LQB-118 Suppresses Migration and Invasion of Prostate Cancer Cells by Modulating the Akt/GSK3ß Pathway and MMP-9/Reck Gene Expression.

BACKGROUND/AIM: Prostate cancer (PCa) is one of the most common malignancies in adult men. LQB-118 is a pterocarpanquinone with antitumor activity toward prostate cancer cells. It inhibits cell proliferation by down-regulating cyclins D1 and B1 and up-regulating p21. However, the effects of LQB-118 on PCa cell migration are still unclear. Herein, the LQB-118 effects on PCa metastatic cell migration/invasion and its mechanism of action were evaluated. MATERIALS AND METHODS: PC3 cells were treated with LQB-118 or Paclitaxel (PTX), and cell migration (wound healing and Boyden chamber assays) and invasion (matrigel assay) were determined. The LQB-118 mechanisms were evaluated by αVßIII protein expression (flow cytometry), protein phosphorylation (Western blot), and mRNA expression (qPCR). RESULTS: LQB-118 impaired PCa cell migration and invasion, down-regulated Akt phosphorylation, and also reduced GSK3ß phosphorylation, through a FAK-independent pathway. Also, it was observed that LQB-118 controlled the invasiveness behavior by reducing matrix metalloproteinase-9 (MMP-9) and up-regulating reversion-inducing cysteine rich protein with Kazal motifs (Reck) mRNA levels. Interestingly, LQB-118 increased integrin αvßIII expression, but this effect was not related to its activation, since the cell adhesion ability was reduced after LQB-118 treatment. CONCLUSION: These data highlight novel LQB-118 mechanisms in prostate cancer cells. LQB-118 acts as a negative regulator of the Akt/GSK3 signaling pathway and can modulate PCa cell proliferation, death, and migration/invasion. The results also support the use of LQB-118 for the treatment of metastatic PCa, alone or combined with another chemotherapeutic agent, due to its demonstrated pleiotropic activities.

Curcumin mediates repulsive guidance molecule B (RGMb) in the treatment mechanism of renal fibrosis induced by unilateral ureteral obstruction.

In this study, we explored the role and mechanism of repulsive guidance molecule B (RGMb, also known as Dragon) in the protective effects of curcumin against renal fibrosis and verified Dragon's effect on renal tubular epithelial cell apoptosis and cell programmability. Unilateral ureteral obstruction (UUO) was surgically induced in rats to establish a model of renal interstitial fibrosis (RIF). The rats were then treated with curcumin. Curcumin prominently decreased the serum creatinine (SCr) and blood urea nitrogen (BUN) levels, and also improved the tubular injury in the UUO-induced rats. Curcumin significantly downregulated the TGF-ß1, P-Smad2/3, cleaved caspase-3, cleaved caspase-8 and Dragon levels. Dragon knockdown also markedly reduced the TGF-ß1, P-Smad2/3, Smad2/3, cleaved caspase-3, cleaved caspase-8, fibronectin, collagen I, collagen IV, vimentin, and α-SMA expression levels. Conversely, Dragon overexpression caused higher expression levels of these proteins, and curcumin reversed this effect. Furthermore, Dragon knockdown increased the E-cadherin levels, whereas Dragon overexpression decreased these levels. Overexpressing Dragon significantly decreased the cell viability, and curcumin reversed this effect. In conclusion, curcumin acted on Dragon and attenuated RIF in UUO rat models. Curcumin downregulated the TGF-ß1/Smad signaling pathway and inhibited Dragon and fibrogenic molecules in both rats and HK-2 cells.

Interactions between maternal fluoxetine exposure, the maternal gut microbiome and fetal neurodevelopment in mice.

