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1.  Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment 
Journal of drug targeting  2013;21(10):956-967.
Engineered nanoparticles are widely used for delivery of drugs but frequently lack proof of safety for cancer patient's treatment. All-in-one covalent nanodrugs of the third generation have been synthesized based on a poly(β-L-malic acid) (PMLA) platform, targeting human triple-negative breast cancer (TNBC). They significantly inhibited tumor growth in nude mice by blocking synthesis of epidermal growth factor receptor, and α4 and β1 chains of laminin-411, the tumor vascular wall protein and angiogenesis marker. PMLA and nanodrug biocompatibility and toxicity at low and high dosages were evaluated in vitro and in vivo. The dual-action nanodrug and single-action precursor nanoconjugates were assessed under in vitro conditions and in vivo with multiple treatment regimens (6 and 12 treatments). The monitoring of TNBC treatment in vivo with different drugs included blood hematologic and immunologic analysis after multiple intravenous administrations. The present study demonstrates that the dual-action nanoconju-gate is highly effective in preclinical TNBC treatment without side effects, supported by hematologic and immunologic assays data. PMLA-based nanodrugs of the Polycefin™ family passed multiple toxicity and efficacy tests in vitro and in vivo on preclinical level and may prove to be optimized and efficacious for the treatment of cancer patients in the future.
doi:10.3109/1061186X.2013.837470
PMCID: PMC4043297  PMID: 24032759
Hematologic; immunogenicity; in vivo treatment; nanoconjugate drugs; polymalic acid; toxicity; triple-negative breast cancer
2.  Polymalic acid nanobioconjugate for simultaneous inhibition of tumor growth and immunostimulation in HER2/neu-positive breast cancer✩ 
Breast cancer remains the second leading cause of cancer death among women in the United States. The breast cancer prognosis is particularly poor in case of tumors overexpressing the oncoprotein HER2/neu. A new nanobioconjugate of the Polycefin family of anti-cancer drugs based on biodegradable and non-toxic polymalic acid (PMLA) was engineered for a multi-pronged attack on HER2/neu-positive breast cancer cells. An antibody cytokine fusion protein consisting of the immunostimulatory cytokine interleukin-2 (IL-2) genetically fused to an antibody specific for human HER2/neu [anti-HER2/neu IgG3-(IL-2)] was covalently attached to the PMLA backbone to target HER2/neu expressing tumors and ensuring the delivery of IL-2 to the tumor microenvironment. Antisense oligonucleotides (AON) were conjugated to the nanodrug to inhibit the expression of vascular tumor protein laminin-411 in order to block tumor angiogenesis. It is shown that the nanobioconjugate was capable of specifically binding human HER2/neu and retaining the biological activity of IL-2. We also showed the uptake of the nanobioconjugate by HER2/neu-positive breast cancer cells and enhanced tumor targeting in vivo. In addition, the nanobioconjugate was capable of eliciting anti-tumor activity in immunocompetent mice bearing D2F2/E2 murine mammary tumors that express human HER2/neu. Both IgG1 and IgG2a levels were significantly increased in animals treated with the PMLA-fusion nanobioconjugate compared to animals treated with the antibody–cytokine fusion protein alone or control animals, indicative of the induction of a humoral (TH2) and cell-mediated (TH1) immune responses. Animal survival in vivo was significantly longer after treatment with leading nanobioconjugate with fusion [anti-HER2/neu IgG3-(IL-2)] antibody, p < 0.05. The combination of these molecules on a single polymeric platform is expected to act through direct elimination of cancer cells, inhibition of tumor angiogenesis, and orchestration of a potent immune response against tumor.
doi:10.1016/j.jconrel.2013.06.001
PMCID: PMC3971991  PMID: 23770212
Polymalic acid; Nanobioconjugate; Nanopolymer; HER2/neu; Antibody fusion protein; IL-2; Laminin-411; Breast cancer
3.  Liver-Specific γ-Glutamyl Carboxylase-Deficient Mice Display Bleeding Diathesis and Short Life Span 
PLoS ONE  2014;9(2):e88643.
