MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential.MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of alphaVbeta3, alpha6beta1 and alpha9beta1 integrins in xenograft cells. Treatment with bicistronic constructs reduced alphaVbeta3, alpha6beta1 and alpha9beta1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments.Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma

Association of pretreatment hippocampal volume with neurocognitive function in patients treated with hippocampal avoidance whole brain radiation therapy for brain metastases: Secondary analysis of NRG Oncology/RTOG 0933

PURPOSE: Hippocampal volume (HV) is an established predicting factor for neurocognitive function (NCF) in neurodegenerative disease. Whether the same phenomenon exists with hippocampal-avoidant whole brain radiation therapy is not known; therefore, we assessed the association of baseline HV with NCF among patients enrolled on RTOG 0933. METHODS AND MATERIALS: Hippocampal volume and total brain volume were calculated from the radiation therapy plan. Hippocampal volume was correlated with baseline and 4-month NCF scores (Hopkins Verbal Learning Test-Revised [HVLT-R] Total Recall [TR], Immediate Recognition, and Delayed Recall [DR]) using Pearson correlation. Deterioration in NCF was defined per the primary endpoint of RTOG 0933(mean 4-month relative decline in HVLT-R DR). Comparisons between patients with deteriorated and nondeteriorated NCF were made using the Wilcoxon test. RESULTS: Forty-two patients were evaluable. The median age was 56.5 years (range, 28-83 years), and 81% had a class II recursive partitioning analysis. The median total, right, and left HVs were 5.4 cm CONCLUSIONS: Larger HV was positively associated with improved performance on baseline and 4-month HVLT-R TR and DR scores in patients with brain metastases undergoing hippocampal-avoidant whole brain radiation therapy but was not associated with a change in NCF

Clinical implications of determination of safe surgical margins by using a combination of CT and 18FDG-positron emission tomography in soft tissue sarcoma

<p>Abstract</p> <p>Background</p> <p>To determine safe surgical margins for soft tissue sarcoma, it is essential to perform a general evaluation of the extent of tumor, responses to auxiliary therapy, and other factors preoperatively using multiple types of diagnostic imaging. 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) is a tool for diagnostic imaging that has recently spread rapidly in clinical use. At present, the roles played by FDG-PET/CT in determination of margins for surgical resection of sarcoma are unclear. The present study was undertaken to explore the roles of FDG-PET/CT in determination of surgical margins for soft tissue sarcoma and to examine whether PET can serve as a standard means for setting the margins of surgical resection during reduced surgery.</p> <p>Methods</p> <p>The study involved 7 patients with sarcoma who underwent surgery in our department and in whom evaluation with FDG-PET/CT was possible. Sarcoma was histologically rated as MFH in 6 cases and leiomyosarcoma in 1 case. In all cases, sarcoma was superficial (T1a or T2a). The tumor border was defined by contrast-enhanced MRI, and SUVs were measured at intervals of 1 cm over a 5-cm long area from the tumor border. Mapping of viable tumor cells was carried out on whole-mount sections of resected tissue, and SUVs were compared with histopathological findings.</p> <p>Results</p> <p>Preoperative maximum SUVs (SUV-max) of the tumor averaged 11.7 (range: 3.8-22.1). Mean SUV-max was 2.2 (range: 0.3-3.8) at 1 cm from the tumor border, 1.1 (0.85-1.47) at 2 cm, 0.83 (0.65-1.15) at 3 cm, 0.7 (0.42-0.95) at 4 cm, and 0.64 (0.45-0.82) at 5 cm. When resected tissue was mapped, tumor cells were absent in the areas where SUV-max was below 1.0.</p> <p>Conclusions</p> <p>Our findings suggest that a safe surgical margin free of viable tumor cells can be ensured if the SUV cut-off level is set at 1.0. FDG-PET/CT is promising as a diagnostic imaging technique for setting of safe minimal margins for surgical resection of soft tissue sarcoma.</p

Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells

<p>Abstract</p> <p>Background</p> <p>uPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts.</p> <p>Methods</p> <p>To investigate whether cholesterol could regulate uPAR and MMP-9 in breast carcinoma, we used MβCD (methyl beta cyclodextrin, which extracts cholesterol from lipid rafts) to disrupt lipid rafts and studied its effect on breast cancer cell migration, invasion, angiogenesis and signaling.</p> <p>Results</p> <p>Morphological evidence showed the association of uPAR with lipid rafts in breast carcinoma cells. MβCD treatment significantly reduced the colocalization of uPAR and MMP-9 with lipid raft markers and also significantly reduced uPAR and MMP-9 at both the protein and mRNA levels. Spheroid migration and invasion assays showed inhibition of breast carcinoma cell migration and invasion after MβCD treatment. <it>In vitro </it>angiogenesis studies showed a significant decrease in the angiogenic potential of cells pretreated with MβCD. MβCD treatment significantly reduced the levels of MMP-9 and uPAR in raft fractions of MDA-MB-231 and ZR 751 cells. Phosphorylated forms of Src, FAK, Cav, Akt and ERK were significantly inhibited upon MβCD treatment. Increased levels of soluble uPAR were observed upon MβCD treatment. Cholesterol supplementation restored uPAR expression to basal levels in breast carcinoma cell lines. Increased colocalization of uPAR with the lysosomal marker LAMP1 was observed in MβCD-treated cells when compared with untreated cells.</p> <p>Conclusion</p> <p>Taken together, our results suggest that cholesterol levels in lipid rafts are critical for the migration, invasion, and angiogenesis of breast carcinoma cells and could be a critical regulatory factor in these cancer cell processes mediated by uPAR and MMP-9.</p

