Genomic structural alteration is common in pediatric cancers, and analysis of data generated by the Pediatric Cancer Genome Project reveals such tumor-related alterations in many Wnt signaling–associated genes. Most pediatric cancers are thought to arise within developing tissues that undergo substantial expansion during early organ formation, growth and maturation, and Wnt signaling plays an important role in this development. We examined three pediatric tumors—medullobastoma, early T-cell precursor acute lymphoblastic leukemia, and retinoblastoma—that show multiple genomic structural variations within Wnt signaling pathways. We mathematically modeled this pathway to investigate the effects of cancer-related structural variations on Wnt signaling. Surprisingly, we found that an outcome measure of canonical Wnt signaling was consistently similar in matched cancer cells and normal cells, even in the context of different cancers, different mutations, and different Wnt-related genes. Our results suggest that the cancer cells maintain a normal level of Wnt signaling by developing multiple mutations.
This study investigated the Jurkat T cell line expresses cytotoxicity when treated with different concentrations of FK506, and analyzed the expression pattern of microRNA when stimulated by FK506 using the microRNAs microarray, as well as the expression pattern of a gene that is related to the differentiation, activation and proliferation of T cells after being affected by the change of microRNAs.
To investigate the effects of FK506 on microRNA expression, we purified total RNA of Jurkat cells treated with 20 µM FK506 for 72 hours and used to analyze microRNA profiling by using Agilent's chip.
These results demonstrated that treatment with FK506 markedly induced the down-regulation of 20 microRNAs as well as the up-regulation of 20 microRNAs in a time-dependent manner. The genes that down-regulated by FK506 include let-7a*, miR-20a*, and miR-487a. Otherwise miR-202, miR-485-5p, and miR-518c* are gradually up-regulated in expression. Sanger Institute and DAVIDs bioinformatics indicated that microRNAs regulated the several transcriptomes including nuclear factor of activated T cell-related, T cell receptor/interleukin-2 signaling, and Ca2+-calmodulin-dependent phosphatase calcineurin pathways.
As a result of treating FK506 to a Jurkat cell line and running the microRNA microarray, it was found that FK506 not only took part in the suppression of T cell proliferation/activation by inhibiting calcineurin in Jurkat apoptosis, but also affected the microRNAs that are involved in the regulation of various signal transduction pathways.
Tacrolimus; MicroRNAs; Jurkat T lymphocyte
Recently, partial ligation of the common carotid artery (CCA) was reported to induce carotid atheromata rapidly in apolipoprotein-E knockout (ApoE-/-) mice. We investigated this new atherosclerosis model by using combined matrix-metalloproteinase (MMP) near-infrared fluorescent (NIRF) imaging and macrophage-tracking luciferase imaging.
Methodology and Principal Findings
Partial ligation of the left CCA was performed in 10-week-old ApoE-/- mice on a high fat diet (n=33); the internal and external carotid arteries and occipital artery were ligated, while the superior thyroid artery was left intact. Two thirds of the animals were treated with either LiCl or atorvastatin. At 1-week, Raw264.7 macrophages modified to express the enhanced firefly-luciferase reporter gene (107 Raw-luc cells) were injected intravenously. At 2-week, NIRF molecular imaging visualized strong MMP-2/9 activity in the ligated area of the left CCA as well as in the aortic arch. Left-to-right ratios of the NIRF signal intensities in the CCA had a decreasing gradient from the highest value in the upper-most ligated area to the lowest value in the lower-most region adjacent to the aortic arch. Luciferase imaging showed that most Raw-luc macrophages were recruited to the ligated area of the CCA rather than to the aortic arch, despite similarly strong MMP-2/9-related NIRF signal intensities in both areas. In addition, LiCl or atorvastatin could reduce MMP-2/9 activity in the aortic arch but not in the ligated area of the CCA.
This is the first molecular imaging study to characterize the partial ligation-induced carotid atherosclerosis model. Molecularly divergent types of atherosclerosis were identified: conventional lipogenic atherosclerosis in the aorta vs. flow-related mechanical atherosclerosis in the partially ligated left system.
