Establishment and characterization of an immortalized renal cell line of the Chinese tree shrew (Tupaia belangeri chinesis).

The Chinese tree shrew holds a great potential as a viable animal model in biomedical research, especially for infectious diseases and neuropsychiatric disorders. A thorough understanding of the innate immunity, which represents the first line that defends the host against viral infection, of the Chinese tree shrew, is needed. However, the progress is hindered by the lack of a proper cell line for research usage. In this study, we established a cell line that is applicable to the study of tree shrew innate immune responses against viral infections. The Chinese tree shrew primary renal cells (TSPRCs) were immortalized by simian virus 40 large T antigen (SV40LT) transduction, and the immortalized cells were termed TSR6 (tree shrew renal cell #6). TSR6 showed a similar morphology to TSPRCs and expressed the epithelial cell-specific marker cytokeratin 18 (KRT18). In addition, TSR6 could be transfected by transfection reagent and was suitable for CRISPR/Cas9-mediated gene editing. Infection of Newcastle disease virus (NDV) or herpes simplex virus 1 (HSV-1) in TSR6 induced the mRNA expression of tree shrew interferon-ß (tIFNB1) and myxovirus resistance protein 1 (tMx1) in a dose- and time-dependent manner. Collectively, we successfully established a tree shrew renal cell line and demonstrated that this cell line was suitable for the study of the innate immune response to viral infections.

Prognostic value and clinicopathological significance of serum- and tissue-based cytokeratin 18 express level in breast cancer: a meta-analysis.

Cytokeratin 18 (CK18), a type I cytokeratin of the intermediate filament family, has been associated with the prognosis of cancer patients for decades. However, its exact role in predicting the clinical outcome of breast cancer remains controversial. To comprehensively investigated the prognostic value of CK18 in breast cancer, a systematically meta-analysis was conducted to explore the association between CK18 expression and overall survival. Literature collection was conducted by retrieving electronic databases Pubmed, Cochrane Library, Web of Science, EMBASE, and OVID completely (up to January 1, 2017). Nine relevant studies with 4857 cases assessing the relationship between CK18 high expression and the outcome of breast cancer patients were enrolled in our analysis. The results indicated that the high level of CK18 expression was significantly associated with overall survival of breast cancer patients via a specimen-depended manner. Reports which used serum to detect the expression of CK18 predicted a poor outcome of breast cancer (HR = 1.24, 95%CI: 1.11-1.38, P<0.0001), while studies which used tissue as specimen indicated a reverse result (HR = 0.71, 95%CI: 0.60-0.84, P<0.00001). Moreover, overexpression of CK18 was highly relevant to advanced clinicopathological parameters of breast cancer, such as progesterone receptor, human epidermal growth factor receptor-2, tumor size, tumor stage, nodal status, and tumor grade. Taken together, the present study demonstrated that CK18 might be served as a novel biomarker to predict clinicopathological features and the outcome of breast cancer.

Cytokeratin 18 is necessary for initiation of TGF-ß1-induced epithelial-mesenchymal transition in breast epithelial cells.

During epithelial-mesenchymal transition (EMT), epithelial cells lose key phenotypic markers (e.g., E-cadherin and cytokeratin 18) and acquire mesenchymal markers (e.g., N-cadherin and vimentin). Although the loss of cytokeratin 18 is a hallmark of EMT, the regulatory role of cytokeratin 18 in EMT is not yet fully understood. Here, we report that cytokeratin 18 is involved in the regulation of transforming growth factor-beta1 (TGF-ß1)-induced EMT in breast epithelial cells. When MCF10A cells were treated with TGF-ß1 for 24 h, considerable morphological changes, indicative of the early stages of EMT (e.g., loss of cell-cell contact), were observed and cytokeratin 18 was downregulated. However, E-cadherin levels were not altered until a later time point. This suggests that cytokeratin 18 may play an active role during the earlier stages of EMT. Consistent with this notion, siRNA-mediated knockdown of cytokeratin 18 delayed TGF-ß1-mediated EMT, and the associated downregulation of E-cadherin reduced the phosphorylation/nuclear localization of smad 2/3 and decreased the expression levels of snail and slug (which inhibit E-cadherin expression in epithelial cells as an early response to TGF-ß1). Taken together, these results suggest that cytokeratin 18 critically contributes to initiating TGF-ß1-induced EMT via the smad 2/3-mediated regulation of snail and slug expression in breast epithelial cells.

