Developmental synapse pathology triggered by maternal exposure to the herbicide glufosinate ammonium.

Environmental and genetic factors influence synapse formation. Numerous animal experiments have revealed that pesticides, including herbicides, can disturb normal intracellular signals, gene expression, and individual animal behaviors. However, the mechanism underlying the adverse outcomes of pesticide exposure remains elusive. Herein, we investigated the effect of maternal exposure to the herbicide glufosinate ammonium (GLA) on offspring neuronal synapse formation in vitro. Cultured cerebral cortical neurons prepared from mouse embryos with maternal GLA exposure demonstrated impaired synapse formation induced by synaptic organizer neuroligin 1 (NLGN1)-coated beads. Conversely, the direct administration of GLA to the neuronal cultures exhibited negligible effect on the NLGN1-induced synapse formation. The comparison of the transcriptomes of cultured neurons from embryos treated with maternal GLA or vehicle and a subsequent bioinformatics analysis of differentially expressed genes (DEGs) identified "nervous system development," including "synapse," as the top-ranking process for downregulated DEGs in the GLA group. In addition, we detected lower densities of parvalbumin (Pvalb)-positive neurons at the postnatal developmental stage in the medial prefrontal cortex (mPFC) of offspring born to GLA-exposed dams. These results suggest that maternal GLA exposure induces synapse pathology, with alterations in the expression of genes that regulate synaptic development via an indirect pathway distinct from the effect of direct GLA action on neurons.

Overexpression of tyrosine hydroxylase in dopaminergic neurons increased sensitivity to methamphetamine.

Drug abuse is one of the great social problems in the world and a major healthcare challenge. It is supposed that sensitivity and reactivity to abuse drugs may vary from person to person, while its molecular basis is largely unknown. Dopaminergic neurons are deeply involved in addiction, and tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step of the biosynthesis of dopamine (DA). We investigated the effects of increased TH expression on the metabolism of DA and reactivity to methamphetamine (METH), a drug of abuse, in mice. Wild-type TH (WT-TH) or the S40E mutant of TH (S40E-TH), which is an active form of TH mimicking phosphorylated TH at the 40th serine, was expressed in midbrain dopaminergic neurons using an adeno-associated virus (AAV) vector. The biochemical analysis showed that the turnover rates of DA in the nerve terminals were increased by the expression of WT-TH and S40E-TH, while there were few changes in the DA contents. Next, we administered METH to TH-overexpressing mice. We found that the S40E-TH-expressing mice responded to lower doses of METH than the control mice and WT-TH mice. The stereotyped behaviors appeared first in S40E-TH mice and then in WT-TH and control mice in this order. These data showed that the TH activity and expression level differentially affect DA metabolism in the nerve terminals from that in the cell bodies and that the TH activity and expression level are one of the determining factors for sensitivity and reactivity to METH. We suggest that TH may be a drug target for ameliorating sensitivity to drugs of abuse.

A replication-competent smallpox vaccine LC16m8Δ-based COVID-19 vaccine.

Viral vectors are a potent vaccine platform for inducing humoral and T-cell immune responses. Among the various viral vectors, replication-competent ones are less commonly used for coronavirus disease 2019 (COVID-19) vaccine development compared with replication-deficient ones. Here, we show the availability of a smallpox vaccine LC16m8Δ (m8Δ) as a replication-competent viral vector for a COVID-19 vaccine. M8Δ is a genetically stable variant of the licensed and highly effective Japanese smallpox vaccine LC16m8. Here, we generated two m8Δ recombinants: one harbouring a gene cassette encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein, named m8Δ-SARS2(P7.5-S)-HA; and one encoding the S protein with a highly polybasic motif at the S1/S2 cleavage site, named m8Δ-SARS2(P7.5-SHN)-HA. M8Δ-SARS2(P7.5-S)-HA induced S-specific antibodies in mice that persisted for at least six weeks after a homologous boost immunization. All eight analysed serum samples displayed neutralizing activity against an S-pseudotyped virus at a level similar to that of serum samples from patients with COVID-19, and more than half (5/8) also had neutralizing activity against the Delta/B.1.617.2 variant of concern. Importantly, most serum samples also neutralized the infectious SARS-CoV-2 Wuhan and Delta/B.1.617.2 strains. In contrast, immunization with m8Δ-SARS2(P7.5-SHN)-HA elicited significantly lower antibody titres, and the induced antibodies had less neutralizing activity. Regarding T-cell immunity, both m8Δ recombinants elicited S-specific multifunctional CD8+ and CD4+ T-cell responses even after just a primary immunization. Thus, m8Δ provides an alternative method for developing a novel COVID-19 vaccine.

