Cold atmospheric nitrogen plasma induces metal-initiated cell death by cell membrane rupture and mitochondrial perturbation.

Cold atmospheric plasma (CAP) is a novel biomedical tool used for cancer therapy. A device using nitrogen gas (N2 CAP) produced CAP that induced cell death through the production of reactive nitrogen species and an increase in intracellular calcium. In this study, we investigated the effect of N2 CAP-irradiation on cell membrane and mitochondrial function in human embryonic kidney cell line 293T. We investigated whether iron is involved in N2 CAP-induced cell death, as deferoxamine methanesulfonate (an iron chelator) inhibits this process. We found that N2 CAP induced cell membrane disturbance and loss of mitochondrial membrane potential in an irradiation time-dependent manner. BAPTA-AM, a cell-permeable calcium chelator, inhibited N2 CAP-induced loss of mitochondrial membrane potential. These results suggest that disruption of intracellular metal homeostasis was involved in N2 CAP-induced cell membrane rupture and mitochondrial dysfunction. Moreover, N2 CAP irradiation generated a time-dependent production of peroxynitrite. However, lipid-derived radicals are unrelated to N2 CAP-induced cell death. Generally, N2 CAP-induced cell death is driven by the complex interaction between metal movement and reactive oxygen and nitrogen species produced by N2 CAP.

Generation of Rat Monoclonal Antibody for Human IQGAP1 by Immunization of Three-Dimensional-Cultured Cancer Cells.

The scaffold protein IQ motif containing GTPase activating protein 1 (IQGAP1) is an adherens junction component in the epithelial tissue that binds many signaling and structural molecules to regulate biological processes. It is known that IQGAP1 is overexpressed in some tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from three-dimensional (3D)-cultured DLD-1 cells to elucidate a characteristic feature of a tumor. In cancer research, 3D-cultured cancer cells are used as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. Our results showed that mAb 7E11 recognized increasing antigen in the lysate of 3D-cultured cells comparing with two-dimensional-cultured cells, and its antigen is the human IQGAP1. Furthermore, we indicated that mAb 7E11 was used in immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful in the analysis of human cancer.

Cell Death via Lipid Peroxidation and Protein Aggregation Diseases.

Lipid peroxidation of cellular membranes is a complicated cellular event, and it is both the cause and result of various diseases, such as ischemia-reperfusion injury, neurodegenerative diseases, and atherosclerosis. Lipid peroxidation causes non-apoptotic cell death, which is associated with cell fate determination: survival or cell death. During the radical chain reaction of lipid peroxidation, various oxidized lipid products accumulate in cells, followed by organelle dysfunction and the induction of non-apoptotic cell death. Highly reactive oxidized products from unsaturated fatty acids are detected under pathological conditions. Pathological protein aggregation is the general cause of these diseases. The cellular response to misfolded proteins is well-known as the unfolded protein response (UPR) and it is partially concomitant with the response to lipid peroxidation. Moreover, the association between protein aggregation and non-apoptotic cell death by lipid peroxidation is attracting attention. The link between lipid peroxidation and protein aggregation is a matter of concern in biomedical fields. Here, we focus on lethal protein aggregation in non-apoptotic cell death via lipid peroxidation. We reviewed the roles of protein aggregation in the initiation and execution of non-apoptotic cell death. We also considered the relationship between protein aggregation and oxidized lipid production. We provide an overview of non-apoptotic cell death with a focus on lipid peroxidation for therapeutic targeting during protein aggregation diseases.

Generation of Rat Monoclonal Antibody for Cytokeratin 18 by Immunization of Three-Dimensional-Cultured Cancer Cells.

Cytokeratin (CK) 18 is an intermediate filament protein that plays a major functional role in the integrity and mechanical stability of cells. Since both CK8 and CK18 are major components of simple epithelia, in the context of tumors, they are expressed in most carcinomas, and have been studied as diagnostic and prognostic markers in tumor pathology. CK18 is also cleaved by some caspases during apoptosis. Three-dimensional (3D)-cultured cancer cells are useful for cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from 3D-cultured DLD-1 cells to elucidate a characteristic feature of a tumor, and our results showed that mAb 2H7 recognized human CK18. Furthermore, we indicated that mAb 2H7 was useful for immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful as a diagnostic tool for evaluating malignancy.

