we apply self organizing maps to classify the toxicants according to their adverse biological response. Presented in absentia on April 27, 2020 at Student Research Day held at MacEwan University in Edmonton, Alberta. (Conference cancelled.) Also presented on June 19-22, 2019 at the Classification Society meeting held at MacEwan University in Edmonton, Alberta. Faculty Mentor: Cristina Anton Department: Statistic

Cytotoxicity of the Bacillus thuringiensis Crystal Protein against Mammalian Cells

The crystal proteins produced by Bacillus thuringiensis subsp, israelensis (Bti) and subsp. coreanensis A1519 strain were examined for the cytotoxicity against MOLT-4 and HeLa cells by MTT assay and LDH assay, The A1519 crystal proteins processed by proteinase K exhibited the specific cell-killing activity toward MOLT-4 with little damage to the cell membrane, On the other hand, the Bti crystal proteins processed by proteinase K caused the substantial damage to the cell membrane of both MOLT-4 and HeLa, leading to the cell lysis. The non-digested crystal proteins of both strains exhibited no cytotoxicity, These data suggested that while the Bti crystal proteins caused the colloid-osmotic swelling and cell lysis of MOLT-4 and HeLa, the proteinase K-digested A1519 crystal proteins induced the specific cell death of MOLT-4 through a mechanism other than that of Bti

Identification of Cytotoxic Flavor Chemicals in Top-Selling Electronic Cigarette Refill Fluids.

We identified the most popular electronic cigarette (EC) refill fluids using an Internet survey and local and online sales information, quantified their flavor chemicals, and evaluated cytotoxicities of the fluids and flavor chemicals. "Berries/Fruits/Citrus" was the most popular EC refill fluid flavor category. Twenty popular EC refill fluids were purchased from local shops, and the ingredient flavor chemicals were identified and quantified by gas chromatography-mass spectrometry. Total flavor chemical concentrations ranged from 0.6 to 27.9 mg/ml, and in 95% of the fluids, total flavor concentration was greater than nicotine concentration. The 20 most popular refill fluids contained 99 quantifiable flavor chemicals; each refill fluid contained 22 to 47 flavor chemicals, most being esters. Some chemicals were found frequently, and several were present in most products. At a 1% concentration, 80% of the refill fluids were cytotoxic in the MTT assay. Six pure standards of the flavor chemicals found at the highest concentrations in the two most cytotoxic refill fluids were effective in the MTT assay, and ethyl maltol, which was in over 50% of the products, was the most cytotoxic. These data show that the cytotoxicity of some popular refill fluids can be attributed to their high concentrations of flavor chemicals

Differential Targeting of Stem Cells and Differentiated Glioblastomas by NK Cells.

We have recently shown that Natural Killer (NK) cells control survival and differentiation of Cancer Stem-like Cells (CSCs) through two distinct phenotypes of cytotoxic and anergic NK cells, respectively. In this report, brain CSCs and their serum and NK cell differentiated counterparts were studied. Serum-differentiated brain CSCs were significantly less susceptible to NK cells and CTL direct cytotoxicity as well as NK cell mediated Antibody Dependent Cellular Cytotoxicity (ADCC), whereas their CSCs were highly susceptible. The levels of CD44 and EGFR were higher in brain tumor CSCs when compared to the serum-differentiated tumors. No differences could be observed for the expression of MHC class I between brain tumor stem cells and their serum-differentiated counterparts. Moreover, supernatants from the combination of IL-2 and anti-CD16mAb treated NK cells (anergized NK cells) induced resistance of brain tumor CSCs to NK cell mediated cytotoxicity. Unlike serum-differentiated CSCs, NK supernatant induced differentiation and resistance to cytotoxicity in brain CSCs correlated with the increased expression of CD54 and MHC class I. The addition of anti-MHC class I antibody moderately inhibited NK mediated cytotoxicity against untreated or serum-differentiated CSCs, whereas it increased cytotoxicity against NK supernatant differentiated tumors. Therefore, two distinct mechanisms govern serum and NK supernatant mediated differentiation of brain tumors

Synthesis, reactivity studies, and cytotoxicity of two trans-Iodidoplatinum(II) complexes. Does photoactivation work?

trans-Platinum complexes have been the landmark in unconventional drugs prompting the development of innovative structures that might exhibit chemical and biological profiles different to cisplatin. Iodido complexes signaled a new turning point in the platinum drug design field when their cytotoxicity was reevaluated and reported. In this new study, we have synthesized and evaluated diodidoplatinum complexes trans-[PtI2(amine)(pyridine)] bearing aliphatic amines (isopropylamine and methylamine) and pyridines in trans configuration. X-ray diffraction data support the structural characterization. Their cytotoxicity has been evaluated in tumor cell lines such as SAOS-2, A375, T-47D, and HCT116. Moreover, we report their solution behavior and reactivity with biological models. Ultraviolet-a (UVA) irradiation induces an increase in their reactivity towards model nucleobase 5′-GMP in early stages, and promotes the release of the pyridine ligand (spectator ligand) at longer reaction times. Density Functional calculations have been performed and the results are compared with our previous studies with other iodido derivatives.MINECO CTQ-2015-68779