Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Fascin is a globular actin cross-linking protein, which plays a major role in forming parallel actin bundles in cell protrusions and is found to be associated with tumor cell invasion and metastasis in various type of cancers including oral squamous cell carcinoma (OSCC). Previously, we have demonstrated that fascin regulates actin polymerization and thereby promotes cell motility in K8-depleted OSCC cells. In the present study we have investigated the role of fascin in tumor progression of OSCC.</p> <p>Methods</p> <p>To understand the role of fascin in OSCC development and/or progression, fascin was overexpressed along with vector control in OSCC derived cells AW13516. The phenotype was studied using wound healing, Boyden chamber, cell adhesion, Hanging drop, soft agar and tumorigenicity assays. Further, fascin expression was examined in human OSCC samples (N = 131) using immunohistochemistry and level of its expression was correlated with clinico-pathological parameters of the patients.</p> <p>Results</p> <p>Fascin overexpression in OSCC derived cells led to significant increase in cell migration, cell invasion and MMP-2 activity. In addition these cells demonstrated increased levels of phosphorylated AKT, ERK1/2 and JNK1/2. Our in vitro results were consistent with correlative studies of fascin expression with the clinico-pathological parameters of the OSCC patients. Fascin expression in OSCC showed statistically significant correlation with increased tumor stage (<it>P </it>= 0.041), increased lymph node metastasis (<it>P </it>= 0.001), less differentiation (<it>P </it>= 0.005), increased recurrence (<it>P </it>= 0.038) and shorter survival (<it>P </it>= 0.004) of the patients.</p> <p>Conclusion</p> <p>In conclusion, our results indicate that fascin promotes tumor progression and activates AKT and MAPK pathways in OSCC-derived cells. Further, our correlative studies of fascin expression in OSCC with clinico-pathological parameters of the patients indicate that fascin may prove to be useful in prognostication and treatment of OSCC.</p

Agnikarma in Ayurveda- A review article

Ayurveda is the everlasting supreme science of medicine because it deals with promotion of health and curing diseases. Sushruta known as father of surgery has described various surgical and para surgical measures. Sushruta has mentioned different methods of management of diseases, such as Bheshaja karma, Ksharkarma, Agnikarma and Shastrakarma. In this fast lifestyle patients need instant result on all pain. Agnikarma is one such procedure and it is believed that disease treayed with this never reoccurs. This Agnikarma is original idea of modern cauterization procedure. This review deals with various aspects of Agnikarma

Prognostic role of Oct4, CD44 and c-Myc in radio-chemo-resistant oral cancer patients and their tumourigenic potential in immunodeficient mice

In the present study, we have investigated the prognostic value of known stem cell-associated molecules such as Oct4, CD44 and c-Myc in patients with oral SCC who had received post-surgery radio- and/or chemotherapy. Immunohistochemistry was performed to analyse the expression of Oct4, CD44 and c-Myc in 87 tumour tissues, and the expression profile obtained was correlated with clinicopathological parameters of the patients with oral cancer. Tumourigenic potential of these molecules was also evaluated by in vivo studies. Our results showed significant correlation of Oct4 (OS, p = 0.003; DFS, p = 0.001) and c-Myc (OS, p = 0.01; DFS, p = 0.03) with overall survival and disease-free survival independently. Furthermore, all the three markers in combinations of two markers each, i.e. Oct4 + CD44 (OS, p = 0.003; DFS, p = 0.001), Oct4 + c-Myc (OS, p = 0.0001; DFS, p = 0.0001), CD44 + c-Myc (OS, p = 0.008; DFS, p = 0.02) and in combinations of three markers each, i.e. Oct4 + CD44 + c-Myc (OS, p = 0.0001; DFS, p = 0.0001) also significantly correlated with overall survival and disease-free survival. Univariate and multivariate analyses further established the independent prognostic value of Oct4. Oct4-, CD44- and c-Myc-enriched populations independently induced sarcomatoid carcinomas whereas primary keratinocytes developed poorly differentiated carcinomas in immunodeficient mice. Oct4 and c-Myc independently as well as in combination with CD44 might be useful for the prediction of local recurrence and poor survival of patients with oral cancer which is the novel finding of this study. Oct4, c-Myc and CD44 can be used to predict local recurrence and the outcome of treatment in oral cancer patients. In addition, these molecules may find use as molecular targets for effective therapy