Selective serotonin reuptake inhibitors (SSRIs) are the most widely used treatment by women experiencing depression during pregnancy. However, the effects of maternal SSRI use on early offspring development remain poorly understood. Recent studies suggest that SSRIs can modify the gut microbiota and interact directly with particular gut bacteria, raising the question of whether the gut microbiome impacts host responses to SSRIs. In this study, we investigate effects of prenatal SSRI exposure on fetal neurodevelopment and further evaluate potential modulatory influences of the maternal gut microbiome. We demonstrate that maternal treatment with the SSRI fluoxetine induces widespread alterations in the fetal brain transcriptome during midgestation, including increases in the expression of genes relevant to synaptic organization and neuronal signaling and decreases in the expression of genes related to DNA replication and mitosis. Notably, maternal fluoxetine treatment from E7.5 to E14.5 has no overt effects on the composition of the maternal gut microbiota. However, maternal pretreatment with antibiotics to deplete the gut microbiome substantially modifies transcriptional responses of the fetal brain to maternal fluoxetine treatment. In particular, maternal fluoxetine treatment elevates localized expression of the opioid binding protein/cell adhesion molecule like gene Opcml in the fetal thalamus and lateral ganglionic eminence, which is prevented by maternal antibiotic treatment. Together, these findings reveal that maternal fluoxetine treatment alters gene expression in the fetal brain through pathways that are impacted, at least in part, by the presence of the maternal gut microbiota.

The exosomes derived from CAR-T cell efficiently target mesothelin and reduce triple-negative breast cancer growth.

Genetically engineered T cells expressing a chimeric antigen receptor (CAR) have rapidly developed into a powerful and innovative therapeutic modality for cancer patients. However, the problem of dose-dependent systemic toxicity cannot be ignored. In this study, exosomes derived from mesothelin (MSLN)-targeted CAR-T cells were isolated, and we found that they maintain most characteristics of the parental T cells, including surface expression of the CARs and CD3. Furthermore, CAR-carrying exosomes significantly inhibited the growth of both endogenous and exogenous MSLN-positive triple-negative breast cancer (TNBC) cells. The expression of the effector molecules perforin and granzyme B may be a mechanism of tumor killing. More importantly, a highly effective tumor inhibition rate without obvious side effects was observed with the administration of CAR-T cell exosomes in vivo. Thus, the use of CAR-T cell exosomes has great therapeutic potential against MSLN-expressing TNBC.

Emerging roles for the GPI-anchored tumor suppressor OPCML in cancers.

OPCML is a highly conserved glycosyl phosphatidylinositol (GPI)-anchored protein belonging to the IgLON family of cell adhesion molecules. OPCML functions as a tumor suppressor and is silenced in over 80% of ovarian cancers by loss of heterozygosity and by epigenetic mechanisms. OPCML inactivation is also observed in many other cancers suggesting a conservation of tumor suppressor function. Although epigenetic silencing and subsequent loss of OPCML expression correlate with poor progression-free and overall patient survival, its mechanism of action is only starting to be fully elucidated. Recent discoveries have demonstrated that OPCML exerts its tumor suppressor effect by inhibiting several cancer hallmark phenotypes in vitro and abrogating tumorigenesis in vivo, by downregulating/inactivating a specific spectrum of Receptor Tyrosine Kinases (RTKs), including EphA2, FGFR1, FGFR3, HER2, HER4, and AXL. This modulation of RTKs can also sensitize ovarian and breast cancers to lapatinib, erlotinib, and anti-AXL therapies. Furthermore, OPCML has also been shown to function in synergy with the tumor suppressor phosphatase PTPRG to inactivate pro-metastatic RTKs such as AXL. Recently, the identification of inactivating point mutations and the elucidation of the crystal structure of OPCML have provided valuable insights into its structure-function relationships, giving rise to its potential as an anti-cancer therapeutic.

Single-cell analyses identify dysfunctional CD16+ CD8 T cells in smokers.

Tobacco smoke exposure contributes to the global burden of communicable and chronic diseases. To identify immune cells affected by smoking, we use single-cell RNA sequencing on peripheral blood from smokers and nonsmokers. Transcriptomes reveal a subpopulation of FCGR3A (CD16)-expressing Natural Killer (NK)-like CD8 T lymphocytes that increase in smokers. Mass cytometry confirms elevated CD16+ CD8 T cells in smokers. Inferred as highly differentiated by pseudotime analysis, NK-like CD8 T cells express markers characteristic of effector memory re-expressing CD45RA T (TEMRA) cells. Indicative of immune aging, smokers' CD8 T cells are biased toward differentiated cells and smokers have fewer naïve cells than nonsmokers. DNA methylation-based models show that smoking dose is associated with accelerated aging and decreased telomere length, a biomarker of T cell senescence. Immune aging accompanies T cell senescence, which can ultimately lead to impaired immune function. This suggests a role for smoking-induced, senescence-associated immune dysregulation in smoking-mediated pathologies.

Opposing Effects of Adenosine and Inosine in Human Subcutaneous Fibroblasts May Be Regulated by Third Party ADA Cell Providers.

