Overcoming multidrug resistance in cancer: Recent progress in nanotechnology and new horizons.

Multidrug resistance (MDR), defined as the ability of cancer cells to gain resistance to both conventional and novel chemotherapy agents, is an important barrier in treating malignancies. Initially, it was discovered that cellular pumps dependent on ATP were the cause of resistance to chemotherapy, and further studies have found that other mechanisms such as increased metabolism of drugs, decreased drug entry, and defective apoptotic pathways are involved in this process. MDR has been the focus of numerous initiatives and countless studies have been undertaken to better understand MDR and formulate strategies to overcome its effects. The current review highlights various nano-drug delivery systems including polymeric/solid lipid/mesoporous silica/metal nanoparticles, dendrimers, liposomes, micelles, and nanostructured lipid carriers to overcome the mechanism of MDR. Nanoparticles are novel gateways to enhance the therapeutic efficacy of anticancer agents at the target site of action due to their tumor-targeting abilities, which can limit the unwanted systemic effects of chemotherapy agents and also reduce drug resistance. Additionally, other innovative strategies including RNA interference as a biological process used to inhibit or silence specific gene expression, natural products as MDR modulators with little systemic toxic effects, which interfere with the functions of proteins involved in drug efflux, and physical approaches such as combination of conventional drug administration with thermal/ultrasound/photodynamic strategies are also highlighted.

Involvement of connexin43 in the acute hyperosmotic stimulus­induced synthesis and release of vasopressin in the supraoptic nucleus of rats.

There is evidence that astroglial connexin43 (Cx43) in the supraoptic nucleus (SON) is required for the hyperosmolarity­induced increase in Fos protein expression in magnocellular neurosecretory cells (MNCs). In the present study, the role of astroglial Cx43 in the synthesis and release of vasopressin (VP) by MNCs in the SON subjected to hyperosmotic stimulus was examined. The results revealed that the VP levels in the SON and plasma were increased following acute hyperosmotic stimulus. Treatment of MNCs with Cx43­specific antisense oligodeoxynucleotides (ASODN), which temporarily reduced Cx43 protein production, limited the VP synthesis and release induced by a hyperosmotic stimulus. Similarly, the addition of gap junction and Cx43 hemichannel blockers also attenuated the VP synthesis and release induced by an acute hyperosmotic stimulus. A high extracellular [Ca2+]([Ca2+]o) has been demonstrated to reduce the gap junction activity or opening probability of Cx54 hemichannels. Notably, it was identified that high [Ca2+]o attenuated the VP synthesis and release induced by acute hyperosmotic stimulus, while low [Ca2+]o had a weak or no effect. These results suggested that Cx43 participates in the VP synthesis and release induced by hyperosmotic stimulation in the SON.

A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition.

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time-consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co-introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic-resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high-throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant-specific DNA herbicides.

Antisense oligodeoxynucleotide inhibition as an alternative and convenient method for gene function analysis in pollen tubes.

Antisense oligodeoxynucleotide (A-ODN) inhibition works well in animal cells. However, there have been few successful examples to date of its application in plants, and more specifically whether the technique can be used in pollen tubes as a model of plant cell growth. NtGNL1 plays an important role in pollen tube development and was thus selected as an indicator to assess the biological effects of A-ODN. An A-ODN inhibition technique was used to down-regulate NtGNL1 expression in tobacco pollen tubes and showed that A-ODNs could quickly enter pollen tubes through the thick wall and cell membrane and effectively block NtGNL1 expression. Phenotype analysis revealed that the down-regulation of NtGNL1 by A-ODNs resulted in abnormalities in endocytosis and subsequent vesicle trafficking, similar to the phenotypes of pollen tubes treated with NtGNL1 RNAi. This investigation confirmed that A-ODNs could specifically inhibit target gene expression, and furthermore demonstrated that A-ODN functioned in a concentration- and duration-dependent manner, because A-ODNs could be degraded when incubated with pollen tubes. Thus, the A-ODN technique was successfully used for gene function analysis in pollen tubes and appears to be an alternative and convenient technique when the in vitro pollen tube is used as the study model. This technique will greatly facilitate investigations on the molecular mechanism(s) underlying pollen tube growth.

Effect of antisense oligodeoxynucleotide targeted against NF-κB/P65 on cell proliferation and tumorigenesis of gastric cancer.

