Assessing abnormal corneal endothelial cells from in vivo confocal microscopy images using a fully automated deep learning system.

BACKGROUND: The goal of this study is to develop a fully automated segmentation and morphometric parameter estimation system for assessing abnormal corneal endothelial cells (CECs) from LASER in vivo confocal microscopy (IVCM) images. METHODS: First, we developed a fully automated deep learning system for assessing abnormal CECs using a previous development set composed of normal images and a newly constructed development set composed of abnormal images. Second, two testing sets, one with 169 normal images and the other with 211 abnormal images, were used to evaluate the clinical validity and effectiveness of the proposed system on LASER IVCM images with different corneal endothelial conditions, particularly on abnormal images. Third, the automatically calculated endothelial cell density (ECD) and the manually calculated ECD were compared using both the previous and proposed systems. RESULTS: The automated morphometric parameter estimations of the average number of cells, ECD, coefficient of variation in cell area and percentage of hexagonal cells were 257 cells, 2648 ± 511 cells/mm2, 32.18 ± 6.70% and 56.23 ± 8.69% for the normal CEC testing set and 83 cells, 1450 ± 656 cells/mm2, 34.87 ± 10.53% and 42.55 ± 20.64% for the abnormal CEC testing set. Furthermore, for the abnormal CEC testing set, Pearson's correlation coefficient between the automatically and manually calculated ECDs was 0.9447; the 95% limits of agreement between the manually and automatically calculated ECDs were between 329.0 and - 579.5 (concordance correlation coefficient = 0.93). CONCLUSIONS: This is the first report to count and analyze the morphology of abnormal CECs in LASER IVCM images using deep learning. Deep learning produces highly objective evaluation indicators for LASER IVCM corneal endothelium images and greatly expands the range of applications for LASER IVCM.

Fully automated grading system for the evaluation of punctate epithelial erosions using deep neural networks.

PURPOSE: The goal was to develop a fully automated grading system for the evaluation of punctate epithelial erosions (PEEs) using deep neural networks. METHODS: A fully automated system was developed to detect corneal position and grade staining severity given a corneal fluorescein staining image. The fully automated pipeline consists of the following three steps: a corneal segmentation model extracts corneal area; five image patches are cropped from the staining image based on the five subregions of extracted cornea; a staining grading model predicts a score for each image patch from 0 to 3, and automated grading score for the whole cornea is obtained from 0 to 15. Finally, the clinical grading scores annotated by three ophthalmologists were compared with automated grading scores. RESULTS: For corneal segmentation, the segmentation model achieved an intersection over union of 0.937. For punctate staining grading, the grading model achieved a classification accuracy of 76.5% and an area under the receiver operating characteristic curve of 0.940 (95% CI 0.932 to 0.949). For the fully automated pipeline, Pearson's correlation coefficient between the clinical and automated grading scores was 0.908 (p<0.01). Bland-Altman analysis revealed 95% limits of agreement between the clinical and automated grading scores of between -4.125 and 3.720 (concordance correlation coefficient=0.904). The average time required for processing a single stained image during pipeline was 0.58 s. CONCLUSION: A fully automated grading system was developed to evaluate PEEs. The grading results may serve as a reference for ophthalmologists in clinical trials and residency training procedures.

A Fully Automated Segmentation and Morphometric Parameter Estimation System for Assessing Corneal Endothelial Cell Images.

PURPOSE: To develop a fully automated segmentation and morphometric parameter estimation system for assessing corneal endothelial cells from in vivo confocal microscopy images. DESIGN: Artificial intelligence (neural network) study. METHODS: First, a fully automated deep learning system for assessing corneal endothelial cells was developed using the development set (from 99 subjects). Second, 184 images (from 97 subjects) were used to construct the testing set to evaluate the clinical validity and usefulness of the automated segmentation and morphometric system. Third, the automatically calculated endothelial cell density (ECD) values, Topcon's cell density, and manually calculated ECD were compared. RESULTS: After slit lamp examination, 88 healthy subjects, 2 Fuchs endothelial dystrophy patients, and 7 corneal endotheliitis patients were identified among the 97 subjects in the testing set. The automatedly estimated morphometric parameters for the testing set were an average number of 234 cells, an ECD of 2592 cells/mm2, a coefficient of variation in the cell area of 32.14%, and a percentage of hexagonal cells of 54.16%. Pearson's correlation coefficient between the automated ECD and Topcon's cell density and between the manually calculated ECD and Topcon's cell density was 0.932 (P < .01) and 0.818 (P < .01), respectively. The Bland-Altman plot of Topcon's cell density and the automated ECD yielded 95% limits of agreement between 271.94 and -572.46 (concordance correlation coefficient = 0.9). CONCLUSIONS: A fully automated method for segmenting corneal endothelial cells and estimating morphometric parameters using in vivo confocal microscopy images is more efficient and accurate for assessing the normal corneal endothelium.

