This study explored the generation site and regulation mechanism of reactive oxygen species(ROS) in the apoptosis of colorectal cancer cells induced by furanodienone(Fur). RKO cells were treated with 200 μmol·L~(-1) of Fur, and the changes in intracellular nicotinamide adenine dinucleotide phosphate oxidase(NOX) activity were detected by the NOX activity detection method. The control group, Fur group, diphenyleneiodonium(DPI) inhibitor group for general NOX, mitochondrial-targeted antioxidant(MitoTEMPO) group, Fur+DPI group, Fur+MitoTEMPO group, and H_2O_2 positive control group were set up. Intracellular ROS levels were detected by the ROS fluorescent staining method, and NOX1-NOX5 protein expressions were detected by Western blot. The NOX1-specific inhibitor ML171 and NOX4-specific inhibitor(GLX351322) were further introduced, and the cell activity was determined by cell counting kit-8(CCK-8) assay. The effects of ROS level change on the protein expressions of NOX4 and peroxiredoxin 1(PRDX1) were measured by Western blot. BAY11-7082, which is an inhibitor of the inhibitor of nuclear factor κB protein α(IκBα), was used to explore the effect of the expression of phosphorylated nuclear factor κB(p-NF-κB) in the nucleus after the Fur treatment on the NOX4 protein level. The lentiviral plasmid and empty plasmid for PRDX1 gene silencing were constructed to transfect RKO cells, and stably transfected strains were screened. The impact of PRDX1 gene knockout on Fur-induced apoptosis was further analyzed using the flow cytometry assay. The findings demonstrate a considerable increase in mitochondrial ROS level in response to Fur treatment, with an increase in intracellular NOX activity. However, the mitochondrial ROS level is significantly reduced in the Fur+DPI group. The results from Western blot and CCK-8 analysis suggest that intracellular NOX1 and NOX4 protein expressions are elevated by Fur treatment, and GLX351322 effectively reverses the pro-apoptotic effect of Fur, while ML171 has a minimal impact on apoptosis rate. Meanwhile, Fur significantly boosts the level of p-NF-κB in the nucleus, whereas the protein levels of p-NF-κB and NOX4 are reduced after the BAY treatment. The regulation of Fur on NOX4 and PRDX1 protein expressions is negatively correlated. In the stably transfected cell strain with PRDX1 gene knockout, the apoptosis rate is considerably higher than that of the negative control group after Fur treatment. The above results indicate that Fur can induce the apoptosis of colorectal cancer cells by promoting the signal transduction of NF-κB in the nucleus and increasing the generation of mitochondrial ROS derived from NOX4 to inhibit the PRDX1 protein expression.
Humans ; Peroxiredoxins/metabolism* ; Apoptosis/drug effects* ; Reactive Oxygen Species/metabolism* ; NADPH Oxidase 4/metabolism* ; Mitochondria/genetics* ; Cell Line, Tumor ; Colorectal Neoplasms/drug therapy* ; NADPH Oxidases/metabolism* ; Furans/pharmacology*

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent and prevalent nitrosamine procarcinogen found in cigarette smoke. The aim of this work is to study alterations in peroxiredoxin (Prx) expression induced by NNK during carcinogenesis. Characterization of Prx genes from hamster was performed using bioinformatics. V79 cells were induced with different concentrations of NNK (0.1-0.4 mg/mL), and the expression levels of six Prx genes (Prx1-Prx6) were measured by qRT-PCR 24 h following NNK treatment. Prx gene expression was induced by NNK stress, and the highest transcription levels were induced by over 20.42-fold relative to that of the control. NNK induced alterations in Prx expression over the course of lung cancer, which means Prxs may play important roles in ROS detoxification under NNK stress and their functions are complementary.
Animals ; Carcinogens ; administration & dosage ; toxicity ; Cell Line ; Cell Survival ; Cricetinae ; Cricetulus ; Dose-Response Relationship, Drug ; Gene Expression Regulation ; drug effects ; Nitrosamines ; administration & dosage ; toxicity ; Peroxiredoxins ; genetics ; metabolism

