Exosomes derived from chemically induced human hepatic progenitors inhibit oxidative stress induced cell death.

The emerging field of regenerative medicine has revealed that the exosome contributes to many aspects of development and disease through intercellular communication between donor and recipient cells. However, the biological functions of exosomes secreted from cells have remained largely unexplored. Here, we report that the human hepatic progenitor cells (CdHs)-derived exosome (EXOhCdHs ) plays a crucial role in maintaining cell viability. The inhibition of exosome secretion treatment with GW4869 results in the acceleration of reactive oxygen species (ROS) production, thereby causing a decrease of cell viability. This event provokes inhibition of caspase dependent cell death signaling, leading to a ROS-dependent cell damage response and thus induces promotion of antioxidant gene expression or repair of cell death of hypoxia-exposed cells. Together, these findings show the effect of exosomes in regeneration of liver cells, and offer valuable new insights into liver regeneration.

Effects of fingerprint development reagents on subsequent DNA analysis.

A variety of evidences are found at crime scenes. Fingerprint and DNA evidences are especially important in the process of identifying personal sources. Among evidences found at crime scenes, cigarette butts are important because they might contain both fingerprints and DNA. In this study, latent fingerprints were detected in cigarette butts using 1,8-diazafluoren-9-one (DFO) and 1,2-Indanedione/zinc chloride (1,2-IND/Zn). Next, DNA extraction and real-time qPCR were performed to quantify and identify the DNA present. Short tandem repeat (STR) profiling was also performed. The results showed that the quantity of DNA recovered was decreased by 16% in DFO-treated cigarettes and by 27% in 1,2-IND/Zn-treated cigarettes when compared to untreated controls. When the STR profiling results were compared with those of the control sample, DFO, and 1,2-IND/Zn reagent-treated DNA samples showed individualized genotyping at several loci. Results of this study showed that when cigarette butts were found, DFO and 1,2-IND/Zn reagents could be used for DNA profiling after fingerprint identification. However, the effect of DFO on STR profiling was less than that of 1,2-IND/Zn. Therefore, we recommend the use of DFO for fingerprinting cigarette butts if further DNA processing is planned.

Improved infrared spectroscopic discrimination between gall bladder (GB) polyps and GB cancer using component-descriptive spectral features of separated phases from bile.

This study demonstrates a unique strategy for enhancing infrared (IR) spectroscopic discrimination between gall bladder (GB) polyps and cancer. This strategy includes the separation of raw bile juice into three sections of organic, aqueous, and amphiphilic phases and a cooperative combination of all IR spectral features of each separated phase for the discrimination. Raw bile juice is viscous and complex in composition because it contains fatty acids, cholesterol, proteins, phospholipids, bilirubin, and other components; therefore, the acquisition of IR spectra providing more component-discernible information is fundamental for improving discrimination. For this purpose, raw bile juice was separated into an aqueous phase, mostly containing bile salts, an organic phase with isolated lipids, and an amphiphilic phase, mainly containing proteins. The subsequent IR spectra of each separated phase were mutually characteristic and complementary to each other. When all the IR spectral features were combined, the discrimination was improved compared to that using the spectra of raw bile juice with no separation. The cooperative integration of more component-specific spectra obtained from each separated phase enhanced the discrimination. In addition, the IR spectra of the major constituents in bile juice, such as bile acids, conjugated bile salts, lecithin, and cholesterol, were recorded to explain the IR features of each separated phase.

Radiosensitizing effect of PSMC5, a 19S proteasome ATPase, in H460 lung cancer cells.