SFRP4 is a prognostic marker and correlated with Treg cell infiltration in pancreatic ductal adenocarcinoma.

Secreted Frizzled-Related Protein 4 (SFRP4), a member of secreted frizzled-related protein family, has been found as a vital modulator in cell proliferation, cell self-renew and apoptosis through Wnt signaling transduction pathway. In the present study, we re-analyzed the expression pattern of SFRPs in Gene Expression Omnibus (GEO) datasets and evaluated the expression of SFRP4 at protein level in both KrasG12D/+; Trp53R172H/+; Pdx1-Cre; (KPC) mice and human pancreatic ductal adenocarcinoma (PDAC) tissue. We found that the expression of SFRP4 increased gradually in PanINs and PDAC lesions in KPC mice and high expression of SFRP4 was much more common in tumor lesions compared to the adjacent non-tumor tissues. Then we performed Kaplan-Meier survival and Cox regression analysis and found that high expression of SFRP4 in the serum and tumor lesions predicted poor prognosis for pancreatic cancer patients. Furthermore, we demonstrated that SFRP4 positively correlated with FOXP3+ Treg cells infiltration while the down-regulation of SFRP4 in tumor cells impaired the production of cytokines and the recruitments of T cells. This study suggested that SFRP4 can be a novel prognostic biomarker and potential therapeutic target for pancreatic cancer.

Developing a safety heatmap of uncontrolled intersections using both conflict probability and severity.

This paper presents a method to assess the safety of uncontrolled intersections considering two major properties of traffic conflicts-conflict probability and severity. This method assesses both the safety level of the entire intersection in addition to the distribution of safety within it. Intersections are modeled by a two-dimensional Cartesian coordinate system and the internal space of intersections is divided into cells. First, the vehicle movement characteristics of an uncontrolled intersection are modeled. Second, the conflict probability of each cell within the intersection is estimated considering the approaching probability and lateral migration probability of vehicles. The quantification of conflict severity is based on kinetic energy loss of potential crashes. Cluster analysis is used to combine conflict probability and severity to model the safety assessment of each cell. Third, the application of the method is discussed, and an overall safety index of intersections is proposed which considers weighted safety level and relative value of areas of different safety levels. Finally, a case study, which includes three different designs, is presented along with safety heatmaps to demonstrate the results. The results not only demonstrate the validity of the model, but also indicate that the proposed method can be applied to: i) safety evaluation of build-up intersections; ii) dangerous position management within an intersection; iii) safety assessment of designed intersections, and iv) safety level comparison among different intersections or various designs for a single intersection. Using this method, engineers and planners can better evaluate and improve the safety of existing or future uncontrolled intersections.

Establishment and characterization of 7 novel hepatocellular carcinoma cell lines from patient-derived tumor xenografts.

Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903) by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR) analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3) was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening.

HZ08 reverse the aneuploidy-induced cisplatin-resistance in Gastric cancer by modulating the p53 pathway.