Silicosis is one of the most serious occupational diseases in China and dates back to centuries ago. In this study, we successfully established a rat model of silicosis by intratracheal silica injection for 28 days and determined hydroxyproline levels to evaluate collagen metabolism in lung homogenates. Oxidative stress status was evaluated by detecting catalase and glutathione peroxidase activities. Expression levels of peroxiredoxins (Prx I and Prx VI) were detected by Western blotting. Pulmonary surfactant protein A (SP-A) levels in rat serum and lung tissue were analyzed by ELISA, and SP-A and Prx expression levels in lung tissues were detected by immunohistochemistry. The results suggest that Prx proteins may be involved in pulmonary fibrosis induced by silica. Downregulation of SP-A expression caused due to silica is an important factor in the occurrence and development of silicosis.
Animals ; Disease Models, Animal ; Humans ; Lung ; enzymology ; metabolism ; Male ; Oxidative Stress ; Peroxiredoxin VI ; genetics ; metabolism ; Peroxiredoxins ; genetics ; metabolism ; Pulmonary Surfactant-Associated Protein A ; genetics ; metabolism ; Rats ; Silicon Dioxide ; toxicity ; Silicosis ; genetics ; metabolism

Redox adaptation is an important concept that explains the mechanisms by which cancer cells survive under persistent endogenous oxidative stress and become resistant to certain anticancer agents. To investigate this concept, we determined the expression levels of peroxiredoxins (Prxs), antioxidant enzymes in drug-resistant non-small cell lung carcinoma cells. Prx II was remarkably increased only in A549/GR (gefitinib-resistant) cells compared with A549 cells, consistent with methylation/demethylation. Prx II was highly methylated in the A549 cells but was demethylated in the A549/GR cells. The elevated expression of Prx II resulted in the downregulation of reactive oxygen species (ROS) and cell death and upregulation of cell cycle progression in the A549/GR cells. When Prx II mRNA in the A549/GR cells was knocked down, the levels of ROS and apoptosis were significantly recovered to the levels of the controls. In addition, signaling molecules involved in apoptosis were increased in the A549/GR-shPrx II cells. There was no difference in the expression of MAPK/ERK between the A549/GR cells and A549/GR-shPrx II cells, but the phosphorylation of JNK was increased in the A549/GR cells and was markedly decreased in the A549/GR-shPrx II cells. Colony number and tumor growth were significantly decreased in the A549/GR-shPrx II cells compared with the A549/GR cells. Our findings suggest that Prx II has an important role in cancer cell survival via the modulation of signaling molecules involved in apoptosis and the phosphorylation of JNK by the downregulation of ROS levels in A549/GR cells.
Animals ; Antineoplastic Agents/*therapeutic use ; Apoptosis/drug effects ; Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics/metabolism/pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Female ; Humans ; Lung/drug effects/metabolism/pathology ; Lung Neoplasms/*drug therapy/genetics/metabolism/pathology ; Mice, Inbred BALB C ; Mice, Nude ; Oxidative Stress/drug effects ; Peroxiredoxins/*genetics/metabolism ; Quinazolines/*therapeutic use ; Reactive Oxygen Species/metabolism

