Regulation of Expression of Hyperalgesic Priming by Estrogen Receptor α in the Rat.

Hyperalgesic priming, a sexually dimorphic model of transition to chronic pain, is expressed as prolongation of prostaglandin E2-induced hyperalgesia by the activation of an additional pathway including an autocrine mechanism at the plasma membrane. The autocrine mechanism involves the transport of cyclic adenosine monophosphate (AMP) to the extracellular space, and its conversion to AMP and adenosine, by ecto-5'phosphodiesterase and ecto-5'nucleotidase, respectively. The end product, adenosine, activates A1 receptors, producing delayed onset prolongation of prostaglandin E2 hyperalgesia. We tested the hypothesis that the previously reported, estrogen-dependent, sexual dimorphism observed in the induction of priming is present in the mechanisms involved in its expression, as a regulatory effect on ecto-5'nucleotidase by estrogen receptor α (EsRα), in female rats. In the primed paw AMP hyperalgesia was dependent on conversion to adenosine, being prevented by ecto-5'nucleotidase inhibitor α,ß-methyleneadenosine 5'-diphosphate sodium salt and A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. To investigate an interaction between EsRα and ecto-5'nucleotidase, we treated primed female rats with oligodeoxynucleotide antisense or mismatch against EsRα messenger RNA. Whereas in rats treated with antisense AMP-induced hyperalgesia was abolished, the A1 receptor agonist N6-cyclopentiladenosine still produced hyperalgesia. Thus, EsRα interacts with this autocrine pathway at the level of ecto-5'nucleotidase. These results demonstrate a sexually dimorphic mechanism for the expression of priming. PERSPECTIVE: This study presents evidence of an estrogen-dependent mechanism of expression of chronic pain in female rats, supporting the suggestion that differential targets must be considered when establishing protocols for the treatment of painful conditions in men and women.

Fluorescence Characterization of Gold Modified Liposomes with Antisense N-myc DNA Bound to the Magnetisable Particles with Encapsulated Anticancer Drugs (Doxorubicin, Ellipticine and Etoposide).

Liposome-based drug delivery systems hold great potential for cancer therapy. The aim of this study was to design a nanodevice for targeted anchoring of liposomes (with and without cholesterol) with encapsulated anticancer drugs and antisense N-myc gene oligonucleotide attached to its surface. To meet this main aim, liposomes with encapsulated doxorubicin, ellipticine and etoposide were prepared. They were further characterized by measuring their fluorescence intensity, whereas the encapsulation efficiency was estimated to be 16%. The hybridization process of individual oligonucleotides forming the nanoconstruct was investigated spectrophotometrically and electrochemically. The concentrations of ellipticine, doxorubicin and etoposide attached to the nanoconstruct in gold nanoparticle-modified liposomes were found to be 14, 5 and 2 µg·mL(-1), respectively. The study succeeded in demonstrating that liposomes are suitable for the transport of anticancer drugs and the antisense oligonucleotide, which can block the expression of the N-myc gene.

Spinal muscular atrophy: diagnosis and management in a new therapeutic era.

Spinal muscular atrophy (SMA) describes a group of disorders associated with spinal motor neuron loss. In this review we provide an update regarding the most common form of SMA, proximal or 5q-SMA, and discuss the contemporary approach to diagnosis and treatment. Electromyography and muscle biopsy features of denervation were once the basis for diagnosis, but molecular testing for homozygous deletion or mutation of the SMN1 gene allows efficient and specific diagnosis. In combination with loss of SMN1, patients retain variable numbers of copies of a second similar gene, SMN2, which produces reduced levels of the survival motor neuron (SMN) protein that are insufficient for normal motor neuron function. Despite the fact that understanding of how ubiquitous reduction of SMN protein leads to motor neuron loss remains incomplete, several promising therapeutics are now being tested in early-phase clinical trials.

[Growth inhibition effection of perlecan anti-sense cDNA on human laryngeal carcinoma xnograft in nude mice].

OBJECTIVE: To observe growth inhibition effect of perlecan anti-sense cDNA (pAP) on human laryngeal carcinoma xnografted in nude mice. To vertify its antitumor effect and mechanism in vivo, and it may be useful as a biomarker in carcinoma of larynx cancer. METHOD: Created the model of human laryngeal carcinoma xnograft in nude mice. To observe growth of those xnografts in nude mice and draw growth curve of xnografted. The expression of perlecan mRNA and portein in xnografts were examined by RT-PCR and immunohistochemistry. RESULT: Volume of xnografts in the group transfected by the plasmids of pAP were significant small as compared with other two groups made by the wild type cells and phpApr-neol cells (P < 0.05). It was showed that the expression of perlecan mRNA and protein were significantly reduced in the tumor of pAP transfected Hep-2 cells as compared with the tumors transfected by the wild type cells and phßApr-neol cells (P < 0.01). CONCLUSION: These data raise the possibility that pAP many play key roles in the growth of those xnografts in nude mice.