To study the inhibitory effect of nuclear transcription factor-kappa B(NF-κB) antisense oligodeoxynucleotide(ASODN) on the growth and tumorgenesis of human gastric cancer. We synthesized and transfected the ASODN of NF-κB/P65 to gastric cancer cell line. The effect of ASODN of NF-κB/P65 on the proliferation of gastric cancer cells was measured by MTT method. The subcutaneous xenograft model of human gastric cancer was established in nude mice, and the tumor growth curve was observed. The cell proliferation was significantly inhibited in P65 ASODN-transfected group in vitro (P<0.05). In vivo, tumor formation test showed that the tumor volume in nude mice in ASODN group was obviously smaller than in other groups (P<0.05); the apoptosis index (AI) was significantly higher (P<0.001). Simultaneously, MVD in ASODN group was markedly lower than in other groups (P<0.01). NF-κB could be used as a new biological therapeutic target of gastric cancer.

Static micromixer-coaxial electrospray synthesis of theranostic lipoplexes.

Theranostic lipoplexes are an integrated nanotherapeutic system with diagnostic imaging capability and therapeutic functions. They hold great promise to improve current cancer treatments; however, producing uniform theranostic lipoplexes with multiple components in a reproducible manner is a highly challenging task. Conventional methods, such as bulk mixing, are not able to achieve this goal because of their macroscale and random nature. Here we report a novel technique, called the static micromixer-coaxial electrospray (MCE), to synthesize theranostic lipoplexes in a single step with high reproducibility. In this work, quantum dots (QD605) and Cy5-labeled antisense oligodeoxynucleotides (Cy5-G3139) were chosen as the model imaging reagent and therapeutic drug, respectively. Compared with bulk mixing, QD605/Cy5-G3139-loaded lipoplexes produced by MCE were highly uniform with polydispersity of 0.024 ± 0.006 and mean diameter by volume of 194 ± 15 nm. MCE also showed higher encapsulation efficiency of QD605 and Cy5-G3139. QD605 and Cy5 also formed the Förster resonance energy transfer pair, and thus the cellular uptake and intracellular fate of theranostic lipoplexes could be visualized by flow cytometry and confocal microscopy. The lipoplexes were efficiently delivered to A549 cells (non-small cell lung cancer cell line) and down-regulated the Bcl-2 gene expression by 48 ± 6%.

Suppression of microRNA-96 expression inhibits the invasion of hepatocellular carcinoma cells.

MicroRNA-96 (miR-96) expression is dysregulated in certain types of cancers. However, the role of miR-96 in hepatocellular carcinoma (HCC) invasion and metastasis remains elusive. miR-96 expression was investigated in a number of HCC cell lines by quantitative reverse transcription­polymerase chain reaction (RT-PCR). Cell invasive ability of metastatic HCCLM6 cells transfected with anti-miR-96 oligonucleotides or miR-96 mimic was determined by Matrigel invasion assay in vitro. In addition, metastasis-associated protein, osteopontin, was evaluated by Western blotting. miR-96 expression was significantly increased in highly metastatic HCCLM6 cells. Decreasing miR-96 expression with anti-miR-96 oligonucleotides led to reduced migration and invasion of HCCLM6 cells in vitro. In particular, down-regulation of miR-96 decreased osteopontin expression in HCCLM6 cells. Suppression of miR-96 expression inhibits the invasion of HCC cells, suggesting that miR-96 may be a therapeutic target for inhibiting HCC invasion and metastasis.

Antisense oligodeoxynucleotide targeting HER2 mRNA sensitized docetaxel in breast cancer treatment.

CONTEXT: Human epidermal growth factor receptor 2 (HER2) is one of the oncogenes closely associated with the development and prognosis of breast carcinoma. Down-regulation of HER2 mRNA by antisense oligodeoxynucleotide (ASO) HER2 has been suggested to be a feasible treatment for patients with breast carcinoma. OBJECTIVE: The antitumor effects of ASO HA6722 were investigated in vitro and in vivo. MATERIALS AND METHODS: In this study, SK-BR-3, a HER2-overexpressing breast carcinoma cell line, was used as the model for in vitro experiments. Inhibitory effects of the ASO HA6722 were detected by methyl-thiazoldiphenyl tetrazolium (MTT) assay. Meanwhile, HER2 mRNA levels were monitored by reverse transcription polymerase chain reaction (RT-PCR). The in vivo antitumor effects were evaluated in nude mice xenograft model. RESULTS: Our results showed that HA6722 alone could inhibit the growth of SK-BR-3 cells in a dose-dependent manner with the IC(50) value of 41.8 ± 8.1 nM. In addition, the antitumor effect of docetaxel (TXT) could be sensitized by low dose of HA6722 both in vitro and in vivo, suggesting that ASO HA6722 could inhibit the growth of breast cancer cells and enhance the cytotoxic effects of TXT. DISCUSSION AND CONCLUSION: The combination treatment of TXT and HA6722 could be a more effective approach for breast cancer treatment. The future study should focus on the antitumor effect in other models.

