Self-assembling anticaries mucosal vaccine containing ferritin cage nanostructure and glucan-binding region of S. mutans glucosyltransferase effectively prevents caries formation in rodents.

Anticaries protein vaccines that induce a mucosal immune response are not effective. Therefore, development of effective and convenient anticaries vaccines is a priority of dental research. Here we generated self-assembling nanoparticles by linking the glucan-binding region of Streptococcus mutans glucosyltransferase (GLU) to the N-terminal domain of ferritin to determine whether these novel nanoparticles enhanced the immunogenicity of an anticaries protein vaccine against GLU in rodents. We constructed the expression plasmid pET28a-GLU-FTH and purified the proteins from bacteria using size-exclusion chromatography. BALB/c mice were used to evaluate the ability of GLU-ferritin (GLU-FTH) nanoparticles to induce GLU-specific mucosal and systemic responses. The protective efficiency of GLU-FTH nanoparticles was compared with that of GLU alone or a mixture of GLU and poly(I:C) after administering an intranasal infusion to Wistar rats. The phagocytosis and maturation of dendritic cells (DCs) exposed in vitro to the nanoparticles were assessed using flow cytometry. The GLU-FTH nanoparticle vaccine elicited significantly higher levels of GLU-specific antibodies compared with GLU or a mixture of GLU and poly(I:C). Immunization with GLU-FTH achieved lower caries scores compared with those of the other vaccines. Administration of GLU-FTH nanoparticles enhanced phagocytosis by DCs and their maturation. Thus, self-assembling GLU-FTH is a highly effective anticaries mucosal vaccine that enhanced antibody production and inhibited S. mutans infection in rodents.

Immunogenicity and prediction of epitopic region of antigen Ag I/II and glucosyltransferase from Streptococcus mutans.

The levels of Streptococcus (S.) mutans infections in saliva were evaluated and a comparison for specific antibody levels among children with different levels of S. mutans infection was made. The promising epitopic regions of antigen AgI/II (PAc) and glucosyltransferase (GTF) for potential vaccine targets related to S. mutans adherence were screened. A total of 94 children aged 3-4 years were randomly selected, including 53 caries-negative and 41 caries-positive children. The values of S. mutans and those of salivary total secretory immunoglobulin A (sIgA), anti-PAc and anti-Glucan binding domain (anti-GLU) were compared to determine the correlation among them. It was found the level of s-IgA against specific antigens did not increase with increasing severity of S. mutans infection, and the complete amino acid sequence of PAc and GTFB was analyzed using the DNAStar Protean system for developing specific anti-caries vaccines related to S. mutans adherence. A significantly positive correlation between the amount of S. mutans and children decayed, missing, and filled teeth index was observed. No significant difference was detected in specific sIgA against PAc or GLU between any two groups. No significant correlation was found between such specific sIgA and caries index. A total of 16 peptides from PAc as well as 13 peptides from GTFB were chosen for further investigation. S. mutans colonization contributed to early children caries as an important etiological factor. The level of sIgA against specific antigens did not increase with increasing severity of S. mutans infection in children. The epitopes of PAc and GTF have been screened to develop the peptide-based or protein-based anti-caries vaccines.

Recurrent ESR1-CCDC170 rearrangements in an aggressive subset of oestrogen receptor-positive breast cancers.

Characterizing the genetic alterations leading to the more aggressive forms of oestrogen receptor-positive (ER+) breast cancers is of critical significance in breast cancer management. Here we identify recurrent rearrangements between the oestrogen receptor gene ESR1 and its neighbour CCDC170, which are enriched in the more aggressive and endocrine-resistant luminal B tumours, through large-scale analyses of breast cancer transcriptome and copy number alterations. Further screening of 200 ER+ breast cancers identifies eight ESR1-CCDC170-positive tumours. These fusions encode amino-terminally truncated CCDC170 proteins (ΔCCDC170). When introduced into ER+ breast cancer cells, ΔCCDC170 leads to markedly increased cell motility and anchorage-independent growth, reduced endocrine sensitivity and enhanced xenograft tumour formation. Mechanistic studies suggest that ΔCCDC170 engages Gab1 signalosome to potentiate growth factor signalling and enhance cell motility. Together, this study identifies neoplastic ESR1-CCDC170 fusions in a more aggressive subset of ER+ breast cancer, which suggests a new concept of ER pathobiology in breast cancer.

RACK1 promotes breast carcinoma migration/metastasis via activation of the RhoA/Rho kinase pathway.

