Nanoscaled biphasic calcium phosphate modulates osteogenesis and attenuates LPS-induced inflammation.

Micron-scale structure biphasic calcium phosphate (BCP) materials have demonstrated promising clinical outcomes in the field of bone tissue repair. However, research on biphasic calcium phosphate materials at the nanoscale level remains limited. In this study, we synthesize granular-shaped biphasic calcium phosphate nanomaterials with multiple desirable characteristics, including negatively charged surfaces, non-cytotoxicity, and the capability to penetrate cells, using a nanogrinding dispersion process with a polymeric carboxylic acid as the dispersant. Our results reveal that treating human osteoblasts with 0.5 µg/mL biphasic calcium phosphate nanomaterials results in a marked increase in alkaline phosphatase (ALP) activity and the upregulation of osteogenesis-related genes. Furthermore, these biphasic calcium phosphate nanomaterials exhibit immunomodulatory properties. Treatment of THP-1-derived macrophages with BCP nanomaterials decreases the expression of various inflammatory genes. Biphasic calcium phosphate nanomaterials also mitigate the elevated inflammatory gene expression and protein production triggered by lipopolysaccharide (LPS) exposure in THP-1-derived macrophages. Notably, we observe that biphasic calcium phosphate nanomaterials have the capacity to reverse the detrimental effects of LPS-stimulated macrophage-conditioned medium on osteoblastic activity and mineralization. These findings underscore the potential utility of biphasic calcium phosphate nanomaterials in clinical settings for the repair and regeneration of bone tissue. In conclusion, this study highlights the material properties and positive effects of biphasic calcium phosphate nanomaterials on osteogenesis and immune regulation, opening a promising avenue for further research on inflammatory osteolysis in patients undergoing clinical surgery.

Tumor suppressor p53 mediates interleukin-6 expression to enable cancer cell evasion of genotoxic stress.

The tumor suppressor p53 primarily functions as a mediator of DNA damage-induced cell death, thereby contributing to the efficacy of genotoxic anticancer therapeutics. Here, we show, on the contrary, that cancer cells can employ genotoxic stress-induced p53 to acquire treatment resistance through the production of the pleiotropic cytokine interleukin (IL)-6. Mechanistically, DNA damage, either repairable or irreparable, activates p53 and stimulates Caspase-2-mediated cleavage of its negative regulator mouse double minute 2 (MDM2) creating a positive feedback loop that leads to elevated p53 protein accumulation. p53 transcriptionally controls the major adenosine triphosphate (ATP) release channel pannexin 1 (Panx1), which directs IL-6 induction via a mechanism dependent on the extracellular ATP-activated purinergic P2 receptors as well as their downstream intracellular calcium (iCa2+)/PI3K/Akt/NF-ĸB signaling pathway. Thus, p53 silencing impairs Panx1 and IL-6 expression and renders cancer cells sensitive to genotoxic stress. Moreover, we confirm that IL-6 hampers the effectiveness of genotoxic anticancer agents by mitigating DNA damage, driving the expression of anti-apoptotic Bcl-2 family genes, and maintaining the migratory and invasive properties of cancer cells. Analysis of patient survival and relevant factors in lung cancer and pan-cancer cohorts supports the prognostic and clinical values of Panx1 and IL-6. Notably, IL-6 secreted by cancer cells during genotoxic treatments promotes the polarization of monocytic THP-1-derived macrophages into an alternative (M2-like) phenotype that exhibits impaired anti-survival activities but enhanced pro-metastatic effects on cancer cells as compared to nonpolarized macrophages. Our study reveals the precise mechanism for genotoxic-induced IL-6 and suggests that targeting p53-mediated IL-6 may improve the responsiveness of cancer cells to genotoxic anticancer therapy.

Action mechanism of snake venom l-amino acid oxidase and its double-edged sword effect on cancer treatment: Role of pannexin 1-mediated interleukin-6 expression.