A simple method for isolation and culture of primary hepatocytes from Salvelinus leucomaenis (White-spotted Charr).

White-spotted charr (Salvelinus leucomaenis, S. I.) is an anadromous cold water-adapted fish, distributed in the Far East. We have previously reported the complete mitochondrial DNA sequences of white-spotted chars (S. l. imbrius and S. l. pluvius) in Japan. In general, fish hepatocytes are useful for cellular and biochemical studies of fish. In this study, we isolated hepatocytes from the liver of white-spotted charr and used basic methods, such as enzyme digestion and low centrifugation, to analyze the molecular mechanisms involved in specific cellular responses. The isolated hepatocytes could be cultured at 5-20 °C but not 37 °C. The morphology of hepatocytes was altered in a temperature-dependent manner. The properties of hepatocyte were similar to those of living fish. Moreover, the proliferation rate and damage of isolated hepatocytes depended on the concentration of fetal bovine serum in the culture medium. Taken together, this study demonstrates that this simple method for isolation and culture of hepatocytes from white-spotted charr may be useful for other biochemical and cellular studies.

Upregulation of nuclear factor (erythroid-derived 2)-like 2 protein level in the human colorectal adenocarcinoma cell line DLD-1 by a heterocyclic organobismuth(III) compound: Effect of organobismuth(III) compound on NRF2 signaling.

An increasing number of metal-based compounds, including arsenic trioxide, auranofin, and cisplatin, have been reported to have antitumor activity. Their beneficial effects are controlled by a transcription factor, nuclear factor (erythroid-derived 2)-like 2 (NRF2). In response to oxidative stress, NRF2 induces the expression of cytoprotective genes. NRF2 protein levels are regulated by Kelch-like ECH-associated protein 1 (KEAP1) via ubiquitination. Bi-chlorodibenzo[c,f][1,5]thiabismocine (compound 3), a bismuth compound, is known for its potent anti-proliferative activity against various cancer cell lines. In the present study, we investigated the effect of compound 3 on NRF2 signaling in the human colorectal adenocarcinoma cell line DLD-1 in terms of cell viability as well as mRNA and protein expression levels of NRF2. Compound 3 upregulated NRF2 protein levels in a time- and concentration-dependent manner, accompanied by a marked increase in heme-oxygenase-1 (HO-1) mRNA and protein levels. We observed that brusatol, an NRF2 inhibitor, as well as small interfering RNA (siRNA)-mediated knockdown of NRF2 in DLD-1 cells suppressed compound 3-induced HO-1 expression. The anticancer activity of compound 3 was enhanced by compounds that downregulate NRF2. These results suggest that compound 3 upregulates HO-1 via NRF2 activation and that the NRF2-HO-1 pathway is the cellular response to compound 3. We also discovered that compound 3 slightly downregulated KEAP1; thus, NRF2 activation may be associated with KEAP1 modification. Collectively, our results indicate that compound 3 simultaneously activates an anti-oxidative stress pathway, such as NRF2 and HO-1, and a pro-cell death signal in DLD-1 cells. Our findings may provide useful information for the development of a potent anticancer organobismuth(III) compound.

Heterocyclic organobismuth(III) compound induces nonapoptotic cell death via lipid peroxidation.