Recently, the roles of SIRT1 and deleted in breast cancer 1 (DBC1) in human cancer have been extensively studied and it has been demonstrated that they are involved in many human carcinomas. However, their clinical significance for soft-tissue sarcomas has not been examined. In this study, we evaluated the expression and prognostic significance of the expression of SIRT1, DBC1, P53, β-catenin, cyclin D1, and KI67 in 104 cases of soft-tissue sarcomas. RESULTS: Immunohistochemical expression of SIRT1, DBC1, P53, β-catenin, and cyclin D1 were seen in 71%, 74%, 53%, 48%, and 73% of sarcomas, respectively. The expression of SIRT1, DBC1, P53, β-catenin, and cyclin D1 were significantly correlated with advanced clinicopathological parameters such as higher clinical stage, higher histological grade, increased mitotic counts, and distant metastasis. The expression of SIRT1, DBC1, P53, β-catenin, cyclin D1, and KI67 were significantly correlated with each other and positive expression of all of these predicted shorter overall survival and event-free survival by univariate analysis. Multivariate analysis revealed the expression of SIRT1 as an independent prognostic indicator for overall survival and event-free survival of sarcoma patients. In conclusion, this study demonstrates that SIRT1- and DBC1-related pathways may be involved in the progression of soft-tissue sarcomas and can be used as clinically significant prognostic indicators for sarcoma patients. Moreover, the SIRT1- and DBC1-related pathways could be new therapeutic targets for the treatment of sarcomas.
A multiplex real-time PCR assay that simultaneously detects the mecA, staphylococcal cassette chromosome (SCCmec)-open reading frame X (orfX) junction, and staphylococcal 16S rRNA genes was developed and evaluated using 444 staphylococcal strains. We demonstrated that this assay resulted in fewer false-positive results than a single-locus real-time PCR assay that amplified the SCCmec-orfX junction. This assay would be useful in a clinical laboratory in a region of high endemicity for methicillin-resistant Staphylococcus aureus (MRSA) infections.
We have analyzed the correlation between the House-Brackmann (HB) scale and Facial Nerve Grading System 2.0 (FNGS 2.0) in patients with Bell palsy, and evaluated the usefulness of the new grading system.
Sixty patients diagnosed with Bell palsy from May 2009 to December 2010 were evaluated using the HB scale and FNGS 2.0 scale during their initial visit, and after 3 and 6 weeks and 3 months.
The overall intraclass correlation coefficient (ICC) was 0.908 (P=0.000) and the Spearman correlation coefficient (SCC) was 0.912 (P<0.05). ICC and SCC displayed differences over time, being 0.604 and 0.626, respectively, at first visit; 0.834 and 0.843, respectively, after 3 weeks; 0.844 and 0.848, respectively, after 6 weeks; and 0.808 and 0.793, respectively, after 3 months. There was a significant difference in full recovery, depending on the scale used (HB, P=0.000; FNGS 2.0, P<0.05). The exact agreements between regional assessment and FNGS 2.0 for the mouth, eyes, and brow were 72%, 63%, and 52%, respectively.
FNGS 2.0 shows moderate agreement with HB grading. Regional assessment, rather than HB grading, yields stricter evaluation, resulting in better prognosis and determination of grade.