The function of PSMC5 (proteasome 26S subunit, ATPase 5) in tumors, particularly with respect to cancer radioresistance, is not known. Here, we identified PSMC5 as a novel radiosensitivity biomarker, demonstrating that radiosensitive H460 cells were converted to a radioresistance phenotype by PSMC5 depletion. Exposure of H460 cells to radiation induced a marked accumulation of cell death-promoting reactive oxygen species, but this effect was blocked in radiation-treated H460 PSMC5-knockdown cells through downregulation of the p53-p21 pathway. Interestingly, PSMC5 depletion in H460 cells enhanced both AKT activation and MDM2 transcription, thereby promoting the degradation of p53 and p21 proteins. Furthermore, specific inhibition of AKT with triciribine or knockdown of MDM2 with small interfering RNA largely restored p21 expression in PSMC5-knockdown H460 cells. Our data suggest that PSMC5 facilitates the damaging effects of radiation in radiation-responsive H460 cancer cells and therefore may serve as a prognostic indicator for radiotherapy and molecular targeted therapy in lung cancer patients.

Knockdown of hepatoma-derived growth factor-related protein-3 induces apoptosis of H1299 cells via ROS-dependent and p53-independent NF-κB activation.

We previously identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistant biomarker in p53 wild-type A549 cells and found that p53-dependent induction of the PUMA pathway was a critical event in regulating the radioresistant phenotype. Here, we found that HRP-3 knockdown regulates the radioresistance of p53-null H1299 cells through a distinctly different molecular mechanism. HRP-3 depletion was sufficient to cause apoptosis of H1299 cells by generating substantial levels of reactive oxygen species (ROS) through inhibition of the Nrf2/HO-1 antioxidant pathway. Subsequent, ROS-dependent and p53-independent NF-κB activation stimulated expression of c-Myc and Noxa proteins, thereby inducing the apoptotic machinery. Our results thus extend the range of targets for the development of new drugs to treat both p53 wild-type or p53-null radioresistant lung cancer cells.

Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation.

Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotype of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer.

Identification of HDAC4 as a target of γ-catenin that regulates the oncogenic K-Ras-mediated malignant phenotype of Rat2 cells.

The mechanisms by which activated Ras accelerates malignant transformation of normal cells are not fully understood. Here, we characterized the role and molecular mechanism of γ-catenin in regulating the malignant phenotype of Rat2 cells induced by codon 12-mutant K-Ras (K-Ras12V). Suppression of γ-catenin signaling by K-Ras12V was an early event and played a crucial role in promoting the acquisition of a highly metastatic phenotype of Rat2 cells. Notably, the gene encoding histone deacetylase 4 (HDAC4) was identified as a target of γ-catenin during this process. The transcription factor, lymphoid enhancer-binding factor-1 (Lef1), was involved in the modulation of HDAC4 transcription, and disruption of this pathway was a key event in promoting the invasion and migration of K-Ras12V-transduced Rat2 cells. Thus, our findings extend the range of targets for the development of new drugs for the therapy of oncogenic K-Ras-driven cancer.

Nonintegrating Direct Conversion Using mRNA into Hepatocyte-Like Cells.

Recently, several researchers have reported that direct reprogramming techniques can be used to differentiate fibroblasts into hepatocyte-like cells without a pluripotent intermediate step. However, the use of viral vectors for conversion continues to pose important challenges in terms of genome integration. Herein, we propose a new method of direct conversion without genome integration with potential clinical applications. To generate hepatocyte-like cells, mRNA coding for the hepatic transcription factors Foxa3 and HNF4α was transfected into mouse embryonic fibroblasts. After 10-12 days, the fibroblasts converted to an epithelial morphology and generated colonies of hepatocyte-like cells (R-iHeps). The generated R-iHeps expressed hepatocyte-specific marker genes and proteins, including albumin, alpha-fetoprotein, HNF4α, CK18, and CYP1A2. To evaluate hepatic function, indocyanine green uptake, periodic acid-Schiff staining, and albumin secretion were assessed. Furthermore, mCherry-positive R-iHeps were engrafted in the liver of Alb-TRECK/SCID mice, and we confirmed FAH enzyme expression in Fah1RTyrc/RJ models. In conclusion, our data suggest that the nonintegrating method using mRNA has potential for cell therapy.

Knockdown of end-binding protein 1 induces apoptosis in radioresistant A549 lung cancer cells via p38 kinase-dependent COX-2 upregulation.