Wingless ligand 5a is a critical regulator of placental growth and survival.

The maternal uterine environment is likely critical for human placental morphogenesis and development of its different trophoblast subtypes. However, factors controlling growth and differentiation of these cells during early gestation remain poorly elucidated. Herein, we provide evidence that the ligand Wnt5a could be a critical regulator of trophoblast proliferation and survival. Immunofluorescence of tissues and western blot analyses of primary cultures revealed abundant Wnt5a expression and secretion from first trimester decidual and villous stromal cells. The ligand was also detectable in decidual glands, macrophages and NK cells. Wnt5a increased proliferation of villous cytotrophoblasts and cell column trophoblasts, outgrowth on collagen I as well as cyclin A and D1 expression in floating explant cultures, but suppressed camptothecin-induced apoptosis. Similarly, Wnt5a stimulated BrdU incorporation and decreased caspase-cleaved cytokeratin 18 neo-epitope expression in primary cytotrophoblasts. Moreover, Wnt5a promoted activation of the MAPK pathway in the different trophoblast models. Chemical inhibition of p42/44 MAPK abolished cyclin D1 expression and Wnt5a-stimulated proliferation. Compared to controls, MAPK phosphorylation and proliferation of cytotrophoblasts declined upon supplementation of supernatants from Wnt5a gene-silenced decidual or villous stromal cells. In summary, non-canonical Wnt5a signalling could play a role in early human trophoblast development by promoting cell proliferation and survival.

The Impact of Liver Cell Injury on Health-Related Quality of Life in Patients with Chronic Liver Disease.

BACKGROUND: Patients with chronic liver disease often suffer from unspecific symptoms and report severe impairment in the quality of life. The underlying mechanisms are multifactorial and include disease-specific but also liver related causes. The current analysis evaluated the association of hepatocellular apoptosis in non-viral chronic liver disease and health-related quality of life (HRQL). Furthermore we examined factors, which influence patient's physical and mental well-being. METHODS: A total of 150 patients with non-infectious chronic liver disease were included between January 2014 and June 2015. The German version of the Chronic Liver Disease Questionnaire (CLDQ-D), a liver disease specific instrument to assess HRQL, was employed. Hepatocellular apoptosis was determined by measuring Cytokeratin 18 (CK18, M30 Apoptosense ELISA). RESULTS: Female gender (5.24 vs. 5.54, p = 0.04), diabetes mellitus type II (4.75 vs. 5.46, p<0.001) and daily drug intake (5.24 vs. 6.01, p = 0.003) were associated with a significant impairment in HRQL. HRQL was not significantly different between the examined liver diseases. Levels of CK18 were the highest in patients with NASH compared to all other disease entities (p<0.001). Interestingly, CK18 exhibited significant correlations with obesity (p<0.001) and hyperlipidemia (p<0.001). In patients with cirrhosis levels of CK18 correlated with the MELD score (r = 0.18, p = 0.03) and were significantly higher compared to patients without existing cirrhosis (265.5 U/l vs. 186.9U/l, p = 0.047). Additionally, CK18 showed a significant correlation with the presence and the degree of hepatic fibrosis (p = 0.003) and inflammation (p<0.001) in liver histology. Finally, there was a small negative association between CLDQ and CK18 (r = -0.16, p = 0.048). CONCLUSION: Different parameters are influencing HRQL and CK18 levels in chronic non-viral liver disease and the amount of hepatocellular apoptosis correlates with the impairment in HRQL in chronic non-viral liver diseases. These findings support the role of liver-protective therapies for the improvement of the quality of life in chronic liver disease.

[DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES INDUCED BY RAT FIBROTIC LIVER TISSUE EXTRACTS].