Snake venom l-amino acid oxidases (svLAAOs) have been recognized as promising candidates for anticancer therapeutics. However, multiple aspects of their catalytic mechanism and the overall responses of cancer cells to these redox enzymes remain ambiguous. Here, we present an analysis of the phylogenetic relationships and active site-related residues among svLAAOs and reveal that the previously proposed critical catalytic residue His 223 is highly conserved in the viperid but not the elapid svLAAO clade. To gain further insight into the action mechanism of the elapid svLAAOs, we purify and characterize the structural, biochemical, and anticancer therapeutic potentials of the Thailand elapid snake Naja kaouthia LAAO (NK-LAAO). We find that NK-LAAO, with Ser 223, exhibits high catalytic activity toward hydrophobic l-amino acid substrates. Moreover, NK-LAAO induces substantial oxidative stress-mediated cytotoxicity with the magnitude relying on both the levels of extracellular hydrogen peroxide (H2O2) and intracellular reactive oxygen species (ROS) generated during the enzymatic redox reactions, but not being influenced by the N-linked glycans on its surface. Unexpectedly, we discover a tolerant mechanism deployed by cancer cells to dampen the anticancer activities of NK-LAAO. NK-LAAO treatment amplifies interleukin (IL)-6 expression via the pannexin 1 (Panx1)-directed intracellular calcium (iCa2+) signaling pathway to confer adaptive and aggressive phenotypes on cancer cells. Accordingly, IL-6 silencing renders cancer cells vulnerable to NK-LAAO-induced oxidative stress together with abrogating NK-LAAO-stimulated metastatic acquisition. Collectively, our study urges caution when using svLAAOs in cancer treatment and identifies the Panx1/iCa2+/IL-6 axis as a therapeutic target for improving the effectiveness of svLAAOs-based anticancer therapies.

Tumor suppressor p53 restrains cancer cell dissemination by modulating mitochondrial dynamics.

Tumor suppressor p53 plays a central role in preventing tumorigenesis. Here, we unravel how p53 modulates mitochondrial dynamics to restrain the metastatic properties of cancer cells. p53 inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling to attenuate the protein level of mitochondrial fission process 1 (MTFP1), which fosters the pro-fission dynamin-related protein 1 (Drp1) phosphorylation. This regulatory mechanism allows p53 to restrict cell migration and invasion governed by Drp1-mediated mitochondrial fission. Downregulating p53 expression or elevating the molecular signature of mitochondrial fission correlates with aggressive tumor phenotypes and poor prognosis in cancer patients. Upon p53 loss, exaggerated mitochondrial fragmentation stimulates the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling resulting in epithelial-to-mesenchymal transition (EMT)-like changes in cell morphology, accompanied by accelerated matrix metalloproteinase 9 (MMP9) expression and invasive cell migration. Notably, blocking the activation of mTORC1/MTFP1/Drp1/ERK1/2 axis completely abolishes the p53 deficiency-driven cellular morphological switch, MMP9 expression, and cancer cell dissemination. Our findings unveil a hitherto unrecognized mitochondria-dependent molecular mechanism underlying the metastatic phenotypes of p53-compromised cancers.

A structure-function approach identifies L-PGDS as a mediator responsible for glucocorticoid-induced leptin expression in adipocytes.

Leptin is an adipokine predominantly secreted by adipocytes and has many physiological roles, including in energy homeostasis. We identified that AM630, a cannabinoid receptor 2 (CB2) antagonist, down-regulated leptin expression in mature adipocytes differentiated from either stromal vascular fractions isolated from inguinal fat pads of C57BL/6J mice or 3T3-L1 preadipocytes. However, the leptin-suppressive effects of AM630 preserved in CB2-deficient adipocytes indicated the off-target activity of AM630 in leptin expression. Pharmacological and genetic studies, cheminformatics, and docking simulation were applied to identify the potential protein target of AM630 that modulates leptin expression in differentiated primary preadipocytes. Screening of the reported off-targets of AM630 identified a synthetic cannabinoid WIN55212-2 exerting the same function. Target deconvolution and docking simulation suggested that AM630 and WIN55212-2 were both inhibitors of lipocalin-type prostaglandin D2 synthase (L-PGDS). Further studies showed that L-PGDS positively regulates leptin expression. Although glucocorticoid and aldosterone were previously reported to induce expression of both L-PGDS and leptin, our data demonstrated that L-PGDS mediates only glucocorticoid-induced leptin expression in differentiated primary preadipocytes. No effect was observed after aldosterone treatment. This newly discovered glucocorticoid - L-PGDS - leptin pathway may provide insights into current clinical use of glucocorticoid and management of their undesired effects such as obesity.