Redox adaptation is an important concept that explains the mechanisms by which cancer cells survive under persistent endogenous oxidative stress and become resistant to certain anticancer agents. To investigate this concept, we determined the expression levels of peroxiredoxins (Prxs), antioxidant enzymes in drug-resistant non-small cell lung carcinoma cells. Prx II was remarkably increased only in A549/GR (gefitinib-resistant) cells compared with A549 cells, consistent with methylation/demethylation. Prx II was highly methylated in the A549 cells but was demethylated in the A549/GR cells. The elevated expression of Prx II resulted in the downregulation of reactive oxygen species (ROS) and cell death and upregulation of cell cycle progression in the A549/GR cells. When Prx II mRNA in the A549/GR cells was knocked down, the levels of ROS and apoptosis were significantly recovered to the levels of the controls. In addition, signaling molecules involved in apoptosis were increased in the A549/GR-shPrx II cells. There was no difference in the expression of MAPK/ERK between the A549/GR cells and A549/GR-shPrx II cells, but the phosphorylation of JNK was increased in the A549/GR cells and was markedly decreased in the A549/GR-shPrx II cells. Colony number and tumor growth were significantly decreased in the A549/GR-shPrx II cells compared with the A549/GR cells. Our findings suggest that Prx II has an important role in cancer cell survival via the modulation of signaling molecules involved in apoptosis and the phosphorylation of JNK by the downregulation of ROS levels in A549/GR cells.
Animals ; Antineoplastic Agents/*therapeutic use ; Apoptosis/drug effects ; Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics/metabolism/pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Female ; Humans ; Lung/drug effects/metabolism/pathology ; Lung Neoplasms/*drug therapy/genetics/metabolism/pathology ; Mice, Inbred BALB C ; Mice, Nude ; Oxidative Stress/drug effects ; Peroxiredoxins/*genetics/metabolism ; Quinazolines/*therapeutic use ; Reactive Oxygen Species/metabolism

OBJECTIVETo screen genes related to peritoneal metastasis of colorectal cancer.

METHODSSpecimens of primary cancer and normal mucosa tissues were collected from 3 patients with peritoneal metastasis of colorectal cancer. The total RNA were extracted and inversely transcribed into cDNA to synthesize aRNA using in vitro RNA synthesis. The synthesized aRNA, after labeling with Cy3, were hybridized with the whole human genome oligo microarray. The Empirical Bayes method was used to screen the differentially expressed genes, followed by confirmation of the selected genes by semi-quantitative RT-PCR.

RESULTSWith a threshold of P≤0.05, a total of 105 differentially expressed genes were identified in primary cancer lesions, including 42 up-regulated and 63 down-regulated genes. Three of the up-regulated genes (S100P, PRDX1 and SLPI) were selected and confirmed by RT-PCR, which yielded results consistent with those from gene microarray.

CONCLUSIONGene microarray technique can provide valuable clues for locating the tumor markers of peritoneal metastasis in colorectal cancer patients.

Adenocarcinoma ; genetics ; secondary ; Adenocarcinoma, Mucinous ; genetics ; secondary ; Adult ; Aged ; Calcium-Binding Proteins ; genetics ; metabolism ; Colorectal Neoplasms ; genetics ; pathology ; Female ; Gene Expression Profiling ; Genome-Wide Association Study ; Humans ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; metabolism ; Oligonucleotide Array Sequence Analysis ; methods ; Peritoneal Neoplasms ; genetics ; secondary ; Peroxiredoxins ; genetics ; metabolism ; Secretory Leukocyte Peptidase Inhibitor ; genetics ; metabolism