The role of end-binding protein 1 (EB1) in lung cancer tumorigenesis and radiotherapy remains poorly understood. In the present study, we observed that EB1 was highly expressed in lung tumor tissues compared with normal non-tumor tissues based on immunohistochemical analysis of lung cancer tissue samples obtained from human tissue microarrays. EB1 was also highly overexpressed in radioresistant lung and cervical cancer cells, which exhibited increased cell death after EB1 silencing. The cytotoxicity induced by EB1 gene knockdown was due to the activation and generation of reactive oxygen species by p38 mitogen-activated protein kinase. Notably, this signaling cascade, however not nuclear factor-κB-mediated signaling, induced the expression of cyclooxygenase-2, a key effector of apoptotic death. Our results provided new molecular evidence supporting the use of EB1 as a novel target in lung cancer therapy, especially in the case of radioresistance.

mHGTD-P mediates hypoxic neuronal cell death via the release of apoptosis-inducing factor.

HGTD-P is a pro-apoptotic target protein of hypoxia-inducible factor 1alpha (HIF-1alpha). It localizes to mitochondria and induces the mitochondrial permeability transition through its interaction with voltage dependent anion channels when overexpressed. However, the molecular mechanisms responsible for its induction and its downstream effector molecules required during cell death, especially in neuronal cell death by hypoxia, are largely unknown. We performed this work to elucidate the effects of the pro-apoptotic protein HGTD-P on neuronal cell death induced by hypoxia and to investigate the cell death mechanisms activated during this process. In this report, we show that mouse HGTD-P (mHGTD-P) is transcriptionally increased by hypoxia and that its overexpression triggers neuronal cell death with affected cells displaying shrunken cytoplasm and condensed pyknotic nuclei in a caspase-independent manner. In addition, suppression of endogenous mHGTD-P expression by siRNA rescues neuronal cells from hypoxic injury. Finally, we show that mHGTD-P induces the mitochondrial release of apoptosis-inducing factor into the cytoplasm. Taken together, our data suggest that mHGTD-P participates in caspase-independent hypoxic neuronal cell death. Future studies will be necessary in order to determine whether hypoxia-induced mHGTD-P expression has any relevance in an ischemic animal model or clinical hypoxia-induced disorders.

GLTSCR2 sensitizes cells to hypoxic injury without involvement of mitochondrial apoptotic cascades.

OBJECTIVE: We attempted to identify novel genes that induce hypoxic cell death to better understand the molecular mechanisms underlying hypoxia-induced cell death. Through this process the GLTSCR2 gene was found. The purpose of this work was to investigate the role of GLTSCR2 in hypoxic cell death pathways. METHODS: This work focuses on an investigation of roles and mechanisms of GLTSCR2 in hypoxic cell death by means of subtractive hybridization, RT-PCR, Western blot, immunocytochemistry, cell death assay, transient gene overexpression, and determination of mitochondrial membrane potential. RESULTS: We found that GLTSCR2 was transcriptionally suppressed by hypoxia, and ectopic expression of GLTSCR2 sensitized cells to hypoxic injury. Interestingly, while the majority of hypoxia-inducible pro-death proteins signal through mitochondrion-dependent pathways, GLTSCR2-overexpressed cells underwent apoptosis in a mitochondrion- and caspase-independent manner. CONCLUSION: Our data categorizes GLTSCR2 as a proapoptotic protein sensitizing cells to hypoxic injury when overexpressed.

Phosphoprotein profiles of candidate markers for early cellular responses to low-dose γ-radiation in normal human fibroblast cells.