We aimed to gain a mechanistic understanding of the role of RACK1 in breast carcinoma migration/metastasis. Migration assays were conducted in breast carcinoma cell lines. siRNA targeting RACK1 as well as the Rho kinase inhibitor were also applied. Immunoprecipitation and immunofluorescence were used to study the RACK1/RhoA interaction. GTP-Rho pull-down assays were performed to assess the activation of RhoA. We also conducted immunohistochemistry in 160 breast carcinoma samples. Experiments in vitro showed that RACK1 promotes migration via interaction with RhoA and activation of the RhoA/Rho kinase pathway. Immunohistochemistry in 160 samples revealed that RACK1 is strongly correlated with accepted tumor spread indicators and RhoA (all P < 0.05). Kaplan-Meier survival analysis indicated a correlation between higher RACK1 expression and shorter survival times (P < 0.001). RACK1 is a prognostic factor that promotes breast carcinoma migration/metastasis by interacting with RhoA and activating the RhoA/Rho kinase pathway.

BCL2L10 protein regulates apoptosis/proliferation through differential pathways in gastric cancer cells.

The reason for and consequences of BCL2L10 down-regulation in gastric carcinoma are poorly understood. Our aim was to investigate the function of the protein BCL2L10 in gastric carcinoma. We investigated BCL2L10 expression using quantitative real-time PCR and immunoblotting. The methylation status of the BCL2L10 gene promoter was examined by bisulphite sequencing in fresh gastric normal and carcinoma tissues. We studied apoptosis and proliferation regulation in gastric cancer cell lines using flow cytometry, fluorescence staining, murine xenografting and immunoblotting. Pathway inhibitors were applied to confirm the major pathways involved in apoptosis or proliferation regulation. We observed significant correlations between lower BCL2L10 expression and CpG island hypermethylation of the BCL2L10 gene promoter in gastric carcinoma, apoptosis induced by over-expressed BCL2L10 through mitochondrial pathways, and proliferation accelerated by BCL2L10 siRNA via the PI3K-Akt signalling pathway in gastric cancer cell lines. The pro-apoptotic effect of BCL2L10 and growth promotion by BCL2L10 siRNA in gastric cancer cells suggest that it may be a tumour suppressor.

Loss of BCL2L10 protein expression as prognostic predictor for poor clinical outcome in gastric carcinoma.

AIMS: BCL2L10 protein is an apoptosis-related member of the Bcl-2 protein family. The clinical significance of its expression in gastric carcinoma is poorly understood. The aim was to investigate BCL2L10 expression and its clinical and prognostic significance in gastric carcinoma patients. METHODS AND RESULTS: Immunohistochemistry, real-time polymerase chain reaction (PCR) and immunoblotting all revealed extensive loss of BCL2L10 expression in gastric cancer cells. The scaled BCL2L10 expression data was categorized into three groups (groups 0-2) to facilitate statistical analysis. A significant correlation was observed between the lower BCL2L10 expression group and shorter disease-free survival (P=1.956×10(-18)). Multivariate regression analysis showed that loss of BCL2L10 protein expression [P=4.883×10(-8), hazard ratio (HR)=0.252] is an independent prognostic predictor of gastric carcinoma. The receiver operator characteristic (ROC) curve showed that the area for BCL2L10 protein was 0.817 (P=8.331×10(-14)), indicating that loss of BCL2L10 protein expression is an excellent prognostic predictor of gastric carcinoma. CONCLUSIONS: Loss of BCL2L10 protein expression predicts poor clinical outcome in gastric carcinoma.

RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo.

A yeast two-hybrid system was utilized to identify novel PI3K p110alpha-interacting proteins, of which receptor of activated protein kinase C1 (RACK1) was chosen for successive detailed analyses. Our aim was to investigate the function(s) of RACK1 and its involvement in mechanisms of breast carcinoma proliferation and invasion/metastasis. Experiments in breast carcinoma cell lines stably transfected with RACK1, as well as nude mouse models, showed that RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo. Conversely, knockdown of RACK1 by siRNA in vitro inhibited proliferation, migration, and invasion. In cell lines stably transfected with RACK1, p-AKT, cyclin D1, cyclin D3, and CD147 expression, as well as MMP2 activity, were elevated. RACK1-induced migration could be inhibited by the addition of Rho-kinase inhibitor. In 160 breast carcinoma cases, survival analyses established that RACK1 is an independent prognostic factor for poor outcome (P < 0.001). In conclusion, RACK1 is an independent prognosis-related factor and promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo.