OBJECTIVE: To investigate the differentiation potential of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes induced by rat fibrotic liver tissue extracts. METHODS: Liver fibrosis was induced in the Sprague Dawley rats (weighting, 180-220 g) by repeated intraperitoneal injections of 3% thioacetamide-saline at a dose of 200 mg/kg twice a week for 4 weeks; fibrotic liver tissues were used to prepare liver homogenate supernatants. The HUCMSCs at passage 3 were cultured in DMEM/F12 with 10% fetal bovine serum (FBS) (control group) and in DMEM/F12 with 10% FBS and 50 g/L liver homogenate supernatants (experimental group) for 7 days. The morphological changes of the cells were recorded; the protein levels of cytokeratin 18 (CK18), alpha fetoprotein (AFP), and CYP3A4 were measured using Western blot. The glycogen storing ability of the cells was detected by periodic acid-schiff (PAS) staining. Furthermore, the synthesis of albumin (ALB) and blood urea nitrogen (BUN) was measured. RESULTS: In experimental group, after 1 day of induction, the stem cells of fusiform shape began to lose sharp edges and progressively shrunk, and then they changed into hepatocyte-like cells with round and irregular shape at 7 days. Positive expressions of AFP, CK18, and CYP3A4 were observed in the experimental group, but negative expression in the control group. The concentrations of BUN and ALB were (0.43 ± 0.07) mmol/L and (8.08 ± 0.41) µg/mL in the control group and were (2.52 ± 0.20) mmol/L and (41.48 ± 4.11) µg/mL in the experimental group, showing significant differences (t=24.160, P = 0.000; t = 19.810, P = 0.000). PAS staining results showed navy blue nucleus and lavender cytoplasm in the control group, but dark purple cell body and visible nucleus in the experimental group. CONCLUSION: HUCMSCs could differentiate into hepatocyte-like cells induced by rat fibrotic liver tissue extracts, which have hepatocyte biomarkers (AFP, CK18, and CYP3A4) and hepatocyte-specific functions of glycogen storage, urea production and ALB secretion, so they could partially replace the function of hepatocytes, that may be one of the therapeutic mechanisms of stem cell transplantation.

Galectin-9 suppresses cholangiocarcinoma cell proliferation by inducing apoptosis but not cell cycle arrest.

Cholangiocarcinoma is the most common biliary malignancy and the second most common hepatic malignancy after hepatocellular carcinoma (HCC). Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has recently been shown to exert antiproliferative effects on cancer cells. Therefore, the present study evaluated the effects of Gal-9 on the proliferation of human cholangiocarcinoma cells in vitro as well as the microRNAs (miRNAs) associated with the antitumor effects of Gal-9. Gal-9 suppressed the proliferation of cholangiocarcinoma cell lines in vitro and the growth of human cholangiocarcinoma cell xenografts in nude mice. Our data further revealed that Gal-9 increased caspase­cleaved keratin 18 (CCK18) levels, and the expression of cytochrome c increased in Gal-9-treated cholangiocarcinoma cell lines. These data suggested that Gal-9 induced cholangiocarcinoma cell apoptosis via the intrinsic apoptosis pathway mediated by caspase-dependent or -independent pathways. In addition, Gal-9 reduced the phosphorylation of the epidermal growth factor receptor (EGFR), insulin-like growth factor and insulin-like growth factor-1 receptor (IGF-1R), hepatocyte growth factor receptor and fibroblast growth factor receptor 3 (FGFR3). These findings suggest that Gal-9 can be a candidate of therapeutic target in the treatment of cholangiocarcinoma.

[Detection of cytokeratin18 and cytokeratin19 gene expression in blood and tumor tissue of nasopharyngeal carcinoma patients by RT-PCR].

UNLABELLED: Abstract OBJECTIVE: To measure the expression of CK18 and CK19 in the cells from peripheral blood and tumor tissue of the nasopharyngeal carcinoma patients,to test whether CK 18 and CK 19 could be biomarkers of nasopharyngeal carcinoma fordiagnosis. METHOD: The mRNA was extracted from the blood and carcinoma tissue of nasopharyngeal carcinoma and was reversed transcription to cDNA. The 3 pairs primers were designed for RT-PCR and the fold value was calculated to evaluated expression by ΔCT. RESULT: There are no statistical differences between the CK18 and CK19 gene expression and the gender, age and metastasis in tumor tissue of 45 nasopharyngeal carcinoma patients (P>0. 05). There are significant differences among 3 pathological stages and 2 genes expressed increase as the grade malignancy (P<0. 05). The detecting of the 2 genes expression from blood cells shows that CK18 and CK19 had a high positive ratio 64% and 75% respectively. Meanwhile this method showed a same detection characteristic in tumor and blood, the positive.rate of CK18 and CK19 genes in metastasis is higher than non-metastasis. The results showed CK18 has a high specificity and CK19 has a high sensitivity for prognosis and all relapsed cases are associated with the expression of CK18 and CK19. CONCLUSION: CK18 and CK19 may be used as biomarkers of nasopharyngeal carcinoma for diagnosis.

