Gambogic acid (GA) is an anticancer agent in phase ‖b clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.
Antineoplastic Agents ; pharmacokinetics ; Apoptosis ; drug effects ; Breast Neoplasms ; drug therapy ; metabolism ; Calcium-Binding Proteins ; genetics ; Cell Line, Tumor ; Cell Migration Assays ; Cell Migration Inhibition ; drug effects ; Cell Proliferation ; drug effects ; Computational Biology ; methods ; Cytoskeleton ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; Flow Cytometry ; Gene Expression ; Humans ; Keratin-18 ; genetics ; Oxidation-Reduction ; Protein Biosynthesis ; drug effects ; Protein Transport ; Proteomics ; methods ; Transcription, Genetic ; drug effects ; Ubiquitin-Specific Proteases ; pharmacokinetics ; Vimentin ; genetics ; Xanthones ; pharmacokinetics

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.
Biomarkers, Tumor ; Carcinoma ; DNA, Complementary ; Gene Expression ; Humans ; Keratin-18 ; biosynthesis ; Keratin-19 ; biosynthesis ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; diagnosis ; metabolism ; pathology ; Neoplasm Metastasis ; Prognosis ; RNA, Messenger ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo characterize the biologic featrues of hepatic oval cells and their protein expression profiles during induced differentiation in vitro.

METHODSRat hepatic oval cells were treated with epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in vitro, followed by morphological and molecular marker assessment by electromicroscopy, immunocytochemistry, RT-PCR and protein expression chip technology.

RESULTSTen weeks after induction, the levels of GST-P mRNA and M2-PK mRNA were significantly reduced, whereas those of ALB and CK18 were elevated. Significant variations of expression was seen in 8 protein species during the course of the induced differentiation.

CONCLUSIONCombined EGF and HGF treatment in vitro induces cell differentiation of hepatic oval cells, a process in which 8 protein species may play some regulatory roles.

Albumins ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Epidermal Growth Factor ; pharmacology ; Glutathione Transferase ; biosynthesis ; genetics ; Hepatocyte Growth Factor ; pharmacology ; Hepatocytes ; cytology ; metabolism ; ultrastructure ; Immunohistochemistry ; Keratin-18 ; metabolism ; Protein Array Analysis ; Pyruvate Kinase ; biosynthesis ; genetics ; RNA, Messenger ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction

Adult ; Aged ; Biomarkers, Tumor ; analysis ; Carcinoma, Non-Small-Cell Lung ; blood ; genetics ; pathology ; Cell Differentiation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Keratin-19 ; genetics ; Leukocytes ; metabolism ; Lung Neoplasms ; blood ; genetics ; pathology ; Male ; Middle Aged ; Neoplasm Metastasis ; Neoplasm Staging ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Survival Analysis

OBJECTIVETo investigate the prognostic significance of micrometastasis (MM) in peripheral blood of patients with non-small cell lung cancer (NSCLC) treated by chemo-radiation therapy.

METHODSPeripheral blood was taken from 67 NSCLC patients before and after definitive chemo-radiation therapy. CK19 mRNA of the peripheral blood was measured by nested RT-PCR and both their relationship with clinicopathological features and prognostic significance were further investigated.

RESULTSThe micrometastasis-positive rates were 65.7% (44/67) and 32.8% (22/67), respectively, before and after the treatment. The micrometastasis-positive rate before treatment was closely in correlation with N-stage (P = 0.014). In contrast, it turned out to be more closely related with histological types (P = 0.019), weight loss (P = 0.01), KPS status (P = 0.027) as well as N-stage (P = 0.032) after chemo-radiation therapy. 4-yr distant metastasis rates (DMR) for micrometastasis-positive and -negative patients were 78.3% and 70.4%, respectively, before the treatment (P = 0.544) while they were 100% and 62.9%, respectively, after the chemoradiation (P < 0.001). The median survival time (MST) and 4-yr overall survival rate (OSR) for pretreatment micrometastasis-positive and -negative patients were 13.8 months and 17.6 months, and 18.2% and 17.4%, respectively (P = 0.619), while for post-treatment micrometastasis-positive and -negative patients they were 7.8 months and 27.6 months and 0 and 26.4%, respectively (P < 0.001). Multivariate analysis showed that the post-treatment positive micrometastasis was an independent unfavorable prognostic factor (P = 0.000).