RACK1: A superior independent predictor for poor clinical outcome in breast cancer.

We aimed to investigate the expression of RACK1 in breast cancer, evaluate its role in predicting prognosis and compare with commonly used biomarkers: Ki67, ER, PR and HER-2 for patients with breast cancer. The RACK1 expression and its clinical significance were examined in 160 breast carcinoma patients using immunohistochemistry. Correlations of RACK1 expression with other commonly used biomarkers and survival analyses were assessed. Immunohistochemistry results showed that the number of RACK1 cases scoring 0, 1, and 2 were 66, 54, and 40, respectively. RACK1 staining was strongly related to clinical stage, histological grade, Ki67, ER, PR and HER-2 (all p < 0.05). Consistently, all of the cases exhibiting RACK1 staining score 0 were survivors, whereas the majority (55.0%) of those exhibiting RACK1 staining score 2 were deaths. Kaplan-Meier survival analysis of 160 cases revealed a correlation between higher RACK1 expression levels and shorter overall survival times (p < 0.001). Univariate and multivariate analyses revealed that RACK1, tumor size, lymph node metastasis, and HER-2 were independent prognostic factors (all p < 0.05). Interestingly, receiver operator characteristic (ROC) curves showed that the ROC areas for RACK1, Ki67, ER, PR and HER-2 were 0.833, 0.766, 0.446, 0.387, and 0.689, respectively, and the superiority of RACK1 in sensitivity and specificity as biomarker was demonstrated. To our knowledge, it is the first time to investigate the expression of RACK1, and identified that RACK1 is a superior independent biomarker for diagnosis and prognosis comparing with currently widely used diagnostic index in breast carcinoma.

Overexpression and involvement of special AT-rich sequence binding protein 1 in multidrug resistance in human breast carcinoma cells.

Special AT-rich sequence binding protein (SATB) 1 has been proposed to act as a determinant for the acquisition of metastatic activity by controlling expression of a specific set of genes that promote metastatic activity. Here we found that SATB1 expression is upregulated in multidrug-resistant breast cancer cells that exhibit higher invasive potential than the parental cells. Apart from accelerating metastasis and inducing epithelial-mesenchymal transition, SATB1 was demonstrated to confer resistance to both P-glycoprotein-related and P-glycoprotein-non-related drugs on MCF7 cells, which was accompanied by decreasing accumulation of adriamycin in SATB1-overexpressing transfectants. SATB1 depletion could partially reverse the multidrug resistance (MDR) phenotype of MCF7/ADR in vitro and in vivo. The SATB1-induced P-glycoprotein-mediated MDR could be reversed by treatment with anti-P-glycoprotein mAb. Moreover, SATB1 plays an important role in anti-apoptotic activity in MCF7/ADR cells in response to adriamycin treatment, which suggests another mechanism contributing to SATB1-related MDR of breast cancers. These data provide new insights into the mode by which breast tumors acquire the MDR phenotype and also imply a role for SATB1 in this process.

DIXDC1 targets p21 and cyclin D1 via PI3K pathway activation to promote colon cancer cell proliferation.

DIXDC1 is the human homolog of Ccd1, a recently identified DIX domain containing protein in zebrafish. It is a positive regulator in the Wnt signaling pathway functioning downstream of Wnt and upstream of Axin. Since Wnt pathway activation is correlated with human colon cancer formation and progression, the biological role of DIXDC1 in human colon cancer was examined. In the current study, up-regulation of DIXDC1 protein was detected in human colorectal adenocarcinoma tissues and was found to be correlated well with high cell proliferation index. Ectopic over-expression of DIXDC1 resulted in increased cell proliferation in vitro and accelerated tumorigenesis on nude mice in vivo. We also showed that DIXDC1 promoted G0/G1 to S phase transition concomitantly with up-regulation of cyclin D1 and down-regulation of p21 protein. DIXDC1 over-expression cells showed activation of the PI3K/AKT pathway. Both siRNA knockdown of DIXDC1 and blocking the PI3K pathway using a specific inhibitor caused G1/S phase arrest, as well as down-regulation of cyclin D1 and up-regulation of p21 in DIXDC1 over-expression colon cancer cells. Collectively, this study demonstrates that over-expression of DIXDC1 might target p21 and cyclin D1 to promote colon cancer cell proliferation and tumorigenesis at least partially through activation of the PI3K/Akt pathway.

Twist1-mediated adriamycin-induced epithelial-mesenchymal transition relates to multidrug resistance and invasive potential in breast cancer cells.