House-Brackmann scale; Facial Nerve Grading System 2.0; Bell palsy
Oocyte cryopreservation has become an essential tool in the treatment of infertility by preserving oocytes for women undergoing chemotherapy. However, despite recent advances, pregnancy rates from all cryopreserved oocytes remain low. The inevitable use of the cryoprotectants (CPAs) during preservation affects the viability of the preserved oocytes and pregnancy rates either through CPA toxicity or osmotic injury. Current protocols attempt to reduce CPA toxicity by minimizing CPA concentrations, or by minimizing the volume changes via the step-wise addition of CPAs to the cells. Although the step-wise addition decreases osmotic shock to oocytes, it unfortunately increases toxic injuries due to the long exposure times to CPAs. To address limitations of current protocols and to rationally design protocols that minimize the exposure to CPAs, we developed a microfluidic device for the quantitative measurements of oocyte volume during various CPA loading protocols. We spatially secured a single oocyte on the microfluidic device, created precisely controlled continuous CPA profiles (step-wise, linear and complex) for the addition of CPAs to the oocyte and measured the oocyte volumetric response to each profile. With both linear and complex profiles, we were able to load 1.5 M propanediol to oocytes in less than 15 min and with a volumetric change of less than 10%. Thus, we believe this single oocyte analysis technology will eventually help future advances in assisted reproductive technologies and fertility preservation.
Atherosclerosis, a chronic and progressive disease characterized by vascular inflammation, is a leading cause of death in diabetes patients. Doinseunggitang (DYSGT), traditional prescription, has been used for promoting blood circulation to remove blood stasis. The aim of this study was to investigate the beneficial effects of DYSGT on endothelial dysfunction in diabetic atherosclerosis animal model. Apolipoprotein E knockout (ApoE KO) mice fed on a Western diet were treated with DYSGT (200 mg/kg/day). DYSGT significantly lowered blood glucose level and glucose tolerance as well as systolic blood pressure. Metabolic parameter showed that DYSGT markedly decreased triglyceride and LDL-cholesterol levels. In the thoracic aorta, the impairment of vasorelaxation response to acetylcholine and atherosclerotic lesion was attenuated by DYSGT. Furthermore, DYSGT restored the reduction of endothelial nitric oxide synthase (eNOS) expression, leading to the inhibition of intracellular adhesion molecule-1 (ICAM-1) and endothelin-1 (ET-1) expression. In conclusion, DYSGT improved the development of diabetic atherosclerosis via attenuation of the endothelial dysfunction, possibly by inhibiting ET-1, cell adhesion molecules, and lesion formation. Therefore, these results suggest that Korean traditional prescription Doinseunggitang may be useful in the treatment and prevention of diabetic vascular complications.
We for the first time reported evidence for the existence of a novel network, a PVS, abovethe epicardium of the rat heart. (1) We were consecutively able to visualize the PVs and the PNs above the epicardial spaces of five rats' hearts by using Cr-Hx spraying or injection. (2) Hematoxylin and eosin (H&E) and toluidine blue staining of the PVs and the PNs showed that they consisted of a basophilic matrix; specifically the PNs contained several mast cells, some of which were degranulating into pericardial space. Also, 4′, 6-diamidino-2 phenylindole (DAPI) images of the PVs and the PNs showed that they contained various kinds of cells. (3) Transmission electron microscopic (TEM) longitudinal image of the PVs showed that the sinuses contained many granules with high-electron-density cores in parallel with putative endothelial cells. (4) TEM images of the PNs demonstrated that they consisted of lumen-containing cells surrounded by fibers and that they had mast cells that were degranulating toward the epicardium of the rat heart. The above data suggest that mast-cells-containing novel network exists above the epicardium of the rat heart.
Hypothesizing that redundant functional ovarian reserve (FOR) at young ages may clinically obfuscate prematurely diminished FOR (PDFOR), we investigated in young oocyte donors genotypes and sub-genotypes of the FMR1 gene, in prior studies associated with specific ovarian aging patterns, and determined whether they already at such young age were associated with variations in ovarian reserve (OR). We also investigated racial as well as FMR1 associations with menarcheal age in these donors.
In a cohort study we investigated 157 oocyte donor candidates and, based on the 95% CI of AMH, divided them into normal age-specific (AMH greater or equal to 2.1 ng/mL; n = 121) and PDFOR (AMH < 2.1 ng/mL; n = 36). We then assessed associations between numbers of trinucleotide repeat (CGGn) on the FMR1 gene and FOR (based on anti-Müllerian hormone, AMH).