Vitamin K is a fat-soluble vitamin that plays important roles in blood coagulation and bone metabolism. One of its functions is as a co-factor for γ-glutamyl carboxylase (Ggcx). Conventional knockout of Ggcx causes death shortly after birth in homozygous mice. We created Ggcx-floxed mice by inserting loxP sequences at the sites flanking exon 6 of Ggcx. By mating these mice with albumin-Cre mice, we generated Ggcx-deficient mice specifically in hepatocytes (GgcxΔliver/Δliver mice). In contrast to conventional Ggcx knockout mice, GgcxΔliver/Δliver mice had very low activity of Ggcx in the liver and survived several weeks after birth. Furthermore, compared with heterozygous mice (Ggcx+/Δliver), GgcxΔliver/Δliver mice had shorter life spans. GgcxΔliver/Δliver mice displayed bleeding diathesis, which was accompanied by decreased activity of coagulation factors II and IX. Ggcx-floxed mice can prove useful in examining Ggcx functions in vivo.
doi:10.1371/journal.pone.0088643
PMCID: PMC3919827  PMID: 24520408
4.  A Novel Acylaminoimidazole Derivative, WN1316, Alleviates Disease Progression via Suppression of Glial Inflammation in ALS Mouse Model 
PLoS ONE  2014;9(1):e87728.
Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron degenerative disease. Given that oxidative stress and resulting chronic neuronal inflammation are thought to be central pathogenic, anti-oxidative agents and modulators of neuronal inflammation could be potential therapies for ALS. We report here that the novel small molecular compound, 2-[mesityl(methyl)amino]-N-[4-(pyridin-2-yl)-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316) selectively suppresses oxidative stress-induced cell death and neuronal inflammation in the late-stage ALS mice. WN1316 has high blood-brain-barrier permeability and water solubility, and boosts both neuronal apoptosis inhibitory protein (NAIP) and NF-E2-related factor 2 (Nrf2) which governed glutathione (GSH)-related anti-oxidation pathway protecting motor neurons against oxidative injuries. Post-onset oral administration of low dose (1–100 µg/kg/day) WN1316 in ALS(SOD1H46R) and ALS(SOD1G93A) mice resulted in sustained improved motor function and post onset survival rate. Immunohistochemical analysis revealed less DNA oxidative damage and motor neuronal inflammation as well as repression of both microgliosis and astrocytosis, concomitant down regulation of interleukin-1β and inducible nitric oxide synthase, and preservation of the motoneurons in anterior horn of lumbar spinal cord and skeletal muscle (quadriceps femoris). Thus, WN1316 would be a novel therapeutic agent for ALS.
doi:10.1371/journal.pone.0087728
PMCID: PMC3909264  PMID: 24498180
5.  BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival 
The Journal of Experimental Medicine  2013;210(8):1529-1544.
BRCA1 deficiency results in impaired Nrf2-mediated antioxidant responses followed by cell death, with estradiol rescuing the effect by inducing Nrf2 stabilization.
Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers.
doi:10.1084/jem.20121337
PMCID: PMC3727320  PMID: 23857982
6.  Bat Lyssaviruses, Northern Vietnam 
Emerging Infectious Diseases  2014;20(1):161-163.
doi:10.3201/eid2001.130813
PMCID: PMC3884725  PMID: 24377728
bats; rabies; lyssaviruses; viruses; serology; Vietnam
7.  MiR-424/503-Mediated Rictor Upregulation Promotes Tumor Progression 
PLoS ONE  2013;8(11):e80300.
mTOR complex 2 (mTORC2) signaling is upregulated in multiple types of human cancer, but the molecular mechanisms underlying its activation and regulation remain elusive. Here, we show that microRNA-mediated upregulation of Rictor, an mTORC2-specific component, contributes to tumor progression. Rictor is upregulated via the repression of the miR-424/503 cluster in human prostate and colon cancer cell lines that harbor c-Src upregulation and in Src-transformed cells. The tumorigenicity and invasive activity of these cells were suppressed by re-expression of miR-424/503. Rictor upregulation promotes formation of mTORC2 and induces activation of mTORC2, resulting in promotion of tumor growth and invasion. Furthermore, downregulation of miR-424/503 is associated with Rictor upregulation in colon cancer tissues. These findings suggest that the miR-424/503–Rictor pathway plays a crucial role in tumor progression.