Uptake and intracellular fate of multifunctional nanoparticles: a comparison between lipoplexes and polyplexes via quantum dot mediated Förster resonance energy transfer.

Lipoplexes and polyplexes represent the two major nanocarrier systems for nucleic acid delivery. Previous studies examining their uptake and intracellular unpacking rely on organic fluorophores fraught with low signal intensity and photobleaching. In this work quantum dot mediated Förster resonance energy transfer (QD-FRET) was first used to study and compare the cellular uptake and the intracellular fate of oligodeoxynucelotide (ODN)-based lipoplexes and polyplexes. QD605-amine and Cy5-labeled ODN (Cy5-GTI2040) were chosen as the FRET pair. By adjusting the lipid/ODN ratio of lipoplexes and the nitrogen/phosphate (N/P) ratio of polyplexes, lipoplexes and polyplexes with comparable physical properties were produced. The biological activities of dual-labeled lipoplexes and polyplexes remained unaltered compared to their unlabeled counterparts as evidenced by their comparable antisense activities against protein R2 in KB cells. Flow cytometry and confocal microscopy revealed similar pattern of uptake for these two types of nanoparticles, although polyplexes had a higher dissociation rate than lipoplexes in KB cells. We demonstrate that QD-FRET is a sensitive tool to study the uptake and intracellular unpacking of lipoplexes and polyplexes, which may help optimize their formulations for various theranostics applications.

IL-6 antisense-mediated growth inhibition in a head and neck squamous cell carcinoma cell line.

The growth of tumor cells can be regulated by a variety of cytokines. To investigate the pathogenesis of head and neck cancer and explore a new therapeutic approach for the carcinoma, the role of interleukin-6 (IL-6) in the growth of a human head and neck squamous cell carcinoma (HNSCC) cell line was examined. Whether or not IL-6 is increased in HNSCC and whether or not IL-6 antisense oligonucleotide treatment could decrease proliferation and angiogenic activity of HNSCC cell lines, was determined. Established human HNSCC cell lines were screened for IL-6 expression at both mRNA and protein levels. By using a 15-mer antisense phosphorothioate oligonucleotide targeting a sequence in the second exon of the IL-6 gene, modulation of IL-6 and vascular endothelial growth factor (VEGF) expression was examined in UMSCC IIA in cell supernatants by capture enzyme-linked immunosorbent assay (ELISA), and in cell lysates by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, cell growth was determined by cell count. Endothelial cell migration was measured using a modified Boyden chamber. IL-6 was identified in the supernatant of the cell culture medium, indicating that these cells secreted IL-6, and the mRNAs of IL-6 were shown to be present in the cell lysates. IL-6 antisense oligonucleotide treatment resulted in a significant reduction of IL-6 protein expression compared to the sense control. The antisense oligonucleotides targeting IL-6 mRNA, also, inhibited cell growth and IL-6 production as well as VEGF expression. The addition of conditioned medium from IL-6 antisense-treated tumor cells resulted in decreased endothelial cell migration and tubule formation. Taken together, these findings indicate that endogenous IL-6 plays an important role in the growth of HNSCC and exerts its action by an autocrine growth mechanism, and that therapeutic trials with antisense oligonucleotides targeted to IL-6 mRNA may have some value for the treatment of HNSCC due to a decrease of neovascularization.

An antisense CAG repeat transcript at JPH3 locus mediates expanded polyglutamine protein toxicity in Huntington's disease-like 2 mice.