PURPOSE: Besides its therapeutic effects, chemotherapeutic agents also enhance the malignancy of treated cancers in clinical situations. Recently, epithelial-mesenchymal transition (EMT) has attracted attention in studies of tumor progression. We aimed to test whether transient Adriamycin treatment induces EMT and apoptosis simultaneously in cancer cells, clarify why the same type of cells responds differentially (i.e., apoptosis, EMT) to Adriamycin treatment, and elucidate the role of Twist1, the master regulator of EMT, in this process. EXPERIMENTAL DESIGN: In unsynchronized MCF7 cells or cells synchronized at different phases, apoptosis, EMT, and concurrent events [multidrug resistance (MDR) and tumor invasion] after Adriamycin or/and Twist1 small interfering RNA treatment were examined in vitro and in vivo. The Adriamycin-induced Twist1 expression and the interaction of Twist1 with p53-Mdm2 were examined by immunoblotting and immunoprecipitation, respectively. RESULTS: We showed in vitro that Adriamycin induced EMT and apoptosis simultaneously in a cell cycle-dependent manner. Only the cells undergoing EMT displayed enhanced invasion and MDR. Twist1 depletion completely blocked the mesenchymal transformation, partially reversed MDR, and greatly abolished invasion induced by Adriamycin. Also, we confirmed in vivo that Twist1 RNA interference improved the efficacy of Adriamycin for breast cancers. Further, Twist1 reduction in Adriamycin-treated cells promoted p53-dependent p21 induction and disrupted the association of p53 with Mdm2. CONCLUSIONS: Our studies show the diverse responses to Adriamycin treatment in cells at different phases, suggest an unrecognized role of EMT in regulating MDR and invasion, and show the efficacy of Twist1 RNA interference in Adriamycin-based chemotherapies for breast cancer.

Hydropericardium prevented diagnosis of constrictive pericarditis: an unusual case of Rosai-Dorfman disease.

The present report concerns an unusual case of extranodal Rosai-Dorfman disease involving the epicardium, mesenterium, pleural cavity and lung. All involved lesions showed characteristics of S100-positive histiocytes exhibiting emperipolesis. The patient was a 51-year-old woman with a 2-year evolution of chest distress, dyspnoea and oedema. Pathological examination indicated that heart failure, which resulted from constrictive pericarditis, led to the fatal outcome of this case. Interference from severe hydropericardium prevented timely diagnosis and appropriate treatment. Therefore it is recommend that, despite being a rare condition, pericardium-involved Rosai-Dorfman disease should be taken into consideration in differential diagnoses, especially in cases of severe hydropericardium.

Over-expression of ubiquitin carboxy terminal hydrolase-L1 induces apoptosis in breast cancer cells.

Ubiquitin carboxy terminal hydrolase-L1 (UCH-L1) belongs to the UCH proteases family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. Previous research showed that UCH-L1 was expressed in mouse retinal cells and testicular germ cells, and its function was associated with apoptosis. But it is still unclear whether UCH-L1 is concerned with apoptosis in tumor cells. In order to clarify the role of UCH-L1 in tumor cells, multi-drug resistance (MDR) human breast carcinoma cell line MCF7/Adr, that expresses relatively high UCH-L1, and its parental cell line MCF7, that expresses relatively low UCH-L1, were chosen for this study. We transfected pcDNA3.1-UCH-L1 plasmid and UCH-L1 siRNA into MCF7 and MCF7/Adr cells, respectively. Using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, western blot, Hoechst 33258 staining assay and flow cytometry, we found that over-expression of UCH-L1 in MCF7 cells induced apoptosis. On the other hand, silencing of UCH-L1 in MCF7/Adr cells led to the opposite effect. Moreover, to explore the mechanism underling these observations, we further investigated the expression of phospho-Akt and its downstream signal phospho-IkB-alpha and other signal molecules including Fas, Fas-L, Trail, DR4, DR5, Bax, cytochrome C, active caspase-3, phospho-p53, phospho-Mdm-2, Bcl-2, Bcl-xL, p21 and p27. The results indicated that the process of apoptosis triggered by UCH-L1 is, at least in part, probably through Phosphoinositide 3-kinase (PI3K)/Akt signal pathway. Our findings suggest that modulating the ubiquitination and deubiquitination pathway could be a novel method for tumor therapy.

Interaction between CD147 and P-glycoprotein and their regulation by ubiquitination in breast cancer cells.