doi:10.1371/journal.pone.0080300
PMCID: PMC3823661  PMID: 24244675
10.  Studies on Therapeutic Effects and Pathological Features of an Antithrombin Preparation in Septic Disseminated Intravascular Coagulation Patients 
Yonsei Medical Journal  2013;54(3):686-689.
Purpose
Few reports have been made on the therapeutic effects as well as pathological features of an antithrombin preparation in patients diagnosed with septic disseminated intravascular coagulation (DIC) by the diagnostic criteria for acute DIC.
Materials and Methods
A total of 88 sepsis patients who had received inpatient hospital care during the period from January 2000 through December 2008 were divided into two groups, an antithrombin group and a non-antithrombin group, to study the outcomes. Furthermore, the relationship between sepsis-related factors and DIC in 44 patients was studied.
Results
The antithrombin group contained 34 patients, and the non-antithrombin group contained 54 patients. The outcomes were significantly better in the antithrombin group. The levels of protein C were low in DIC patients.
Conclusion
Our results suggest that early administration of antithrombin might improve outcomes of septic DIC patients in the diagnostic criteria for Japanese Association for Acute Medicine acute DIC.
doi:10.3349/ymj.2013.54.3.686
PMCID: PMC3635623  PMID: 23549815
Antithrombin; sepsis; DIC
11.  Clinical Significance of Amyloid Precursor Protein in Patients with Testicular Germ Cell Tumor 
Advances in Urology  2013;2013:348438.
Introduction. The biological role of amyloid precursor protein (APP) is not well understood, especially in testicular germ cell tumors (TGCTs). Therefore, we aimed to investigate the immunoreactivity (IR) and expression of APP in TGCTs and evaluated its clinical relevance. Materials and Methods. We performed an analysis of immunohistochemistry and mRNA expression of APP in 64 testicular specimens and 21 snap-frozen samples obtained from 1985 to 2004. We then evaluated the association between APP expression and clinicopathological status in TGCTs. Results. Positive APP IR was observed in 9.8% (4/41) of seminomatous germ cell tumors (SGCTs) and 39.1% (9/23) of nonseminomatous germ cell tumors (NGCTs). NGCTs showed significantly more cases of positive IR (P = 0.00870) and a higher mRNA expression level compared with those of SGCTs (P = 0.0140). Positive APP IR was also significantly associated with α-fetoprotein (αFP) elevation (P = 0.00870) and venous invasion (P = 0.0414). Conclusion. We observed an elevated APP expression in TGCTs, especially in NGCTs. APP may be associated with a more aggressive cancer in TGCTs.
doi:10.1155/2013/348438
PMCID: PMC3639667  PMID: 23662100
12.  Genetic Diversity and Geographic Distribution of Genetically Distinct Rabies Viruses in the Philippines 
Background
Rabies continues to be a major public health problem in the Philippines, where 200–300 human cases were reported annually between 2001 and 2011. Understanding the phylogeography of rabies viruses is important for establishing a more effective and feasible control strategy.
Methods
We performed a molecular analysis of rabies viruses in the Philippines using rabied animal brain samples. The samples were collected from 11 of 17 regions, which covered three island groups (Luzon, Visayas, and Mindanao). Partial nucleoprotein (N) gene sequencing was performed on 57 samples and complete glycoprotein (G) gene sequencing was performed on 235 samples collected between 2004 and 2010.