Arsenite-induced apoptosis can be attenuated via depletion of mTOR activity to restore autophagy.

Arsenic and its compounds are toxic environmental pollutants and known carcinogens. We investigated here the mechanism of arsenite-induced damage in renal cells. Treating human embryonic kidney cells (HEK293) with sodium arsenite reduces cell viability in a dose- and time-dependent manner. The decline of cell viability is due to apoptotic death since arsenite treatment reduces Akt activity and the Bcl2 level but increases caspase 3 activity and the cytochrome c level. These effects can be reverted by the addition of an apoptosis inhibitor. PTEN, the upstream negative regulator of Akt activity, was also reduced with arsenite treatment. Noticeably, PTEN markedly increased in the insoluble fraction of the cells, suggesting a cell failure in removing the damaged proteins. Arsenite treatment activates a variety of signaling factors. Among them, ERK and JNK are associated with autophagy via regulating the levels of LC3 and p62. With arsenite administration, the LC3 and p62 levels increased. However, lysosomal activity was decreased and led to the decline of autophagic activity. The addition of rapamycin, the mTOR inhibitor, activated the autophagic pathway that accelerated the removal of damaged proteins. The recovery of autophagy increased the viability of arsenite-treated cells. Similar to rapamycin treatment, the knockdown of mTOR expression also enhanced the viability of arsenite-treated cells. Both rapamycin treatment and mTOR knockdown enhanced ERK activity further, but reduced JNK activity and the p62 level in arsenite-treated cells. Lysosomal activity increased with the depletion of mTOR, indicating an increase of autophagic activity. These results reveal the critical role of mTOR in regulating the cell fate of arsenite-exposed renal cells.

The conserved basic residues and the charged amino acid residues at the α-helix of the zinc finger motif regulate the nuclear transport activity of triple C2H2 zinc finger proteins.

Zinc finger (ZF) motifs on proteins are frequently recognized as a structure for DNA binding. Accumulated reports indicate that ZF motifs contain nuclear localization signal (NLS) to facilitate the transport of ZF proteins into nucleus. We investigated the critical factors that facilitate the nuclear transport of triple C2H2 ZF proteins. Three conserved basic residues (hot spots) were identified among the ZF sequences of triple C2H2 ZF proteins that reportedly have NLS function. Additional basic residues can be found on the α-helix of the ZFs. Using the ZF domain (ZFD) of Egr-1 as a template, various mutants were constructed and expressed in cells. The nuclear transport activity of various mutants was estimated by analyzing the proportion of protein localized in the nucleus. Mutation at any hot spot of the Egr-1 ZFs reduced the nuclear transport activity. Changes of the basic residues at the α-helical region of the second ZF (ZF2) of the Egr-1 ZFD abolished the NLS activity. However, this activity can be restored by substituting the acidic residues at the homologous positions of ZF1 or ZF3 with basic residues. The restored activity dropped again when the hot spots at ZF1 or the basic residues in the α-helix of ZF3 were mutated. The variations in nuclear transport activity are linked directly to the binding activity of the ZF proteins with importins. This study was extended to other triple C2H2 ZF proteins. SP1 and KLF families, similar to Egr-1, have charged amino acid residues at the second (α2) and the third (α3) positions of the α-helix. Replacing the amino acids at α2 and α3 with acidic residues reduced the NLS activity of the SP1 and KLF6 ZFD. The reduced activity can be restored by substituting the α3 with histidine at any SP1 and KLF6 ZFD. The results show again the interchangeable role of ZFs and charge residues in the α-helix in regulating the NLS activity of triple C2H2 ZF proteins.

Cadmium Activates Multiple Signaling Pathways That Coordinately Stimulate Akt Activity to Enhance c-Myc mRNA Stability.