We evaluated the influence of DNA aneuploidy on chemotherapy-resistance in human Gastric cancer cell MKN45; we also evaluated the reversal effects of HZ08 on these cells and then preliminary investigated the possible involved pathway. We made use of a pair of human Gastric cancer cell dip-MKN45 (diploid MKN45) and aneu-MKN45 (aneuploid MKN45). Growth inhibition in response to chemotherapeutic drugs was evaluated by CellTiter-Glo Luminescent Cell Viability assay and clone formation assay. Flow cytometry and immuno-assay were applied to evaluate apoptosis and the expression of relative signaling molecules. MKN45 xenograft was generated to evaluate in vivo action. Aneu-MKN45 developed a resistance to cisplatin which could be reversed by HZ08; Flow cytometry and western-blot indicates that HZ08-combination could induce apoptosis and increase the expression of apoptosis-related biomarkers on aneu-MKN45; in vivo study also reflect the same correlation between aneuploidy and cisplatin-resistance, which could be antagonized by HZ08 combination; When investigating the involved pathway, in anue-MKN45, the expression of molecules in p53 pathway was decreased; HZ08 could increase the expression of p53 down-stream molecules as well as elevate the activity of p53, while inhibiting Mdm2, the major negative regulator of p53; p53 inhibitor Pifithrin-α could completely abrogate HZ08's synergism effects, and mimic cisplatin-resistance on dip-MKN45.Lower p53 pathway expression that attenuates cisplatin-induced apoptosis might be at least partly the reason of cisplatin-resistance occurred in aneuploid MKN45 both in vitro and in vivo; Combination of HZ08 could sensitize cisplatin-induced apoptosis through the activation of the p53 pathway, therefore represented a synergism effect on aneuploid MKN45 cells.

Multi-drug-resistant cells enriched from chronic myeloid leukemia cells by Doxorubicin possess tumor-initiating-cell properties.

Multiple drug resistance (MDR) occurring during chemotherapy is a major obstacle for treatment of cancers using chemotherapeutic drugs; thus, the mechanisms underlying MDR have attracted intensive attention. Many studies have shown that tumor-initiating cells exhibit a chemotherapeutic tolerance characteristic. However, whether the MDR cells possess tumor-initiating cells properties and its underlying mechanisms remain to be fully elucidated. In this study, we utilized a well-established MDR cell line K562/A02 enriched by doxorubicin from K562 cells to determine if the K562/A02 cells possess tumor-initiating properties and investigated its potential molecular mechanisms. We observed that the expressions of Oct4, Sox2, and Nanog, all of which are well-characterized stem cell markers, in K562/A02 cells were elevated in comparison to parental K562 cells; in addition, we found that K562/A02 cells exhibited more potent in vitro and in vivo tumor-initiating properties, as revealed by sphere assay, self-renewal assay, soft agar assay, and animal studies. Furthermore, our data suggest that snail and twist1, two well known transcriptional factors for the epithelial-mesenchymal transition (EMT) program, may be potentially involved in the acquisition of tumor-initiating properties of K562/A02 cells. Thus, our study demonstrates that MDR K562/A02 cells possess tumor-initiating properties, most likely due to the elevated expressions of snail and twist1.

Transactivation of the human NME5 gene by Sp1 in pancreatic cancer cells.

Non-metastatic cells 5 (NME5), a recently found gene belonging to the NDPK-like molecules gene family, is highly expressed in testis and some types of human cancer. Current studies have revealed diverse potential functions of NME5 and we have reported that NME5 is associated with innate resistance to gemcitabine in human pancreatic cancer cells in previous study. However, the mechanism underlying the transcriptional regulation of NME5 has not been elucidated yet. In this study, we analyzed the 5'-flanking region of the human NME5 gene and revealed its transcription start site (TSS) at -35 bp relative to its translation start codon ATG. Using 5' unidirectional deletion analysis, we demonstrated that the proximal promoter of NME5 is located within -1051 bp to +35 bp. Two functional GC-boxes (-300 bp and -323 bp) were identified within the promoter region. Mutation of either GC-box led to significant reduction in NME5 promoter activity, whereas overexpression of Sp1 activated NME5 promoter activity in MIA PaCa-2 and 293T cells. In silico analysis predicted that transcription factor Sp1 binds to both GC-boxes, which were confirmed by EMSA and ChIP. In addition, we found that compared with MIA PaCa-2, Sp1 was highly expressed in PAXC002, a well characterized human pancreatic cancer cell line with innate gemcitabine resistance where NME5 was reported to be highly expressed, indicating that Sp1 induces NEM5 expression in PAXC002 cells. In conclusion, our study characterized for the first time the human NME5 promoter which is controlled by Sp1 transcription factor in pancreatic cancer.

Interleukin-8, a promising predictor for prognosis of pancreatic cancer.