Results
The Philippine strains of rabies viruses were included in a distinct phylogenetic cluster, previously named Asian 2b, which appeared to have diverged from the Chinese strain named Asian 2a. The Philippine strains were further divided into three major clades, which were found exclusively in different island groups: clades L, V, and M in Luzon, Visayas, and Mindanao, respectively. Clade L was subdivided into nine subclades (L1–L9) and clade V was subdivided into two subclades (V1 and V2). With a few exceptions, most strains in each subclade were distributed in specific geographic areas. There were also four strains that were divided into two genogroups but were not classified into any of the three major clades, and all four strains were found in the island group of Luzon.
Conclusion
We detected three major clades and two distinct genogroups of rabies viruses in the Philippines. Our data suggest that viruses of each clade and subclade evolved independently in each area without frequent introduction into other areas. An important implication of these data is that geographically targeted dog vaccination using the island group approach may effectively control rabies in the Philippines.
Author Summary
Rabies continues to be a major public health problem in the Philippines. We conducted a molecular epidemiological study of rabies using the complete glycoprotein (G) gene from 235 animal brain samples collected in the Philippines between 2004 and 2010. We identified three major clades and two distinct genogroups in the Philippines. The three major clades L, V, and M were found specifically in the Luzon, Visayas, and Mindanao island groups, respectively. Additionally, two minor genogroups were located in the Luzon island group. These data suggest that although human mediated transmission may have occurred, these virus clades evolved independently after a single introduction into each island group. All of the analyzed Philippine strains were clustered into Asian 2b, which diverged from the Chinese strain Asian 2a. No recent introduction of rabies into the Philippines from other countries was apparent. The elimination of rabies by 2020 is a national goal in the Philippines, necessitating urgent development of a more effective and feasible strategy for controlling rabies. Our findings indicate that a geographically targeted dog vaccination campaign may effectively control rabies in island nations such as the Philippines.
doi:10.1371/journal.pntd.0002144
PMCID: PMC3617229  PMID: 23593515
13.  Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form 
BMC Research Notes  2012;5:483.
Background
In 2009, a novel influenza A/H1N1 virus (H1N1pdm) quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1). Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs).
Findings
Human single-fold scFv libraries (Tomlinson I + J) underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA). After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity.
Discussion
Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display libraries was highly advantageous for the rapid development of molecules to detect target antigens. However, our results also indicated that this strategy might not have been effective for selecting H1N1pdm-specific antibodies during the 2009 pandemic, where the co-circulating sH1N1 virus shared similar antigenic properties. This suggests that it might be advisable to use a synthetic scFv phage display library by strategically considering the characteristics of target antigens and the potential situations.
doi:10.1186/1756-0500-5-483
PMCID: PMC3492028  PMID: 22943792
Influenza; Pandemic; Diagnosis; Single-chain variable fragment (scFv); Altered specificity
14.  Polymalic Acid–Based Nanobiopolymer Provides Efficient Systemic Breast Cancer Treatment by Inhibiting both HER2/neu Receptor Synthesis and Activity 
Cancer research  2011;71(4):1454-1464.
Biodegradable nanopolymers are believed to offer great potential in cancer therapy. Here, we report the characterization of a novel, targeted, nanobiopolymeric conjugate based on biodegradable, nontoxic, and nonimmunogenic PMLA [poly(β-l-malic acid)]. The PMLA nanoplatform was synthesized for repetitive systemic treatments of HER2/neu-positive human breast tumors in a xenogeneic mouse model. Various moieties were covalently attached to PMLA, including a combination of morpholino antisense oligonucleotides (AON) directed against HER2/neu mRNA, to block new HER2/neu receptor synthesis; anti-HER2/neu antibody trastuzumab (Herceptin), to target breast cancer cells and inhibit receptor activity simultaneously; and transferrin receptor antibody, to target the tumor vasculature and mediate delivery of the nanobiopolymer through the host endothelial system. The results of the study showed that the lead drug tested significantly inhibited the growth of HER2/neu-positive breast cancer cells in vitro and in vivo by enhanced apoptosis and inhibition of HER2/neu receptor signaling with suppression of Akt phosphorylation. In vivo imaging analysis and confocal microscopy demonstrated selective accumulation of the nanodrug in tumor cells via an active delivery mechanism. Systemic treatment of human breast tumor-bearing nude mice resulted in more than 90% inhibition of tumor growth and tumor regression, as compared with partial (50%) tumor growth inhibition in mice treated with trastuzumab or AON, either free or attached to PMLA. Our findings offer a preclinical proof of concept for use of the PMLA nanoplatform for combination cancer therapy.