Heterocyclic organobismuth compounds, such as N-tert-butyl-bi-chlorodibenzo[c,f][1,5]azabismocine (compound 1) and bi-chlorodibenzo[c,f ][1,5]thiabismocine (compound 3), exert potent antiproliferative activities in vitro in human cancer cell lines. We showed that compound 3 induced both apoptotic and nonapoptotic cell death via reactive oxygen species production and mitotic arrest in a dose-dependent manner. The mechanisms underlying the dose-dependent effect of these organobismuth compounds were not clear. In the present study, we examined the dose-dependent mechanism underlying cell death induced by compound 1 in a human pancreatic cancer cell line, SUIT-2, and a human colorectal cancer cell line, DLD-1. Compound 1 inhibited cell growth in a dose-dependent manner and induced cell death. Treatment with the pan-caspase inhibitor zVAD-fmk reduced cell death induced by compound 1, whereas the inhibitory effect of zVAD-fmk was limited. Moreover, compound 1 significantly induced lipid peroxidation with concomitant induction of caspase-independent cell death. Our results suggested that eight-membered ring organobismuth compounds induce nonapoptotic cell death via lipid peroxidation.

Different morphologies of human embryonic kidney 293T cells in various types of culture dishes.

Human embryonic kidney 293T (HEK293T) cells are used in various biological experiments and researches. In this study, we investigated the effect of cell culture environments on morphological and functional properties of HEK293T cells. We used several kinds of dishes made of polystyrene or glass for cell culture, including three types of polystyrene dishes provided from different manufacturers for suspension and adherent cell culture. In addition, we also investigated the effect of culturing on gelatin-coated surfaces on the cell morphology. We found that HEK293T cells aggregated and formed into three-dimensional (3-D) multicellular spheroids (MCS) when non-coated polystyrene dishes were used for suspension culture. In particular, the non-coated polystyrene dish from Sumitomo bakelite is the most remarkable characteristic for 3-D MCS among the polystyrene dishes. On the other hand, HEK293T cells hardly aggregated and formed 3-D MCS on gelatin-coated polystyrene dishes for suspension culture. HEK293T cells adhered on the non- or gelatin-coated polystyrene dish for adherent culture, but they did not form 3-D MCS. HEK293T cells also adhered to non- or gelatin-coated glass dishes and did not form 3-D MCS in serum-free medium. These results suggest that HEK293T cells cultured on non-coated polystyrene dish may be useful for the tool to analyze the characteristics of 3D-MCS.

Oxidized unsaturated fatty acids induce apoptotic cell death in cultured cells.

Polyunsaturated fatty acids are oxidized by non­enzymatic or enzymatic reactions. The oxidized products are multifunctional. In this study, we investigated how oxidized fatty acids inhibit cell proliferation in cultured cells. We used polyunsaturated and saturated fatty acids, docosahexaenoic acid (DHA; 22:6), eicosapentaenoic acid (EPA; 20:5), linoleic acid (LA; 18:2), and palmitic acid (16:0). Oxidized fatty acids were produced by autoxidation of fatty acids for 2 days in the presence of a gas mixture (20% O2 and 80% N2). We found that oxidized polyunsaturated fatty acids (OxDHA, OxEPA and OxLA) inhibited cell proliferation much more effectively compared with un­oxidized fatty acids (DHA, EPA and LA, respectively) in THP­1 (a human monocytic leukemia cell line) and DLD­1 (a human colorectal cancer cell line) cells. In particular, OxDHA markedly inhibited cell proliferation. DHA has the largest number of double bonds and is most susceptible to oxidation among the fatty acids. OxDHA has the largest number of highly active oxidized products. Therefore, the oxidative levels of fatty acids are associated with the anti­proliferative activity. Moreover, caspase­3/7 was activated in the cells treated with OxDHA, but not in those treated with DHA. A pan­caspase inhibitor (zVAD­fmk) reduced the cell death induced by OxDHA. These results indicated that oxidized products from polyunsaturated fatty acids induced apoptosis in cultured cells. Collectively, the switch between cell survival and cell death may be regulated by the activity and/or number of oxidized products from polyunsaturated fatty acids.

Cold atmospheric-pressure nitrogen plasma induces the production of reactive nitrogen species and cell death by increasing intracellular calcium in HEK293T cells.