Low-intensity ultrasound inhibits melanoma cell proliferation in vitro and tumor growth in vivo.

PURPOSE: To determine the effect of low-intensity ultrasound on cancer cell proliferation in vitro and tumor growth in vivo. METHODS: In vitro, several cancer cell lines were exposed to low-intensity ultrasound at 0.11 W/cm2 for 2 min. Of the cell lines screened, melanoma C32 is one of the cell lines that showed sensitivity to growth inhibition by ultrasound and was therefore used in succeeding experiments. In vivo, under the same ultrasound conditions used in vitro, C32 tumors in mice were exposed to ultrasound daily for 2 weeks, and the tumor volumes were monitored weekly using sonography. RESULTS: In vitro, C32 cell growth was inhibited, attaining 43.2% inhibition on the 3rd day. In vivo, tumor growth was significantly inhibited, with the treated tumors exhibiting 2.7-fold slowed tumor growth vs. untreated tumors at week 2. Such inhibition was not associated with increased cell death. Several genes related to the cell cycle and proliferation were among those significantly regulated. CONCLUSION: These findings highlight the potential of low-intensity ultrasound to inhibit tumor growth in a noninvasive, safe, and easy-to-administer way. In addition, this may suggest that the mechanical stress induced by ultrasound on C32 cells may have affected the intrinsic biomolecular mechanism related to the cell growth of this particular cell line. Further research is needed to identify which of the regulated genes played key roles in growth inhibition.

Effects of orally administered Euglena gracilis and its reserve polysaccharide, paramylon, on gastric dysplasia in A4gnt knockout mice.

Euglena gracilis is widely utilized as food or supplement to promote human and animal health, as it contains rich nutrients. In this study, we administered spray-dried powder of E. gracilis and paramylon, ß-glucan stored in E. gracilis cells, to A4gnt knockout (KO) mice. A4gnt KO mice are a mutant mouse model that spontaneously develops gastric cancer through hyperplasia-dysplasia-adenocarcinoma sequence in the antrum of the stomach, and we observed the effects of E. gracilis and paramylon on the early involvements of A4gnt KO mice. Male and female 10-week-old A4gnt KO mice and their age-matched wildtype C57BL/6J mice were orally administered with 50 mg of E. gracilis or paramylon suspended in saline or saline as a control. After 3-week administration, animals were euthanatized and the stomach was examined histopathologically and immunohistochemically. Gene expression patterns of the stomach, which have been reported to be altered with A4gnt KO, and IgA concentration in small intestine were also analyzed with real-time PCR and ELISA, respectively. Administration of Euglena significantly reduced the number of stimulated CD3-positive T-lymphocytes in pyloric mucosa of A4gnt KO mice and tend to reduce polymorphonuclear leukocytes infiltration. Euglena administration further downregulated the expression of Il11 and Cxcl1 of A4gnt KO mice. Euglena administration also affected IgA concentration in small intestinal contents of A4gnt KO mice. Paramylon administration reduced the number of CD3-positive lymphocytes in pyloric mucosa of A4gnt KO mice, and downregulated the expressions of Il11 and Ccl2 of A4gnt KO mice. Although we found no significant effects on gross and microscopic signs of gastric dysplasia and cell proliferation, the present study suggests that the administration of Euglena and paramylon may ameliorate the early involvements of A4gnt mice through the effects on inflammatory reactions in the gastric mucosa. The cancer-preventing effects should be studied with long-term experiments until actual gastric cancer formation.