doi:10.1158/0008-5472.CAN-10-3093
PMCID: PMC3428373  PMID: 21303974
15.  In Vitro Activity of E1210, a Novel Antifungal, against Clinically Important Yeasts and Molds▿ 
Antimicrobial Agents and Chemotherapy  2011;55(10):4652-4658.
E1210 is a new antifungal compound with a novel mechanism of action and broad spectrum of antifungal activity. We investigated the in vitro antifungal activities of E1210 compared to those of fluconazole, itraconazole, voriconazole, amphotericin B, and micafungin against clinical fungal isolates. E1210 showed potent activities against most Candida spp. (MIC90 of ≤0.008 to 0.06 μg/ml), except for Candida krusei (MICs of 2 to >32 μg/ml). E1210 showed equally potent activities against fluconazole-resistant and fluconazole-susceptible Candida strains. E1210 also had potent activities against various filamentous fungi, including Aspergillus fumigatus (MIC90 of 0.13 μg/ml). E1210 was also active against Fusarium solani and some black molds. Of note, E1210 showed the greatest activities against Pseudallescheria boydii (MICs of 0.03 to 0.13 μg/ml), Scedosporium prolificans (MIC of 0.03 μg/ml), and Paecilomyces lilacinus (MICs of 0.06 μg/ml) among the compounds tested. The antifungal action of E1210 was fungistatic, but E1210 showed no trailing growth of Candida albicans, which has often been observed with fluconazole. In a cytotoxicity assay using human HK-2 cells, E1210 showed toxicity as low as that of fluconazole. Based on these results, E1210 is likely to be a promising antifungal agent for the treatment of invasive fungal infections.
doi:10.1128/AAC.00291-11
PMCID: PMC3186989  PMID: 21825291
16.  Efficacy of Oral E1210, a New Broad-Spectrum Antifungal with a Novel Mechanism of Action, in Murine Models of Candidiasis, Aspergillosis, and Fusariosis▿ 
Antimicrobial Agents and Chemotherapy  2011;55(10):4543-4551.
E1210 is a first-in-class, broad-spectrum antifungal with a novel mechanism of action—inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and reference antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater reduction in the number of oral CFU than the control treatment (P < 0.05). In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to determine E1210's potential for the treatment of disseminated fungal infections are indicated.
doi:10.1128/AAC.00366-11
PMCID: PMC3187015  PMID: 21788462
17.  Single-nucleotide polymorphism in the hyaluronan and proteoglycan link protein 1 (HAPLN1) gene is associated with spinal osteophyte formation and disc degeneration in Japanese women 
European Spine Journal  2010;20(4):572-577.
Spinal osteoarthritis including disc degeneration is a very common condition in the axial skeletons of aged people. Recently, spinal osteoarthritis has been shown to be influenced by specific genetic risk factors. Vertebral osteophytes, endplate sclerosis, and intervertebral disc narrowing are recognized as radiographic features of spinal disc degeneration. HAPLN1 is a key component of the cartilage extracellular matrix; thus, variations in this gene may affect the pathogenesis of cartilage-related diseases such as spinal degeneration. Here, we examine the association between an HAPLN1 gene polymorphism and the radiographic features of spinal degeneration. We evaluated the degree of endplate sclerosis, osteophyte formation, and disc space narrowing in 622 Japanese postmenopausal women. Four SNPs in the HAPLN1 gene—in the 5′ flanking region, intron 1, intron 2, and intron 4—were analyzed using the TaqMan polymerase chain reaction method. We found that compared to subjects with the CC or CT genotype, those with the TT genotype for an SNP at intron 2 (rs179851) were significantly overrepresented among the subjects with higher scores for osteophyte formation (P = 0.0001; odds ratio 2.12; 95% confidence interval 1.45–3.11, as determined by logistic regression analysis) and disc space narrowing (P = 0.0057; odds ratio 1.83; 95% confidence interval 1.19–2.83). Consistent with the involvement of the HAPLN1 gene in cartilage metabolism, a variation in a specific HAPLN1 gene locus may be associated with spinal degeneration.