Cold atmospheric-pressure plasma (CAP) has been emerging as a promising tool for cancer therapy in recent times. In this study, we used a CAP device with nitrogen gas (N2CAP) and investigated the effect of the N2CAP on the viability of cultured cells. Moreover, we investigated whether N2CAP-produced hydrogen peroxide (H2O2) in the medium is involved in N2CAP-induced cell death. Here, we found that the N2CAP irradiation inhibited cell proliferation in the human embryonic kidney cell line HEK293T and that the N2CAP induced cell death in an irradiation time- and distance-dependent manner. Furthermore, the N2CAP and H2O2 increased intracellular calcium levels and induced caspase-3/7 activation in HEK293T cells. The N2CAP irradiation induced a time-dependent production of H2O2 and nitrite/nitrate in PBS or culture medium. However, the amount of H2O2 in the solution after N2CAP irradiation was too low to induce cell death. Interestingly, carboxy-PTIO, a nitric oxide scavenger, or BAPTA-AM, a cell-permeable calcium chelator, inhibited N2CAP-induced morphological change and cell death. These results suggest that the production of reactive nitrogen species and the increase in intracellular calcium were involved in the N2CAP-induced cell death.

Induction of oxidative stress by anticancer drugs in the presence and absence of cells.

Reactive oxygen species (ROS) are generated in the cell through multiple mechanisms. Intracellular ROS are rapidly detoxified by various enzymatic and non-enzymatic mechanisms; however, disruption of the oxidant-antioxidant balance causes oxidative stress and elicits cell damage. The oxidative stress induced by chemotherapy is known to cause side effects in patients with cancer. However, few studies have examined whether anticancer drugs induce oxidative stress in cancer cells. Furthermore, the precise mechanism by which anticancer drugs induce the generation of ROS remains unclear. In the present study, to investigate whether anticancer drugs induce oxidative stress, DLD-1 human colorectal cancer cells were treated with 20 different anticancer drugs and then stained with CellROX® ROS detection reagent. Furthermore, an oxygen radical absorbance capacity assay in the presence of copper was performed to estimate the oxidative activities of the anticancer drugs in the absence of cells. The data of the present study using assay methods in the presence and absence of cells suggest that nimustine, actinomycin D, doxorubicin, mitomycin C, mitoxantrone, carmofur, gemcitabine, mercaptopurine, camptothecin, paclitaxel, vinblastine, and vinorelbine are able to induce oxidative stress.

Interleukin-8 enhances the effect of colchicine on cell death.

Pro-inflammatory cytokines are known to be generated in tumors and play important roles in angiogenesis, mitosis, and tumor progression. However, few studies have investigated the synergistic effects of pro-inflammatory cytokines and anticancer drugs on cell death. In the present study, we examined the combined effects of pro-inflammatory cytokines and colchicine on cell death of cancer cells. Colchicine induces G2/M arrest in the cell cycle by binding to tubulin, one of the main constituents of microtubules. SUIT-2 human pancreatic cancer cell line cells overexpressing pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α, were treated with colchicine. The effect of colchicine on cell death was enhanced in cells overexpressing IL-8. Moreover, the effect of colchicine on cell death was enhanced in cells overexpressing two IL-8 up-regulators, NF-κB and IL-6, but not in cells overexpressing an IL-8 down-regulator, splicing factor proline/glutamine-rich (SFPQ). Synergistic effects of IL-8 and colchicine were also observed in cells overexpressing IL-8 isoforms lacking the signal peptide. Therefore, IL-8 appeared to function as an enhancer of cell death in cancer cells treated with colchicine. The present results suggest a new role for IL-8 related to cell death of cancer cells.

Alternative splicing isoform in succinate dehydrogenase complex, subunit C causes downregulation of succinate-coenzyme Q oxidoreductase activity in mitochondria.