Cadmium is a known environmental carcinogen. Exposure of Cd leads to the activation of several proto-oncogenes in cells. We investigated here the mechanism of c-Myc expression in hepatic cells under Cd treatment. The c-Myc protein and mRNA levels increased in dose- and time-dependent manners in HepG2 cells with Cd treatment. This increase was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated by Cd. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. We conclude that Cd-induced accumulation of c-Myc requires the activation of several signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins.

Coordinative modulation of human zinc transporter 2 gene expression through active and suppressive regulators.

Zinc transporter 2 (ZnT2) is one of the cellular factors responsible for Zn homeostasis. Upon Zn overload, ZnT2 reduces cellular Zn by transporting it into excretory vesicles. We investigated the molecular mechanism that regulates human ZnT2 (hZnT2) gene expression. Zn induces hZnT2 expression in dose- and time-dependent manners. Overexpression of metal-responsive transcription factor 1 (MTF-1) increases hZnT2 transcription, whereas depletion of MTF-1 reduces hZnT2 expression. There are five putative metal response elements (MREs) within 1kb upstream of the hZnT2 gene. A serial deletion of the hZnT2 promoter region (from 5' to 3') shows that the two MREs proximal to the gene are essential for Zn-induced promoter activity. Further mutation analysis concludes that the penultimate MRE (MREb) supports the metal-induced promoter activity. The hZnT2 promoter has also a zinc finger E-box binding homeobox (ZEB) binding element. Mutation or deletion of this ZEB binding element elevates the basal and Zn-induced hZnT2 promoter activities. Knockdown of ZEB1 mRNA enhances the hZnT2 transcript level in HEK-293 cells. In MCF-7 (ZEB-deficient) cells, expression of ZEB proteins attenuates the Zn-induced hZnT2 expression. However, expressions of MTF-1 target genes such as human ZnT1 and metallothionein IIA were not affected. Our study shows the expression of the hZnT2 gene is coordinately regulated via active and suppressive modulators.

Determination of urine cofilin-1 level in acute kidney injury using a high-throughput localized surface plasmon-coupled fluorescence biosensor.

The actin-depolymerizing factor (ADF)/cofilin protein family has been reported to be associated with ischemia-induced renal disorders. We examine whether cofilin-1 is associated with acute kidney injury (AKI) using human urine samples. We exploited a 96-well based high-throughput biosensor that uses gold nanoparticles and a sandwich immunoassay to detect the urine cofilin-1 level of AKI patients. The mean urine cofilin-1 level of the AKI patients (n=37 from 47 cases analyzed) was twofold higher than that of healthy adults (n=21 from 29 cases analyzed). The receiver operating characteristic (ROC) curve showed that cofilin-1 was acceptable for discriminating AKI patients from healthy adults. However, an increase of the sample size is required to conclude the importance of urine cofilin-1 on AKI diagnosis, and the high-throughput ultrasensitive biosensor used in this study would greatly accelerate the measurement of urine cofilin-1 in an increased sample size.

Association of Asef and Cdc42 expression to tubular injury in diseased human kidney.

BACKGROUND: Cdc42 is a small guanosine-5'-triphosphatase of the Rho family and plays essential roles in the establishment of cellular polarity and tight junctions in epithelial cells. Adenomatous polyposis coli-associated exchange factor (Asef) is a canonical guanine nucleotide exchange factor of Cdc42 and renders Cdc42 to be guanosine-5'-triphosphate bound and activated. The expression patterns and their significance in human renal diseases are unknown. METHODS: We examined the expression of Cdc42 and Asef in kidney biopsy specimens of 15 patients and in normal kidney tissue using immunofluorescence and correlated the expression patterns with the clinical characteristics. We also analyzed the coexpression pattern of Ki-67, a marker indicating cell division, and Asef in selected patients. RESULTS: Expression of Asef and Cdc42 together was associated with tubular injury with 100% specificity. Expression of Asef, regardless of Cdc42, also showed a significant diagnostic odds ratio for the presence of the injury. Expression of Asef was associated with lower estimated glomerular filtration rate at the time of biopsy and larger area of interstitial fibrosis. All Ki-67-expressing tubular cells expressed Asef. CONCLUSIONS: Induction of Asef and Cdc42 in the renal tubules is a cellular response to injury. Asef induction seems a necessary step for injured tubular cells to enter cell cycle.