Culturing Chinese hamster ovary cells on cyclo olefin polymer triggers epithelial-mesenchymal transition and spheroid formation, which increases the foreign gene expression driven by the Moloney murine leukemia virus long terminal repeat promoter.

Chinese hamster ovary (CHO) cells are frequently used for recombinant protein production (RPP) as a host. While the RPP has been proven successful, there is still a compelling need for further improvement. Cyclo olefin polymer (COP) is a plastic material widely utilized due to its properties including its low protein absorption. We applied this as a raw material for RPP cell culture to see if the COP is suitable. A recombinant CHO cell line expressing the human erythropoietin (hEPO) gene under the control of the Moloney murine leukemia virus-long terminal repeat (MMLV-LTR) was established. When the cells were cultured in a dish made from COP, the cells attached to the bottom, and then started to float and form spheroids. RNASeq data analysis suggested the epithelial-mesenchymal transition (EMT) was triggered with receptor tyrosine kinase activation shortly after cultivation. It coincided with the hEPO transcription increase. After the cell floating, though EMT marker gene expression subsided, a hEPO expression increase sustained. When fibronectin was applied to COP dish surface, the cell floating was suppressed and hEPO expression decreased. We then treated cells with MßCD, a drug that destroys the lipid raft, eliminating molecules in the raft. This facilitated cell floating and spheroid formation coincided with hEPO expression enhancement. These results suggest interactions between a cell and COP surface might trigger the EMT and the subsequent event, both of which activated the MMLV-LTR promoter. Thus, employing COP for culturing cells, a potent RPP system could be established with its advantage for efficient protein purification.

Real-time visualization of intratumoral necrosis using split-luciferase reconstitution by protein trans-splicing.

Necrosis, a form of cell death, occurs not only with the development of various diseases but also with a tumor tissue response to cancer treatment. Therefore, pursuing progress for cancer therapy through induction of necrosis may be one of the most effective approaches for cancer eradication. We herein describe the development of a real-time imaging system to visualize intratumoral necrosis. The system is composed of two types of cells expressing either one of two necrosis imaging reporters that consist of a DnaE intein sequence linking to one of two split-luciferase fragments. When necrosis occurs in a tumor composed of both of the cells, the two types of leaked reporters can reconstitute the enzymatic activity as a result of protein trans-splicing and thereby emit bioluminescence in the presence of the substrate. This system, which was constructed with shrimp-derived luciferase, allowed in vitro imaging of necrosis. We further confirmed real-time imaging of intratumoral necrosis caused by physical or chemical tissue disruption, validating its application in in vivo necrosis imaging. Thus, the constructed imaging system could be a powerful tool for the optimization of the therapeutic condition for cancer therapy and for the evaluation of novel anticancer drugs targeting necrosis.

Characterization of the Gene Expression Patterns in the Murine Liver Following Intramuscular Administration of Baculovirus.

Intramuscular administration of wild-type baculovirus is able to both protect against Plasmodium sporozoite challenge and eliminate liver-stage parasites via a Toll-like receptor 9-independent pathway. To investigate its effector mechanism(s), the gene expression profile in the liver of baculovirus-administered mice was characterized by cDNA microarray analysis. The ingenuity pathway analysis gene ontology module revealed that the major gene subsets induced by baculovirus were immune-related signaling, such as interferon signaling. A total of 40 genes commonly upregulated in a Toll-like receptor 9-independent manner were included as possible candidates for parasite elimination. This gene subset consisted of NT5C3, LOC105246895, BTC, APOL9a/b, G3BP3, SLC6A6, USP25, TRIM14, and PSMB8 as the top 10 candidates according to the special unit. These findings provide new insight into effector molecules responsible for liver-stage parasite killing and, possibly, the development of a new baculovirus-mediated prophylactic and therapeutic biopharmaceutical for malaria.

