Komgrid Charngkaew

Gene expression profiling of cholangiocarcinoma-derived fibroblast reveals alterations related to tumor progression and indicates periostin as a poor prognostic marker

BackgroundFibroblasts play important roles in several cancers. It was hypothesized that cholangiocarcinoma (CCA)-associated fibroblasts (Cfs) differ from non-tumorigenic liver fibroblasts (Lfs) in their gene expression profiles resulting in the capability to promote cancer. Periostin (PN) is a multi-functional protein and has emerged as a promising marker for tumor progression. The role of PN in CCA, however, has not yet been explored.ResultsIn this study, the gene expression profile of Cfs in comparison to Lfs was performed using oligonucleotide microarrays. The common- and unique-expressed genes in Cfs and the promising roles in cancer promotion and progression were determined. PN was markedly over-expressed in Cfs confirmed by real time RT-PCR and western blot analysis. Immunohistochemistry examination of a number of patients with intrahepatic CCA showed the expression of PN solely in stromal fibroblasts, but was expressed neither in cancer cells nor immune cells. Low to no expression of PN was observed in tissues of benign liver disease and hepatocellular carcinoma. CCA patients with high levels of PN had significantly shorter survival time than those with low levels (P = 0.026). Multivariate analysis revealed high levels of PN (P = 0.045) and presence of lymph node metastasis (P = 0.002) as independent poor prognostic factors. The in vitro study revealed that recombinant PN induced CCA cell proliferation and invasion. Interestingly, interference RNA against integrin α5 significantly reduced the cellular response to PN-stimulated proliferation and invasion.ConclusionThe gene expression profile of fibroblasts in CCA is apparently explored for the first time and has determined the genes involving in induction of this cancer progression. High PN can be used to distinguish CCA from other related liver diseases and is proposed as a prognostic factor of poor survival. Regulation of fibroblast-derived PN in CCA proliferation and invasion may be considered as an alternative therapeutic approach.

Melatonin attenuates methamphetamine-induced disturbances in mitochondrial dynamics and degeneration in neuroblastoma SH-SY5Y cells

Methamphetamine (METH) is a psychostimulant drug that can cause toxicity and degeneration in the brain. The toxicity due to METH involves multiple pathways, including the mitochondrial‐dependent death pathway. Several pieces of evidence have emphasized that the fragmentation of mitochondria into smaller structures plays some role in the cell‐death process. In this study, we investigated the role of mitochondrial dynamics in METH‐induced toxicity in human dopaminergic neuroblastoma SH‐SY5Y cultured cell lines. In addition, the protective effect of melatonin against METH‐induced toxicity was investigated. Our results show that METH significantly decreased cell viability and increased the levels of the mitochondrial fission protein, Fis1 and the Drp1 oligomer. However, the levels of the mitochondrial fusion proteins OPA1 and Mfn1 did not change in METH‐treated cells. Melatonin can reverse the toxic effects of the METH‐induced reduction in cell viability and the production of the Fis1 protein and the Drp1 oligomer. Moreover, the morphological alteration of mitochondria was investigated in METH‐treated cells in the presence of melatonin using transmission electron microscopy (TEM). At 24 hr after METH exposure, typical cell shrinkage was observed in SH‐SY5Y cells. Mitochondria were fragmented into small globular structures in a large proportion of METH‐treated cells, but tubular networks of mitochondria were present in large proportions of control‐untreated cells and METH‐treated cells in the presence of melatonin. The results of the present study demonstrate the potential of melatonin to reduce cell death and restore mitochondrial function in neurons affected by METH‐induced toxicity.

Autophagic degradation of aquaporin-2 is an early event in hypokalemia-induced nephrogenic diabetes insipidus

Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.

Increased ETV4 expression correlates with estrogen-enhanced proliferation and invasiveness of cholangiocarcinoma cells

BackgroundCholangiocarcinoma (CCA) is one of the worst prognosis cancer. The survival time of CCA patients is related to serum estrogen levels and estrogen has been found to enhance the proliferation and invasiveness of CCA cells in vitro. This has led to the suggestion that estrogen may play an important role in the progression of CCA. This study tests the relevance of the previous in vitro findings in vivo using a mouse xenograft model of CCA, and investigates possible signaling mechanisms involved.MethodsKKU-213 and KKU-139 CCA cell lines were used in the experiments, xenografted to nude mice and treated with a potent estrogenic agent, 17β-estradiol (E2), and/or with tamoxifen (TAM), an estrogen antagonist.ResultsThe results demonstrated that E2 could accelerate growth of the xenograft-tumor and the effect was inhibited by TAM. PCR array screening of E2 responsive genes suggested ETV4 as a promising candidate intracellular mediator. ETV4-knockdown CCA cells were generated and these showed a diminished responsiveness to E2 in both cell and spheroid proliferation assays, and in invasion tests. These results point to ETV4 as a possible mediator of E2-activated CCA progression and as a potential target of TAM-mediated inhibition.ConclusionsFinally, TAM may be suggested as an adjunctive treatment of CCA to improve the conventional cytotoxic method with more patient toleration.