Attracting and retaining health workers in rural areas: investigating nurses’ views on rural posts and policy interventions

<p>Abstract</p> <p>Background</p> <p>Kenya has bold plans for scaling up priority interventions nationwide, but faces major human resource challenges, with a lack of skilled workers especially in the most disadvantaged rural areas.</p> <p>Methods</p> <p>We investigated reasons for poor recruitment and retention in rural areas and potential policy interventions through quantitative and qualitative data collection with nursing trainees. We interviewed 345 trainees from four purposively selected Medical Training Colleges (MTCs) (166 pre-service and 179 upgrading trainees with prior work experience). Each interviewee completed a self-administered questionnaire including likert scale responses to statements about rural areas and interventions, and focus group discussions (FGDs) were conducted at each MTC.</p> <p>Results</p> <p>Likert scale responses indicated mixed perceptions of both living and working in rural areas, with a range of positive, negative and indifferent views expressed on average across different statements. The analysis showed that attitudes to working in rural areas were significantly positively affected by being older, but negatively affected by being an upgrading student. Attitudes to living in rural areas were significantly positively affected by being a student at the MTC furthest from Nairobi.</p> <p>During FGDs trainees raised both positive and negative aspects of rural life. Positive aspects included lower costs of living and more autonomy at work. Negative issues included poor infrastructure, inadequate education facilities and opportunities, higher workloads, and inadequate supplies and supervision. Particular concern was expressed about working in communities dominated by other tribes, reflecting Kenya’s recent election-related violence.</p> <p>Quantitative and qualitative data indicated that students believed several strategies could improve rural recruitment and retention, with particular emphasis on substantial rural allowances and the ability to choose their rural location. Other interventions highlighted included provision of decent housing, and more rapid career advancement. However, recently introduced short term contracts in named locations were not favoured due to their lack of pension plans and job security.</p> <p>Conclusions</p> <p>This study identified a range of potential interventions to increase rural recruitment and retention, with those most favored by nursing students being additional rural allowances, and allowing choice of rural location. Greater investment is needed in information systems to evaluate the impact of such policies.</p

Cystatin E/M suppresses legumain activity and invasion of human melanoma

<p>Abstract</p> <p>Background</p> <p>High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied.</p> <p>Methods</p> <p>A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay.</p> <p>Results</p> <p>Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay.</p> <p>Conclusions</p> <p>These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells.</p

Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches

<p>Abstract</p> <p>Background</p> <p>Drawing from previous studies, the traditional routine diagnostic microbiology evaluation of samples from chronic respiratory conditions may provide an incomplete picture of the bacteria present in airways disease. Here, the aim was to determine the extent to which routine diagnostic microbiology gave a different assessment of the species present in sputa when analysed by using culture-independent assessment.</p> <p>Methods</p> <p>Six different media used in routine diagnostic microbiology were inoculated with sputum from twelve patients. Bacterial growth on these plates was harvested and both RNA and DNA extracted. DNA and RNA were also extracted directly from the same sample of sputum. All nucleic acids served as templates for PCR and reverse transcriptase-PCR amplification of "broad range" bacterial 16S rRNA gene regions. The regions amplified were separated by Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling and compared to assess the degree of overlap between approaches.</p> <p>Results</p> <p>A mean of 16.3 (SD 10.0) separate T-RF band lengths in the profiles from each sputum sample by Direct Molecular Analysis, with a mean of 8.8 (SD 5.8) resolved by DNA profiling and 13.3 (SD 8.0) resolved by RNA profiling. In comparison, 8.8 (SD 4.4) T-RF bands were resolved in profiles generated by Culture-derived Molecular Analysis. There were a total of 184 instances of T-RF bands detected in the direct sputum profiles but not in the corresponding culture-derived profiles, representing 83 different T-RF band lengths. Amongst these were fifteen instances where the T-RF band represented more than 10% of the total band volume (with a mean value of 23.6%). Eight different T-RF band lengths were resolved as the dominant band in profiles generated directly from sputum. Of these, only three were detected in profiles generated from the corresponding set of cultures.</p> <p>Conclusion</p> <p>Due to their focus on isolation of a small group of recognised pathogens, the use of culture-dependent methods to analyse samples from chronic respiratory infections can provide a restricted understanding of the bacterial species present. The use of a culture-independent molecular approach here identifies that there are many bacterial species in samples from CF and COPD patients that may be clinically relevant.</p