Understanding the Role of Keratins 8 and 18 in Neoplastic Potential of Breast Cancer Derived Cell Lines

<div><h3>Background</h3><p>Breast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER), progesterone receptor (PGR) and epidermal growth factor receptor 2 (HER2) in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K) are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood.</p> <h3>Methodology/Principal Findings</h3><p>In this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, <em>in-vitro</em> invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion <em>in-vitro</em>, while MCF10A did not show any changes in cell transformation assays.</p> <h3>Conclusions/Significance</h3><p>These results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer.</p> </div

Analysis of change in <i>in-vitro</i> invasion on K8 up−/down-regulation.

<p>Representative 10X images of H&E stained membrane showing invaded cells. (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing number of invaded cells of MDA MB 435 K8 overexpressed (K8C1 and C2) and vector control (K8Vc) clones (lower panel). (<b>B</b>) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. Histogram showing number of invaded cells of MDA MB 468 K8 down-regulated (ShC1, C2 and C3) and vector control (Vc) clones (lower panel). (<b>C</b>) K8 down-regulated MCF10A (MShC1) and vector control (MVc) clones. Histogram showing number of invaded cells of MCF10A K8 down-regulated (MShC1, C2 and C3) and vector control (MVc) clones (lower panel). Results are mean of ± SE of three independent experiments performed <b>Note:</b> Decreased invasion in K8 over-expressed MDA MB 435 clones (K8C1 and C2) and increased invasion in K8 down-regulated MDA MB 468 clones (ShC1, C2, and C3) as compared to their respective vector controls (K8Vc) and (Vc).</p

Analysis of vimentin expression in K8 (over-expressed) MDA-MB-435 and K8 (down-regulated) MDA-MB-468 and MCF10A clones.

<p>Representative confocal images of vimentin filaments (<b>A</b>) K8 over-expressed MDA MB 435 (K8C1) and vector control (K8Vc) clones. (<b>B</b>) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. (<b>C</b>) MCF10A K8 down-regulated (MshC1) and vector control (MVc) clones. Scale bar: 10 µm. For measuring the intensity of the cells the 4% laser with emission filter band pass of 505–550 was used. All the scanning conditions of gain offset and laser percentage were kept same and applied for all images of vector and their respective clones with secondary control as threshold. The mean fluorescence intensity of 20 cells was calculated and the average of three independent experiments was calculated. Results are mean of ± SE of three independent experiments performed <b>Note:</b> No change in vimentin expression or filament formation on K8 up−/down-regulation.</p

Analysis of change in motility on K8 up−/down-regulation.

<p>Representative 10X Phase contrast images of time lapse microscopy at 0 hour and 20 hours showing wound healing (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing % wound closure at the end of 20 hours of MDA MB 435 K8 over-expressed (K8C1 and C2) and vector control (K8Vc) clones (lower panel). (<b>B</b>) MDAMB 468 K8 down-regulated (ShC1) and vector control (Vc) clone. Histogram showing % wound closure at the end of 20 hours of MDA MB 468 K8 down-regulated (ShC1, C2and C3) and vector control (Vc) clones (lower panel). (<b>C</b>) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clone. Histogram showing % wound closure at the end of 20 hours of MCF10A K8 down-regulated (MShC1, C2and C3) and vector control (MVc) clones (lower panel). Results are mean of ± SE of three independent experiments performed. Migration rate was calculated by AxioVision software. <b>Note:</b> Significant Increase in motility in K8 down-regulated MDA MB 468 clones as compared to vector control. Motility of K8 up−/down-regulated clones by transwell assay: Representative images of H &E stained migrated cells (<b>D</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing number of migrated cells at the end of 16 hours of MDA MB 435K8 over-expressed (K8C1 and C2) and vector control (K8Vc) clones (lower panel). (<b>E</b>) MDAMB 468 K8 down-regulated (ShC1) and vector control (Vc) clone. Histogram showing number of migrated cells at the end of 16 hours of MDA MB 468 K8 knockdown clones (ShC1, C2 and C3) and vector control (Vc) clones (lower panel). (<b>F</b>) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clone. Histogram showing number of migrated cells of MCF10A K8 down-regulated (MShC1, C2 and C3) and vector control (MVc) clones (lower panel). Results are mean of ± SE of three independent experiments performed <b>Note:</b> Significant decrease in motility in K8 over-expressed MDA MB 435 clones and significant increase in motility in K8 down-regulated MDA MB 468 clones (**p<0.01 by students t-test).</p