Cyclin D1 depletion interferes with oxidative balance and promotes cancer cell senescence

ABSTRACT Expression of cyclin D1 (CCND1) is required for cancer cell survival and proliferation. This is presumably due to the role of cyclin D1 in inactivation of the RB tumor suppressor. Here, we investigated the pro-survival function of cyclin D1 in a number of cancer cell lines. We found that cyclin D1 depletion facilitated cellular senescence in several cancer cell lines. Senescence triggered by cyclin D1 depletion was more extensive than that caused by the prolonged CDK4 inhibition. Intriguingly, the senescence caused by cyclin D1 depletion was independent of RB status of the cancer cell. We identified a build-up of intracellular reactive oxygen species in the cancer cells that underwent senescence upon depletion of cyclin D1 but not in those cells where CDK4 was inhibited. The higher ROS levels were responsible for the cell senescence, which was instigated by the p38-JNK-FOXO3a-p27 pathway. Therefore, expression of cyclin D1 prevents cancer cells from undergoing senescence, at least partially, by keeping the level of intracellular oxidative stress at a tolerable sub-lethal level. Depletion of cyclin D1 promotes the RB-independent pro-senescence pathway and the cancer cells then succumb to the endogenous oxidative stress levels. This article has an associated First Person interview with the first author of the paper. Summary: The cell cycle protein cyclin D1, independently of its role in cell cycle regulation, acts as a controller of oxidative balance in cancer cells. This function is required for cancer cell survival.

More complete polarization of renal tubular epithelial cells by artificial urine

Cell polarization using Transwell is a common method employed to study renal tubular epithelial cells. However, this conventional protocol does not precisely recapitulate renal tubular epithelial cell phenotypes. In this study, we simulated renal physiological microenvironment by replacing serum-containing culture medium in upper chamber of the Transwell with physiologic artificial urine (AU) (to mimic renal tubular fluid), whereas the lower chamber still contained serum-containing medium (to mimic plasma-enriched renal interstitium). Comparing to the conventional protocol (control), the AU-assisted protocol offered more complete polarization of MDCK renal tubular cells as indicated by higher transepithelial electrical resistance (TER) and greater levels of tight junction (TJ) proteins (ZO-1 and occludin). Transmission electron microscopy (TEM) showed greater densities of TJ and desmosome, narrower intercellular spaces, greater cell height, and longer microvilli in the AU-treated cells. Secretome analysis revealed that the AU-treated cells secreted greater proportion of the proteins matched to normal human urinary proteome via both classical and non-classical secretory pathways. Finally, modifying/omitting each component of AU (one at a time) followed by validation revealed that urea was responsible for such property of AU to improve cell polarization. These data indicate that replacing AU on the upper chamber of Transwell can improve or optimize renal cell polarization for more precise investigations of renal physiology and cell biology in vitro.

Microparticle and anti-influenza activity in human respiratory secretion

Respiratory secretions, such as saliva and bronchoalveolar fluid, contain anti-influenza activity. Multiple soluble factors have been described that exert anti-influenza activity and are believed to be responsible for the anti-influenza activity in respiratory secretions. It was previously shown that a bronchial epithelial cell culture could produce exosome-like particles with anti-influenza activity. Whether such extracellular vesicles in respiratory secretions have anti-influenza activity is unknown. Therefore, we characterized bronchoalveolar lavage fluid and found microparticles, which mostly stained positive for epithelial cell markers and both α2,3- and α2,6-linked sialic acid. Microparticles were purified from bronchoalveolar lavage fluid and shown to exhibit anti-influenza activity by a hemagglutination inhibition (HI) assay and a neutralization (NT) assay. In addition, physical binding between influenza virions and microparticles was demonstrated by electron microscopy. These findings indicate that respiratory microparticles containing viral receptors can exert anti-viral activity by probably trapping viral particles. This innate mechanism may play an important role in the defense against respiratory viruses.

γ-COPI mediates the retention of kAE1 G701D protein in Golgi apparatus – a mechanistic explanation of distal renal tubular acidosis associated with the G701D mutation

Mutations of the solute carrier family 4 member 1 (SLC4A1) gene encoding kidney anion (chloride/bicarbonate ion) exchanger 1 (kAE1) can cause genetic distal renal tubular acidosis (dRTA). Different SLC4A1 mutations give rise to mutant kAE1 proteins with distinct defects in protein trafficking. The mutant kAE1 protein may be retained in endoplasmic reticulum (ER) or Golgi apparatus, or mis-targeted to the apical membrane, failing to display its function at the baso-lateral membrane. The ER-retained mutant kAE1 interacts with calnexin chaperone protein; disruption of this interaction permits the mutant kAE1 to reach the cell surface and display anion exchange activity. However, the mechanism of Golgi retention of mutant kAE1 G701D protein, which is otherwise functional, is still unclear. In the present study, we show that Golgi retention of kAE1 G701D is due to a stable interaction with the Golgi-resident protein, coat protein complex I (COPI), that plays a role in retrograde vesicular trafficking and Golgi-based quality control. The interaction and co-localization of kAE1 G701D with the γ-COPI subunit were demonstrated in human embryonic kidney (HEK-293T) cells by co-immunoprecipitation and immunofluorescence staining. Small interference RNA (siRNA) silencing of COPI expression in the transfected HEK-293T cells increased the cell surface expression of transgenic kAE1 G701D, as shown by immunofluorescence staining. Our data unveil the molecular mechanism of Golgi retention of kAE1 G701D and suggest that disruption of the COPI-kAE1 G701D interaction could be a therapeutic strategy to treat dRTA caused by this mutant.