BACKGROUND: Multidrug-resistant cancer cells overexpressing P-glycoprotein (P-gp) display variations in invasive and metastatic ability through the upregulation of the extracellular matrix metalloproteinase (MMP) inducer (CD147). However, the direct linkage between these two proteins is still unclear. METHODS: We used immunoprecipitation, immunofluorescence analysis, migration and invasion assays, drug sensitivity assay and Western blot to measure the physical and functional interaction between P-gp and CD147. Then we transfected vectors carrying ubiquitin C-terminal hydrolase L1 (UCH-L1) or UCH-L1 siRNA into MCF7 and MCF7/Adr cells, respectively, and investigated the role of UCH-L1 in the regulation of the expression and degradation of P-gp, CD147 and MMP-1, MMP-2, and MMP-9 by quantitative real-time polymerase chain reaction, Western blot and immunoprecipitation. RESULTS: In this paper, we showed that P-gp and CD147 interacted with each other, and that the ubiquitin-proteasome pathway played an important role in the turnover of them. In addition, we found that inhibition of N-glycosylation increased the ubiquitination and degradation of P-gp and CD147, and affected their function. UCH-L1 not only regulated the expression of P-gp, CD147 and MMP-1, MMP-2, and MMP-9, but also the ubiquitination and degradation of P-gp and CD147 in breast cancer cells. CONCLUSION: Our results demonstrate a mechanism underlying the linkage between multidrug resistance and tumor metastasis, and suggest for the first time that modulating the ubiquitination of P-gp and CD147 might be a novel method for tumor therapy.

Cervical actinomycosis with spinal cord compression. Case report and literature review.

Cervical actinomycosis with spinal cord compression is extremely rare. The clinical presentation of spinal actinomycosis may be nonspecific and back pain is the most consistent early symptom. Here, we present such a case with fever, pain in the neck and upper back, progressive weakness and numbness in all 4 limbs with difficulty ambulating, constipation and uroschesis. Correct diagnosis is difficult because the clinical and radiological findings of actinomycosis closely resemble metastatic tumors and other infectious processes. Timely surgical debridement and decompression contributed to the prompt improvement of the patient's conditions, and histopathological demonstration of the inflammatory granulation tissue and Gram-positive sulfur-containing filamentous bacteria led to the correct diagnosis of actinomycosis. The diagnosis must be made promptly because delayed treatment can result in irreversible neurologic damage or death. Timely and long-term antibacterial therapy is essential for the complete recovery of the patient with actinomycosis.

[Effect of Smad7 on transforming growth factor-beta1-induced alveolar epithelial to mesenchymal transition].

OBJECTIVE: To investigate whether transforming growth factor beta1 (TGF-beta1) can induce in vitro alveolar epithelial-mesenchymal transition (EMT), and whether Smad7 gene transfer can block this transition and the possible signaling mechanism. METHODS: Rat alveolar type II epithelial cells of the line RLE-6TN were cultured. TGF beta1 (3 ng/mL) was added into the culture fluid. Lipofectamine 2000 was used to transfect Smad7 gene to the RLE-6TN cells. The expression of the markers of the epithelial cells, including E-cadherin and cytokeratin-19 (CK19), and markers of mesenchymal cells, including fibronectin (FN), vimentin, and alpha-smooth muscle actin (alpha-SMA) were assayed using Western blotting and real-time PCR. The morphological alterations were examined by phase-contrast microscope while the ultrastructure changes were examined by electron microscope. RESULTS: Smad7 was successfully transfected to the RLE-6TN cells. Before transfection TGF-beta1 treatment could lead to the expression upregulation of the mesenchymal markers and downregulation of the epithelial markers at the levels of both mRNA and protein, and after transfection, the mesenchymal makers were downregulated while the epithelial markers were upregulated. Before the transfection, TGF-beta1 treatment could lead to the expression upregulation of phosphorylated Smad2/3 which did not obvious change following transfection. TGF beta1 treatment could induce the EMT process of the RLE-6TN cells. Smad7 gene transfected into the RLE-6TN cells could block the process of EMT. Exposed of the RLE-6TN cells to TGF beta1 resulted in degeneration, tumefaction, and gradual disappearance of the osmiophilic multilamellar bodies, markers of type II alveolar epithelial cells. CONCLUSION: Under TGF beta1 treatment, RLE-6TN undergoes a conversion process into myofibroblasts in vitro with the conversion mechanism related to Smad signaling pathway, and transfection of Smad7 gene can partly reverse this process.

Up-regulation of CD147 and matrix metalloproteinase-2, -9 induced by P-glycoprotein substrates in multidrug resistant breast cancer cells.

Treatment of animals bearing multidrug resistant (MDR) tumor cells with P-glycoprotein (P-gp) substrates could worsen host survival. It is assumed that this is due to increased tumor metastasis. To clarify the mechanism(s) underlying this observation, the MDR human breast cancer cell line, MCF-7/AdrR, and its sensitive parental line, MCF-7, was treated with various concentrations of P-gp substrate drugs (vincristine, paclitoxel, adriamycin) and a P-gp non-substrate drug (bleomycin) in serum-free media. Increased production of CD147, and matrix metalloproteinases (MMP)-2, -9 was observed only in MDR cancer cells exposed to P-gp substrates, as determined using real-time polymerase chain reaction, western blotting and zymography. Correspondingly, P-gp substrates significantly enhanced the in vitro invasion abilities of MCF-7/Adr cells. It was also found that the drug-induced promotion of CD147, and MMP-2, -9 was consistent with increased expression of epidermal growth factor receptor (EGFR) and that inhibition of either EGFR or P-gp activity could significantly interrupt the downstream effects, and so inhibit in vitro invasion abilities motivated by P-gp substrates. These results imply that treatment of MDR tumors with P-gp substrates could adversely affect therapeutic outcomes through modulating the production of CD147, MMP-2, -9, and EGFR, and suggest that this effect may be initiated by the transporter function of P-gp.

[CD147 and matrix metallo-proteinase (MMP) 2 and MMP9 expression in multidrug resistant breast cancer cells treated with P-glycoprotein substrate drugs].

OBJECTIVE: To investigate effects of P-glycoprotein (gp) substrate drugs on the expression of CD147 and MMP2 and 9 in multidrug resistant breast cancer cells. METHODS: MDR human breast cancer cell line, MCF7/AdrR, and its sensitive parental line, MCF7, were treated with various concentrations of P-gp substrate drugs, including paclitoxel and vincristine, and P-gp nonsubstrate drugs, bleomycin, in serum-free media. At the end of the treatment, expressions of CD147 and MMP2 and 9 were determined by real-time PCR and western blot. RESULTS: Increased expressions of CD147 and MMP2 and 9 were observed in multidrug resistant cancer cells compared with their parental MCF7 cells. After treatment with bleomycin, the expression of CD147 and MMP2 and 9 in both MCF7 and MCF7/AdrR cells remained unchanged (P > 0.05). However, treatment with paclitoxel and vincristine resulted in a remarkable over-expression of CD147 and MMP2 and 9 at both transcription and protein levels in MCF7/AdrR cell line (P < 0.05), while MCF7 cells failed to show similar response. CONCLUSIONS: P-gp substrate drugs can greatly upregulate the expression of CD147 and MMP2 and 9 in multidrug resistant breast cancer cells, therefore enhancing the tumor metastatic capability.

Involvement of CD147 in regulation of multidrug resistance to P-gp substrate drugs and in vitro invasion in breast cancer cells.

Multidrug resistant (MDR) cancer cells overexpressing P-glycoprotein (P-gp) display variations in invasive and metastatic behavior. We aimed to clarify the mechanism(s) underlying this observation and transfected vectors carrying CD147, a glycoprotein enriched on the surface of tumor cells that stimulates the production of matrix metalloproteinases (MMPs), and specific shCD147 into MCF7 and MCF7/Adr cells, respectively. Using quantitative real-time polymerase chain reaction and Western blot, we found that overexpression of CD147 in MCF7 cells up-regulated MDR1, MMP2, and MMP9 on both transcription and expression levels, which promoted tumor cells metastasis and conferred them multidrug resistance to P-gp substrate drugs, as determined by in vitro invasion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. On the other hand, silencing of CD147 in MCF7/Adr cells led to the opposite effect. Moreover, Erk1/2 in CD147-overexpressing clones were observed to be highly activate and after treatment with U0126, an Erk1/2-specific inhibitor, the expression of MDR1, MMP2 and MMP9 were decreased significantly. Thus, CD147 may assume a dual role, since it had intrinsic stimulative effects on tumor invasion in vitro as well as increasing resistance to P-gp substrate drugs.

The effect of TGF-ß1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells.

The aim of this study was to investigate whether transforming growth factor-ß1 (TGF-ß1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-ß1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-ß1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-ß1 treatment had no effect on the morphology of the RLE-6TN. TGF-ß1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-ß1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-ß1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-ß1 can induce alveolar epithelial-mesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process.