Human subcutaneous fibroblasts (HSCF) challenged with inflammatory mediators release huge amounts of ATP, which rapidly generates adenosine. Given the nucleoside's putative relevance in wound healing, dermal fibrosis, and myofascial pain, we investigated the role of its precursor, AMP, and of its metabolite, inosine, in HSCF cells growth and collagen production. AMP (30 µM) was rapidly (t½ 3 ± 1 min) dephosphorylated into adenosine by CD73/ecto-5'-nucleotidase. Adenosine accumulation (t½ 158 ± 17 min) in the extracellular fluid reflected very low cellular adenosine deaminase (ADA) activity. HSCF stained positively against A2A and A3 receptors but were A1 and A2B negative. AMP and the A2A receptor agonist, CGS21680C, increased collagen production without affecting cells growth. The A2A receptor antagonist, SCH442416, prevented the effects of AMP and CGS21680C. Inosine and the A3 receptor agonist, 2Cl-IB-MECA, decreased HSCF growth and collagen production in a MRS1191-sensitive manner, implicating the A3 receptor in the anti-proliferative action of inosine. Incubation with ADA reproduced the inosine effect. In conclusion, adenosine originated from extracellular ATP hydrolysis favors normal collagen production by HSCF via A2A receptors. Inhibition of unpredicted inosine formation by third party ADA cell providers (e.g., inflammatory cells) may be a novel therapeutic target to prevent inappropriate dermal remodeling via A3 receptors activation.

Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression.

Minocycline, a tetracycline antibiotic, is known to exert vasculoprotective effects independent of its anti-bacterial properties; however the underlying molecular mechanisms are not completely understood. Reversion Inducing Cysteine Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored protein, and its overexpression inhibits cancer cell migration. We hypothesized that minocycline inhibits platelet-derived growth factor (PDGF)-induced human aortic smooth muscle cell (SMC) proliferation and migration via RECK upregulation. Our data show that the BB homodimer of recombinant PDGF (PDGF-BB) induced SMC migration and proliferation, effects significantly blunted by pre-treatment with minocycline. Further investigations revealed that PDGF-BB induced PI3K-dependent AKT activation, ERK activation, reactive oxygen species generation, Nuclear Factor-κB and Activator Protein-1 activation, microRNA (miR)-221 and miR-222 induction, RECK suppression, and matrix metalloproteinase (MMP2 and 9) activation, effects that were reversed by minocycline. Notably, minocycline induced RECK expression dose-dependently within the therapeutic dose of 1-100 µM, and silencing RECK partially reversed the inhibitory effects of minocycline on PDGF-BB-induced MMP activation, and SMC proliferation and migration. Further, targeting MMP2 and MMP9 blunted PDGF-BB-induced SMC migration. Together, these results demonstrate that minocycline inhibits PDGF-BB-induced SMC proliferation and migration by restoring RECK, an MMP inhibitor. These results indicate that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.

Engineered CAR T cells targeting mesothelin by piggyBac transposon system for the treatment of pancreatic cancer.

Patients with pancreatic cancer have a poor prognosis largely due to the poor efficacy of the available treatment modalities. In this study, we engineered mesothelin-targeting chimeric antigen receptor T cells (mesoCAR T) using the piggyBac transposon based plasmid electroporation technique for specific targeting of pancreatic cancer cells expressing mesothelin. In vitro, mesoCAR T cells exhibited rapid and robust killing effect against ASPC1 cells with high expression levels of mesothelin with high production of IFN-γ; the cytotoxic effect on PANC1 cells with low expressions of mesothelin was relatively attenuated. In the ASPC1 xenograft mice model, mesoCAR T cells significantly suppressed the tumor growth accompanied with higher-level IFN-γ secretion as compared to control T cells. Besides, more mesoCAR T cells differentiated into memory T cells after tumor remission, whilst causing minimal lesions in major organs. Our study suggests promising efficacy of piggyBac transposon-based mesoCAR T cell therapy for pancreatic cancer, which is a potential candidate for clinical translation.

Recombinant immunotoxins with albumin-binding domains have long half-lives and high antitumor activity.