Oblongifolin C inhibits metastasis by up-regulating keratin 18 and tubulins.

Tumor metastasis is the main cause of cancer-related patient death. In this study, we performed a wound healing migration screen to search for a metastatic inhibitor within our library of natural compounds. We found that oblongifolin C (OC), a natural compound extracted from Garcinia yunnanensis Hu, is an effective inhibitor of metastasis in human esophageal squamous carcinoma Eca109 cells. The transwell migration and matrigel invasion assay results also showed that OC inhibits the migration of Eca109 cells and HepG2 cells. OC can increase the expression of tubulin, indicating that OC inhibits metastasis via tubulin aggregation. In addition, the Western blotting, real-time PCR, and immunostaining results indicated that OC increases the expression of keratin18. Furthermore, the knockdown of keratin 18 by small interfering RNAs inhibited the expression of tubulin and increased the metastasis of cancer cells, suggesting that keratin 18 is the upstream signal of tubulin and plays a vital role in metastasis. A subsequent study in a tail vein injection metastasis model showed that OC can significantly inhibit pulmonary metastasis, as revealed by immunohistochemistry staining. Taken together, our results suggest that OC inhibits metastasis through the induction of the expression of keratin 18 and may be useful in cancer therapy.

Absence of keratins 8 and 18 in rodent epithelial cell lines associates with keratin gene mutation and DNA methylation: Cell line selective effects on cell invasion.

Epithelial-mesenchymal transition (EMT) in carcinoma is associated with dramatic up-regulation of vimentin and down-regulation of the simple-type keratins 8 and 18 (K8/K18), but the mechanisms of these changes are poorly understood. We demonstrate that two commonly-studied murine (CT26) and rat (IEC-6) intestinal cell lines have negligible K8/K18 but high vimentin protein expression. Proteasome inhibition led to a limited increase in K18 but not K8 stabilization, thereby indicating that K8/K18 absence is not due, in large part, to increased protein turnover. CT26 and IEC-6 cells had <10% of normal K8/K18 mRNA and exhibited decreased mRNA stability, with K8 mRNA levels being higher in IEC-6 versus CT26 and K18 being higher in CT26 versus IEC-6 cells. Keratin gene sequencing showed that KRT8 in CT26 cells had a 21-nucleotide deletion while K18 in IEC-6 cells had a 9-amino acid in-frame insertion. Furthermore, the KRT8 promoter in CT26 and the KRT18 promoter in IEC-6 are hypermethylated. Inhibition of DNA methylation using 5-azacytidine increased K8 or K18 in some but all the tested rodent epithelial cell lines. Restoring K8 and K18 by lentiviral transduction reduced CT26 but not IEC-6 cell matrigel invasion. K8/K18 re-introduction also decreased E-cadherin expression in IEC-6 but not CT26 cells, suggesting that the effect of keratin expression on epithelial to mesenchymal transition is cell-line dependent. Therefore, some commonly utilized rodent epithelial cell lines, unexpectedly, manifest barely detectable keratin expression but have high levels of vimentin. In the CT26 and IEC-6 intestinal cell lines, keratin expression correlates with keratin gene insertion or deletion and with promoter methylation, which likely suppress keratin transcription and mRNA or protein stability.

The M30 assay does not detect apoptosis in epithelial-derived cancer cells expressing low levels of cytokeratin 18.