doi:10.1007/s00586-010-1598-0
PMCID: PMC3065599  PMID: 20953637
Single-nucleotide polymorphism (SNP); Hyaluronan and proteoglycan link protein gene (HAPLN1); Osteoarthritis; Cartilage; Disc degeneration
18.  Nanobiopolymer for Direct Targeting and Inhibition of EGFR Expression in Triple Negative Breast Cancer 
PLoS ONE  2012;7(2):e31070.
Treatment options for triple negative breast cancer (TNBC) are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR) therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid) (PMLA) nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb) 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR) antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON) to inhibit EGFR synthesis. The nanobioconjugates variants were: (1) P (BioPolymer) with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR), and (2) P with AON and 2C5 (P/AON/2C5). Controls included (3) P with 2C5 but without AON (P/2C5), (4) PBS, and (5) P with PEG and leucine ester (LOEt) for endosomal escape (P/mPEG/LOEt). Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging) and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting.
In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1) [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2)]. Lead nanobioconjugate (1) also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate represents a new generation of nanodrugs for treatment of TNBC.
doi:10.1371/journal.pone.0031070
PMCID: PMC3280290  PMID: 22355336
20.  Cellular Delivery of Doxorubicin via pH-Controlled Hydrazone Linkage Using Multifunctional Nano Vehicle Based on Poly(β-L-Malic Acid) 
Doxorubicin (DOX) is currently used in cancer chemotherapy to treat many tumors and shows improved delivery, reduced toxicity and higher treatment efficacy when being part of nanoscale delivery systems. However, a major drawback remains its toxicity to healthy tissue and the development of multi-drug resistance during prolonged treatment. This is why in our work we aimed to improve DOX delivery and reduce the toxicity by chemical conjugation with a new nanoplatform based on polymalic acid. For delivery into recipient cancer cells, DOX was conjugated via pH-sensitive hydrazone linkage along with polyethylene glycol (PEG) to a biodegradable, non-toxic and non-immunogenic nanoconjugate platform: poly(β-l-malic acid) (PMLA). DOX-nanoconjugates were found stable under physiological conditions and shown to successfully inhibit in vitro cancer cell growth of several invasive breast carcinoma cell lines such as MDA-MB-231 and MDA-MB- 468 and of primary glioma cell lines such as U87MG and U251.
doi:10.3390/ijms130911681
PMCID: PMC3472769  PMID: 23109877
polymalic acid; doxorubicin; nanoconjugate; pH-controlled hydrazine linkage; brain and breast cancer
21.  Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by RIG-I 
Cell host & microbe  2009;5(5):439-449.
SUMMARY
TRIM25 mediates Lys 63-linked ubiquitination of the N-terminal CARDs of the viral RNA sensor RIG-I, leading to type I interferon (IFN) production. Here, we report that the influenza A virus non-structural protein 1 (NS1) specifically inhibits TRIM25-mediated RIG-I CARD ubiquitination, thereby suppressing RIG-I signal transduction. A novel domain in NS1 comprising E96/E97 residues mediates its interaction with the coiled-coil domain of TRIM25, thus blocking TRIM25 multimerization and RIG-I CARD ubiquitination. Furthermore, a recombinant influenza A virus expressing an E96A/E97A NS1 mutant is defective in blocking TRIM25-mediated anti-viral IFN response and loses virulence in mice. Our findings reveal a novel mechanism of influenza virus to inhibit host IFN response and also emphasize the vital role of TRIM25 in modulating viral infections.
doi:10.1016/j.chom.2009.04.006
PMCID: PMC2737813  PMID: 19454348
22.  Phosphodiesterase Type 5 Inhibitors Increase Herceptin Transport and Treatment Efficacy in Mouse Metastatic Brain Tumor Models 
PLoS ONE  2010;5(4):e10108.