Combination of 5-aminosalicylic acid and hyperthermia synergistically enhances apoptotic cell death in HSC-3 cells due to intracellular nitric oxide/peroxynitrite generation.

The repurposing of existing FDA-approved non-cancer drugs is a potential source of new treatment options for cancer patients. An anti-inflammatory drug, 5-aminosalicylic acid (5-ASA), has been clinically used to treat inflammatory bowel disease. Hyperthermia (HT) is widely applicable addendum therapy with the existing cancer treatment modalities. Here, we addressed how 5-ASA combined with HT induces lethal effects in human oral squamous cell carcinoma (OSCC) HSC-3 cells. We found that 5-ASA/HT combination significantly inhibited the viability of HSC-3 cells, while cytotoxic effects in primary human dermal fibroblast cells were minor. Apoptotic endpoints were significantly increased by the 5-ASA/HT combined treatment, as evidenced by presence of Annexin V-FITC/PI positive cells, loss of MMP, Bcl-2/Bax ratio alteration, and increased Fas, cleaved Bid, and caspase expression. Interestingly, the enhancement of apoptosis was reversed in the presence of ON/ONOO- scavengers. These findings indicate that the combination treatment enhances apoptosis via ON/ONOO- mediated ER stress-Ca2+-mitochondria signaling and caspase-dependent apoptotic pathways. Our findings provide novel evidence that the combination of 5-ASA and HT is a promising approach for the enhancement of apoptosis; it may serve as an effective strategy for treating human OSCC.

Roles of intracellular and extracellular ROS formation in apoptosis induced by cold atmospheric helium plasma and X-irradiation in the presence of sulfasalazine.

Sulfasalazine (SSZ) is a well-known anti-inflammatory drug and also an inhibitor of the cystine-glutamate antiporter that is known to reduce intracellular glutathione (GSH) level and increase cellular oxidative stress, indicating its anti-tumor potential. However, the combination of SSZ with other physical modalities remains unexplored. Here, the effects of SSZ on cold atmospheric helium plasma (He-CAP), which produces approximately 24 x higher concentration of hydroxyl radicals (. OH) compared to X-irradiation (IR) in aqueous solution, and on IR-induced apoptosis in human leukemia Molt-4 cells were studied to elucidate the mechanism of apoptosis enhancement. Both the Annexin V-FITC/PI and DNA fragmentation assay revealed that pre-treatment of cells with SSZ significantly enhanced He-CAP and IR-induced apoptosis. Similar enhancement was observed during the loss of mitochondrial membrane potential, intracellular Ca2+ ions, and mitochondria- and endoplasmic reticulum-related proteins. The concentration of intracellular reactive oxygen species (ROS) was much higher in He-CAP treated cells than in X-irradiated cells. On the other hand, strong enhancement of Fas expression and caspase-8 and -3 activities were only observed in X-irradiated cells. It might be possible that the higher concentration of intracellular and extracellular ROS suppressed caspase activities and Fas expression in He-CAP-treated cells. Notably, pretreating the cells with an antioxidant N-acetyl-L-cysteine (NAC) dramatically decreased apoptosis in cells treated by He-CAP, but not by IR. These results suggest that IR-induced apoptosis is due to specific and effective ROS distribution since intracellular ROS formation is marginal and the high production of ROS inside and outside of cells plays unique roles in He-CAP induced apoptosis. We conclude that our data provides efficacy and mechanistic insights for SSZ, which might be helpful for establishing SSZ as a future sensitizer in He-CAP or IR therapy for cancer.

Baculovirus-Induced Fast-Acting Innate Immunity Kills Liver-Stage Plasmodium.