Huntington's disease-like-2 (HDL2) is a phenocopy of Huntington's disease caused by CTG/CAG repeat expansion at the Junctophilin-3 (JPH3) locus. The mechanisms underlying HDL2 pathogenesis remain unclear. Here we developed a BAC transgenic mouse model of HDL2 (BAC-HDL2) that exhibits progressive motor deficits, selective neurodegenerative pathology, and ubiquitin-positive nuclear inclusions (NIs). Molecular analyses reveal a promoter at the transgene locus driving the expression of a CAG repeat transcript (HDL2-CAG) from the strand antisense to JPH3, which encodes an expanded polyglutamine (polyQ) protein. Importantly, BAC-HDL2 mice, but not control BAC mice, accumulate polyQ-containing NIs in a pattern strikingly similar to those in the patients. Furthermore, BAC mice with genetic silencing of the expanded CUG transcript still express HDL2-CAG transcript and manifest polyQ pathogenesis. Finally, studies of HDL2 mice and patients revealed CBP sequestration into NIs and evidence for interference of CBP-mediated transcriptional activation. These results suggest overlapping polyQ-mediated pathogenic mechanisms in HD and HDL2.

Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivo tumor growth.

INTRODUCTION: MicroRNAs (miRNAs) are a class of small non-coding RNAs (20 to 24 nucleotides) that post-transcriptionally modulate gene expression. A key oncomir in carcinogenesis is miR-21, which is consistently up-regulated in a wide range of cancers. However, few functional studies are available for miR-21, and few targets have been identified. In this study, we explored the role of miR-21 in human breast cancer cells and tissues, and searched for miR-21 targets. METHODS: We used in vitro and in vivo assays to explore the role of miR-21 in the malignant progression of human breast cancer, using miR-21 knockdown. Using LNA silencing combined to microarray technology and target prediction, we screened for potential targets of miR-21 and validated direct targets by using luciferase reporter assay and Western blot. Two candidate target genes (EIF4A2 and ANKRD46) were selected for analysis of correlation with clinicopathological characteristics and prognosis using immunohistochemistry on cancer tissue microrrays. RESULTS: Anti-miR-21 inhibited growth and migration of MCF-7 and MDA-MB-231 cells in vitro, and tumor growth in nude mice. Knockdown of miR-21 significantly increased the expression of ANKRD46 at both mRNA and protein levels. Luciferase assays using a reporter carrying a putative target site in the 3' untranslated region of ANKRD46 revealed that miR-21 directly targeted ANKRD46. miR-21 and EIF4A2 protein were inversely expressed in breast cancers (rs = -0.283, P = 0.005, Spearman's correlation analysis). CONCLUSIONS: Knockdown of miR-21 in MCF-7 and MDA-MB-231 cells inhibits in vitro and in vivo growth as well as in vitro migration. ANKRD46 is newly identified as a direct target of miR-21 in BC. These results suggest that inhibitory strategies against miR-21 using peptide nucleic acids (PNAs)-antimiR-21 may provide potential therapeutic applications in breast cancer treatment.

Therapeutic potential of antisense oligodeoxynucleotides in downregulating p53 oncogenic mutations in cancers.

PURPOSE OF WORK: mutation of the p53 gene is the most common genetic alteration in human cancers. Our study proposes to rationally design a p53 antisense oligonucleotide (ASO) repository, which contains a series of ASOs containing single nucleotide differences to discriminate between each mutant and wild type (WT) p53. The Sfold software was used to predict target-accessibility and we designed an initial series of antisense oligonucleotides (ASO) that target the p53 mutants A161T, R175H and R249S. Western-blot analysis indicated that ASOs strongly inhibited the expression of p53 mutants in a panel of human tumor cell lines (SNU-449, SK-BR-3 and PLC/PRF/5) while having little effect on the expression of WT p53 (HepG2 cells). In three cancer lines harboring each of the p53 mutations, mutant-specific ASO treatment led to a dose-dependent inhibition of cell growth, cell viability, colony formation and invasion, and expression of mutant p53-dependent survival proteins. Our preliminary results indicate that a single nucleotide difference in ASOs can discriminate between mutant and WT p53. These observations support the hypothesis that a p53 ASO repository can be a potentially valuable tool to knock down oncogenic mutant p53 and warrant the testing of a p53 ASO repository in in vivo settings.

Role of midbrain periaqueductal gray P2X3 receptors in electroacupuncture-mediated endogenous pain modulatory systems.