BACKGROUND/AIMS: Oxidative stress results in protein oxidation and is implicated in carcinogenesis. Sulfiredoxin (Srx) is responsible for the enzymatic reversal of inactivated peroxiredoxin (Prx). Nuclear factor E2-related factor 2 (Nrf2) binds to antioxidant responsive elements and upregulates the expression of Srx and Prx during oxidative stress. We aimed to elucidate the biological functions and potential roles of Srx in lung cancer. METHODS: To study the roles of Srx and Prx III in lung cancer, we compared the protein levels of Nrf2, Prxs, thioredoxin, and Srx in 40 surgically resected human lung cancer tissues using immunoblot and immunohistochemical analyses. Transforming growth factor-beta1, tumor necrosis factor-alpha, and camptothecin treatment were used to examine Prx III inactivation in Mv1Lu mink lung epithelial cells and A549 lung cancer cells. RESULTS: Prx I and Prx III proteins were markedly overexpressed in lung cancer tissues. A significant increase in the oxidized form of a cysteine sulfhydryl at the catalytic site of Prxs was found in carcinogenic lung tissue compared to normal lung tissue. Densitometric analyses of immunoblot data revealed significant Srx expression, which was higher in squamous cell carcinoma tissue (60%, 12/20) than in adenocarcinoma (20%, 4/20). Also, Nrf2 was present in the nuclear compartment of cancer cells. CONCLUSIONS: Srx and Prx III proteins were markedly overexpressed in human squamous cell carcinoma, suggesting that these proteins may play a protective role against oxidative injury and compensate for the high rate of mitochondrial metabolism in lung cancer.
Adenocarcinoma/*enzymology/genetics/mortality/pathology ; Animals ; Antineoplastic Agents, Phytogenic/pharmacology ; Blotting, Western ; Camptothecin/pharmacology ; Carcinoma, Squamous Cell/*enzymology/genetics/mortality/pathology ; Cell Line, Tumor ; Humans ; Immunohistochemistry ; Lung Neoplasms/*enzymology/genetics/mortality/pathology ; Mink ; NF-E2-Related Factor 2/*metabolism ; Oxidoreductases Acting on Sulfur Group Donors/genetics/*metabolism ; Peroxiredoxin III/*metabolism ; Peroxiredoxins/metabolism ; Prognosis ; RNA Interference ; Reactive Oxygen Species/metabolism ; Transfection ; Transforming Growth Factor beta1/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Up-Regulation

Alzheimer's disease (AD) pathogenesis is characterized by senile plaques in the brain and evidence of oxidative damage. Oxidative stress may precede plaque formation in AD; however, the link between oxidative damage and plaque formation remains unknown. Presenilins are transmembrane proteins in which mutations lead to accelerated plaque formation and early-onset familial Alzheimer's disease. Presenilins physically interact with two antioxidant enzymes thiol-specific antioxidant (TSA) and proliferation-associated gene (PAG) of the peroxiredoxin family. The functional consequences of these interactions are unclear. In the current study we expressed a presenilin transgene in Drosophila wing and sensory organ precursors of the fly. This caused phenotypes typical of Notch signaling loss-of-function mutations. We found that while expression of TSA or PAG alone produced no phenotype, co-expression of TSA and PAG with presenilin led to an enhanced Notch loss-of-function phenotype. This phenotype was more severe and more penetrant than that caused by the expression of Psn alone. In order to determine whether these phenotypes were indeed affecting Notch signaling, this experiment was performed in a genetic background carrying an activated Notch (Abruptex) allele. The phenotypes were almost completely rescued by this activated Notch allele. These results link peroxiredoxins with the in vivo function of Presenilin, which ultimately connects two key pathogenetic mechanisms in AD, namely, antioxidant activity and plaque formation, and raises the possibility of a role for peroxiredoxin family members in Alzheimer's pathogenesis.
Amino Acid Sequence ; Animals ; Drosophila ; metabolism ; physiology ; Drosophila Proteins ; metabolism ; Molecular Sequence Data ; Peroxiredoxins ; chemistry ; genetics ; metabolism ; Presenilins ; chemistry ; metabolism ; Receptors, Notch ; metabolism ; Sequence Alignment ; Signal Transduction

BACKGROUND AND OBJECTIVEDJ-1, a suppressor of PTEN, promotes metastasis of different tumors, but its function and mechanisms in glioma metastasis remain unclear. This study aimed to investigate the effect of the DJ-1 protein on the migration and invasion of human glioma cells, and to explore potential mechanisms.

METHODSThe eukaryotic expression vector pEGFP/DJ-1 and small interfering RNA (siRNA) were constructed and transfected into human glioma SWO-38 cells. The expression of DJ-1 and PTEN in SWO-38 cells were detected by Western blot. Cell migration and invasion were detected by transwell assay.