Recombinant immunotoxins (RITs) are chimeric proteins consisting of a Fv that binds to a cancer cell and a portion of a protein toxin. One of these, Moxetumomab pasudotox, was shown to be effective in treating patients with some leukemias, where the cells are readily accessible to the RIT. However, their short half-life limits their efficacy in solid tumors, because penetration into the tumors is slow. Albumin and agents bound to albumin have a long half-life in the circulation. To increase the time tumor cells are exposed to RITs, we have produced and evaluated variants that contain either an albumin-binding domain (ABD) from Streptococcus or single-domain antibodies from Llama. We have inserted these ABDs into RITs targeting mesothelin, between the Fv and the furin cleavage site. We find that these proteins can be produced in large amounts, are very cytotoxic to mesothelin-expressing cancer cell lines, and have a high affinity for human or mouse serum albumin. In mice, the RIT containing an ABD from Streptococcus has a longer half-life and higher antitumor activity than the other two. Its half-life in the circulation of mice ranges from 113 to 194 min compared with 13 min for an RIT with no ABD. Cell uptake studies show the RIT enters the target cell bound to serum albumin. We conclude that RITs with improved half-lives and antitumor activity should be evaluated for the treatment of cancer in humans.

Insights into Pathomechanisms and Treatment Development in Heritable Ectopic Mineralization Disorders: Summary of the PXE International Biennial Research Symposium-2016.

Pseudoxanthoma elasticum is a prototype of heritable ectopic mineralization disorders, with phenotypic overlap with generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Recent observations have suggested that the reduced inorganic pyrophosphate/phosphate ratio is the cause of soft connective tissue mineralization in these disorders. PXE International, a patient advocacy organization, supports research in part by sponsoring biennial research symposia on these disorders; the latest meeting was held in September 2016 at Thomas Jefferson University, Philadelphia. This report summarizes the progress in pseudoxanthoma elasticum and other ectopic mineralization disorders, as presented in the symposium, with focus on translational aspects of precision medicine toward improved diagnostics and treatment development for these currently intractable disorders.

Mesothelin Immunotherapy for Cancer: Ready for Prime Time?

Mesothelin is a tumor antigen that is highly expressed in many human cancers, including malignant mesothelioma and pancreatic, ovarian, and lung adenocarcinomas. It is an attractive target for cancer immunotherapy because its normal expression is limited to mesothelial cells, which are dispensable. Several antibody-based therapeutic agents as well as vaccine and T-cell therapies directed at mesothelin are undergoing clinical evaluation. These include antimesothelin immunotoxins (SS1P, RG7787/LMB-100), chimeric antimesothelin antibody (amatuximab), mesothelin-directed antibody drug conjugates (anetumab ravtansine, DMOT4039A, BMS-986148), live attenuated Listeria monocytogenes-expressing mesothelin (CRS-207, JNJ-64041757), and chimeric antigen receptor T-cell therapies. Two antimesothelin agents are currently in multicenter clinical registration trials for malignant mesothelioma: amatuximab in the first-line setting and anetumab ravtansine as second-line therapy. Phase II randomized clinical trials of CRS-207 as a boosting agent and in combination with immune checkpoint inhibition for pancreatic cancer are nearing completion. These ongoing studies will define the utility of mesothelin immunotherapy for treating cancer.

Prospective evaluation of the molecular effects of metformin on the endometrium in women with newly diagnosed endometrial cancer: A window of opportunity study.

OBJECTIVE: Metformin reduces cancer incidence and improves overall survival in diabetic patients. In preclinical studies, metformin decreases endometrial cancer (EC) cell growth by activation of AMPK/mTOR inhibition. We sought to determine the effects of metformin on serum/tumor biomarkers in women with EC. METHODS: In this prospective trial, newly diagnosed EC patients underwent pre-treatment blood draw/endometrial biopsy, were administered oral metformin 850mg daily for ≥7days, and underwent post-treatment blood draw/definitive surgery. Pre- and post- serum analyses were performed. Tumor samples were evaluated for changes in AMPK, PI3K/AKT pathway, proliferation, and apoptosis by immunohistochemistry. RESULTS: Twenty patients completed the trial. Median age and BMI were 57years (range: 27-67) and 34.5kg/m2 (range: 21.9-50.0). Median duration of metformin was 9.5days (range: 7-24). A majority of women had endometrioid adenocarcinomas (90%) and were early stage (85%). After metformin, there were significant decreases in serum IGF-1 (p=0.046), omentin (p=0.007), insulin (p=0.012), C-peptide (p=0.018), and leptin (p=0.0035). Compared to baseline, post-treatment tissue showed decreased phospho-AKT in 18/20 patients (90%, p=0.0002), decreased phospho-S6rp in 14/20 patients (70%, p=0.057), and decreased phospho-p44/42MAPK in 15/18 patients (83.3%, p=0.0038). There was no difference in Ki67, phospho-ACC, or caspase 3. Changes did not correlate with BMI, grade, or KRAS mutation. CONCLUSION: In this prospective window of opportunity study, we demonstrated that relevant serum and molecular changes occur in patients with newly diagnosed EC after a short course of metformin. Ongoing clinical trials will help determine the appropriate role for metformin in the treatment of women with EC.