Extracellular ATP facilitates pain transmission at peripheral and spinal sites via the P2X receptors and the P2X3 subtype is an important candidate for this effect. Electroacupuncture (EA) has been clinically utilized to manage chronic pain. In this study, with neuropathic pain model of Sprague-Dawley (SD) rats, the P2X3 receptor protein level and expression location in the midbrain periaqueductal gray (PAG), a crucial site in endogenous pain modulatory system, were evaluated by Western blotting and immunohistochemistry. The results showed (1) pain thresholds were decreased while P2X3 receptor expression was up-regulated in the lateral PAG (lPAG) when neuropathic pain occurred. When the lPAG was pretreated with P2X3 receptors, antagonist A-317491 attenuated the antinociceptive effect produced by intra-lPAG injection of alpha,beta-methylene-ATP (alpha, beta-meATP), an agonist for P2X3 receptor. (2) Multiple EA treatments begot enhanced pain thresholds and increased P2X3 receptor immunoreactivity in the lPAG in neuropathic pain rats. Conversely, the down-regulated P2X3 receptor expression in the lPAG with antisense oligodeoxynucleotide (ODN) for P2X3 gene significantly attenuated the antinociceptive effect of EA treatment. These results suggest that P2X3 receptors in the lPAG play an inhibitory role in pain modulation and EA exerts a marked therapeutic effect in relieving neuropathic pain in CCI rats, which may be related to its regulative effect on the expression of P2X3 receptors in the lPAG. In conclusion, P2X3 receptors in the lPAG are involved in the supraspinal antiociception effect of EA treatment.

Repetitive hypoxic preconditioning attenuates renal ischemia/reperfusion induced oxidative injury via upregulating HIF-1 alpha-dependent bcl-2 signaling.

BACKGROUND: In response to ischemic/hypoxic preconditioning, tissues/organs exhibit protective responses to subsequent and severe ischemic stress. We hypothesized that repetitive hypoxic preconditioning (RHP) may provide long-lasting protection than single preconditioning against ischemia/reperfusion injury in rat kidneys through hypoxia-induced factor (HIF)-1-dependent pathway. METHODS: For RHP induction, female Wistar rats were subjected to intermittent hypoxic exposure (380 Torr) 15 hr/day for 28 days. RESULTS: RHP increased renal HIF-1 alpha mRNA and protein expression and triggered HIF-1 alpha-dependent renal Bcl-2 protein expression in a time-dependent manner. When returning to normoxia, increased RHP exposure prolonged renal Bcl-2 expression. Forty-five minutes of renal ischemia with 4 hr of reperfusion enhanced O2- levels and proapoptotic mechanisms, including enhanced cytosolic Bax translocation to mitochondria, release of cytochrome c to cytosol, activation of caspase 3, poly-(ADP-ribose)-polymerase fragments, tubular apoptosis, blood urea nitrogen, and creatinine level. RHP treatment depressed renal O2- production, mitochondrial Bax translocation and cytochrome c release, and tubular apoptosis. In the primary tubular cultures from RHP-treated kidneys, antisense oligodeoxyribonucleotides of bcl-2 abrogated this protection. CONCLUSIONS: RHP activates an HIF-1 alpha-dependent signaling cascade leading to an increase in Bcl-2 protein expression, an inhibition in cytosolic Bax and mitochondrial cytochrome c translocation, and a hypoxic/ischemia tolerance against renal ischemia/reperfusion injury.

Egr-1, a central and unifying role in cardioprotection from ischemia-reperfusion injury?

AIMS: Our previous studies have shown that N-n-butyl haloperidol iodide (F(2)) can antagonize myocardial ischemia/reperfusion (I/R) injury by blocking intracellular Ca(2+) overload and suppressing Egr-1 overexpression. The present study is to investigate the relation between the reduction of Ca(2+) overload and the inhibition of Egr-1 overexpression. METHODS: The Sprague-Dawley rat myocardial I/R model and cultured cardiomyocyte hypoxia-reoxygenation (H/R) model were established. Administration of Egr-1 antisense oligodeoxyribonucleotide (AS-ODN) only or combining with F(2), Egr-1 protein expression was examined by Western-blot analyses. Hemodynamic parameters, creatine kinase (CK) and lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA), myeloperoxidase (MPO), cardiac troponin I (cTnI), and tumor necrosis factor-alpha (TNF-alpha) were measured to assess the degree of injury and inflammation of myocardial tissues and cells. RESULTS: Treatment with Egr-1 AS-ODN significantly reduced Egr-1 protein expression and attenuated injury and inflammation of myocardium caused by I/R or H/R evidenced by the amelioration of hemodynamics, the decrease in leakage of CK, LDH, cTnI, the increase in MDA generation, the decrease in SOD activity, the reduction of MPO activity in myocardial tissues and release of TNF-alpha from cultured cardiomyocytes. Treatment with F(2) combined with Egr-1 AS-ODN, the inhibition of Egr-1 protein expression and inflammation (MPO activity and TNF-alpha level) were not enhanced, but the protection from myocardial I/R (or H/R) injury was significantly increased in hemodynamics and cytomembrane permeability relative to the using of Egr-1 AS-ODN only. CONCLUSION: These data suggest that the inhibition of Egr-1 overexpression cannot involve all mechanisms of cardioprotection from I/R injury.