A novel cell-penetrating peptide derived from human eosinophil cationic protein.

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP(32-41), derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP(32-41) entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP(32-41) to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP(32-41) internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP(32-41) attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP(32-41) internalization routes. ECP(32-41) was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.

Expression and characterization of SUMO-conjugated metal-responsive transcription factor 1: SIM-dependent cross-interaction and distinct DNA binding activity.

Metal-responsive transcription factor 1 (MTF-1) regulates a variety of genes involving in metal homeostasis and oxidative stress. We have shown that MTF-1 can be conjugated by small ubiquitin-like modifier (SUMO) and forms complexes with cellular factor(s) in a SUMO-interacting motif (SIM)-dependent manner. To investigate whether the interaction of MTF-1 and its SUMO conjugate occurs, we expressed and isolated MTF-1 and sumoylated MTF-1 (S-MTF-1) for functional studies. Various conditions were examined to optimize the expressions of MTF-1 and S-MTF-1. Results from affinity column chromatography demonstrated that the unmodified MTF-1 consistently co-eluted with the S-MTF-1. Mutations at the SIM did not reduce the level of MTF-1 sumoylation but the sumoylated product can then be purified to homogeneity. The presence of MTF-1 cross-interaction was further supported by in vitro pull-down assays. The ability of the purified proteins in binding metal-responsive element (MRE) was assessed with electrophoretic mobility shift assay. Noticeably, MTF-1 required the presence of cell extracts to render the binding activity. However, S-MTF-1 binds MRE in void of other cellular factors. The same characteristic was found for MTF-1 with SUMO fusion at the carboxyl terminus. These results indicate that the presence of SUMO moiety allows the protein to interact directly with MRE.

Single-walled carbon nanotube coated antibacterial paper: preparation and mechanistic study.

Development of carbon nanotubes toward commercial antibacterial applications warrants the understanding of their interaction mechanism with bacterial cells. The antibacterial activity and mechanism of acid-functionalized single-walled carbon nanotube (AFSWCNT) coated paper was assessed for gram-positive Staphylococcus aureus and gram-negative Escherichia coli models of bacteria. Better activity towards gram-positive bacteria was observed, whereas the presence of an outer membrane makes gram-negative bacteria more resistant to cell membrane damage caused by AFSWCNTs. Based on measured cytoplasmic efflux materials of bacteria, X-ray photoelectron spectroscopy, and scanning transmission electron microscopy combined with electron energy-loss spectroscopy imaging studies, we found that the better antibacterial activity of AFSWCNTs toward gram-positive bacteria is attributed to not only direct physical contact and piercing action, but also molecular-scale interaction with surface functional groups of bacteria. The novel antibacterial mechanism of AFSWCNTs might bring a promising strategy to design new antibacterial materials against drug-resistant bacteria species.

PTEN interacts with metal-responsive transcription factor 1 and stimulates its transcriptional activity.

MTF-1 (metal-responsive transcription factor 1) is an essential mammalian protein for embryonic development and modulates the expression of genes involving in zinc homoeostasis and responding to oxidative stress. We report in the present paper that PTEN (phosphatase and tensin homologue deleted on chromosome 10) associates with MTF-1 in the cells. These two proteins interact via the acidic domain of MTF-1 and the phosphatase/C2 domain of PTEN. Depletion of PTEN reduced MT (metallothionein) gene expression and increased cellular sensitivity to cadmium toxicity. PTEN did not alter the nuclear translocation, protein stability or DNA-binding activity of MTF-1. Zinc increased MTF-1-PTEN interaction in a dose-dependent manner. The interaction elevated within 2 h of zinc addition and declined afterwards in the cells. The enhanced binding activity occurred mainly in the cytoplasm and reduced after translocating the MTF-1 into the nucleus. Blocking signalling through the PI3K (phosphoinositide 3-kinase) pathway did not alter the zinc-induced MT expression. Analysis of enzymatically inactive PTEN mutants demonstrated that protein but not lipid phosphatase activity of PTEN was involved in the regulation of MTF-1 activity. The same regulatory role of PTEN was also noted in the regulation of ZnT1 (zinc transporter 1), another target gene of MTF-1.