FMR1 did not associate with AMH overall. Amongst 36 donors with PDFOR, 17 (42%) presented with at least one low (CGGn < 26 ) allele. Remaining donors with normal FOR presented with significantly more CGGn greater or equal to 26 (73.6% vs. 26.4%; P = 0.024) and higher AMH (P = 0.012). This finding was mostly the consequence of interaction between FMR1 (CGGn < 26 vs. CGGn greater or equal to 26) and race (P = 0.013), with Asians most responsible (P = 0.009). Menarcheal age was in donors with normal FOR neither associated with race nor with FMR1 status. In donors with PDFOR race was statistically associated with CGGn (P = 0.018), an association primarily based on significantly delayed age of menarche in African donors with CGGn < 26 in comparison to African donors with CGGn greater or equal to 26 (P = 0.019), and Caucasian (P = 0.017) and Asian donors (P = 0.025) with CGGn < 26.
CGGn on FMR1 already at young ages affects FOR, but is clinically apparent only in cases of PDFOR. Screening for low FMR1 CGGn < 26 at young age, thus, appears predictive of later PDFOR.
Fragile X gene; FMR1 gene; Ovarian reserve; Anti-müllerian hormone (AMH); Oocyte donor
Most patients with non–small cell lung cancer (NSCLC) have responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). We investigated the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in primary resistance to EGFR TKIs and the molecular determinants of resistance to IGF-1R TKIs.
Phosphorylated IGF-1R/insulin receptor (pIGF-1R/IR) was immunohistochemically evaluated in a NSCLC tissue microarray. We analyzed the antitumor effects of an IGF-1R TKI (PQIP or OSI-906), either alone or in combination with a small-molecular inhibitor (PD98059 or U0126) or with siRNA targeting K-Ras or MAPK/extracellular signal-regulated kinase kinase (MEK), in vitro and in vivo in NSCLC cells with variable histologic features and EGFR or K-Ras mutations.
pIGF-1R/IR expression in NSCLC specimens was associated with a history of tobacco smoking, squamous cell carcinoma histology, mutant (mut) K-Ras, and wild-type (wt) EGFR, all of which have been strongly associated with poor response to EGFR TKIs. IGF-1R TKIs exhibited significant antitumor activity in NSCLC cells with wt EGFR and wt K-Ras but not in those with mutations in these genes. Introduction of mut K-Ras attenuated the effects of IGF-1R TKIs on NSCLC cells expressing wt K-Ras. Conversely, inactivation of MEK restored sensitivity to IGF-TKIs in cells carrying mut K-Ras.
The mutation status of both EGFR and K-Ras could be predictive markers of response to IGF-1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC cells to IGF-1R TKIs.
EGFR; K-Ras; IGF-1R; lung cancer; TKI
Recent legislation established a requirement for nutrition education in federal assistance programs to be evidence-based. Recruitment of low-income persons to participate and evaluate nutrition education activities can be challenging and costly. Facebook has been shown to be a cost-effective strategy to recruit this target audience to a nutrition program.
The purpose of our study was to examine Facebook as a strategy to recruit participants, especially Supplemental Nutrition Assistance Program Education (SNAP-Ed) eligible persons, to view and evaluate an online nutrition education program intended to be offered as having some evidence base for SNAP-Ed programming.
English-speaking, low-income Pennsylvania residents, 18-55 years with key profile words (eg, Supplemental Nutrition Assistance Program, Food bank), responded to a Facebook ad inviting participation in either Eating Together as a Family is Worth It (WI) or Everyone Needs Folic Acid (FA). Participants completed an online survey on food-related behaviors, viewed a nutrition education program, and completed a program evaluation. Facebook set-up functions considered were costing action, daily spending cap, and population reach.
Respondents for both WI and FA evaluations were similar; the majority were white, <40 years, overweight or obese body mass index, and not eating competent. A total of 807 Facebook users clicked on the WI ad with 73 unique site visitors and 47 of them completing the program evaluation (ie, 47/807, 5.8% of clickers and 47/73, 64% of site visitors completed the evaluation). Cost per completed evaluation was US $25.48; cost per low-income completer was US $39.92. Results were similar for the FA evaluation; 795 Facebook users clicked on the ad with 110 unique site visitors, and 73 completing the evaluation (ie, 73/795, 9.2% of ad clickers and 73/110, 66% of site visitors completed the evaluation). Cost per valid completed survey with program evaluation was US $18.88; cost per low-income completer was US $27.53.