AIM: To investigate the value of interleukin-8 (IL-8), a pro-inflammatory chemokine, in predicting the prognosis of pancreatic cancer. METHODS: Expression of IL-8 and its receptor CXCR1 was assessed by immunohistochemistry in pancreatic cancer and chronic pancreatitis samples. Enzyme-linked immunosorbent assay was used to detect the serum IL-8 levels in pancreatic cancer patients. Human pancreatic cancer tissues were heterotopically transplanted to the immune-deficiency mice to evaluate the effect of serum IL-8 on the tumorigenesis of the cancer samples. RESULTS: IL-8 and CXCR1 proteins were both over-expressed in pancreatic adenocarcinoma samples (55.6% and 65.4%, respectively) compared with the matched para-cancer tissues (25.9% and 12.3%, P < 0.01), or chronic pancreatitis (0% and 25%, P < 0.05). Serum IL-8 levels in pancreatic cancer patients (271.1 ± 187.7 ng/mL) were higher than in other digestive system tumors, such as gastric cancer (41.77 ± 9.11 ng/mL, P = 0.025), colorectal carcinoma (78.72 ± 80.60 ng/mL, P = 0.032) and hepatocellular carcinoma (59.60 ± 19.80 ng/mL, P = 0.016). In vivo tumorigenesis analysis further proved that tumor tissues from patients with higher serum IL-8 levels grew faster than those with lower IL-8 levels. CONCLUSION: IL-8 can be a fine serum marker for predicting the prognosis pancreatic cancer.

Identification of NME5 as a contributor to innate resistance to gemcitabine in pancreatic cancer cells.

The limited therapeutic effect of gemcitabine on pancreatic cancer is largely attributed to pre-existing or acquired resistance of the tumor cells. This study was aimed at screening for candidate resistance-related gene(s) and elucidating the underlying mechanisms. NME5 was found to be highly expressed in an innate gemcitabine-resistant human pancreatic cancer sample and the cell line PAXC002 derived from the sample. Downregulation of NME5 significantly reversed gemcitabine resistance in PAXC002 cells, whereas NME5 overexpression induced gemcitabine resistance in the pancreatic cancer cell line BxPC-3. NME5 attenuated the induction of apoptosis and cell cycle arrest induced by gemcitabine, probably accounting for the blunted sensitivity to gemcitabine. Furthermore, NME5 was demonstrated to play its role in a nuclear factor kappaB (NF-κB)-dependent manner. NME5 was capable of directly binding NF-κB, and possibly regulated its expression level in PAXC002 cells. Our results also suggest that NF-κB is a key executor of NME5 in regulating apoptosis and cell cycle. All of these data suggest that NME5 is a promising target for relieving innate gemcitabine resistance in pancreatic cancer cells.

Intrinsic gemcitabine resistance in a novel pancreatic cancer cell line is associated with cancer stem cell-like phenotype.

Pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal malignancies in the world, often diagnosed at an advanced stage, resistant to conventional chemotherapy and having high invasive and metastatic potential. The mechanism of drug resistance of PDA is still not clear. In the present study, we established two novel pancreatic cancer cell lines PAXC-002 and PAXC-003 from human primary xenograft models. The cell lines were characterized by morphology, karyotype, pancreatic cancer marker and short tandem repeat (STR) analysis, and growth kinetics and tumorigenicity. The in vitro anti-proliferation test revealed that PAXC-002 cell was intrinsically resistant to the standard of care chemotherapy-gemcitabine, compared with that of PAXC-003 and other widely used pancreatic cancer cell lines. Interestingly, the gemcitabine resistant PAXC-002 cell line was more potent in forming colonies in 3-Dimensional matrigel culture conditions and had a higher percentage of CD133 positive cells, which is recognized as a cancer stem cell marker, compared to the gemcitabine-sensitive PAXC-003 cell line. In this study, we present two novel pancreatic cancer cell lines which could be used for gemcitabine resistance investigation, mechanism identification of pancreatic cancer and anticancer drug screening. The preliminary data indicate that the drug resistance of pancreatic carcinoma cells is associated with a cancer stem cell-like phenotype.

Recombinant adenovirus carrying the hepatocyte nuclear factor-1alpha gene inhibits hepatocellular carcinoma xenograft growth in mice.