Development and antitumor activity of a BCL-2 targeted single-stranded DNA oligonucleotide.

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

Overview of the role of macrophage migration inhibitory factor (MIF) in inflammatory bowel disease.

An array of investigations has revealed that macrophage migration inhibitory factor (MIF) plays an important role in the exacerbation of a wide range of inflammatory diseases. For the past two decades, we have extensively studied MIF's pathophysiological roles in human diseases, and have accumulated evidence elucidating its molecular mechanisms in the pathogenesis of immune disorders and inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel diseases (IBD). In a study of IBD, we demonstrated for the first time that anti-MIF antibody suppressed the degree of dextran-sulfate sodium (DSS)-induced colitis, indicating its potential therapeutic use for IBD patients. Following that report, a number of researchers, including us, clarified that MIF was profoundly involved in various gastrointestinal disorders, such as hepatitis and pancreatitis. We recently revealed that a MIF-deficient mouse was resistant to a challenge of DSS, and showed few clinical and pathological signs. Currently, we are developing new therapeutic approaches targeting MIF in inflammatory disorders, particularly IBD. We here overview MIF's pathophysiological function, mainly in IBD, and introduce two therapeutic approaches, anti-MIF antibody treatment and MIF-antisense therapy, via a drug delivery system using 1,3-beta glucan.

Intratumoral epidermal growth factor receptor antisense DNA therapy in head and neck cancer: first human application and potential antitumor mechanisms.

PURPOSE: Squamous cell carcinoma of the head and neck (SCCHN) is characterized by upregulation of the epidermal growth factor receptor (EGFR). We developed a novel strategy to target EGFR by using a therapeutic gene that consisted of an EGFR antisense (AS) gene sequence under U6 promoter control. A phase I clinical trial was conducted to evaluate the safety and biologic effects of EGFR AS. PATIENTS AND METHODS: Patients with advanced SCCHN who were refractory to standard therapies and who had at least one assessable and accessible lesion were enrolled. The EGFR AS dose was escalated in successive cohorts (six dose levels; 60 to 1,920 microg/injection). Patients received four weekly intratumoral EGFR AS injections. Tumor biopsies were performed before and after completion of therapy. Treatment response was assessed by tumor volume measurements (positron emission tomography/computed tomography), and levels of target proteins were assessed by immunohistochemistry. RESULTS: Seventeen assessable patients were treated. No grades 3 to 4 or dose-limiting toxicities were noted, and a maximum-tolerated dose was not reached. Five patients (29%) achieved a clinical response, which included two complete responses (CRs) and three partial responses (PRs); two additional patients had stable disease (SD) as the best response. Patients with disease control (CR + PR + SD) had tumors with higher EGFR and lower STAT3 expression at baseline compared with patients who had progressive disease (P = .0312 and P = .095, respectively). CONCLUSION: Intratumoral EGFR AS was safe and resulted in antitumor activity in patients with advanced SCCHN. Baseline levels of high EGFR and low STAT3 may be associated with antitumor effects.

Custirsen (OGX-011): a second-generation antisense inhibitor of clusterin for the treatment of cancer.

BACKGROUND: Clusterin is a stress-induced cytoprotective chaperone protein, regulated by HSF1, and functions similarly to a small heat-shock protein. Clusterin is expressed in a variety of cancers and associated with broad-spectrum treatment resistance. Custirsen (OGX-011) is a 2'-methoxyethyl modified phosphorothioate antisense oligonucleotide that is complementary to clusterin mRNA; it is currently in clinical trials for patients with cancer. OBJECTIVE/METHODS: To review the literature on the role of clusterin in cancer progression and treatment resistance, and to summarize completed and ongoing clinical trials with custirsen. RESULTS/CONCLUSIONS: Custirsen is well tolerated in humans and biologically active in inhibiting expression of clusterin in patients with cancer. Randomized trials of custirsen in combination with chemotherapy are planned in patients with castration-resistant prostate cancer.

Recombinant adeno-associated virus-mediated delivery of antisense angiotensin II receptor 1 gene attenuates hypertension development.