The primary aim of this study was to compare measurement of apoptosis by M30 immunoreactivity (a biomarker for apoptosis) to other apoptosis assays (morphological assessment of nuclei, Annexin-V-FITC staining, DNA fragmentation and PARP cleavage) in vitro. Caspase-cleaved cytokeratin 18 (M30, ccK18) is only produced in epithelial cells and is regarded as a pharmacodynamic biomarker of apoptotic cell death because it is released from cells during apoptosis induced by chemotherapeutic agents. However, we have observed false negative results using this assay in clinical samples. Therefore, we tested its ability to accurately detect apoptosis in a panel of lung cancer cell lines with a range of clinically approved chemotherapeutic drugs. Three different non-small cell lung cancer (NSCLC) cell lines (A549, H1299, PC3) were used to correlate M30 levels with alternate apoptosis assays. Following successful induction of apoptosis, the A549 cell line showed an increase in M30 levels along with other well-known features of apoptosis, whilst H1299 and PC3 cell lines did not show an increase in M30 levels, even when apoptosis was detected by other means. Further analysis showed that H1299 and PC3 cell lines expressed much lower levels of cytokeratin 18 protein compared to the A549 cell line. Our results suggest that reliable detection of apoptosis via the M30 assay only works when sufficient levels of cytokeratin 18 are present in the cells. This means that the M30 assay may result in false negative results for apoptosis, and as such, the ELISA should be used in conjunction with other assays.

The effects of human serum to the morphology, proliferation and gene expression level of the respiratory epithelium in vitro.

The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. The present study focused on investigating the effects of human serum (HS) on the qualitative and quantitative properties of the human respiratory epithelium compared to the fetal bovine serum (FBS), as a supplement in culture. Respiratory epithelial (RE) cells derived from human nasal turbinate were co-cultured with fibroblasts, subsequently separated at 80-90% confluency by differential trypsinization. RE cells were then sub-cultured into 2 different plates containing 5% allogenic HS and FBS supplemented media respectively up to passage 1 (P1). Cell morphology, growth rate, cell viability and population doubling time were assessed under light microscope, and levels of gene expression were measured via real time reverse transcriptase-polymerase chain reaction (qRT-PCR). RE cells appeared as polygonal shape and expanded when cultured in HS whereas RE cells in FBS were observed to be easily matured thus limit the RE cells expansion. Proliferation rate of RE cells in HS supplemented media (7673.18 ± 1207.15) was 3 times higher compared to RE in FBS supplemented media (2357.68 ± 186.85). Furthermore, RE cells cultured in HS-supplemented media required fewer days (9.15 ± 1.10) to double in numbers compared to cells cultured in FBS-supplemented media (13.66 ± 0.81). Both the differences were significant (p<0.05). However, there were no significant differences in the viability of RE cells in both groups (p=0.105). qRT-PCR showed comparable expressions of gene Cytokeratin-14 (CK-14), Cytokeratin-18 (CK-18) and Mucin-5 subtype B (MUC5B) in RE cells cultured in both groups (p>0.05). In conclusion, HS is a comparatively better choice of media supplement in accelerating growth kinetics of RE cells in vitro thus producing a better quality of respiratory epithelium for future tracheal reconstruction.

Differentially expressed proteins in human breast cancer cells sensitive and resistant to paclitaxel.

The resistance of cancer cells to chemotherapeutic drugs represents a major problem in cancer treatment. Despite all efforts, mechanisms of resistance have not yet been elucidated. To reveal proteins that could be involved in resistance to taxanes, we compared protein expression in whole cell lysates of SK-BR-3 breast cancer cells sensitive to paclitaxel and in lysates of the same line with acquired resistance to paclitaxel. The resistant SK-BR-3 cell line was established in our lab. Protein separation was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectro-metry. With these techniques we identified four proteins with different expression in resistant SK-BR-3 cells, i.e., serpin B3, serpin B4, heat shock protein 27 (all three upregulated) and cytokeratin 18 (downregulated). Observed changes were confirmed using western blot analysis. This study suggests new directions worthy of further study in the effort to reveal the mechanism of resistance to paclitaxel in breast cancer cells.

Carcinoma initiation via RB tumor suppressor inactivation: a versatile approach to epithelial subtype-dependent cancer initiation in diverse tissues.