Cytoprotective effects of melatonin on zoledronic acid-treated human mesenchymal stem cells in vitro.

OBJECTIVE: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a common clinical complication in patients receiving bisphosphonate therapy. Furthermore, melatonin has been proposed as a therapeutic drug for the oral cavity due to its antioxidant properties. This study aimed to evaluate the cytoprotective effects of melatonin on zoledronic acid (ZA)-treated human mesenchymal stem cells from periodontal ligament (PDLSCs) and bone marrow (BMMSCs). METHODS: PDLSCs and BMMSCs were exposed to ZA, melatonin or ZA + melatonin for 72 h. Cell proliferation was measured by a colorimetric assay, whereas their mesenchymal phenotype was analyzed by flow cytometry. RESULTS: Proliferation assays showed that BMMSCs presented higher ZA resistance than PDLSCs, as well as a difference in response to the simultaneous treatment of ZA + melatonin. Using PDLSCs, high doses of melatonin significantly increased their proliferation, whereas lower concentrations were enough to enhance ZA-treated BMMSC proliferation. Moreover, PDLSCs displayed a CD90/CD105 downregulation and CD73 upregulation in response to ZA, which was more pronounced in response to melatonin. Furthermore, ZA or ZA + low doses of melatonin induced a decrease of expression of CD90/CD105/CD73 on BMMSCs, while a higher concentration recovered CD73 levels. CONCLUSION: These results suggest that melatonin has a cytoprotective effect on ZA-treated PDLSCs and BMMSCs. Thus, it could be used for BRONJ prevention.

Effects of 5-aza-2'deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma.

Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2'deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECK gene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.

Omentin-1 levels are reduced by pharmacologic doses of leptin, but remain unaffected by energy deprivation and display no day-night variation.

OBJECTIVE: To study the day-night variation of omentin-1 levels and assess whether leptin and/or short- and long-term energy deprivation alter circulating omentin-1 levels via cytokines. DESIGN AND METHODS: Omentin-1 levels were measured hourly in serum samples from six healthy men to evaluate for day-night variation. To study effects of acute energy deprivation and of leptin administration, eight healthy subjects were studied in the fasting state for 72 h with administration of either placebo or metreleptin (recombinant human leptin) in physiologic replacement doses. We evaluated the effect of leptin in pharmacologic doses on serum omentin-1 and cytokine levels, as well as on omentin-1 levels in ex vivo omental adipose tissue, in 15 healthy volunteers. To study the effect of chronic energy deprivation and weight loss on omentin-1 levels, we followed 18 obese subjects for 12 months who underwent bariatric surgery. RESULTS: There is no day-night variation in omentin-1 levels. Short-term and chronic energy deprivation, as well as ex vivo leptin administration and physiologic replacement doses of leptin, do not alter omentin-1 levels; pharmacologic doses of metreleptin reduce omentin-1 levels, whereas levels of tumor necrosis factor-α receptor II and interleukin-6 tend to increase. CONCLUSIONS: Omentin-1 levels are reduced by pharmacologic doses of metreleptin independent of effects on cytokine levels.

Differential TNF production by monocyte subsets under physical stress: blunted mobilization of proinflammatory monocytes in prehypertensive individuals.