CONCLUSIONDetection of micrometastasis in peripheral blood may possess a prognostic significance after definitive chemo-radiation therapy. Micrometastasis-negative patients have better prognosis compared to those with positive micrometastasis.

Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; therapy ; Cisplatin ; administration & dosage ; Combined Modality Therapy ; Female ; Follow-Up Studies ; Gene Expression Regulation, Neoplastic ; drug effects ; radiation effects ; Humans ; Keratin-19 ; genetics ; Lung Neoplasms ; genetics ; pathology ; therapy ; Male ; Middle Aged ; Neoplasm Metastasis ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Neoplastic Cells, Circulating ; drug effects ; pathology ; radiation effects ; Prognosis ; RNA, Messenger ; biosynthesis ; genetics ; Radiotherapy, High-Energy ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; Survival Analysis

OBJECTIVETo induce the differentiation of human bone marrow mesenchymal stem cells (HMSCs) into hepatocyte-like cells with hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) in vitro.

METHODSHMSCs were induced to differentiate into hepatocyte-like cells by HGF (group B), FGF-4 (group C) and HGF+FGF-4 (group D) in vitro. Undifferentiated HMSCs and L-02 cells were used as the negative (group A) and positive (group E) controls, respectively. The changes of cell morphology were observed microscopically. The expressions of hepatic markers, alpha fetoprotein (AFP) and CK-18, were detected by immunocytochemical staining at different times after induction, and the differentiation ratios of the various groups of HMSCs were calculated on the basis of image analysis. The expressions of AFP and ALB were detected by immunofluorescence assay in each group at different times after induction, and the expressions of AFP and ALB mRNA by RT-PCR.

RESULTSHMSCs gradually transformed into spindle-shaped, round, polygonal or irregular cells after induction. Immunocytochemical staining revealed positive AFP and CK18 expressions in groups B, C, and D after induction as well as in group E. The positive units (PU) of AFP and CK18 in group D calculated according to image analysis were significantly higher than that of groups A, B, and C. The expressions of AFP and ALB detected by immunofluorescence were both positive after induction in all groups except group A, similar to the findings of the expressions of AFP and ALB mRNA by RT-PCR.

CONCLUSIONHMSCs can be induced to differentiate into hepatocyte-like cells by HGF, FGF-4 and their combination at certain concentrations, and the hepatocyte-like cells can express some hepatic markers such as AFP, ALB, CK18, etc. HGF+FGF-4 may achieve more effective induction of HMSC differentiation into hepatocyte-like cells, and the efficiency of HGF is greater than that of FGF-4.

Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Fibroblast Growth Factor 4 ; pharmacology ; Hepatocyte Growth Factor ; pharmacology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Immunohistochemistry ; Keratin-18 ; biosynthesis ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo study the cytokeratin 18 and 13 and their gene (CK) expression in post-operation maxillary cyst linings with metaplastic epithelium.

METHODSCK expressions were examined with immunohistochemistry in 46 post-operative maxillary cyst (POMC) which were lined with pseudostriated columnar cells only (13 cases), both kinds of columnar and squamous cells (30 cases) and squamous cells only (3 cases).

RESULTSThe expressions of CK8, CK13 and CK18 were observed in 39, 9 and all of the 43 columnar epithelial linings, respectively. Metaplastic squamous epithelia expressed more CK13 and less CK18 and CK8. Of the 33 metaplastic linings, 24 expressed CK8, 23 CK13 and 26 linings expressed CK18. The expression of CK13- and CK18-mRNA was generally correlated with the protein expression level. By in situ hybridization, CK18-mRNA expression was observed not only in 26 metaplastic linings which were positive for CK18 protein but also in five of the seven metaplastic linings which did not express CK18 protein. In addition, RT-PCR revealed an expression of CK18-mRNA in all metaplastic squamous linings although the expression level was weaker than that in the columnar epithelial linings. The CK13-mRNA was expressed in a fashion inverse to the CK18-mRNA.

CONCLUSIONSThese results indicate that CK18-mRNA is preserved through metaplasia although the protein expression decreases and metaplastic squamous cells differentiate with a decrease of CK18 and an increase of CK13 expression.