Carcinomas arise in a complex microenvironment consisting of multiple distinct epithelial lineages surrounded by a variety of stromal cell types. Understanding cancer etiologies requires evaluating the relationship among cell types during disease initiation and through progression. Genetically engineered mouse (GEM) models facilitate the prospective examination of early oncogenic events, which is not possible in humans. Since most solid tumors harbor aberrations in the RB network, we developed an inducible GEM approach for the establishment and assessment of carcinoma initiation in a diverse range of epithelial tissues and subtypes upon inactivation of RB-mediated tumor suppression (RB-TS). The system allows independent assessment of epithelial subtypes that express either cytokeratins (K) 18 or 19. By Cre-dependent expression of a protein that dominantly inactivates RB and functionally redundant proteins p107 and p130, neoplasia could be initiated in either K18 or K19 expressing cells of numerous tissues. By design, because only a single pathway aberration was engineered, carcinomas developed stochastically only after long latency. Hence, this system, which allows for directed cell type-specific carcinoma initiation, facilitates further definition of events that can progress neoplasms to aggressive cancers via engineered, carcinogen-induced and/or spontaneous evolution.

The predictive ability of a CK5/p63/CK8/18 antibody cocktail in stratifying breast papillary lesions on needle biopsy: an algorithmic approach works best.

OBJECTIVES: Immunohistochemical markers have been shown to assist in the stratification of breast papillary lesions. We evaluated the ability of different cytokeratin (CK) and p63 expression profiles on needle biopsy specimens to predict excision diagnoses. METHODS: A CK5/p63/CK8/18 antibody cocktail was applied to 58 needle biopsy specimens (32 papillomas, 7 atypical papillomas, 19 papillary carcinomas on excision). RESULTS: p63 expression was greater in papillomas than in atypical papillomas (P = .044) and papillary carcinomas (P< .0001). Papillary carcinomas and atypical papillomas showed greater CK8/18 expression (and conversely less CK5 expression) than did papillomas (P < .0001). Negative or focal p63 expression was 96% sensitive for diagnosing any atypical lesion (atypical papilloma or papillary carcinoma) on excision, whereas CK8/18 predominant expression (≥80% cells) was 100% sensitive. In contrast, the sensitivity of the original diagnosis was only 81%. The greatest accuracy for the diagnosis of atypical papillary lesions (95%) was achieved when both p63 and cytokeratins were used in combination in an algorithmic fashion. This method also correctly identified all cases that had papillary carcinoma (100% sensitivity) on excision. CONCLUSIONS: Although a single stain or combination cannot independently stratify papillary lesions, a CK5/p63/CK8/18 antibody cocktail is a useful adjunct to morphology for evaluating breast papillary lesions in needle biopsy specimens.

The clinical significance of apoptosis and M30 expression in colonic cancer progression.

BACKGROUND/AIM: The aim of this study is to identify the significance of M30, an early apoptosis indicator, in colorectal cancer and its liver metastasis. PATIENTS AND METHODS: The expression of M30 was immunohistochemically estimated at colonic and liver metastatic tissues of 66 patients. The results were correlated to clinical and pathological features of the tumors. RESULTS: High expression of M30 was observed in 15.5% of cases. No metastatic tissue showed expression of M30, while stage D tumors (metastasis included) showed a statistic significant lower expression of M30, when compared to earlier tumor stages. CONCLUSION: Low expression of M30 implies the development of resistance mechanisms against apoptosis, facilitating the progression of colon cancer.

Effect of erythropoietin on mesenchymal stem cell differentiation and secretion in vitro in an acute kidney injury microenvironment.