Ionizing radiation causes biological damage that leads to severe health effects. However, the effects and subsequent health implications caused by exposure to low-dose radiation are unclear. The objective of this study was to determine phosphoprotein profiles in normal human fibroblast cell lines in response to low-dose and high-dose γ-radiation. We examined the cellular response in MRC-5 cells 0.5 h after exposure to 0.05 or 2 Gy. Using 1318 antibodies by antibody array, we observed ≥1.3-fold increases in a number of identified phosphoproteins in cells subjected to low-dose (0.05 Gy) and high-dose (2 Gy) radiation, suggesting that both radiation levels stimulate distinct signaling pathways. Low-dose radiation induced nucleic acid-binding transcription factor activity, developmental processes, and multicellular organismal processes. By contrast, high-dose radiation stimulated apoptotic processes, cell adhesion and regulation, and cellular organization and biogenesis. We found that phospho-BTK (Tyr550) and phospho-Gab2 (Tyr643) protein levels at 0.5 h after treatment were higher in cells subjected to low-dose radiation than in cells treated with high-dose radiation. We also determined that the phosphorylation of BTK and Gab2 in response to ionizing radiation was regulated in a dose-dependent manner in MRC-5 and NHDF cells. Our study provides new insights into the biological responses to low-dose γ-radiation and identifies potential candidate markers for monitoring exposure to low-dose ionizing radiation.

Estimation of low-dose radiation-responsive proteins in the absence of genomic instability in normal human fibroblast cells.

PURPOSE: Low-dose radiation has various biological effects such as adaptive responses, low-dose hypersensitivity, as well as beneficial effects. However, little is known about the particular proteins involved in these effects. Here, we sought to identify low-dose radiation-responsive phosphoproteins in normal fibroblast cells. MATERIALS AND METHODS: We assessed genomic instability and proliferation of fibroblast cells after γ-irradiation by γ-H2AX foci and micronucleus formation analyses and BrdU incorporation assay, respectively. We screened fibroblast cells 8 h after low-dose (0.05 Gy) γ-irradiation using Phospho Explorer Antibody Microarray and validated two differentially expressed phosphoproteins using Western blotting. RESULTS: Cell proliferation proceeded normally in the absence of genomic instability after low-dose γ-irradiation. Phospho antibody microarray analysis and Western blotting revealed increased expression of two phosphoproteins, phospho-NFκB (Ser536) and phospho-P70S6K (Ser418), 8 h after low-dose radiation. CONCLUSIONS: Our findings suggest that low-dose radiation of normal fibroblast cells activates the expression of phospho-NFκB (Ser536) and phospho-P70S6K (Ser418) in the absence of genomic instability. Therefore, these proteins may be involved in DNA damage repair processes.

Adrenomedullin regulates cellular glutathione content via modulation of gamma-glutamate-cysteine ligase catalytic subunit expression.

Adrenomedullin (AM) participates in a wide range of physiological and pathological processes including vasorelaxation, angiogenesis, cancer promotion, and apoptosis. Recently, it has been reported that AM protects a variety of cells against oxidative stress induced by stressors such as hypoxia, ischemia/reperfusion, and hydrogen peroxide through the phosphatidylinositol 3-kinase (PI3K)-dependent pathway. However, the molecular mechanisms underlying the pathway of cell survival against hypoxic injury are largely unknown. In an effort to investigate the survival mechanism against hypoxic injury, we studied the effects of AM on cellular levels of reactive oxygen species, well-known mediators of cell death after oxidative stress, and the mechanism involved in the regulation of reactive oxygen species levels. Here, we show that AM increases gamma-glutamate-cysteine ligase (gamma-GCL) activity under both hypoxic and normoxic conditions, resulting in an up-regulation of cellular glutathione levels to more than 2-fold higher than basal expression. In addition, we demonstrate that AM induces concentration-dependent expression of the catalytic subunit of gamma-GCL (gamma-GCLC) at the mRNA and protein levels through the activation of the gamma-GCLC promoter fragment sequence from -597 to -320. However, when treated with the PI3K inhibitors, the effects of AM on gamma-GCLC expression were completely abrogated, suggesting that a PI3K pathway linked AM with the transcriptional activation of the gamma-GCLC promoter. Taken together, our data suggests that AM participates in the regulation of cellular redox status via glutathione synthesis. These results may explain, in part, the mechanism by which AM protects cells against oxidative stress.