The role of small ubiquitin-like modifier-interacting motif in the assembly and regulation of metal-responsive transcription factor 1.

Metal-responsive transcription factor 1 (MTF-1) is an essential protein required for mouse embryonic development. We report here the occurrence of sumoylation on MTF-1. Mutational studies demonstrated that sumoylation occurs on the lysine residue at position 627 (Lys(627)) of mouse MTF-1. Small ubiquitin-like modifier (SUMO)-1 was fused to the C terminus of MTF-1 to mimic the sumoylated form of the protein and it suppressed the transcriptional activity of MTF-1. The nuclear translocation activity, DNA-binding activity, and protein stability of SUMO-fused MTF-1 are similar to that of wild type MTF-1. The level of sumoylation was reduced by metal in a dose- and time-dependent manner. The fact that zinc reduces MTF-1 sumoylation makes the suppressive role of sumoylated MTF-1 in transcription physiologically less significant because the SUMO moiety of MTF-1 is removed when MTF-1 translocates into nucleus. We further identified a SUMO-interacting motif (SIM) on MTF-1. Remarkably, MTF-1 binds sumoylated MTF-1 and/or other cellular factors in a SIM-dependent manner. This interaction was disrupted by treating cells with zinc. Gel permeation chromatography demonstrated that MTF-1 forms SIM-dependent complexes. This cross-interaction transpires in the cytoplasm and markedly reduces upon nuclear translocation. It can therefore be concluded that SUMO conjugation and the SIM on MTF-1 do not play a critical role in suppressing transcriptional activity. Instead, MTF-1 forms complexes with cellular factors through SIM and SUMO moiety in the cytoplasm. The result explores a new understanding for the mode of MTF-1 assembly and regulation in cells.

Water-soluble germanium nanoparticles cause necrotic cell death and the damage can be attenuated by blocking the transduction of necrotic signaling pathway.

Water-soluble germanium nanoparticles (wsGeNPs) with allyamine-conjugated surfaces were fabricated and emit blue fluorescence under ultraviolet light. The wsGeNP was physically and chemically stable at various experimental conditions. Cytotoxicity of the fabricated wsGeNP was examined. MTT assay demonstrated that wsGeNP possessed high toxicity to cells and clonogenic survival assay further indicated that this effect was not resulted from retarding cell growth. Flow cytometric analysis indicated that wsGeNP did not alter the cell cycle profile but the sub-G1 fraction was absent from treated cells. Results from DNA fragmentation and propidium iodide exclusion assays also suggested that apoptotic cell death did not occur in cells treated with wsGeNP. Addition of a necrosis inhibitor, necrostatin-1, attenuated cell damage and indicated that wsGeNP caused necrotic cell death. Cell signaling leads to necrotic death was investigated. Intracellular calcium and reactive oxygen species (ROS) levels were increased upon wsGeNP treatment. These effects can be abrogated by BAPTA-AM and N-acetyl cysteine respectively, resulting in a reduction in cell damage. In addition, wsGeNP caused a decrease in mitochondrial membrane potential (MMP) which could be recovered by cyclosporine A. The cellular signaling events revealed that wsGeNP increase the cellular calcium level which enhances the production of ROS and leads to a reduction of MMP, consequentially results in necrotic cell death.

Comparison of organic and inorganic germanium compounds in cellular radiosensitivity and preparation of germanium nanoparticles as a radiosensitizer.

