Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects

p21Waf1 expression is regulated by nuclear intermediate filament vimentin in neuroblastoma

<p>Abstract</p> <p>Background</p> <p>Human neuroblastoma (NB) cell lines may present with either one of the so-called S-and N-subtypes. We have previously reported a strong correlation between protein expression levels of vimentin, an S-subtype marker, and the p21^Waf1cyclin-dependent kinase inhibitor. We here investigated whether this correlation extend to the mRNA level in NB cell lines as well as in patients' tumors. We also further explored the relationship between expression of vimentin and p21, by asking whether vimentin could regulate p21 expression.</p> <p>Methods</p> <p>Vimentin and p21 mRNA levels in NB cell lines as well as in patients' tumors (<it>n </it>= 77) were quantified using Q-PCR. Q-PCR data obtained from tumors of high risk NB patients (<it>n </it>= 40) were analyzed in relation with the overall survival using the Log-rank Kaplan-Meier estimation. siRNA-mediated depletion or overexpression of vimentin in highly or low expressing vimentin cell lines, respectively, followed by protein expression and promoter activation assays were used to assess the role of vimentin in modulating p21 expression.</p> <p>Results</p> <p>We extend the significant correlation between vimentin and p21 expression to the mRNA level in NB cell lines as well as in patients' tumors. Overall survival analysis from Q-PCR data obtained from tumors of high risk patients suggests that lower levels of p21 expression could be associated with a poorer outcome. Our data additionally indicate that the correlation observed between p21 and vimentin expression levels results from p21 transcriptional activity being regulated by vimentin. Indeed, downregulating vimentin resulted in a significant decrease in p21 mRNA and protein expression as well as in p21 promoter activity. Conversely, overexpressing vimentin triggered an increase in p21 promoter activity in cells with a nuclear expression of vimentin.</p> <p>Conclusion</p> <p>Our results suggest that p21 mRNA tumor expression level could represent a refined prognostic factor for high risk NB patients. Our data also show that vimentin regulates p21 transcription; this is the first demonstration of a gene regulating function for this type III-intermediate filament.</p

<Book Reviews> Ingemar Fagerlind and Lawrence J Saha Education and National Development : A Comparative Perspective

textabstractVarious modeling methods have been proposed to estimate the potential predictive ability of polygenic risk variants that predispose to various common diseases. However, it is unknown whether differences between them affect their conclusions on predictive ability. We reviewed input parameters, assumptions and output of the five most common methods and compared their estimates of the area under the receiver operating characteristic (ROC) curve (AUC) using hypothetical data representing effect sizes and frequencies of genetic variants, population disease risk and number of variants. To assess the accuracy of the estimated AUCs, we aimed to reproduce the AUCs of published empirical studies. All methods assumed that the combined effect of genetic variants on disease risk followed a multiplicative risk model of independent genetic effects, but they either assumed per allele, per genotype or dominant/recessive effects for the genetic variants. Modeling strategy and input parameters differed. Methods used simulation analysis or analytical formulas with effect sizes quantified by odds ratios (ORs) or relative risks. Estimated AUC values were similar for lower ORs (0.7) due to variants with strong effects, differences in estimated AUCs between methods increased. The simulation methods accurately reproduced the AUC values of empirical studies, but the analytical methods did not. We conclude that despite differences in input parameters, the modeling methods estimate similar AUC for realistic values of the ORs. When one or more variants have stronger effects and AUC values are higher, the simulation methods tend to be more accurate

Calpain-mediated vimentin cleavage occurs upstream of MT1-MMP membrane translocation to facilitate endothelial sprout initiation

Endothelial cells normally line the vasculature and remain quiescent. However, these cells can be rapidly stimulated to undergo morphogenesis and initiate new blood vessel formation given the proper cues. This study reports a new mechanism for initiating angiogenic sprout formation that involves vimentin, the major intermediate filament protein in endothelial cells. Initial studies confirmed vimentin was required for sphingosine 1-phosphate (S1P)- and growth factor (GF)-induced endothelial cell invasion, and vimentin was cleaved by calpains during invasion. Calpains were predominantly activated by GF and were required for sprout initiation. Because others have reported membrane type 1-matrix metalloproteinase (MT1-MMP) is required for endothelial sprouting responses, we tested whether vimentin and calpain acted upstream of MT1-MMP. Both calpain and vimentin were required for successful MT1-MMP membrane translocation, which was stimulated by S1P. In addition, vimentin complexed with MT1-MMP in a manner that required both the cytoplasmic domain of MT1-MMP and calpain activation, which increased the soluble pool of vimentin in endothelial cells. Altogether, these data indicate that pro-angiogenic signals converge to activate calpain-dependent vimentin cleavage and increase vimentin solubility, which act upstream to facilitate MT1-MMP membrane translocation, resulting in successful endothelial sprout formation in three-dimensional collagen matrices. These findings help explain why S1P and GF synergize to stimulate robust sprouting in 3D collagen matrices

Quantitative evaluation of vimentin expression in tumour stroma of colorectal cancer

Recent studies have identified vimentin, a type III intermediate filament, among genes differentially expressed in tumours with more invasive features, suggesting an association between vimentin and tumour progression. The aim of this study, was to investigate whether vimentin expression in colon cancer tissue is of clinical relevance. We performed immunostaining in 142 colorectal cancer (CRC) samples and quantified the amount of vimentin expression using computer-assisted image analysis. Vimentin expression in the tumour stroma of CRC was associated with shorter survival. Overall survival in the high vimentin expression group was 71.2% compared with 90.4% in the low-expression group (P=0.002), whereas disease-free survival for the high-expression group was 62.7% compared with 86.7% for the low-expression group (P=0.001). Furthermore, the prognostic power of vimentin for disease recurrence was maintained in both stage II and III CRC. Multivariate analysis suggested that vimentin was a better prognostic indicator for disease recurrence (risk ratio=3.5) than the widely used lymph node status (risk ratio=2.2). Vimentin expression in the tumour stroma may reflect a higher malignant potential of the tumour and may be a useful predictive marker for disease recurrence in CRC patients

Do African-American men need separate prostate cancer screening guidelines?

BACKGROUND: In 2012, the United States Preventative Services Task Force issued new guidelines recommending that male U.S. residents, irrespective of race, no longer be screened for prostate cancer. In African American men, the incidence of prostate cancer is almost 60 % higher and the mortality rate is two to three times greater than in Caucasians. The purpose of this study is to reduce African American men's prostate cancer burden by demonstrating they need separate screening guidelines. METHODS: We performed a PubMed search using the keywords: African American, Prostate cancer, Outcomes, Molecular markers, Prostate-specific Antigen velocity, PSA density, and to derive data relevant to our hypothesis. RESULTS: In our literature review, we identified several aspects of prostate cancer that are different in Caucasian and African American men. These included prostate cancer incidence and outcome, the clinical course of the disease, serum PSA levels, genetic differences, and social barriers. It's also important to note that the USPSTF guidelines were based on two studies, one of which reported that only 4 % of its participants were African American. The other did not report demographic information, but used participants from seven European countries with small African American populations. CONCLUSION: Given the above, we conclude that separate prostate cancer screening guidelines are greatly necessary to help save the lives of African Americans