Epithelial Cells ; metabolism ; pathology ; Humans ; Jaw Cysts ; etiology ; metabolism ; pathology ; Keratin-13 ; biosynthesis ; genetics ; Keratin-18 ; biosynthesis ; genetics ; Maxillary Diseases ; etiology ; metabolism ; pathology ; Metaplasia ; metabolism ; pathology ; Postoperative Complications ; RNA, Messenger ; genetics

The purpose of this study is to characterize and compare the ultrastructural changes occurring during the in vivo cultivation of corneal epithelium on amniotic membrane (AM) at several different time points. Corneal burn patients (n=7) with a corneal epithelial defect and severe limbal damage were selected. Initially, AM transplantation with limbal autograft was performed at the acute stage of corneal burn to reconstruct the damaged ocular surface. One to six (mean interval; 3.3+/-1.2) months later, the central part of AM containing an in vivo expanded corneal epithelium was excised and retransplanted in adjacent lesions. The excised epithelium with AM was examined by electron microscopy and immunohistochemical study. By electron microscopy, one and two months after expansion, cultivated epithelium on AM showed an undifferentiated epithelium and an incomplete basement membrane (BM). But, after three months, the cultivated epithelium began to differentiate into a multilayered epithelium with a continuous BM with increased hemidesmosomes. These findings were further confirmed by immunohistochemical study, that cytokeratin K3 was expressed in the cultivated corneal epithelium and newly formed BM was partially positive of collagen IV at three months. At least 3 months may be needed for the proliferation and differentiation of in vivo cultivated corneal epithelium on AM.
Stem Cells/cytology ; Stem Cell Transplantation/*methods ; Middle Aged ; Microscopy, Electron ; Male ; Keratin-3/biosynthesis ; Immunohistochemistry ; Humans ; Epithelium, Corneal/cytology/*metabolism/*pathology/*transplantation ; Corneal Diseases/*therapy ; Burns/*surgery/therapy ; Biological Dressings ; Amnion/*ultrastructure ; Adult

This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcinoma (CRC) patients and 30 from healthy volunteers were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and cytokeratin 19 (CK19) mRNA. CEA mRNA was detected in 35.8% of patients and 3.3% of controls, CK20 mRNA in 28.3% of patients and 6.7% of controls, and CK19 mRNA in 41.9% of patients and 3.3% of controls. CEA and CK20 mRNA positive ratio increased with the advancing Dukes stages, but there was no significant difference in positive ratio between any two stages (P>0.05). Also, relatively high positive ratio of CEA, CK20 and CK19 mRNA expression was observed in some CRC patients with earlier Dukes stages. A higher positive ratio was obtained when two or three detection markers were combined compared to a single marker. Our study indicates that quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood is a valuable tool for monitoring early stage dissemination of CRC cells in blood circulation.
Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; biosynthesis ; blood ; genetics ; Carcinoembryonic Antigen ; biosynthesis ; blood ; genetics ; Carcinoma ; blood ; genetics ; Colorectal Neoplasms ; blood ; genetics ; Female ; Humans ; Keratin-20 ; Keratins ; biosynthesis ; blood ; genetics ; Male ; Middle Aged ; RNA, Messenger ; blood ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity

Recently, the rearrangement of RET proto-oncogene has been reported to be the most common genetic change in papillary thyroid carcinoma (PTC). However, its prevalence has been reported variably and its relation to clinical outcome has been controversial. The characteristic nuclear features of PTC usually render the diagnosis, but problem arises with equivocal cytologic features that are present focally. Although there remains some controversy, CK19 has been reported to be a useful ancillary tool for diagnosis of PTC. To evaluate the expression rate of RET/PTC rearrangement and CK19 in PTCs in a Korean population, we studied 115 papillary thyroid carcinomas in 3 mm-core tissue microarray based immunohistochemical analysis. The prevalence of Ret protein expression was 62.6% and the CK19 immunoreactivity was 80.9%. There was no statistically significant asso-ciation between the Ret positivity and CK19 immunoreactivity, although the percent agreement of the two was relatively high. The clinicopathological variables did not correlate with the expression of Ret. In conclusion, the prevalence of Ret protein expression and its clinicopathological implications in a Korean population are not much different from those reported in previous studies. However, its detection via immunohistochemistry can be a useful diagnostic tool for diagnosing papillary thyroid carcinoma in conjunction with CK19.
Adenocarcinoma, Papillary/*metabolism ; Adult ; Carcinoma/pathology ; Cell Line, Tumor ; Cytoplasm/metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Keratin/*biosynthesis ; Korea ; Lymphatic Metastasis ; Male ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; Oncogene Proteins/*biosynthesis ; Receptor Protein-Tyrosine Kinases/*biosynthesis ; Thyroid Neoplasms/*metabolism/pathology