UNLABELLED: Hepatocyte nuclear factor-1alpha (HNF1α) is one of the key transcription factors of the HNF family, which plays a critical role in hepatocyte differentiation. Substantial evidence has suggested that down-regulation of HNF1α may contribute to the development of hepatocellular carcinoma (HCC). Herein, human cancer cells and tumor-associated fibroblasts (TAFs) were isolated from human HCC tissues, respectively. A recombinant adenovirus carrying the HNF1α gene (AdHNF1α) was constructed to determine its effect on HCC in vitro and in vivo. Our results demonstrated that HCC cells and HCC tissues revealed reduced expression of HNF1α. Forced reexpression of HNF1α significantly suppressed the proliferation of HCC cells and TAFs and inhibited the clonogenic growth of hepatoma cells in vitro. In parallel, HNF1α overexpression reestablished the expression of certain liver-specific genes and microRNA 192 and 194 levels, with a resultant increase in p21 levels and induction of G(2)/M arrest. Additionally, AdHNF1α inhibited the expression of cluster of differentiation 133 and epithelial cell adhesion molecule and the signal pathways of the mammalian target of rapamycin and transforming growth factor beta/Smads. Furthermore, HNF1α abolished the tumorigenicity of hepatoma cells in vivo. Most interestingly, intratumoral injection of AdHNF1α significantly inhibited the growth of subcutaneous HCC xenografts in nude mice. Systemic delivery of AdHNF1α could eradicate the orthotopic liver HCC nodules in nonobese diabetic/severe combined immunodeficiency mice. CONCLUSION: These results suggest that the potent inhibitive effect of HNF1α on HCC is attained by inducing the differentiation of hepatoma cells into mature hepatocytes and G(2)/M arrest. HNF1α might represent a novel, promising therapeutic agent for human HCC treatment. Our findings also encourage the evaluation of differentiation therapy for tumors of organs other than liver using their corresponding differentiation-determining transcription factor.

Downregulation of Dickkopf-1 is responsible for high proliferation of breast cancer cells via losing control of Wnt/beta-catenin signaling.

AIM: To investigate the role of DKK-1/Wnt/beta-catenin signaling in high proliferation of LM-MCF-7 breast cancer cells, a sub-clone of MCF-7 cell line. METHODS: Two cell lines (MCF-7 and LM-MCF-7) with different proliferation abilities were used. LM-MCF-7 cells were transiently transfected with the pcDNA3-DKK-1 plasmid encoding the DKK-1 gene (or MCF-7 cells were transfected siRNA targeting DKK-1 mRNA). Flow cytometry analysis and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay were applied to detect the cell proliferation. The expression levels of beta-catenin, phosphorylated beta-catenin, c-Myc, cyclin D1 and Survivin were examined by Western blot analysis. The regulation of Survivin was investigated by Luciferase reporter gene assay. RESULTS: Western blot and RT-PCR analysis showed that the expression level of DKK-1 was downregulated in LM-MCF-7 relative to MCF-7 cells. Flow cytometry and BrdU incorporation assay showed DKK-1 could suppress growth of breast cancer cells. Overexpression of DKK-1 was able to accelerate phosphorylation-dependent degradation of beta-catenin and downregulate the expression of beta-catenin, c-Myc, cyclin D1 and Survivin. Luciferase reporter gene assay demonstrated that Survivin could be regulated by beta-catenin/TCF4 pathway. CONCLUSION: We conclude that the downregulation of DKK-1 is responsible for the high proliferation ability of LM-MCF-7 breast cancer cells via losing control of Wnt/beta-catenin signaling pathway, in which c-Myc, cyclinD1 and Survivin serve as essential downstream effectors. Our finding provides a new insight into the mechanism of breast cancer cell proliferation.

Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine.

The actin-myosin interaction of vascular smooth muscle cells (VSMCs) is regulated by myosin light chain kinase (MLCK), which is a fusion protein of the central catalytic domain with the N-terminal actin-binding and C-terminal myosin-binding domains. In addition to the regulatory role of kinase activity mediated by the catalytic domain, nonkinase activity that derives from both terminals is able to exert a regulatory role as reviewed by Nakamura et al. (32). We previously showed that nonkinase activity mediated the filopodia upon the stimulation by sphingosylphosphorylcholine (SPC) (25). To explore the regulatory role of nonkinase activity in chemotaxis, we constructed VSMCs where the expression of MLCK was totally abolished by using a lentivirus-mediated RNAi system. We hypothesized that the MLCK-downregulated VSMCs were unable to form filopodia and to migrate upon SPC stimulation and confirmed the hypothesis. We further constructed a kinase-inactive mutant from bovine cDNA coding wild-type (WT) MLCK by mutating the ATP-binding sites located in the catalytic domain, followed by confirming the presence (absence) of the kinase activity of WT (kinase-inactive mutant). We transfected WT and the mutant into MLCK-downregulated VSMCs. We expected that the transfected VSMCs will recover the ability to induce filopodia and chemotaxis toward SPC and found both constructs rescued the ability. Because they share the actin- and myosin-binding domains, we concluded nonkinase activity plays a major role for SPC-induced migration.