PURPOSE: The aim of this work is to compare the radiosensitizing effect between organic and inorganic germanium compounds and to investigate whether nanometer-sized germanium particles can act as radiosensitizers. MATERIALS AND METHODS: Bis (2-carboxyethylgermanium) sesquioxide (Ge-132), germanium oxide (GeO(2)) and germanium nanoparticles were used in this study. Cell viability was determined by clonogenic survival assay. Cellular DNA damage was evaluated by alkaline comet assay, confocal microscopy and the cellular level of phospho-histone H2AX (gamma-H2AX). RESULTS: Nanometer-sized germanium particles were fabricated. They have a similar radiosensitizing effect as that of GeO(2). Conversely, Ge-132 did not enhance the radiosensitivity of cells. Comet assay was employed to evaluate the level of DNA damage and confirmed that inorganic germanium compounds enhanced cellular radiosensitivity. Notably, the comet assay indicated that the nanoparticle itself caused a higher level of DNA damage. The possibility that germanium nanoparticles per se caused DNA damage was ruled out when the cellular level of gamma-H2AX was examined. CONCLUSIONS: We demonstrated that inorganic but not organic germanium compounds exerted radiosensitizing effect in cells. Nanometer-sized germanium particles were fabricated and were able to enhance the radiosensitivity of cells. Confounding effect may occur when comet assay is used to estimate the level of DNA damage in the presence of germanium nanoparticles.

Attenuation of cadmium-induced necrotic cell death by necrostatin-1: potential necrostatin-1 acting sites.

Cadmium (Cd) induces necrotic death in Chinese hamster ovary (CHO) K1 cells and we have established the responsible signaling pathway. Reportedly, necrostatin-1 (Nec-1) rescues cells from necrotic death by mediating through the death domain receptor (DR) signaling pathway. We show here that Nec-1 also effectively attenuates necrotic death triggered by Cd. Two other treatments that cause necrotic cell death, one can (z-VAD-fmk/TNF-alpha on U937 cells) and the other cannot (etherynic acid (EA) on DLD-1 cells) be rescued by Nec-1, were also studied in parallel for comparison. Results show that Nec-1 is ineffectual in modulating intracellular calcium contents, calpain activity (a downstream protease), or reactive oxygen species production. It can counteract the reduction in mitochondrial membrane potential (MMP) caused by treating CHO K1 or U937 cells with necrosis-inducing agent. However, this effect was not found in EA-treated DLD-1 cells. Notably, Nec-1 elevates NF-kappaB activity in the presence or absence of necrosis-inducing agents. Our study shows that, in addition to DR-mediated necrosis, Nec-1 is effective in attenuating Cd-induced necrosis. It rescues cells with reduced MMP implying that mitochondrion is its major acting site.

Cadmium induces Ca2+-dependent necrotic cell death through calpain-triggered mitochondrial depolarization and reactive oxygen species-mediated inhibition of nuclear factor-kappaB activity.

This study investigates the mechanism of cell death induced by cadmium (Cd) in Chinese hamster ovary (CHO) cells. Cells exposed to 4 microM Cd for 24 h did not show signs of apoptosis, such as DNA fragmentation and caspase-3 activation. The pro-apoptotic (Bax) or anti-apoptotic (Bcl-2 and Bcl-xL) protein levels in the Bcl-2 family were not altered. However, an increase in propidium iodide uptake and depletion of ATP, characteristics of necrotic cell death, were observed. Cd treatment increased the intracellular calcium (Ca2+) level. Removal of the Ca2+ by a chelator, BAPTA-AM, efficiently inhibited Cd-induced necrosis. The increased Ca2+ subsequently mediated calpain activation and intracellular ROS production. Calpains then triggered mitochondrial depolarization resulting in cell necrosis. Cyclosporin A, an inhibitor of mitochondrial permeability transition, recovered the membrane potential and reduced the necrotic effect. The generated ROS reduced basal NF-kappaB activity and led cells to necrosis. An increase of NF-kappaB activity by its activator, PMA, attenuated Cd-induced necrosis. Calpains and ROS act cooperatively in this process. The calpain inhibitor and the ROS scavenger synergistically inhibited Cd-induced necrosis. Results in this study suggest that Cd stimulates Ca2+-dependent necrosis in CHO cells through two separate pathways. It reduces mitochondrial membrane potential by activating calpain and inhibits NF-kappaB activity by increasing the ROS level.