AIM: The renin-angiotensin system plays a crucial role in the development and establishment of hypertension, and the pharmacological blockade of the system results in a reduction in blood pressure. In the present study, we investigated whether the effects of a novel, double-stranded, recombinant adeno-associated virus vector (rAAV)-mediated antisense angiotensin II receptor 1 (AT1R) gene efficiently prevents the development of hypertension induced by a high-salt diet in adult, male Sprague-Dawley (SD) rats. METHODS: A rAAV was prepared with a cassette containing a cytomegalovirus promoter and partial cDNA (660 base pairs) for the AT1R inserted in the antisense direction (rAAV-AT1-AS). A single tail vein injection of the rAAV-AT1-AS or rAAV-GFP (green fluorescent protein, a reporter gene) was performed in adult, male SD rats. Two weeks after injection, the animals were fed a diet containing 8% NaCl, and the systolic blood pressure was measured weekly using the tail-cuff method for 12 weeks. RESULTS: The high-salt diet induced a significant rise in systolic blood pressure in the rAAV-GFP-treated animals; however, the rAAV-AT1-AS treatment attenuated the rise in blood pressure (142.7+/-4.5 mmHg vs 117+/-3.8 mmHg, P<0.01), and the hypotensive effect was maintained until the experiments ended at 12 weeks. In the rAAV-GFP-treated animals AT1 was overexpressed in various tissues, especially in the aorta and kidney at mRNA levels; in contrast, rAAV-AT1-AS treatment markedly attenuated AT1 expression. Furthermore, rAAV-AT1-AS treatment prevented target organ damages from hypertension, including cardiac dysfunction and renal injury compared to the rAAV-GFP group. CONCLUSION: These results suggest that rAAVmediated anti-AT1 delivery attenuates the development of hypertension and protects against renal injury and cardiac remodeling.

Specific apoptosis induction in human papillomavirus-positive cervical carcinoma cells by photodynamic antisense regulation.

Human papillomavirus type 18 (HPV18) is frequently detected in cervical cancer cells. The viral proteins E6 and E7 are expressed consistently and have oncogenic activities. The E7 protein binds to a tumor suppressor, the retinoblastoma gene product (pRB), however, leading to the stabilization of tumor suppressor, p53 protein. On the other hand, another viral product, E6, forms complexes with p53 and abrogates its function, resulting in tumor progression. These facts imply that the E6 oncogene is one of the ideal targets for directed gene therapy in HPV-positive cervical cancer. In this study, we tried photodynamic antisense regulation of the antiapoptotic E6 expression using a photocross-linking reagent, 4,5',8-trimethylpsoralen, conjugated oligo(nucleoside phosphorothioate) (Ps-S-Oligo). This photodynamic antisense strategy effectively elicited the apoptotic death of HPV18-positive cervical cancer cells through the selective repression of E6 mRNA and consequent stabilization of p53 protein. E7-mediated signals potentially activated the p53 function and mobilized the p53 pathway to deliver pro-apoptotic signals to the cancer cells, leading to the suppression of in vivo tumorigenesis. An extremely low concentration of cisplatin in addition to Ps-S-Oligos further up-regulated p53 activity, provoking massive apoptotic induction. These results suggest that the photodynamic antisense strategy has the great therapeutic potential in HPV-positive cervical cancers.

Evaluation of combination gene therapy with PTEN and antisense hTERT for malignant glioma in vitro and xenografts.

Telomerase activation is a critical event in cell immortalization, and an increase in human telomerase reverse transcriptase (hTERT) expression is the key step in activating telomerase. The phosphatase and tensin homolog (PTEN) gene encodes a double-specific phosphatase that induces cell cycle arrest, inhibits cell growth, and causes apoptotic cell death. Here, we evaluated a combined PTEN and antisense hTERT gene therapy for experimental glioma in vitro and in vivo. We demonstrated that infection with antisense-hTERT and wild-type-PTEN adenoviruses significantly inhibited human U251 glioma cell proliferation in vitro and glioma growth in a xenograft mouse model. The efficacy of therapy was obviously higher in the tumor xenografts infected with both PTEN and antisense hTERT than in the gliomas infected with either agent alone at the same total viral dose. Consistent with these results, we showed that telomerase activity and hTERT protein levels were markedly reduced in the glioma cells following adenovirus infection. In contrast, the levels of PTEN protein expression were dramatically increased in these cells. Our data indicate that combination treatment with antisense hTERT and wild-type PTEN effectively suppresses the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach in glioma gene therapy that warrants further investigation.