Stimulation of the immune system by therapeutic antisense oligodeoxynucleotides and small interfering RNAs via nucleic acid receptors.

Most of the therapeutic applications for synthetic oligodeoxynucleotides (ODN) and oligoribonucleotides (ORN) relate to mechanisms of manipulating gene expression based on Watson-Crick base pairing to endogenous nucleic acids. However, in recent years it has become apparent that the immune system has evolved defense mechanisms against infections that are based on the detection of infecting viral and bacterial nucleic acids. In some cases, synthetic ODN and ORN can trigger these defenses and, therefore, can interfere with or distort the mechanism of action of antisense ODN or small interfering RNAs.

TRPC1 and TRPC6 channels cooperate with TRPV4 to mediate mechanical hyperalgesia and nociceptor sensitization.

The transient receptor potential vanilloid 4 (TRPV4) contributes to mechanical hyperalgesia of diverse etiologies, presumably as part of a mechanoreceptor signaling complex (Alessandri-Haber et al., 2008). To investigate the hypothesis that a functional interaction between TRPV4 and stretch-activated ion channels (SACs) is involved in this mechanical transduction mechanism, we used a selective SACs inhibitor, GsMTx-4. Intradermal injection of GsMTx-4 in the rat hindpaw reversed the mechanical hyperalgesia induced by intradermal injection of inflammatory mediators. In vivo single fiber recordings showed that GsMTx-4 reversed inflammatory mediator-induced decrease in mechanical threshold in half of sensitized C-fibers. Furthermore, GsMTx-4 reduced hyperalgesia to both mechanical and hypotonic stimuli in different models of inflammatory and neuropathic pain, although it had no effect on baseline mechanical nociceptive thresholds. TRPC1 and TRPC6, two GsMTx-4-sensitive SACs, are expressed in dorsal root ganglion (DRG) neurons. Single-cell reverse transcription-PCR showed that messenger RNAs for TRPV4, TRPC1, and TRPC6 are frequently coexpressed in DRG neurons. Spinal intrathecal administration of oligodeoxynucleotides antisense to TRPC1 and TRPC6, like that to TRPV4, reversed the hyperalgesia to mechanical and hypotonic stimuli induced by inflammatory mediators without affecting baseline mechanical nociceptive threshold. However, antisense to TRPC6, but not to TRPC1, reversed the mechanical hyperalgesia induced by a thermal injury or the TRPV4-selective agonist 4alpha-PDD (4 alpha-phorbol 12,13-didecanoate). We conclude that TRPC1 and TRPC6 channels cooperate with TRPV4 channels to mediate mechanical hyperalgesia and primary afferent nociceptor sensitization, although they may have distinctive roles.

Arachidonic acid: a key molecule for astrocyte survival to peroxynitrite.

Nontoxic concentrations of peroxynitrite (ONOO(-)) nevertheless commit rat astrocytes to mitochondrial permeability transition-dependent toxicity, however prevented by a signaling response driven by arachidonic acid (ARA). The lipid messenger was released upon ONOO(-)-dependent activation of cytosolic phospholipase A(2) and its pharmacological inhibition, or knock-down, was invariably associated with a prompt apoptotic response sensitive to exogenous ARA, but insensitive to other polyunsaturated fatty acids, as eicosapentaenoic or linoleic acid. Interestingly, while microglia also used ARA to cope with ONOO(-), cerebellar granule cells were killed by the same concentrations of ONOO(-) employed in astrocyte/microglia experiments via a mechanism sensitive to inhibition of ARA release. These results collectively support the notion that resistance of glial cells to ONOO(-), a species extensively produced under neuroinflammatory conditions, is largely based on a critical survival signaling triggered by the inflammatory product ARA. In remarkable contrast with these results, the lipid messenger appears to mediate toxicity in neuronal cells.