We investigated the effect of erythropoietin (EPO) on differentiation and secretion of bone marrow-derived mesenchymal stem cells in an acute kidney injury microenvironment. Acute kidney injury mouse models were prepared. Both renal cortices were then immediately collected to produce the ischemia/reperfusion kidney homogenate supernatant. The morphological and ultrastructural changes in the cells were observed using an inverted microscope and a transmission electron microscope. Cytokeratin-18 was detected using flow cytometry. Bone morphogenetic protein-7 levels, hepatocyte growth factor, and vascular endothelial growth factor in the culture medium were detected using an enzyme-linked immunosorbent assay. The cells had high CD29 and CD44 expression, as well as low CD34 and CD45 expression. More round and oval cells with cobble-like appearances were observed after EPO treatment. In addition, an increase in the number of rough endoplasmic reticula, lysosomes, and mitochondria was observed in the cytoplasm; the intercellular junction peculiar to epithelial cells was also seen on the cell surface. After treatment with ischemia/reperfusion kidney homogenate supernatant, cytokeratin-18 expression increased significantly and EPO could magnify its expression. Bone morphogenetic protein-7 levels, hepatocyte growth factor, and vascular endothelial growth factor levels after treatment with ischemia/reperfusion kidney homogenate supernatant significantly decreased, whereas EPO increased the cytokine secretion. The acute kidney injury microenvironment can induce the bone marrow-derived mesenchymal stem cells to partially differentiate into renal tubular epithelium-shaped cells, but weaken their secretion function. EPO intervention can boost up their differentiation function and reverse their low secretion effect.

The prognostic importance of changing serum M30 and M65 values after chemotherapy in patients with advanced-stage non-small-cell lung cancer.

Although oncological treatments are improving, the prognosis of non-small-cell lung cancer (NSCLC) patients has not. Several biomarkers related to prognosis have been evaluated, and M30 and M65 have been reported to be higher in patients with NSCLC than in healthy people. In the current study, we evaluated the clinical importance of the change in serum M30 and M65 values after chemotherapy in patients with NSCLC. Serum M30 and M65 values were measured before and 48 h after chemotherapy in thirty-two patients with advanced NSCLC. The importance of the change in the levels of these markers after chemotherapy was analyzed by univariate analysis. The median serum M65 and M30 values increased significantly after chemotherapy (p < 0.001). The median M30 value after chemotherapy was an important prognostic factor for both overall survival (OS) (p = 0.002) and progression-free survival (PFS) (p = 0.002). Stage and histopathological type were significant both for PFS and OS. Multivariate analysis showed that the median M30 value after chemotherapy was the only independent prognostic factor for PFS (p = 0.04, HR 5.4) and OS (p = 0.02, HR 11.49). Our results indicated that both serum M30 and M65 values increased after chemotherapy in patients with advanced NSCLC, and an elevated serum M30 value was an independent prognostic factor for both PFS and OS.

Keratin 8 and 18 loss in epithelial cancer cells increases collective cell migration and cisplatin sensitivity through claudin1 up-regulation.

Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression.

Regulation of DCIS to invasive breast cancer progression by Singleminded-2s (SIM2s).

Singleminded-2s (SIM2s) is a member of the bHLH/PAS family of transcription factors and a key regulator of mammary epithelial cell differentiation. SIM2s is highly expressed in mammary epithelial cells and downregulated in human breast cancer. Loss of Sim2s causes aberrant mouse mammary ductal development, with features suggestive of malignant transformation, whereas overexpression of SIM2s promotes precocious alveolar differentiation in nulliparous mouse mammary glands, suggesting that SIM2s is required for establishing and enhancing mammary gland differentiation. To test the hypothesis that SIM2s regulates tumor cell differentiation, we analyzed SIM2s expression in human primary breast ductal carcinoma in situ (DCIS) samples and found that SIM2s is lost with progression from DCIS to invasive ductal cancer (IDC). Using a MCF10DCIS.COM progression model, we have shown that SIM2s expression is decreased in MCF10DCIS.COM cells compared with MCF10A cells, and reestablishment of SIM2s in MCF10DCIS.COM cells significantly inhibits growth and invasion both in vitro and in vivo. Analysis of SIM2s-MCF10DCIS.com tumors showed that SIM2s promoted a more differentiated tumor phenotype including the expression of a broad range of luminal markers (CSN2 (ß-casein), CDH1 (E-cadherin), and KER18 (keratin-18)) and suppressed genes associated with stem cell maintenance and a basal phenotype (SMO (smoothened), p63, SLUG (snail-2), KER14 (keratin-14) and VIM (vimentin)). Furthermore, loss of SIM2s expression in MCF10DCIS.COM xenografts resulted in a more invasive phenotype and increased lung metastasis likely due to an increase in Hedgehog signaling and matrix metalloproteinase expression. Together, these exciting new data support a role for SIM2s in promoting human breast tumor differentiation and maintaining epithelial integrity.