Impact of flavonoids on matrix metalloproteinase secretion and invadopodia formation in highly invasive A431-III cancer cells.

Metastasis is a major cause of mortality in cancer patients. Invadopodia are considered to be crucial structures that allow cancer cells to penetrate across the extracellular matrix (ECM) by using matrix metalloproteinases (MMPs). Previously, we isolated a highly invasive A431-III subline from parental A431 cells by Boyden chamber assay. The A431-III cells possess higher invasive and migratory abilities, elevated levels of MMP-9 and an enhanced epithelial-mesenchymal transition (EMT) phenotype. In this study, we discovered that A431-III cells had an increased potential to form invadopodia and an improved capacity to degrade ECM compared with the original A431 cells. We also observed enhanced phosphorylation levels of cortactin and Src in A431-III cells; these phosphorylated proteins have been reported to be the main regulators of invadopodia formation. Flavonoids, almost ubiquitously distributed in food plants and plant food products, have been documented to exhibit anti-tumor properties. Therefore, it was of much interest to explore the effects of flavonoid antioxidants on the metastatic activity of A431-III cells. Exposure of A431-III cells to two potent dietary flavonoids, namely luteolin (Lu) and quercetin (Qu), caused inhibition of invadopodia formation and decrement in ECM degradation. We conclude that Lu and Qu attenuate the phosphorylation of cortactin and Src in A431-III cells. As a consequence, there ensues a disruption of invadopodia generation and the suppression of MMP secretion. These changes, in concert, bring about a reduction in metastasis

Automated Detection System Based on Convolution Neural Networks for Retained Root, Endodontic Treated Teeth, and Implant Recognition on Dental Panoramic Images

For a daily dental practice, the Panoramic (PANO) X-ray film is one of the most commonly used dental X-rays. One of its important advantages is the coverage of most anatomic structures and clinical findings in a single film. Important information about clinical treatment and diagnosis can be provided from the expert analysis of the PANO. Combined with the assistance of artificial intelligence, the application has great potential. The purpose of this study was to propose an automated detection system based on several modern convolutional neural networks (CNNs) for the classification of retained roots, endodontic treated teeth, and implants. In order to meet the standards of practical clinical application, the database used in this study is provided by dentists with more than three years of practical experience. The contributions of this work are given as follows: 1) proposed more advanced techniques for image segmentation and image position in dental radiographs; 2) a better image enhancement is proposed, which improves the accuracy of the five CNNs to more than 96%; and 3) combined with the fuzzy operation to achieve more powerful and accurate anomaly detection. The final result has an accuracy rate of up to 98.75%. It is about 20% higher than previous techniques. This research designed to identify and document each specific finding automatically could help dentists obtain an objective treatment evaluation and provide dentists more precious clinical time for dental operations and communication with patients

MMPs, especially MMP-9, were responsible for the invadopodia and degrading ability of A431-III cells.

<p>A, A431-III cells were plated on gelatin or Oregon Green® 488-conjugated gelatin and treated with DMSO or 25 µM GM6001 for 5 h to observe the formation of invadopodia and the matrix degrading ability. Tks5, invadopodia component protein, was used as a marker. B, Quantification of cells associated with matrix degradation (left panel). Quantification of degradation area normalized against cell number (right panel). C, Effect of GM6001 on MMPs’ activities and TIMPs’ expression were measured by zymography and western blot. D, The cells were treated with 40 nM MMP-9 siRNA or control siRNA. Knockdown efficiency was measured by qPCR (left) or gelatin zymography (right). E, A431-III cells (expressing control or MMP-9 knockdown siRNA) were plated on gelatin or Oregon Green® 488-conjugated gelatin to investigate the formation of invadopodia and the matrix degrading ability. F, Quantification of cells associated with matrix degradation (left panel) and degradation area normalized against cell number (right panel).*<i>p</i><0.05. Error bars present the standard error of the mean. Scale bar are 22 µm.</p

Effects of SU6656 on invadopodia formation and functioning.

<p>A, A431-III cells were plated on gelatin or Oregon Green® 488-conjugated gelatin and treated with DMSO or 5 µM SU6656 for 5 h to investigate the formation of invadopodia and matrix degradation. B, Quantification of cells associated with matrix degradation (upper panel). Quantification of the degradation area normalized against cell number (lower panel). C, Total cell lysates were prepared for immunoblotting analysis. Active Src and downstream target cortactin (Y421) were analyzed. D, Invasion assays were performed. *<i>p</i><0.05. P values are compared with control A431-III. Error bars present the standard error of the mean. Scale bar are 22 µm.</p

Src kinase activity and phosphorylation of cortactin were responsible for invadopodia formation in A431-III cells.

<p>A, Expression of invadopodia regulators, core components and MMPs/TIMPs in A431-P and A431-III were analyzed by microarray. B, Expression of invadopodia regulators, components and MMPs/TIMPs were validated by qPCR. C, Total cell lysates were subjected for immunoblotting analysis. The active status of Src kinase and the phosphorylation of cortactin were determined.</p

A431-III forms invadopodia and exhibits higher ability to degrade gelatin than A431-P.

<p>A, Upper panel: A431-P and A431-III cells were stained with cortactin (red), F-actin (green), and DAPI (blue). Arrowheads, examples of invadopodia that are identified as cortactin and actin-positive dots. Representative images taken of both cells. Lower panel: Both cells were plated on Oregon Green® 488-conjugated gelatin. Degraded ECM was identified as a dark area on the gelatin. B, Upper panel: Quantification of cells associated with matrix degradation. Lower panel: Quantification of the degradation area normalized against cell number. C, Invasion assays were performed. *<i>p</i><0.05. Error bars present the standard error of the mean. Scale bar are 22 µm. P (A431-P); III (A431-III).</p