Resveratrol inhibits hypoxia-induced metastasis potential enhancement by restricting hypoxia-induced factor-1 alpha expression in colon carcinoma cells.

Resveratrol has been shown recently to exhibit antimetastatic effect on various human solid tumors. However, the possible molecular mechanism for its antimetastatic action needs to be elucidated. In this study, we investigated the effect of resveratrol on metastasis potential of colon carcinoma cells under normoxia and hypoxia in vitro. These results showed that, resveratrol can restrict the migration, adhesion, invasion and MMP-9 and MMP-2 secretion in Lovo cells cultured under normoxia and hypoxia. Hypoxia and iron chelator 2,2'-dipyridyl treatment can stimulate the invasion and migration enhancement of Lovo cells, while resveratrol exhibited substantial resistance on the metastasis potential stimulation by inhibiting the mRNA expression of VEGF and MMP-9 in colon carcinoma cells under normoxia and hypoxia, reducing HIF-1 alpha protein expression under hypoxia. Also, iron chelator 2,2'-dipyridyl treatment showed approximately the same effect on metastasis potential as Lovo cells cultured under hypoxia. These data demonstrated that, the antimetastatic effect of resveratrol under hypoxia were associated with the restriction of HIF-1 alpha protein expression and stabilization, which could be a promising drug target for resveratrol in the development of an effective chemopreventive and anticancer therapy in human tumors.

Blebbistatin inhibits the chemotaxis of vascular smooth muscle cells by disrupting the myosin II-actin interaction.

Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (IC(50) = 26.1 +/- 0.2 and 27.5 +/- 0.5 microM for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC(50) = 32.3 +/- 0.9 and 31.6 +/- 1.3 muM for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the Mg(2+)-ATPase activity of the unphosphorylated (IC(50) = 12.6 +/- 1.6 and 4.3 +/- 0.5 microM for gizzard and bovine stomach, respectively) and phosphorylated (IC(50) = 15.0 +/- 0.6 microM for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the Mg(2+)-ATPase activities of gizzard myosin II fragments, heavy meromyosin (IC(50) = 14.4 +/- 1.6 microM) and subfragment 1 (IC(50) = 5.5 +/- 0.4 microM), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II.

Proliferation and migration mediated by Dkk-1/Wnt/beta-catenin cascade in a model of hepatocellular carcinoma cells.

Beta-catenin is a multifunctional protein acting as a key factor in the cadherin-mediated cell-cell adhesion system and in the Wnt signaling pathway. To demonstrate the molecular mechanisms of metastasis of hepatocellular carcinoma (HCC) cells, we established a metastatic subclone of human HCC H7402 cells, termed M-H7402, by isolating from transplantation of H7402 cells into severe combined immunodeficient (SCID) mice. Based on the 2 parallel cell lines, we investigated the roles of dickkopf-1 (Dkk-1) and Wnt/beta-catenin pathway in proliferation and migration of HCC cells. cDNA microarray showed that 24 genes were related to tumor metastasis differentially expressed between H7402 and M-H7402 cells. Western blot analysis revealed that the expression levels of beta-catenin, c-Myc, and cyclin D1 were upregulated, but Dkk-1 and nm23 were dramatically downregulated in M-H7402 cells, which suggests that the 2 cell lines were remarkably different in molecular events associated with metastasis. Furthermore, we found that overexpression of Dkk-1 by transfection was able to downregulate the expression of c-Myc and cyclin D1, and it also inhibited the growth and migration in M-H7402 cells. Although reduction of Dkk-1 expression by RNA interference was able to upregulate the expression of beta-catenin, c-Myc, and cyclin D1 in H7402 cells, it also promoted beta-catenin translocation from cytoplasm into nuclei and increased the migration of the cells. Therefore, we conclude that Dkk-1/Wnt/beta-catenin cascade may mediate the proliferation and migration of HCC cells during the metastasis process.