Elevated blood pressure (BP) and infiltration of the vasculature by monocytes contribute to vascular pathology; but, monocyte migratory characteristics based on differing inflammatory potential under adrenergic activation remains unclear. We compared nonclassical (CD14(+)CD16(++); HLA-DR(+)), intermediate (CD14(++)CD16(+); HLA-DR(++)), and classical (CD14(++)CD16(-); HLA-DR(+/-)) monocyte trafficking and their LPS-stimulated TNF production in response to a physical stressor (20-min treadmill exercise at 65-70% VO(2peak)) in participants with high prehypertension (PHT), mild PHT or normal BP (NBP). To determine adrenergic receptor (AR) sensitivity, pre-exercise cells were also treated with isoproterenol (Iso). When cells were stimulated with LPS, the CD16 molecules were downregulated, and monocyte subsets were differentiated based on HLA-DR expression. Monocyte subpopulations (as % of total monocytes) and intracellular TNF production were evaluated by flow cytometry. TNF production in all subsets decreased post-exercise and with ex-vivo incubation with Iso, irrespective of BP (p<0.001), with nonclassical and intermediate monocytes being a major source of TNF production. Overall, % nonclassical monocytes increased, % intermediate did not change, whereas % classical decreased post-exercise (p<0.001). However, % increase in nonclassical monocytes under exercise-induced adrenergic activation was blunted in high PHT individuals (p<0.05), but not in individuals with mild PHT and NBP. These findings extend our previous reports by showing that the mobilization of proinflammatory monocytes under physical stress is attenuated with even mild BP elevation. This may be indicative of monocytic AR desensitization and/or greater adhesion of "proinflammatory" monocytes to the vascular endothelium in hypertension with potential clinical implications of vascular pathology.

Treatment with a coinducer of the heat shock response delays muscle denervation in the SOD1-G93A mouse model of amyotrophic lateral sclerosis.

We undertook a longitudinal study of the histological and biochemical changes at the neuromuscular junction (NMJ) in muscles of SOD1-G93A mice. We also assessed these functions in mice treated with a known heat shock protein inducer, arimoclomol. Tissue samples of treated and untreated mSOD mice were analysed for AChE and ChAT enzyme activities as markers of neuromuscular function. Sections of hindlimb muscles (TA, EDL and soleus) were also stained for succinate dehydrogenase and silver cholinesterase activities as well as for immunohistochemistry. Hsp70 levels were also measured from muscle samples using ELISA. Results showed that denervation and nerve sprouting were present at symptom onset in fast muscles, although slow muscles remained fully innervated. Cholinergic enzyme activities were reduced prior to denervation and declined further with disease progression. Reduction of endplate size, a slow to fast shift in muscle phenotype was also observed. Treatment with arimoclomol delayed the appearance of these changes, increased innervation, cholinergic enzyme activities and endplate size and reversed muscle fibre transformation. These beneficial effects of arimoclomol in muscles were accompanied by an increase in Hsp70 expression. In conclusion, our results indicate that pharmacological targeting of muscles at early stages of disease may be a successful strategy to ameliorate disease progression in ALS.

A novel DNA/peptide combined vaccine induces PSCA-specific cytotoxic T-lymphocyte responses and suppresses tumor growth in experimental prostate cancer.

OBJECTIVE: To develop a completely novel DNA peptide-combined vaccine and determine whether it can efficiently improve tumor-specific cytotoxic T lymphocyte (CTL) responses and inhibit tumor progression in experimental prostate cancer models. METHODS: The DNA/peptide combined vaccine was prepared by the self-assembly of a cationic peptide ([K]18P9) containing 18 lysines and a CTL epitope peptide, prostate stem cell antigen (PSCA (14-22)) (HLA-A2 restricted) with a recombinant plasmid encoding human full-length PSCA gene (pcDNA3.1(+)-PSCA) through electrostatic interactions. The formation of a DNA/peptide complex was examined by DNA retardation assay, DNase I protection assay, and transmission electron microscopy. The efficacy of vaccination using this complex was demonstrated in terms of the PSCA-specific CTL activity and antitumor immunity to PSCA(+) tumors in a murine model. RESULTS: This form of DNA/peptide complex could efficiently transfer the plasmid encoding full-length PSCA gene into mammalian cells and induced potent CTLs cytotoxicity against a human prostate carcinoma cell line established from the left supraclavicular lymph node metastasis from a 50-year-old man with prostate carcinoma in 1977. Expressing PSCA compared with pcDNA3.1(+)-PSCA, [K]18P9 peptide, or pcDNA3.1(+). Moreover, the vaccination of mice with this complex induced a potent antitumor immunity to prostate carcinomas in a xenograft tumor model in nude mice. CONCLUSION: This study suggests that a specific antitumor immune response can be induced by this DNA/peptide combined vaccine, which represents a new strategy for use in the immunotherapy of prostate cancer.