Background
Chemotherapeutic drugs and newly developed therapeutic monoclonal antibodies are adequately delivered to most solid and systemic tumors. However, drug delivery into primary brain tumors and metastases is impeded by the blood-brain tumor barrier (BTB), significantly limiting drug use in brain cancer treatment.
Methodology/Principal Findings
We examined the effect of phosphodiesterase 5 (PDE5) inhibitors in nude mice on drug delivery to intracranially implanted human lung and breast tumors as the most common primary tumors forming brain metastases, and studied underlying mechanisms of drug transport. In vitro assays demonstrated that PDE5 inhibitors enhanced the uptake of [14C]dextran and trastuzumab (Herceptin®, a humanized monoclonal antibody against HER2/neu) by cultured mouse brain endothelial cells (MBEC). The mechanism of drug delivery was examined using inhibitors for caveolae-mediated endocytosis, macropinocytosis and coated pit/clathrin endocytosis. Inhibitor analysis strongly implicated caveolae and macropinocytosis endocytic pathways involvement in the PDE5 inhibitor-enhanced Herceptin uptake by MBEC. Oral administration of PDE5 inhibitor, vardenafil, to mice with HER2-positive intracranial lung tumors led to an increased tumor permeability to high molecular weight [14C]dextran (2.6-fold increase) and to Herceptin (2-fold increase). Survival time of intracranial lung cancer-bearing mice treated with Herceptin in combination with vardenafil was significantly increased as compared to the untreated, vardenafil- or Herceptin-treated mice (p<0.01). Log-rank survival analysis of mice bearing HER2-positive intracranial breast tumor also showed a significant survival increase (p<0.02) in the group treated with Herceptin plus vardenafil as compared to other groups. However, vardenafil did not exert any beneficial effect on survival of mice bearing intracranial breast tumor with low HER2 expression and co-treated with Herceptin (p>0.05).
Conclusions/Significance
These findings suggest that PDE5 inhibitors may effectively modulate BTB permeability, and enhance delivery and therapeutic efficacy of monoclonal antibodies in hard-to-treat brain metastases from different primary tumors that had metastasized to the brain.
doi:10.1371/journal.pone.0010108
PMCID: PMC2856671  PMID: 20419092
23.  Competition or complement: relationship between judo therapists and physicians for elderly patients with musculoskeletal disease 
Objectives
Among the complementary and alternative medical services available in Japan, only judo therapists are covered under the national health-insurance program without a referral from a physician. Many orthopedists claim that judo therapists deprive them of potential patients. If such competition exists, fewer patients would be expected to visit orthopedists in areas where many patients visit judo therapists. Therefore, we examined the correlation between the number of patients visiting judo therapists and those visiting physicians for musculoskeletal diseases.
Methods
In a cross-sectional study covering each prefecture in Japan (n = 47), we obtained figures for the numbers of judo therapist facilities and elderly patients (over 70 years old) who visited them and the numbers of orthopedists and patients who visited physicians for musculoskeletal diseases. Correlations between the numbers of practitioners per 100,000 population and the numbers of their patients per 100,000 population were examined by prefecture.
Results
There were positive correlations between the numbers of judo therapist facilities and elderly patients who visited them (r = 0.72, P < 0.01, n = 47), and between the numbers of orthopedists and elderly patients who visited physicians for musculoskeletal diseases (r = 0.32, P = 0.03). However, there was no significant correlation between the numbers of elderly patients who visited judo therapist facilities and those who visited physicians (r = 0.06, P = 0.68) for musculoskeletal diseases.
Conclusions
This study did not find a negative correlation between the numbers of patients visiting judo therapists and patients visiting physicians for musculoskeletal diseases. Thus, these results do not support the orthopedists’ claim that the two services compete for patients.
doi:10.1007/s12199-007-0021-x
PMCID: PMC2698256  PMID: 19568896
Distribution; Health-care service; Judo therapist; Elderly patient; Orthopedist
24.  Antitumor activity and down-regulation of pro-angiogenic molecules in human prostate cancer cells by a novel thiazolidione compound 
The Prostate  2006;66(4):430-438.