Analysis of changes in soft agar colony forming potential and cell proliferation on K8 up−/down-regulation.

<p>Representative phase contrast images (10X) of colonies formed in soft agar (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing number of colonies of MDA MB 435 K8 overexpressed (K8C1, and C2) and vector control (K8Vc) clones (right hand side). (<b>C</b>) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. Histogram showing number of colonies of MDA MB 468 K8 down-regulated (ShC1, C2and C3) and vector control (Vc) clones (right hand side). (<b>E</b>) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clones. Histogram showing number of colonies of MCF10A K8 down-regulated (MshC1, C2 and C3) and vector control (MVc) clones (right hand side). <b>Note:</b> Increase in soft agar colonies formed in K8 down-regulated MDA MB 468 clones (*p<0.05 by students t-test). Cell proliferation curves of (<b>B</b>) MDA MB 435 K8 overexpressed (K8C1, C2 and C3) and vector control (K8Vc) clones. (<b>D</b>) MDA MB 468 K8 down-regulated (ShC1, C2 and C3) and vector control (Vc) clones. (<b>F</b>) MCF10A K8 down-regulated (MShC1, C2 and C3) and vector control (MVc) clones. Cell proliferation was plotted against time. Results are mean ± SE of three independent experiments performed in triplicate. <b>Note:</b> Decrease in proliferation in K8 over-expressed clones of MDA MB 435 (*p<0.05, **p<0.01 by students t-test).</p

Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression

<div><p>To study multistep tumorigenesis process, there is a need of <i>in-vitro</i> 3D model simulating <i>in-vivo</i> tissue. Present study aimed to reconstitute <i>in-vitro</i> tissue models comprising various stages of neoplastic progression of tongue tumorigenesis and to evaluate the utility of these models to investigate the role of stromal fibroblasts in maintenance of desmosomal anchoring junctions using transmission electron microscopy. We reconstituted <i>in-vitro</i> models representing normal, dysplastic, and malignant tissues by seeding primary keratinocytes on either fibroblast embedded in collagen matrix or plain collagen matrix in growth factor-free medium. The findings of histomorphometry, immunohistochemistry, and electron microscopy analyses of the three types of 3D cultures showed that the stratified growth, cell proliferation, and differentiation were comparable between co-cultures and their respective native tissues; however, they largely differed in cultures grown without fibroblasts. The immunostaining intensity of proteins, viz., desmoplakin, desmoglein, and plakoglobin, was reduced as the disease stage increased in all co-cultures as observed in respective native tissues. Desmosome-like structures were identified using immunogold labeling in these cultures. Moreover, electron microscopic observations revealed that the desmosome number and their length were significantly reduced and intercellular spaces were increased in cultures grown without fibroblasts when compared with their co-culture counterparts. Our results showed that the major steps of tongue tumorigenesis can be reproduced <i>in-vitro</i>. Stromal fibroblasts play a role in regulation of epithelial thickness, cell proliferation, differentiation, and maintenance of desmosomalanchoring junctions in <i>in-vitro</i> grown tissues. The reconstituted co-culture models could help to answer various biological questions especially related to tongue tumorigenesis.</p></div