With Facebook we successfully recruited low-income Pennsylvanians to online nutrition program evaluations. Benefits using Facebook as a recruitment strategy included real-time recruitment management with lower costs and more efficiency compared to previous data from traditional research recruitment strategies reported in the literature. Limitations prompted by repeated survey attempts need to be addressed to optimize this recruitment strategy.
nutrition education; folic acid; family meals; low-income; food security; Facebook; SNAP-Ed
Metastasis is a critical event in the progression of head and neck squamous cell carcinoma (HNSCC) and closely correlates with clinical outcome. We previously showed that the farnesyl transferase inhibitor SCH66336 has antitumor activities in HNSCC by inducing insulin-like growth factor binding protein 3 (IGFBP-3) secretion, which in turn inhibits tumor growth and angiogenesis. In this study, we found that SCH66336 at a sublethal dose for HNSCC inhibited the migration and invasion of HNSCC cells. The inhibitory effect of SCH66336 was associated with the blockade of the IGF-1 receptor (IGF-1R) pathway via suppressing IGF-1R itself and Akt expression. Consistent with previous work, induction of IGFBP-3 by SCH66336 also contributed in part to the anti-invasive effect. SCH66336 treatment also reduced the expression and activity of the urokinase-type plasminogen activator (uPA) and matrix metalloproteinase 2 (MMP-2), both important regulators of tumor metastasis. The effect of SCH66336 on uPA activity was inhibited partly by knockdown of IGFBP-3 using siRNA. The inhibitory effect of SCH66336 on migration or invasion was attenuated partly or completely by knockdown of IGFBP-3, Akt, or IGF-1R expression, respectively. Our results demonstrate that the IGF-1R pathway plays a major role in the proliferation, migration, and invasion of HNSCC cells, suggesting that therapeutic obstruction of the IGF-1R pathway would be a useful approach to treating patients with HNSCC.
Insulin-like growth factor binding protein-3; insulin-like growth factor-1 receptor; farnesyl transferase inhibitor; head and neck squamous cell carcinoma; invasion; SCH66336
We propose a protocol that provides a systematic definition of reaction coordinate and related free-energy profile as the function of temperature for the protein-folding simulation. First, using action-derived molecular dynamics (ADMD), we investigate the dynamic folding pathway model of a protein between a fixed extended conformation and a compact conformation. We choose the pathway model to be the reaction coordinate, and the folding and unfolding processes are characterized by the ADMD step index, in contrast to the common a priori reaction coordinate as used in conventional studies. Second, we calculate free-energy profile as the function of temperature, by employing the replica-exchange molecular dynamics (REMD) method. The current method provides efficient exploration of conformational space and proper characterization of protein folding/unfolding dynamics from/to an arbitrary extended conformation. We demonstrate that combination of the two simulation methods, ADMD and REMD, provides understanding on molecular conformational changes in proteins. The protocol is tested on a small protein, penta-peptide of met-enkephalin. For the neuropeptide met-enkephalin system, folded, extended, and intermediate sates are well-defined through the free-energy profile over the reaction coordinate. Results are consistent with those in the literature.
molecular dynamics; free energy; reaction coordinate
Uterine rhabdomyosarcoma (RMS) typically presents as a mixed epithelial and mesenchymal tumors. Pure RMSs of the female genital tract are uncommon. Spindle cell variant of RMS is a rare morphologic subtype of embryonal RMS and mostly occurs in the paratesticular region of children. Here, we present a case of uterine spindle cell RMS in a 76-year-old woman. The tumor, 20×15×7 cm in size, was highly necrotic and adherent to the colon and rectum. Tumor cells were mostly spindle-shaped, and isolated rhabdomyoblasts were scattered. Immunohistochemical stains for myoglobin and myo-D1 showed diffuse positivity for tumor cells. The patient died only of disease three months after diagnosis.