BACKGROUND
Current treatments for prostate cancer are effective in many patients with locally advanced disease, but many of these patients eventually have recurrence. It is therefore important to develop alternative therapeutic agents with improved efficacy and tolerability. We recently identified a synthetic thiazolidin compound, 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidione (DBPT), that induces apoptosis in human colon cancer cells, independent of p53 and P-glycoprotein status. Here, we investigated the antitumor properties and mechanisms of action of this compound in human prostate cancer cell lines.
METHODS
The effect of DBPT on cell-cycle progression and apoptosis in LNCaP and DU145 cells was examined by flow cytometry and Western blotting. The effect of DBPT on pro-angiogenic molecules was analyzed by Western blotting and by an enzyme-linked immunosorbent assay.
RESULTS
DBPT inhibited the growth of LNCaP and DU145 cells with 50% inhibitory concentrations ranging from 1.6 to 5.9 μM. Treating LNCaP and DU145 cells with DBPT led to a time-dependent cell-cycle arrest in the G2/M phase and increased levels of G2/M checkpoint proteins, such as cyclin B1, cdc25C, phosphorylated histone H3, and MPM-2. DBPT induced the phosphorylation of Bcl-xL and Bim, and induced apoptosis, as evidenced by cleavage of caspase and poly (ADP-ribose) polymerase. DBPT also effectively induced apoptosis in Bcl-2-overexpressing DU145 cells. Furthermore, DBPT decreased hypoxia-inducible factor 1α and vascular endothelial growth factor expression in LNCaP cells under both normoxia and hypoxia.
CONCLUSIONS
DBPT can suppress proliferation, induce apoptosis, and down-regulate pro-angiogenic molecules in prostate cancer cells, and might be useful in treating prostate cancer.
doi:10.1002/pros.20373
PMCID: PMC1351358  PMID: 16302260
novel compound; prostate cancer; apoptosis; G2/M-phase arrest; VEGF
25.  Rabies Virus Stimulates Nitric Oxide Production and CXC Chemokine Ligand 10 Expression in Macrophages through Activation of Extracellular Signal-Regulated Kinases 1 and 2 
Journal of Virology  2004;78(17):9376-9388.
Macrophages represent an essential part of innate immunity, and the viral infection of macrophages results in the release of multiple proinflammatory mediators, such as nitric oxide (NO), cytokines, and chemokines. This study was undertaken to define the molecular mechanism of macrophage activation in response to rabies virus (RV) infection. In RAW264 murine macrophage cells, a well-characterized macrophage model, RV replication was strictly restricted, whereas cell proliferation was significantly enhanced upon RV inoculation. Transcriptional analyses for the expression of inducible forms of NO synthase (iNOS), cytokines, and chemokines revealed that RV virions potentiate the gene expression of iNOS and CXC chemokine ligand 10 (CXCL10), a major chemoattractant of T helper cell type 1. However, RV stimulation had little or no effect on the expression profiles of proinflammatory cytokines and other types of chemokines. In macrophages stimulated with UV-inactivated RV virions, as well as infectious viruses, the phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, members of the mitogen-activated protein kinase family, was significantly induced. Specific inhibitors of MAPK/ERK kinase reduced the RV-induced production of NO and CXCL10. Furthermore, the RV-induced activation of the ERK1/2 pathway was severely impaired by the neutralization of the endosomal and lysosomal pH environment with lysosomotropic agents, indicating that endocytosis is a key step leading to the activation of ERK1/2 signaling. Taken together, these results suggest that the ERK1/2-mediated signaling pathway plays a cardinal role in the selective activation of macrophages in response to RV virions, thereby regulating cellular functions during virus infection.
doi:10.1128/JVI.78.17.9376-9388.2004
PMCID: PMC506932  PMID: 15308732

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