Baculovirus (BV), an enveloped insect virus with a circular dsDNA genome, possesses unique characteristics that induce strong innate immune responses in mammalian cells. In this study, we show that BV administration in BALB/c mice not only provides complete protection against a subsequent Plasmodium berghei sporozoite infection for up to 7 d after the injection but also eliminates existing liver-stage parasites completely. The elimination of sporozoites by BV was superior to that by primaquine, and this effect occurred in a TLR9-independent manner. At 6 h after BV administration, IFN-α and IFN-γ were robustly produced in the serum, and RNA transcripts of IFN-stimulated genes were markedly upregulated in the liver compared with control mice. The in vivo passive transfer of serum after BV administration effectively eliminated liver-stage parasites, and IFN-α neutralization abolished this effect, indicating that the BV liver-stage parasite-killing mechanism is downstream of the type I IFN signaling pathway. These findings provide evidence that BV-induced, fast-acting innate immunity completely kills liver-stage parasites and, thus, may lead to new malaria drug and vaccine strategies.

Potential hazards of fenvalerate in massive pollution influence the apoptosis sensitivity.

Fenvalerate (Fen), a synthetic pyrethroid insecticide, is widely used in agricultural, domestic and veterinary applications. Fen induces abnormal cell proliferation and apoptosis, which are linked to its hazardous effects. However, this view is controversial and the underlying molecular mechanisms remain elusive. In the present study, the effects of Fen on cadmium (Cd)-induced apoptosis and the associated molecular mechanisms were investigated in human myeloid leukemia U937 cells. U937 cells were treated with 50 µm cadmium chloride (CdCl2 ) with or without Fen pretreatment at 1-50 µm. Apoptosis was evaluated by externalization of phosphatidylserine on the plasma membrane. The expression levels of apoptosis-related proteins, including Bcl-2 family members were determined by western blot analysis. The results revealed that pretreatment with Fen at 20 µm for 12 hours significantly inhibited Cd-induced apoptosis. Decreased expression of pro-apoptotic Bcl-2 family proteins (Noxa and Bid) and increased expression of anti-apoptotic proteins (Bcl-xL, Mcl-1 and XIAP) were observed after combined treatment with Fen and CdCl2 . Phosphorylation of ERK and AKT was increased, while phosphorylation of JNK was decreased by the combined treatment, compared with CdCl2 treatment alone. In conclusion, Fen decreased apoptotic sensitivity induced by Cd in U937 cells. This effect was associated with activation of ERK and AKT, suppression of JNK and changes in expression of Bcl-2 family proteins and XIAP. The present findings suggest a potential influence of Fen on Cd toxicity via suppression of apoptosis. Fen decreased apoptotic sensitivity induced by Cd, and thus it may contribute carcinogenic risk and influence on cancer therapy.

A Simple Method for Constructing Artificial Promoters Activated in Response to Ultrasound Stimulation.

It has been pointed out that ultrasound could be used as a controller for bioprocesses including gene expression since its energy can noninvasively reach deep in the body. Gene expression may be timely and spatially controlled by ultrasound, thus providing necessary bioactive proteins for the targeted tissue in a timely fashion. Although there are many processes involved in gene expression control, one of the most important processes is transcription, and the promoter plays an essential role in it. There are several promoters known to be activated in response to ultrasound irradiation . However, in our opinion, an artificial promoter is more suitable for clinical use. We herein describe simple methods to construct promoters that are responsive to ultrasound irradiation by randomly combining cis-elements (transcription factor binding motifs) and thereby improve its reactivity to ultrasound irradiation .

Biologically Safe Poly(l-lactic acid) Blends with Tunable Degradation Rate: Microstructure, Degradation Mechanism, and Mechanical Properties.