Rhabdomyosarcoma; Spindle cell variant; Uterus
To investigate the effect on pain reduction and strengthening of the whole body vibration (WBV) in chronic knee osteoarthritis (OA).
Patients were randomly divided into two groups: the study group (WBV with home based exercise) and control group (home based exercise only). They performed exercise and training for 8 weeks. Eleven patients in each group completed the study. Pain intensity was measured with the Numeric Rating Scale (NRS), functional scales were measured with Korean Western Ontario McMaster score (KWOMAC) and Lysholm Scoring Scale (LSS), quadriceps strength was measured with isokinetic torque and isometric torque and dynamic balance was measured with the Biodex Stability System. These measurements were performed before training, at 1 month after training and at 2 months after training.
NRS was significantly decreased in each group, and change of pain intensity was significantly larger in the study group than in the control group after treatment. Functional improvements in KWOMAC and LSS were found in both groups, but no significant differences between the groups after treatment. Dynamic balance, isokinetic strength of right quadriceps and isometric strengths of both quadriceps muscles improved in both groups, but no significant differences between the groups after treatment. Isokinetic strength of left quadriceps did not improve in both groups after treatment.
In chronic knee OA patients, WBV reduced pain intensity and increased strength of the right quadriceps and dynamic balance performance. In comparison with the home based exercise program, WBV was superior only in pain reduction and similarly effective in strengthening of the quadriceps muscle and balance improvement.
Knee; Muscle strength; Osteoarthritis; Pain; Vibration
A 55-year-old female was diagnosed with L5-S1 degenerative disc disease (DDD). Initial scores by the visual analogue scale (VAS) were 5 (back) and 9 (leg) and the Oswestry disability index (ODI) was 32. Arthroplasty was performed. Clinical and radiographic monitoring took place thereafter at one month, three months, six months and annually. At one month, VAS scores were 2 (back) and 3 (leg), ODI was 12 and ROM was 2.1° by radiographs. At two years, VAS scores were 1 (back) and 2 (leg), ODI was 6 and ROM was approaching 0. Five years after surgery, the entire operated segment (L5-S1) was solidly fused. A malpositioned disc implant may impair normal spinal movement, culminating in heterotopic ossification or complete fusion of the operated segment.
Total disc replacement; Lumbar spine; Heterotopic ossification; Fusion, Arthroplasty; Solid fusion
Talin-mediated integrin activation drives integrin-based adhesions. A simple binary switch—vinculin competitively displacing RIAM from talin—is found to play a central role in the maturation and evolving functions of integrin-based adhesions.
Talin-mediated integrin activation drives integrin-based adhesions. Here we examine the roles of two proteins that induce talin–integrin interactions—vinculin and Rap1-GTP-interacting adaptor molecule (RIAM)—in the formation and maturation of integrin-based adhesions. RIAM-containing adhesions are primarily in the lamellipodium; RIAM is subsequently reduced in mature focal adhesions due to direct competition with vinculin for talin-binding sites. We show that vinculin binding to talin induces Rap1-independent association of talin with integrins and resulting integrin activation, in sharp contrast to Rap1-dependent RIAM-induced activation. Vinculin stabilizes adhesions, increasing their ability to transmit force, whereas RIAM played a critical role in lamellipodial protrusion. Thus displacement of RIAM by vinculin acts as a molecular switch that mediates the transition of integrin-based adhesions from drivers of lamellipodial protrusion to stable, force-bearing adhesions. Consequently changes in the abundance of two multiprotein modules within maturing adhesions, one regulated by Rap1 and one by tension, result in the temporal evolution of adhesion functions.