Novel therapies blocking the renin-angiotensin-aldosterone system in the management of hypertension and related disorders.

Although significant advances have been made in the therapeutic blockade of the renin-angiotensin-aldosterone system (RAAS) using angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers and non-selective aldosterone receptor antagonists, there is a clear need for both additional blocking strategies and enhancements of current therapeutic approaches. Vasopeptidase inhibition may still find a role despite the small incremental value of this approach and the obvious issue of kinin-mediated adverse effects still to be fully addressed. Blockade of the RAAS upstream using renin inhibitors as well as the greater selectivity of aldosterone blockade using selective aldosterone blockers such as eplerenone are also novel approaches. Not yet in clinical use but certainly an attractive therapeutic target is angiotensin II growth factor receptor transactivation, with selective inhibitors having been developed for various specific kinase pathways. Finally, ACE2 augmentation, antisense gene strategies, and vaccination against the renin-angiotensin system should still be considered experimental, but have significant appeal as additional approaches to the blockade of this system.

Long-term inhibition of the central alpha(2B)-adrenergic receptor gene via recombinant AAV-delivered antisense in hypertensive rats.

BACKGROUND: Salt-induced hypertension is mediated via the alpha(2B)-adrenergic receptor (AR) subtype. In alpha(2B)-AR gene knockout mice, blood pressure (BP) does not rise with salt loading, and in rats with salt-induced hypertension, BP decreases transiently with antisense (AS) treatment targeting the alpha(2B)-AR gene. The present experiments were designed to explore the possibility of gene transfection in the brain by intracerebroventricular (ICV) delivery of AS-DNA via adeno-associated virus (AAV) to prolong alpha(2B)-AR inhibition and hence reversal of salt-dependent hypertension. METHODS: A recombinant AAV (rAAV) vector preparation encoding the alpha(2B)-AS fragment (previously tested in vitro for inhibition of alpha(2B)-AR protein production in cells) and containing green fluorescence protein (GFP) for visualization was injected ICV into subtotally nephrectomized, salt-fed rats. Control rats received rAAV-GFP (n = 8 per group). RESULTS: We observed that BP rose from a baseline of 120 +/- 10 to 184 +/- 12 mm Hg. Injection of rAAV-alpha(2B)-AS produced a 35 +/- 12 mm Hg fall in BP, lasting without evidence of diminishing for at least 16 days, whereas rAAV-GFP-injected rats showed a continued rise in BP. Rats treated with rAAV-alpha(2B)-AS treated had a 45% to 65% decrease in alpha(2B)-AR protein levels in key regulatory regions of the brain. Neither group had signs of immunologic response to the virus injection. CONCLUSIONS: These results indicate that our construct, when given by ICV means, could reach multiple sites of the central nervous system relevant to BP regulation and could safely inhibit the central alpha(2B)-adrenergic receptor, thereby achieving prolonged reversal of salt-induced hypertension.

Antisense angiopoietin-1 inhibits tumorigenesis and angiogenesis of gastric cancer.

AIM: To investigate the effect of angiopoietin-1 (Ang-1) on biological behaviors in vitro and tumorigenesis and angiogenesis in vitro of human gastric cancer cells. METHODS: Human full-length Ang-1 gene was cloned from human placental tissues by RT-PCR method. Recombinant human Ang-1 antisense eukaryotic expression vector was constructed by directional cloning, and transfected by lipofectin method into human gastric cancer line SGC7901 with high Ang-1 expression level. Inhibition efficiency was confirmed by semi- quantitative PCR and Western blot method. Cell growth curve and cell cycle were observed with MTT assays and flow cytometry, respectively. Nude mice tumorigenicity test was employed to compare in vitro tumorigenesis of cells with Ang-1 suppression. Microvessel density (MVD) of implanted tumor tissues was analyzed by immunohistochemistry for factor VIII staining. RESULTS: Full-length Ang-1 gene was successfully cloned and stable transfectants were established, namely 7Ang1- for antisenseìand 7901P for empty vector transfected. 7Ang1- cells showed down-regulated Ang-1 expression, while its in vitro proliferation and cell cycle distribution were not significantly changed. In contrast, xenograft of 7Ang1- cells in nude mice had lower volume and weight than those of 7901P after 30 days' implantation (P<0.01, 293.00+/-95.54 mg vs. 624.00+/-77.78 mg) accompanied with less vessel formation with MVD 6.00+/-1.73 compared to 7901P group 8.44+/-1.33 (P<0.01). CONCLUSION: Ang-1 may play an important role in tumorigenesis and angiogenesis of gastric cancer, and targeting its expression may be beneficial for the therapy of gastric cancer.