Although poly(l-lactic acid) (PLLA) is reputed to be biodegradable in the human body, its hydrophobic nature lets it persist for ca. 5.5 years. This study demonstrates that biologically safe lactide copolymers, poly(aspartic acid-co-l-lactide) (PAL) and poly(malic acid-co-l-lactide) (PML), dispersed in the PLLA function as detonators (triggers) for its hydrolytic degradation under physiological conditions. The copolymers significantly enhance hydrolysis, and consequently, the degradation rate of PLLA becomes easily tunable by controlling the amounts of PAL and PML. The present study elucidates the effects of uniaxial drawing on the structural development, mechanical properties, and hydrolytic degradation under physiological conditions of PLLA blend films. At initial degradation stages, the mass loss was not affected by uniaxial drawing; however, at late degradation stages, less developed crystals as well as amorphous chains were degradable at low draw ratio (DR), whereas not only highly developed crystals but also the oriented amorphous chains became insensitive to hydrolysis at high DR. Our work provides important molecular level results that demonstrate that biodegradable materials can have superb mechanical properties and also disappear in a required time under physiological conditions.

Development of a real-time imaging system for hypoxic cell apoptosis.

Hypoxic regions within the tumor form due to imbalances between cell proliferation and angiogenesis; specifically, temporary closure or a reduced flow due to abnormal vasculature. They create environments where cancer cells acquire resistance to therapies. Therefore, the development of therapeutic approaches targeting the hypoxic cells is one of the most crucial challenges for cancer regression. Screening potential candidates for effective diagnostic modalities even under a hypoxic environment would be an important first step. In this study, we describe the development of a real-time imaging system to monitor hypoxic cell apoptosis for such screening. The imaging system is composed of a cyclic luciferase (luc) gene under the control of an improved hypoxic-responsive promoter. The cyclic luc gene product works as a caspase-3 (cas-3) monitor as it gains luc activity in response to cas-3 activation. The promoter composed of six hypoxic responsible elements and the CMV IE1 core promoter drives the effective expression of the cyclic luc gene in hypoxic conditions, enhancing hypoxic cell apoptosis visualization. We also confirmed real-time imaging of hypoxic cell apoptosis in the spheroid, which shares properties with the tumor. Thus, this constructed system could be a powerful tool for the development of effective anticancer diagnostic modalities.

Effect of platinum nanoparticles on cell death induced by ultrasound in human lymphoma U937 cells.

In this study, we report on the potential use of platinum nanoparticles (Pt-NPs), a superoxide dismutase (SOD)/catalase mimetic antioxidant, in combination with 1MHz ultrasound (US) at an intensity of 0.4 W/cm(2), 10% duty factor, 100 Hz PRF, for 2 min. Apoptosis induction was assessed by DNA fragmentation assay, cell cycle analysis and Annexin V-FITC/PI staining. Cell killing was confirmed by cell counting and microscopic examination. The mitochondrial and Ca(2+)-dependent pathways were investigated. Caspase-8 expression and autophagy-related proteins were detected by spectrophotometry and western blot analysis, respectively. Intracellular reactive oxygen species (ROS) elevation was detected by flow cytometry, while extracellular free radical formation was assessed by electron paramagnetic resonance spin trapping spectrometry. The results showed that Pt-NPs exerted differential effects depending on their internalization. Pt-NPs functioned as potent free radical scavengers when added immediately before sonication while pre-treatment with Pt-NPs suppressed the induction of apoptosis as well as autophagy (AP), and resulted in enhanced cell killing. Dead cells displayed the features of pyknosis. The exact mode of cell death is still unclear. In conclusion, the results indicate that US-induced AP may contribute to cell survival post sonication. To our knowledge this is the first study to discuss autophagy as a pro-survival pathway in the context of US. The combination of Pt-NPs and US might be effective in cancer eradication.

Ultrasound up-regulates expression of heme oxygenase-1 gene in endothelial cells.