RESULTSAfter transfection of pEGFP/DJ-1, the expression of DJ-1 (1.28 ± 0.15 vs. 0.89 ± 0.04, P < 0.05) and focal adhesion kinase (FAK) phosphorylation (0.76 ± 0.12 vs. 0.51 ± 0.04, P < 0.05) were increased, whereas the expression of PTEN (0.74 ± 0.2 vs. 1.04 ± 0.14, P < 0.05) was suppressed. After transfection of DJ-1 siRNA, both DJ-1 (0.33 ± 0.04 vs. 0.88 ± 0.06, P < 0.05) and p-FAK levels (0.33 ± 0.01 vs. 0.44 ± 0.05, P < 0.05) were decreased, but PTEN expression (1.1 ± 0.06 vs. 0.81 ± 0.12, P < 0.05) was increased. Transwell assay data showed that pEGFP/DJ-1 transfection promoted SWO-38 cell migration (57.2 ± 6.50 vs. 40.4 ± 5.0, P < 0.05) and invasion (54.6 ± 4.9 vs. 27 ± 6.7, P < 0.05), whereas DJ-1 siRNA transfection inhibited SWO-38 cells migration (54.4 ± 6.9 vs. 73.4 ± 7.6, < 0.05) and invasion (44.6 ± 5.8 vs. 69.2 ± 9.2, P < 0.05).

CONCLUSIONOver-expression of DJ-1 promotes SWO-38 cell migration and invasion possibly through the DJ-1 and the PTEN/FAK pathway.

Cell Line, Tumor ; Cell Movement ; Down-Regulation ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Genetic Vectors ; Glioma ; metabolism ; pathology ; Humans ; Neoplasm Invasiveness ; Oncogene Proteins ; genetics ; metabolism ; physiology ; PTEN Phosphohydrolase ; genetics ; metabolism ; Peroxiredoxins ; Phosphorylation ; Plasmids ; Protein Deglycase DJ-1 ; RNA, Small Interfering ; Signal Transduction ; Transfection

OBJECTIVESTo study the biological function and its possible underlying mechanism of peroxiredoxin II (PrxII) in liver cancer cell line Hep3B.

METHODSTwo pairs of double-stranded small interfering RNA (siRNA) targeted on PrxII gene were transfected into Hep3B cells using LipofectamineTM 2000. After confirming the inhibited effects of these siRNAs through Quant SYBR Green polymerase chain reaction and Western blot, the biological characters of Hep3B cell were analyzed by flow cytometry analysis, MTT and colony formation assays. Furthermore, dichlorodihydrofluorescein diacetate (DCFH-DA) and thiobarbituric acid (TBA) assays, for measuring the products of oxidative reaction, such as the reactive oxygen species (ROS) and malondialdehyde (MDA), were applied to explore whether the antioxidant mechanism was involved in the effects of PrxII functioning on Hep3B cell.

RESULTSThe two pairs of siRNA significantly inhibited PrxII mRNA and protein expression. Compared to the mock and blank control groups, the two PrxII-silent groups showed decreased rates of cell growth and clone formation and increased rates of cell apoptosis. The numbers of the formed colonies were 42.0+/-2.8 and 40.5+/-0.7 respectively in the two PrxII-silent groups, while they were 121.5+/-2.1 and 130.0+/-1.4 in the mock and blank control groups (P less than 0.05). The levels of endogenous ROS and MDA were significantly higher in the two PrxII-silent groups than those in the mock and blank control groups (P less than 0.05).

CONCLUSIONPrxII might play an important role in the hepatocarcinogenesis, possibly through an antioxidant function which may provide a favorable microenvironment for cancer cell survival and progression.

Cell Line, Tumor ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Oxidative Stress ; Peroxiredoxins ; genetics ; RNA, Small Interfering ; Reactive Oxygen Species ; Signal Transduction ; Transfection