Anaplastic large cell lymphoma (ALCL) is a rare T-cell lymphoma composed of CD30-positive lymphoid cells. ALCL arising in the dura matter of the brain is even more infrequent, in which only one case has been reported worldwide so far. We report a case of a 30-year-old immunocompetent male with a dura-based mass, radiographically consistent with meningioma. However, the excised mass via a left parieto-occipital craniotomy was composed of large, pleomorphic lymphoid cells to be immunopositive for CD3, CD30, anaplastic lymphoma kinase protein-1 (ALK-1) and epithelial membrane antigen (EMA), and immunonegative for CD20, CD15 and CD68. Multiple ALK gene fusion signals in the ALK locus were detected by fluorescence in situ hybridization (FISH) analysis. The patient was treated with CHOP chemotherapy and intrathecal methotrexate along with brain radiation therapy, which resulted in a complete remission. In an analysis of 25 previously reported primary CNS ALCLs, ALK-1 positivity was shown to be prevalent in younger age, as ALCL occurs outside the brain. Patient less than 23 years, ALK-1 positivity and unifocal tumor may be associated with a better prognosis. However, sex, dural or leptomeningeal involvement, immune status, and tumor necrosis do not appear to have any influence on survival.
Anaplastic large cell lymphoma; ALK-positive; dura; prognosis; primary tumor
Background: Modified UDP-Gal donor substrates with 5-formylthienyl and 5-phenyl substituents on the uracil base exhibit differential inhibition patterns for glycosyltransferases.
Results: Structural studies reveal a new enzyme loop folding mode for the 5-formylthienyl analogue.
Conclusion: Differential inhibition is attributed to alternate enzyme conformational changes and interactions with the respective inhibitors.
Significance: The conformational plasticity of glycosyltransferases can be exploited in designing novel inhibitors.
Glycosyltransferases (GTs) are enzymes that are involved, as Nature's “glycosylation reagents,” in many fundamental biological processes including cell adhesion and blood group biosynthesis. Although of similar importance to that of other large enzyme families such as protein kinases and proteases, the undisputed potential of GTs for chemical biology and drug discovery has remained largely unrealized to date. This is due, at least in part, to a relative lack of GT inhibitors and tool compounds for structural, mechanistic, and cellular studies. In this study, we have used a novel class of GT donor analogues to obtain new structural and enzymological information for a representative blood group GT. These analogues interfere with the folding of an internal loop and the C terminus, which are essential for catalysis. Our experiments have led to the discovery of an entirely new active site folding mode for this enzyme family, which can be targeted in inhibitor development, similar to the DFG motif in protein kinases. Taken together, our results provide new insights into substrate binding, dynamics, and utilization in this important enzyme family, which can very likely be harnessed for the rational development of new GT inhibitors and probes.
Crystallography; Enzyme Inhibitors; Glycosyltransferases; Kinetics; Molecular Dynamics
Angiogenesis, the process by which new blood vessels are recruited to existing ones, is essential for tumor development. Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), which modulates bioavailability of IGF, has been studied for its potential role in angiogenesis during tissue regeneration and cancer development. In this study, we assessed the role of IGFBP-3 in tumor angiogenesis in head and neck squamous cell carcinoma (HNSCC) and human umbilical endothelial cells (HUVECs) using adenoviral (Ad-BP3) and recombinant (rBP3) IGFBP-3. Utilizing an in vivo orthotopic tongue tumor model, we confirmed that both Ad-BP3 and rBP3 suppress the growth of UMSCC38 HNSCC cells in vivo. Ad-BP3 inhibited vascularization in tongue tumors and chorio-allantoic membrane, and suppressed angiogenesis-stimulating activities in UMSCC38 cells. In HUVECs, Ad-BP3 decreased migration, invasion, and tube formation. rBP3 also suppressed production of VEGF in HUVECs and UMSCC38 cells. IGFBP-3-GGG, a mutant IGFBP-3 with loss of IGF binding capacity, suppressed VEGF production. In addition, we found that IGFBP-3 suppressed VEGF expression, even in mouse embryonic fibroblasts from an IGF-1R-null mouse. Finally, we demonstrated that IGFBP-3-GGG inhibits tumor angiogenesis and growth to the same degree as wild-type IGFBP-3. Taken together, these results support the hypothesis that IGFBP-3 has antiangiogenic activity in HNSCC, at least in part due to IGF-independent suppression of VEGF production from vascular endothelial cells and cancer cells.