PURPOSE: We found heme oxygenase-1 (HO-1) gene up-regulation in response to ultrasound stimulation in endothelial cells. This project was started to explore the mechanism since it could be applied clinically. METHODS: Quantitative real-time PCR experiments were conducted to determine the appropriate condition to up-regulate HO-1 gene expression. A promoter assay experiment and a microarray analysis were done to analyze signal transduction pathways to be activated in endothelial cells by ultrasound stimulation. RESULTS: Approximately, 33-fold up-regulation of HO-1 mRNA was observed when endothelial cells were exposed to 1-MHz ultrasound at 0.3 W/cm(2) and DF 10 % for 20 s. Data obtained from promoter analysis and microarray analysis suggested that the Nrf2/StRE signal pathway was involved in the up-regulation. In addition, ultrasound stimulation to endothelial cells changed its gene expression profile so that it could modify the anti-inflammatory, antioxidant, and vasodilatory activities in addition to pro-angiogenic and vascular reconstitution activities of endothelial cells. CONCLUSION: It was suggested that ultrasound stimulation of endothelial cells up-regulated HO-1 expression through intracellular oxidative stress, which subsequently changed the gene expression pattern so that it could modify vascular functions.

Insight into the molecular mechanism of heme oxygenase-1 induction by docosahexaenoic acid in U937 cells.

Heme oxygenase-1 (HO-1) has anti-inflammatory effects on myeloid cells in response to various stimuli. To date, little is known about whether fatty acids can affect HO-1 induction. Here, we report the induction of HO-1 by docosahexaenoic acid (DHA) and the associated molecular mechanisms in human myelomonocytic lymphoma U937 cells. When U937 cells were treated with DHA, eicosapentaenoic acid, palmitic acid or oleic acid, DHA was the most effective inducer of HO-1. The activation of AKT and glycogen synthase kinase-3ß did not significantly change after DHA treatment. However, DHA increased the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), but not of other mitogen-activated protein kinases such as p38 and JNK. The increase in HO-1 expression was significantly inhibited by U0126, an ERK1/2 inhibitor. Nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) and its binding to the HO-1 promoter significantly increased upon DHA treatment. An increase in intracellular reactive oxygen species was detected by dichlorofluorescein diacetate, but not by hydroethidium or 2-[6-(4-hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid after DHA treatment. Pretreatment with NAC dramatically inhibited the ERK1/2 activation, binding of Nrf-2 to antioxidant response elements (AREs) located in the HO-1 promoter and the induction of HO-1 by DHA. In conclusion, DHA increased HO-1 expression in U937 cells via activation of ERK1/2 and increased Nrf-2 binding to ARE in the HO-1 promoter. These findings will help develop better strategies for treating inflammatory disorders with DHA.

Selective enhancement of hypoxic cell killing by tempol-regulated suicide gene expression.

The presence of hypoxic regions within solid tumors is caused by an imbalance between cell proliferation and angiogenesis. Such regions may facilitate the onset of recurrence after radiation therapy and chemotherapy, as hypoxic cells show resistance to these treatments. We found that tempol, a nitroxide, strongly induces the accumulation of hypoxia-inducible factor (HIF)-1α, particularly under conditions of hypoxia. We, therefore, evaluated whether tempol enhances the gene expression via HIF-1α, potentially leading to various applications for cancer gene therapy targeting hypoxic cells. Consequently, following treatment with tempol under hypoxia, the luciferase (Luc) activity in the cells transfected with the plasmid containing the luc gene with the oxygen-dependent degradation domain and a promoter composed of hypoxia-responsive elements increased up to approximately 10-fold compared to that observed in cells treated identically with the exception of tempol. The plasmid constructed by replacing the luc gene with the fcy::fur fusion gene as a suicide gene, strongly induced the accumulation of the Fcy::Fur fusion protein, only when incubated in the presence of the hypoxic mimic CoCl2 and tempol. The transfected cells were successfully killed with the addition of 5-fluorocytosine to the cell culture according to the fcy::fur fusion gene expression. As similar but lesser enhancement of the Luc activity was also observed in solid tumor tissues in nude mice, this strategy may be applied for hypoxic cancer eradication.

The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference.

The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment.