BACKGROUNDCalreticulin (CRT) is major Ca 2+ -binding chaperone mainly resident in the endoplasmic reticulum (ER) lumen. Recently, it has been shown that non-ER CRT regulates a wide array of cellular responses. We previously found that CRT was up-regulated during hypoxia/reoxygenation (H/R) and this study was aimed to investigate whether CRT nuclear translocation aggravates ER stress (ERS)-associated apoptosis during H/R injury in neonatal rat cardiomyocytes.

METHODSApoptosis rate and lactate dehydrogenase (LDH) leakage in culture medium were measured as indices of cell injury. Immunofluorescence staining showed the morphological changes of ER and intracellular translocation of CRT. Western blotting or reverse transcription polymerase chain reaction was used to detect the expression of target molecules.

RESULTSCompared with control, H/R increased apoptosis rate and LDH activity. The ER became condensed and bubbled, and CRT translocated to the nucleus. Western blotting showed up-regulation of CRT, Nrf2, activating transcription factor 4 (ATF4), CHOP and caspase-12 expression after H/R. Exogenous CRT overexpression induced by plasmid transfection before H/R increased cell apoptosis, LDH leakage, ER disorder, CRT nuclear translocation and the expression of ERS-associated molecules. However, administration of the ERS inhibitor, taurine, or CRT siRNA alleviated cell injury, ER disorder, and inhibited ERS-associated apoptosis.

CONCLUSIONSOur results indicated that during H/R stress, CRT translocation increases cell apoptosis and LDH leakage, aggravates ER disorder, up-regulates expression of nuclear transcription factors, Nrf2 and ATF4, and activates ERS-associated apoptosis.

Animals ; Apoptosis ; genetics ; physiology ; Calreticulin ; genetics ; metabolism ; Cell Hypoxia ; genetics ; physiology ; Cell Survival ; genetics ; physiology ; Cells, Cultured ; Endoplasmic Reticulum Stress ; physiology ; Myocytes, Cardiac ; cytology ; metabolism ; RNA Interference ; Rats

OBJECTIVEThe present study was conducted to understand the effects of PrP in different octapeptide repeats on proliferation of HeLa cells.

METHODS AND RESULTSMutant PrPs with octapeptide repeat insertion were transiently expressed in HeLa cells and their results of MTT assay showed stronger cytotoxic effect on the proliferation of cells than wild-type PrP. Annexin V/PI assay also demonstrated that the expression of mutant PrPs was much easier to induce apoptosis than wild-type in HeLa cells. The percentage of both early and late stage apoptosis in mutant groups were significantly higher than that of wild type.

CONCLUSIONThese data suggest that the expression of mutant PrPs associated with familial CJD is much easier to induce apoptosis in cultured cells than expression of wild type PrP.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Proliferation ; Cell Survival ; genetics ; physiology ; Colorimetry ; HeLa Cells ; Humans ; Mutation ; Oligopeptides ; genetics ; Plasmids ; genetics ; Prion Proteins ; Prions ; genetics ; metabolism ; physiology ; Transfection

In order to study the physicochemical characteristics of cytosolic PrP (CytoPrP) and evaluate its possible influence on cell viability, a recombinant plasmid expressing human CytoPrP eukaryoticly was constructed and transfected into human neuroblastoma cell line SH-SY5Y transiently. Proteinase-resistant activities of CytoPrP were evaluated by a proteinase K (PK) digestion and cytotoxic effects of CytoPrP were tested by MTT assay and Trypan Blue cell-counting. The presence of CytoPrP in cytoplasm after transfection was controlled by the presence of protease inhibitor. Compared with wild-type PrP, CytoPrP possessed relatively stronger PK-resistant activities. Obvious cytotoxic effects were observed in the cells after inducement of CytoPrP in cytoplasm by protease inhibitor, showing a dose-dependent manner. The results provide useful scientific evidences for further studies of potential role of CytoPrP in pathological mechanism of prion disease.
Cell Line, Tumor ; Cell Survival ; Cytosol ; chemistry ; Endopeptidase K ; pharmacology ; Humans ; Prions ; genetics ; physiology ; Transfection

Alveolar Epithelial Cells ; metabolism ; pathology ; Animals ; Apoptosis ; physiology ; Cell Survival ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; physiology ; Genes, bcl-2 ; genetics ; Genes, p53 ; genetics ; Oxidative Stress ; genetics ; Rats

BACKGROUNDAstrocyte elevated gene-1 (AEG-1), primarily identified as a late response gene induced by HIV-1 infection, plays multiple roles in the process of oncogenesis. This novel gene has been demonstrated to be involved in the several potent carcinogenic pathways, including PI3K/Akt pathway, nuclear factor (NF)-κB pathway, and Wnt/κ-catenin pathway. Although the function of AEG-1 has been intensively investigated in recent years, the molecular mechanism underlying its oncogenic role is largely unknown. The aim of this research was to explore the potential function of AEG-1 in breast cancer development and progression.

METHODSAEG-1 was ectopically overexpressed in breast cancer MCF-7 cells and its biological effects on the proliferation and invasion of MCF-7 cells were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and invasion assays. The expression of HER2/neu, a crucial oncogene involving in breast cancer carcinogenesis, was also determined.

RESULTSOverexpression of the AEG-1 promoted the proliferation and invasion ability of breast cancer cells, and upregulated the expression of HER2/neu, a crucial oncogene involving in breast cancer carcinogenesis.

CONCLUSIONAEG-1 might facilitate the proliferation and invasion of breast cancer cells by upregulating HER2/neu expression, which provides a potential target for breast cancer therapy.

Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; Cell Adhesion Molecules ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; genetics ; physiology ; Humans ; Neoplasm Invasiveness ; genetics ; Real-Time Polymerase Chain Reaction ; Receptor, ErbB-2 ; genetics ; metabolism ; Signal Transduction ; genetics ; physiology

Protein tyrosine phosphatases (PTPs) play an important role in regulating cell signaling events in coordination with tyrosine kinases to control cell proliferation, apoptosis, survival, migration, and invasion. Receptor-type protein tyrosine phosphatases (PTPRs) are a subgroup of PTPs that share a transmembrane domain with resulting similarities in function and target specificity. In this review, we summarize genetic and epigenetic alterations including mutation, deletion, amplification, and promoter methylation of PTPRs in cancer and consider the consequences of PTPR alterations in different types of cancers. We also summarize recent developments using PTPRs as prognostic or predictive biomarkers and/or direct targets. Increased understanding of the role of PTPRs in cancer may provide opportunities to improve therapeutic approaches.
Apoptosis ; Cell Proliferation ; Cell Survival ; Humans ; Neoplasm Invasiveness ; Neoplasms ; enzymology ; Receptor-Like Protein Tyrosine Phosphatases ; genetics ; physiology

BACKGROUNDThe Notch-regulated ankyrin repeat protein (NRARP) is recently found to promote proliferation of breast cancer cells. The role of NRARP in carcinogenesis deserves extensive investigations. This study attempted to investigate the expression of NRARP in thyroid cancer tissues and assess the influence of NRARP on cell proliferation, apoptosis, cell cycle, and invasion in thyroid cancer.

METHODSThirty-four cases with thyroid cancer were collected from the Department of General Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine between 2011 and 2012. Immunohistochemistry was used to detect the level of NRARP in cancer tissues. Lentivirus carrying NRARP-shRNA (Lenti-NRARP-shRNA) was applied to down-regulate NRARP expression. Cell viability was tested after treatment with Lenti-NRARP-shRNA using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis and cell cycle distribution were determined by flow cytometry. Cell invasion was tested using Transwell invasion assay. In addition, expressions of several cell cycle-associated and apoptosis-associated proteins were examined using Western blotting after transfection. Student's t-test, one-way analysis of variance (ANOVA), or Kaplan-Meier were used to analyze the differences between two group or three groups.

RESULTSNRARP was highly expressed in thyroid cancer tissues. Lenti-NRARP-shRNA showed significantly inhibitory activities against cell growth at a multiplicity of infection of 10 or higher (P < 0.05). Lenti-NRARP-shRNA-induced G1 arrest (BHT101: 72.57% ± 5.32%; 8305C: 75.45% ± 5.26%) by promoting p21 expression, induced apoptosis by promoting bax expression and suppressing bcl-2 expression, and inhibited cell invasion by suppressing matrix metalloproteinase-9 expression.

CONCLUSIONDownregulation of NRARP expression exerts significant antitumor activities against cell growth and invasion of thyroid cancer, that suggests a potential role of NRARP in thyroid cancer targeted therapy.

Adult ; Aged ; Animals ; Apoptosis ; genetics ; physiology ; Cell Cycle ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; genetics ; physiology ; Cell Survival ; genetics ; physiology ; Female ; Humans ; In Vitro Techniques ; Kaplan-Meier Estimate ; Male ; Mice ; Mice, Nude ; Middle Aged ; Neoplasm Proteins ; genetics ; metabolism ; Proteins ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Thyroid Neoplasms ; genetics ; metabolism ; mortality ; pathology

OBJECTIVETo evaluate the therapeutic effect and the expression level of a tumor-specific replication-competent adenovirus and a replication-defective adenovirus expression mouse recombinant IL-12 (mIL-12) gene on hepatocellular carcinoma (HCC) in vitro.

METHODSThe cytotoxicity of replication-competent adenovirus with E1B-55 000 attenuated CNHK200-mIL12 and ONYX-015 (dl1520), and replication-defective adenovirus Adv-mIL12 were evaluated by MTT and replication assay in two HCC cell lines (HepG2 and Hep3B) and human normal hepatocyte line (LO2). Western blot and ELISA were used to determine the expression level of mIL-12.

RESULTSCNHK200-mIL12 replicated in HepG2 and Hep3B with an increase of 3,160-fold and 630-fold respectively in 96 h post-infection. CNHK200-mIL12 could kill HepG2 and Hep3B cells at a very low MOI (Multiplicity of Infection) and in short time course (HepG2:MOI = 0.2, on day 4; Hep3B:MOI = 0.005, on day 2), while it had no significant effect on LO2. Furthermore, the expressing level of mIL-12 in CNHK200-mIL12 treated HCC cell lines was much higher than that in Adv-mIL12 treated one (HepG2 101-fold, Hep3B 20-fold respectively).

CONCLUSIONReplication-competent adenovirus is more effective than replication-defective adenovirus in both cytotoxicity and efficiency of gene transfer in HCC, and holds great promise in the area of HCC therapy.

Adenoviridae ; genetics ; physiology ; Carcinoma, Hepatocellular ; pathology ; therapy ; Cell Line, Tumor ; Cell Survival ; Genetic Therapy ; Genetic Vectors ; Humans ; Interleukin-12 ; biosynthesis ; genetics ; Liver Neoplasms ; pathology ; therapy ; Virus Replication

OBJECTIVETo study the expression of recombinant human SCF-TPO fusion protein and its biological function.

METHODSFour primers were designed according to known sequences of TPO and SCF. The functional amino acid domains of TPO and SCF were amplified by RT-PCR from fetus hepatocytes, respectively. The expression plasmid pET32a/SCF-TPO was constructed by VOE gene fusion technique and expressed in E. coli BL21(DE3) plysS as inclusion body after isopropyl-beta-D-1-thiogalactopyranoside (IPTG) induction. The fusion protein was tested by SDS-PAGE and Western blot. The biological functions of SCF-TPO fusion protein in MO7e cells was investigated by MTT method after purification with metal chelating chromatography.

RESULTSThe high expression SCF-TPO fusion protein was obtained, reaching up to 30% of the total cellular protein. Western blot verified the correct fusion expression and MTT results showed the growth promoting effect of the SCF-TPO fusion protein on MO7e cells, with a higher promoting activity at 100 ng/ml.

CONCLUSIONSExpressed SCF-TPO fusion protein after renaturation has biological activity in promoting the proliferation of MO7e cells.

Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; genetics ; physiology ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Genetic Vectors ; Humans ; Recombinant Fusion Proteins ; genetics ; metabolism ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; genetics ; metabolism ; physiology ; Thrombopoietin ; genetics ; metabolism ; physiology

OBJECTIVETo investigate the effects of hepatocyte growth factor (HGF) on adriamycin-induced apoptosis of gastric cancer cells in vitro.

METHODSAn eukaryotic expression plasmid (pIRES2-EGFP) containing HGF (pIRES2-EGFP-HGF) was constructed. Human gastric cancer cell line MKN-45 cells were transfected in vitro with pIRES2-EGFP containing HGF or not. RT-PCR and Western blot were used to determine the target gene expression. Function of HGF was determined by MDCK cell scattering assay. Cell viability was tested by MTT assay. Apoptosis was evaluated by DNA fragmentation assay as well as flow cytometry using PI staining.

RESULTSThe HGF transfected MKN45 cells could stably express HGF mRNA, and secrete HGF protein to the cell culture median which was detected to exhibit normal function. The cell inhibition rate induced by adriamycin in HGF-transfected cells was decreased as compared to that of parental and mock transfected cells. When treated with adriamycin at 0.1 microg/ml, the parental and mock transfected cells present typical apoptotic ladder on DNA electrophoresis while HGF transfected cell did not. The apoptotic rate was decreased in HGF transfected cells as compared with that of parental and mock transfected cells.

CONCLUSIONHGF gene transfection may suppress adriamycin-induced apoptosis of gastric cancer cells.

Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; genetics ; physiology ; DNA Fragmentation ; drug effects ; Dose-Response Relationship, Drug ; Doxorubicin ; pharmacology ; Flow Cytometry ; Green Fluorescent Proteins ; genetics ; metabolism ; Hepatocyte Growth Factor ; genetics ; metabolism ; physiology ; Humans ; Plasmids ; genetics ; RNA, Messenger ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transfection