Mitochondrial succinate dehydrogenase (SDH) is localized to the inner mitochondrial membrane and is responsible for the redox of succinic acid. SDH is a tetrameric iron-sulfur flavoprotein of the tricarboxylic acid cycle and respiratory chain. The SDH complex, subunit C (SDHC) transcript has deletion-type alternative splicing sites. Generally, alternative splicing produces variant proteins and expression patterns, as products of different genes. In certain cases, specific alternative splicing variants (ASVs) have been associated with human disease. Due to a frameshift mutation causing loss of the heme binding region, the SDHC Δ5 isoform (lacking exon 5) exhibits no SDHC activity. To investigate whether the SDHC splicing variants can function as dominant-negative inhibitors, SDHC ASVs were overexpressed in HCT-15 human colorectal cancer cells. Using real-time reverse transcription-polymerase chain reaction, a dominant-negative effect of the Δ5 isoform on SDHC mRNA was shown. In addition, Δ5 overexpression increased the levels of reactive oxygen species. Furthermore, in the Δ5 isoform-overexpressing cells, SDH activity was reduced. SDHC activation is a significant event during the electron transport chain, and the function of the SDHC Δ5 variant may be significant for the differentiation of tumor cells.

Mitochondrial DNA reduced by hypoxic conditions in three-dimensional (3D) spheroid cell cultures.

Three-dimensional (3D) cell culture reflects many of the important properties of solid tumors, such as the inadequate diffusion of oxygen that results in hypoxia. To understand the mitochondrial states in cancer, we performed comparisons of the levels of mitochondrial DNA (mtDNA), fusion- and fission-related mitochondrial messenger RNA (mRNA), and mitochondrial protein expression between monolayer (2D)- and 3D-cultured cancer cells. The mtDNA levels were observed to be significantly lower in the 3D cells compared with the monolayer cells. In contrast, the differences in expression of the mitochondrial fusion- and fission-related mRNAs and mitochondrial proteins between 2D- and 3D-cultured cancer cells were not significant, as shown by real-time PCR and immunoblot analysis. Therefore, although mtDNA levels decrease as a whole during 3D culture, this does not appear to affect the fusion and fission of individual mitochondria. Indeed, the factors regulating mitochondrial dynamics during 3D cell culture remain unclear. This study provides the basis for future, more detailed studies on the regulation of mtDNA.

Lipogenic gene expression profile in patients with gastric cancer.

Numerous types of cancer exhibit increased lipogenesis and expression of lipogenic enzymes and transcription factors, including sterol regulatory element-binding protein-1. Lipogenic gene expression is upregulated at the mRNA level, in concert with metabolic pathways associated with changes in expression and/or activity of lipogenic transcription factors. However, this expression pattern in human gastric carcinoma has not been elucidated. In this study, lipogenic gene expression in cancer tissues was investigated using quantitative PCR. In patients with gastric cancer, carnitine O-palmitoyltransferase type I mRNA and miR-33b were significantly downregulated, suggesting that miR-33b downregulation is mediated by conditions that also affect the expression and/or activity of transcription factors involved in lipogenic gene expression. Consequently, the association between miR-33b and gastric cancer may provide a novel strategy for the genetic diagnosis of gastric cancer. However, additional studies including a larger number of samples are required to confirm these results.

The C allele of JAK2 rs4495487 is an additional candidate locus that contributes to myeloproliferative neoplasm predisposition in the Japanese population.

BACKGROUND: Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are myeloproliferative neoplasms (MPNs) characterized in most cases by a unique somatic mutation, JAK2 V617F. Recent studies revealed that JAK2 V617F occurs more frequently in a specific JAK2 haplotype, named JAK2 46/1 or GGCC haplotype, which is tagged by rs10974944 (C/G) and/or rs12343867 (T/C). This study examined the impact of single nucleotide polymorphisms (SNPs) of the JAK2 locus on MPNs in a Japanese population. METHODS: We sequenced 24 JAK2 SNPs in Japanese patients with PV. We then genotyped 138 MPN patients (33 PV, 96 ET, and 9 PMF) with known JAK2 mutational status and 107 controls for a novel SNP, in addition to two SNPs known to be part of the 46/1 haplotype (rs10974944 and rs12343867). Associations with risk of MPN were estimated by odds ratios and their 95% confidence intervals using logistic regression. RESULTS: A novel locus, rs4495487 (T/C), with a mutated T allele was significantly associated with PV. Similar to rs10974944 and rs12343867, rs4495487 in the JAK2 locus is significantly associated with JAK2-positive MPN. Based on the results of SNP analysis of the three JAK2 locus, we defined the "GCC genotype" as having at least one minor allele in each SNP (G allele in rs10974944, C allele in rs4495487, and C allele in rs12343867). The GCC genotype was associated with increased risk of both JAK2 V617F-positive and JAK2 V617F-negative MPN. In ET patients, leukocyte count and hemoglobin were significantly associated with JAK2 V617F, rather than the GCC genotype. In contrast, none of the JAK2 V617F-negative ET patients without the GCC genotype had thrombosis, and splenomegaly was frequently seen in this subset of ET patients. PV patients without the GCC genotype were significantly associated with high platelet count. CONCLUSIONS: Our results indicate that the C allele of JAK2 rs4495487, in addition to the 46/1 haplotype, contributes significantly to the occurrence of JAK2 V617F-positive and JAK2 V617F-negative MPNs in the Japanese population. Because lack of the GCC genotype represents a distinct clinical-hematological subset of MPN, analyzing JAK2 SNPs and quantifying JAK2 V617F mutations will provide further insights into the molecular pathogenesis of MPN.