Biofeedback therapy (BFT) can be unsuccessful in constipated patients, even those with pelvic floor dysfunction. Electrical stimulation therapy (EST) has been introduced as a novel therapeutic modality in patients with chronic constipation, especially those who have rectal hyposensitivity. We evaluated the efficacy of EST based on five years' clinical experience.
From January 2002 to February 2007, 159 patients underwent EST. After exclusion of 12 drop-outs, 147 (M:F = 61:86, 49 ± 17 years) finished all treatment sessions. Among them, 88 (M:F = 29:59, 49 ± 17 years) were refractory to BFT without rectal hyposensitivity (RH), and 59 (M:F = 32:27, 54 ± 17 years) were those with RH.
The overall response to EST was 59.2% (87/147) by per-protocol analysis. In the EST-responsive group, overall satisfaction improved significantly (from 7.3 ± 3.0 to 4.3 ± 2.5, P < 0.05). Subgroup analysis showed that the response rate was 64.8% (57/88) in patients refractory to BFT without RH, and 50.8% (30/59) in those with RH.
EST may have additional therapeutic efficacy in patients who are refractory to BFT. EST may also be effective in patients with RH, including restoration of rectal sensation. Therefore, EST could be considered as an alternative choice in patients refractory to BFT and with or without RH.
Biofeedback; Constipation; Electric stimulation therapy
The purpose of this study was to investigate whether selective cyclooxygenase (COX) inhibitors promote paclitaxel-induced apoptosis in taxane-resistant ovarian cancer cells by suppressing MDR1/P-glycoprotein (P-gp) expression.
Taxane-resistant ovarian cancer cells were cultured with paclitaxel alone or combined with a selective COX inhibitors. The expression patterns of MDR1/P-gp and the ability of COX inhibitors to inhibit growth of taxane-resistant ovarian cancer cells were measured. The efficacy of prostaglandin E2 (PGE2) supplementation was measured to evaluate the mechanisms involved in suppressing MDR1 gene expression.
P-gp was upregulated in taxane-resistant ovarian cancer cells compared to paired paclitaxel-sensitive ovarian cancer cells. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that selective COX inhibitors significantly enhanced the cytotoxic effects of paclitaxel in taxane-resistant ovarian cancer cells via a prostaglandin-independent mechanism. These increased apoptotic effects were further verified by measuring an increased percentage of cells in sub-G1 stage using flow cytometry. Selective COX inhibitors suppressed MDR1 and P-gp expression. Moreover, combined treatment with paclitaxel and selective COX inhibitors increased poly (ADP-ribose) polymerase (PARP) cleavage in taxane-resistant ovarian cancer cells.
Selective COX inhibitors significantly promote paclitaxel-induced cell death in taxane-resistant ovarian cancer cells in a prostaglandin-independent manner. COX inhibitors could be potent therapeutic tools to promote paclitaxel sensitization of taxane-resistant ovarian cancers by suppressing MDR1/P-gp, which is responsible for the efflux of chemotherapeutic agents.
Chemosensitizer; Cyclooxygenase inhibitor; Ovarian cancer; Paclitaxel; P-glycoprotein
A perivascular epithelioid cell (PEC) tumor is a rare mesenchymal tumor characterized by abundant cytoplasmic Periodic acid-Schiff positive glycogen (also called sugar tumor or clear cell tumor of the lung for this characteristic) and is mostly benign. We report a case of a 63-year-old man who presented with an enlarging mass on chest radiograph. After a thorough workup, diagnosis of malignant pulmonary PEC tumor with lung to lung metastases was established. Herein, the difficulties of diagnosis and management we confronted are described.
Perivascular epithelioid cell; Pecoma; Clear cell sugar tumor; Lung tumor