Sirt6 attenuates hypoxia-induced tubular epithelial cell injury via targeting G2/M phase arrest

Acute kidney injury (AKI) is a condition that has a high incidence and death rate. Unfortunately, the kidney may not recover completely after AKI, which then develops to chronic kidney disease (CKD). Therefore, it is necessary to identify potential curative targets to avoid its development to CKD. As an NAD⁺-dependent deacetylase, sirtuin 6 (Sirt6) has been linked to different types of biological processes. In the present work, our group investigated the role of Sirt6 in tubular epithelial cells (TECs) under hypoxic stress. Sirt6 expression was examined in mouse kidney following ischemia/reperfusion (IR) injury and hypoxia-challenged TECs. Using Sirt6 plasmid and small interfering RNA, we also investigated how, in regard to inflammation and epithelial-to-mesenchymal transition, Sirt6 affects hypoxia-triggered injury. In addition, cell cycle was detected in hypoxia-challenged TECs. Sirt6 was downregulated in the kidney of mice with IR injury and hypoxia-challenged TECs. Consequently, Sirt6 depletion aggravated hypoxia-induced injury and G2/M phase arrest. Sirt6 overexpression attenuated hypoxia-triggered damage and G2/M phase arrest in TECs. Sirt6 prevented hypoxia-triggered TEC damage via suppressing G2/M phase arrest. Thus, Sirt6 is a possible candidate for alleviating the effects of kidney injury.     

Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis

Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.     

Hepatic stellate cell activation in vitro: cell cycle arrest at G2/M and modification of cell motility

Hepatic fibrosis is a common response to chronic liver injury and is characterized by increased production of extracellular matrix components, whose major part is produced by hepatic stellate cells activated by inflammatory mediators to proliferate and migrate into the injured regions. GRX cells are a model of hepatic stellate cells characterized as myofibroblasts by morphological and biochemical criteria. We have recently shown that they respond to inflammatory mediators and cytokines present in the concanavalin A-activated spleen cell supernatant (SCS) by quantitative changes in the expression of intermediate filaments. The present study investigated the effects of SCS and TNF-alpha on the GRX cell proliferation and on the organization of the actin cytoskeleton. SCS and TNF-alpha diminished the culture cell density, with an increase of cell [(3)H]thymidine incorporation and of cellular protein content, indicating an arrest in the G2/M phase of the cell cycle, which was reversible 48 h after removal of SCS. This effect was abrogated by dibutiryl-cAMP. Actin cytoskeleton reorganization was observed after 24 h treatment, indicating increased cell motility. Our results suggest that inflammation-dependent activation of stellate cells occurs in ordered interaction and coordination of proinflammatory agents. The increase of cAMP levels activates the conversion of lipocytes into myofibroblasts and increases the number of cells that can participate in repair. Since cAMP retains cells in the G1 phase, cytokines of the TNF-alpha group are required for cell proliferation inducing the entry into the S phase. The progression through the G2/M checkpoint is mediated again by increased cAMP levels.     

Persistent activation of autophagy in kidney tubular cells promotes renal interstitial fibrosis during unilateral ureteral obstruction

Renal fibrosis is the final, common pathway of end-stage renal disease. Whether and how autophagy contributes to renal fibrosis remains unclear. Here we first detected persistent autophagy in kidney proximal tubules in the renal fibrosis model of unilateral ureteral obstruction (UUO) in mice. UUO-associated fibrosis was suppressed by pharmacological inhibitors of autophagy and also by kidney proximal tubule-specific knockout of autophagy-related 7 (PT-Atg7 KO). Consistently, proliferation and activation of fibroblasts, as indicated by the expression of ACTA2/α-smooth muscle actin and VIM (vimentin), was inhibited in PT-Atg7 KO mice, so was the accumulation of extracellular matrix components including FN1 (fibronectin 1) and collagen fibrils. Tubular atrophy, apoptosis, nephron loss, and interstitial macrophage infiltration were all inhibited in these mice. Moreover, these mice showed a specific suppression of the expression of a profibrotic factor FGF2 (fibroblast growth factor 2). In vitro, TGFB1 (transforming growth factor β 1) induced autophagy, apoptosis, and FN1 accumulation in primary proximal tubular cells. Inhibition of autophagy suppressed FN1 accumulation and apoptosis, while enhancement of autophagy increased TGFB1-induced-cell death. These results suggest that persistent activation of autophagy in kidney proximal tubules promotes renal interstitial fibrosis during UUO. The profibrotic function of autophagy is related to the regulation on tubular cell death, interstitial inflammation, and the production of profibrotic factors.     