Glial fibrillary acidic protein is one of the key factors underlying neuron-like elongation in PC12 cells.

Nerve growth factor (NGF) normally induces the differentiation of PC12 cells into a neuron-like phenotype. In this study, we found that exposure of PC12 cells to a temperature of 42°C for 24 h significantly decreased NGF-induced neurite elongation. Glial fibrillary acidic protein (GFAP) levels were decreased when PC12 cells were exposed to the heat stress, while PC12 cells overexpressing the gene encoding GFAP showed resistance. Mock-transfected PC12 cells exposed to NGF could be cultured for 72 h at 37°C, whereas GFAP-transfected PC12 cells exposed to NGF could be cultured for over 100 h. Our results suggest that GFAP is necessary for the long-term maintenance of cells with a neuron-like phenotype.

Alternative RNA splicing in expression of the glutathione synthetase gene in human cells.

Ubiquitous free radical production occurs continuously in cells and tissues. Glutathione is the most abundant mammalian antioxidant, and is synthesized by glutathione synthetase (GSS). Therefore, GSS plays an important role in defending the cell against reactive oxygen species. The expression of GSS has been studied in human cells; however, sequence information about alternative splicing variants of GSS mRNA has not been reported. In the present study, we identified a novel alternative splicing variant (ASV) of the GSS gene in 10 human normal tissues and five human cancer cell lines. The deleted transcript of GSS was characterized by an in-frame deletion of 333 bp, corresponding to the complete loss of exons 4 and 5. Thus this GSS ASV causes protein truncation. We quantified the mRNA of GSS ASV in human normal tissues using real-time PCR. The ASV was detected in colon, kidney, lung, liver, placenta, peripheral blood and uterus, but not in heart, skeletal muscle and spleen tissue. Our results provide a basis for more detailed studies on the regulation of GSS, and for further evaluation of this and other possible roles of GSS. Understanding the regulation of GSS expression is very important for the development of new strategies for controlling the development of GSH-based redox homeostasis.

Poloxamer 188 enhances apoptosis in a human leukemia cell line.

Poloxamer block copolymers have been studied in multiple applications as drug delivery systems (DDS). These A-B-A amphiphilic block copolymers up-regulate the expression of selected genes in cells and alter genetic responses to antineoplastic agents in cancer. One example is poloxamer 188, also known as pluronic F68, which may be promising as a carrier in DDS. To clarify the possible mechanistic role of pluronic F68 in several leukemia cell lines, we examined whether pluronic F68-inducible factors were capable of causing apoptosis. The influence of pluronic F68 on the cell lines was examined using a comprehensive analysis. It was found that treatment of K562 cells with 6% pluronic F68 resulted in G2/M phase arrest of the cell cycle, followed by caspase activation and the accumulation of apoptotic cells. When used as a carrier in a DDS, pluronic F68 may provide a synergistic effect on the drug of interest. Although the mechanisms behind the function of pluronic F68 are not fully understood, the results suggests that pluronic F68 may act as a useful carrier in DDS for the purpose of leukemia therapy.