Circulating plasma vascular endothelial growth factor in mice bearing human ovarian carcinoma xenograft correlates with tumor progression and response to therapy.

Vascular endothelial growth factor (VEGF) performs as an angiogenic and permeability factor in ovarian cancer, and its overexpression has been associated with poor prognosis. However, models to study its role as a marker of tumor progression are lacking. We generated xenograft variants derived from the A2780 human ovarian carcinoma (1A9), stably transfected with VEGF(121) in sense (1A9-VS-1) and antisense orientation (1A9-VAS-3). 1A9, 1A9-VS-1, and 1A9-VAS-3 disseminated in the peritoneal cavity of nude mice, but only 1A9-VS-1, the VEGF(121)-overexpressing tumor variant, produced ascites. Tumor biopsies from 1A9-VS-1 showed alterations in the vascular pattern and caused an angiogenic response in the chorioallantoic membrane assay. A significant level of soluble VEGF was detectable in the plasma of mice bearing 1A9-VS-1 even at an early stage of tumor growth. Plasma VEGF correlated positively with tumor burden in the peritoneal cavity and ascites accumulation. Cisplatin reduced the tumor burden and ascites in mice bearing 1A9-VS-1; the response was associated with a significant decrease of VEGF in plasma. This 1A9-VS-1 xenograft model reproduces the behavior of human ovarian cancer by growing in the peritoneal cavity, being highly malignant, and producing ascites. Plasma VEGF as a marker of tumor progression offers a valuable means of detecting early tumor response and following up treatments in an animal model.

Future therapies in hormone-refractory prostate cancer.

Hormone-refractory prostate cancer (HRPC) remains true to its name: it is largely refractory to attempts to delay its progression. Although the number of men presenting with metastatic prostate cancer has decreased significantly over the last several years, the death rate for those men is essentially unchanged. To alter the currently inevitable progression of HRPC to death, new targets and new therapies are needed. This article reviews investigational therapies directed against standard targets (eg, the hypothalamic-pituitary-gonadal axis) as well as novel targets (eg, the endothelin axis).

Targeted therapy in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an incurable malignancy. Recent insights into its biology has allowed the use of novel therapies targeting not only the deregulated intracellular signaling in MM cells but also its interaction with the bone marrow microenvironment that confers drug resistance, growth, and survival advantage to the malignant cells. METHODS: We review and summarize the recent advances in our knowledge of myeloma biology as well as the mechanism of action and clinical efficacy for novel therapeutic agents in clinical trials. RESULTS: Several novel therapeutic agents are currently in clinical trials. Thalidomide is already established for both initial and salvage treatment. Bortezomib is being tested alone and in combination with conventional chemotherapy in various settings. Other agents are less effective in producing response but have been able to stabilize disease in patients with relapsed and/or refractory disease, such as arsenic trioxide, farnesyltransferase inhibitors, 2-methoxyestradiol, and vascular endothelial growth factor receptor inhibitors. Insights into drug resistance mechanism have also led to the development of novel agents that sensitize myeloma cells to chemotherapy (Bcl-2 antisense). Gene expression studies have in many instances identified pathways other than the intended target of the drug and have provided insights into the therapeutic mechanisms. CONCLUSIONS: In the future, patients with MM will have more therapeutic options available than ever before. The challenge will be to identify patient subgroups that will benefit most from the different therapies and then determine how these biologically based therapies could be combined and incorporated into the overall management of patients.

Involvement of cathepsins in the invasion, metastasis and proliferation of cancer cells.

Tumor cell invasion and metastasis are associated with the proteolytic activity of various types of proteinases. Among them, cathepsins, which are lysosomal proteinases, have received more attention recently. Since elevated expressions of cathepsins and diminished levels of their inhibitors have been observed in several human cancers, including breast, gastric and prostate cancer, especially in aggressive cancer cells, cathepsins have been suggested to be biological markers of malignant tumors and have proved useful for prognosis of the disease. Furthermore, cathepsins have various roles in cancer progression. Cathepsin D has a mitogenic activity independent of its proteolytic activity and it attenuates the anti-tumor immune response of decaying chemokines to inhibit the function of dendritic cells. Cathepsins B and L have been shown to play an important role in matrix degradation and cell invasion. The administration of their inhibitors prevents the invasion and metastasis of cancer cells. These results indicate that cancer cells orchestrate various cathepsins to progress malignant diseases. Cathepsins may be a potential target for cancer therapy.