Interaction of pro-apoptotic protein HGTD-P with heat shock protein 90 is required for induction of mitochondrial apoptotic cascades.

HGTD-P is a hypoxia-responsive pro-apoptotic protein that transmits hypoxic signals directly to mitochondria. When overexpressed, HGTD-P induces cell death via typical mitochondrial apoptotic cascades. However, much is unknown about post-transcriptional modification and signaling networks of HGTD-P in association with cell death-regulating proteins. We performed yeast two-hybrid screening to identify the molecules involved in HGTD-P-mediated cell death pathways. In this study, we show that heat shock protein 90 physically interacts with HGTD-P and that suppression of Hsp90 activity by low concentrations of geldanamycin reduced HGTD-P-induced mitochondrial catastrophe through inhibition of mitochondrial translocation of HGTD-P.

Radiotherapy diagnostic biomarkers in radioresistant human H460 lung cancer stem-like cells.

Tumor cell radioresistance is a major contributor to radiotherapy failure, highlighting the importance of identifying predictive biomarkers for radioresistance. In this work, we established a radioresistant H460 (RR-H460) cell line from parental radiosensitive H460 lung cancer cells by exposure to fractionated radiation. The radiation-resistant, anti-apoptotic phenotype of RR-H460 cell lines was confirmed by their enhanced clonogenic survival and increased expression of the radioresistance genes Hsp90 and Her-3. RR-H460 cells displayed characteristics of cancer stem-like cells (CSCs), including induction of the surface marker CD44 and stem cell markers Nanog, Oct4, and Sox2. RR-H460 cells also exhibited sphere formation and malignant behavior, further supporting a CSC phenotype. Using proteomic analyses, we identified 8 proteins that were up-regulated in RR-H460 CSC lines and therefore potentially involved in radioresistance and CSC-related biological processes. Notably, 4 of these-PAI-2, NOMO2, KLC4, and PLOD3-have not been previously linked to radioresistance. Depletion of these individual genes sensitized RR-H460 cells to radiotoxicity and additively enhancing radiation-induced apoptosis. Our findings suggest the possibility of integrating molecular targeted therapy with radiotherapy as a strategy for resolving the radioresistance of lung tumors.

Depletion of end-binding protein 1 (EB1) promotes apoptosis of human non-small-cell lung cancer cells via reactive oxygen species and Bax-mediated mitochondrial dysfunction.

Although end-binding protein 1 (EB1) is well known to regulate microtubule dynamics, the role of EB1 in apoptosis of non-small cell lung cancer (NSCLC) is poorly understood. Here, we investigated the molecular mechanism by which EB1 regulates apoptosis in H460, A549, and H1299 cells. Depletion of EB1 in A549 and H1299 cells, which express high levels of EB1, induced cell death in a p53-independent manner through over-production of reactive oxygen species (ROS) and Bax induction. This phenomenon was potentiated in radiation-treated EB1-knockdown cells and was largely blocked by N-acetyl-L-cysteine, a scavenger of ROS. ROS accelerated the activation of nuclear factor-kappa B (NF-κB) to promote transcriptional activity of Bax, an action that was accompanied by cytochrome c translocation and apoptosis-inducing factor (AIF) release. The NF-κB inhibitor, BAY 11-7082, potently inhibited the apoptosis induced by EB1 knockdown and radiation treatment, in association with diminished activity of the mitochondrial death pathway. Conversely, ectopic overexpression of EB1 in H460 cells, which express low levels of EB1, remarkably abrogated radiation-induced apoptosis and NF-κB-mediated mitochondrial dysfunction. Our data provide the first demonstration that down-regulation of EB1 promotes NSCLC cell death by inducing ROS-mediated, NF-κB-dependent Bax signaling cascades, a process in which cytochrome c and AIF play important roles, indicating a potential therapeutic benefit of EB1 in lung cancer.