An Atg10-like E2 enzyme is essential for cell cycle progression but not autophagy in Schizosaccharomyces pombe

Many proteins involved in autophagy have been identified in the yeast Saccharomyces cerevisiae. For example, Atg3 and Atg10 are two E2 enzymes that facilitate the conjugation of the ubiquitin-like proteins (Ubls) Atg8 and Atg12, respectively. Here, we describe the identification and characterization of the predicted Atg10 homolog (SpAtg10) of the evolutionarily distant Schizosaccharomyces pombe. Unexpectedly, SpAtg10 is not essential for autophagy. Instead, we find that SpAtg10 is essential for normal cell cycle progression, and for responses to various stress conditions that perturb the cell cycle, independently of Atg12 conjugation. Taken together, our data indicate that autophagic Ubl conjugation pathways differ between eukaryotes and, furthermore, that enzymes such as Atg10 may have additional functions in controlling key cellular processes such as cell cycle progression. Atg10-related proteins are found from yeast to humans, and, thus, this study has implications for understanding the functions of this protein family in Ubl conjugation in eukaryotes.     

Adenosine induces G2/M cell‐cycle arrest by inhibiting cell mitosis progression

Cellular adenosine accumulates under stress conditions. Few papers on adenosine are concerned with its function in the cell cycle. The cell cycle is the essential mechanism by which all living things reproduce and the target machinery when cells encounter stresses, so it is necessary to examine the relationship between adenosine and the cell cycle. In the present study, adenosine was found to induce G₂/M cell‐cycle arrest. Furthermore, adenosine was found to modulate the expression of some important proteins in the cell cycle, such as cyclin B and p21, and to inhibit the transition of metaphase to anaphase in mitosis.     

Reversine suppresses oral squamous cell carcinoma via cell cycle arrest and concomitantly apoptosis and autophagy

Background

The effective therapies for oral cancer patients of stage III and IV are generally surgical excision and radiation combined with adjuvant chemotherapy using 5-Fu and Cisplatin. However, the five-year survival rate is still less than 30% in Taiwan. Therefore, evaluation of effective drugs for oral cancer treatment is an important issue. Many studies indicated that aurora kinases (A, B and C) were potential targets for cancer therapies. Reversine was proved to be a novel aurora kinases inhibitor with lower toxicity recently. In this study, the potentiality for reversine as an anticancer agent in oral squamous cell carcinoma (OSCC) was evaluated.

Methods

Effects of reversine on cell growth, cell cycle progress, apoptosis, and autophagy were evaluated mainly by cell counting, flow cytometry, immunoblot, and immunofluorescence.

Results

The results demonstrated that reversine significantly suppressed the proliferation of two OSCC cell lines (OC2 and OCSL) and markedly rendered cell cycle arrest at G2/M stage. Reversine also induced cell death via both caspase-dependent and -independent apoptosis. In addition, reversine could inhibit Akt/mTORC1 signaling pathway, accounting for its ability to induce autophagy.

Conclusions

Taken together, reversine suppresses growth of OSCC via multiple mechanisms, which may be a unique advantage for developing novel therapeutic regimens for treatment of oral cancer in the future.     

Single-strand breaks, cell cycle arrest and apoptosis in HL-60 and LLCPK1 cells exposed to 1,2-dibromo-3-chloropropane

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK₁, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30–300 µmol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK₁ cells (100 µmol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 µmol/L. Flow cytometric analysis showed that DBCP (1–10 µmol/L) exposure for 24 h caused an accumulation of LLCPK₁ cells in the G₂/M-phase. In HL-60 cells the accumulation in G₂/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK₁ cells exposed to 100–500 µmol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G₁ cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK₁ cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G₂/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.     

Effect of potassium on cell volume regulation in renal straight proximal tubules

Summary The present study was designed to assess for the influence of extracellular potassium and of inhibitors of potassium transport on cell volume regulatory decrease in isolated perfused straight proximal tubules of the mouse kidney. Volume regulatory decrease is virtually unaffected when bath potassium concentration is elevated from 5 to 20 mmol/liter, and still persists, albeit significantly retarded, in the presence of the potassium channel blocker barium on both sides of the epithelium and during virtually complete dissipation of the transmembrane potassium gradient by increasing extracellular potassium concentration to 40 mmol/liter. As evident from electrophysiologic observations, barium blocks the potassium conductance of the basolateral cell membrane. Reduction of bicarbonate concentration and increase of H⁺ concentration in the bath solution cannot compensate for enhanced potassium concentration and cell volume regulatory decrease is not affected in the presence of the K/H exchange inhibitor omeprazole. Similarly cell volume regulatory decrease is not affected by ouabain. In conclusion, potassium movements through potassium channels in the basolateral cell membrane are important determinants of cell volume and may participate in cell volume regulatory decrease. However, a powerful component of cell volume regulatory decrease in straight proximal tubules of the mouse kidney is apparently independent of potassium conductive pathways, K/H exchange and Na⁺/K⁺-ATPase.     

p~(38)MAPK在IL-18诱导肾小管上皮细胞转分化中的作用

目的:白细胞介素18(IL-18)可诱导肾小管上皮细胞转分化,本研究探讨其是否是通过p38MAPK途径而起作用。方法:应用不同浓度的p38MAPK通路特异性阻断剂SB203580(0、5、10、20μmol/L)预孵育人近端肾小管上皮细胞(HK-2细胞)30min后,加入IL-18(100ng/ml)共培养24、48、72h。应用RT-PCR法检测α-平滑肌肌动蛋白(α-SMA)mRNA的表达水平;应用ELISA法测定细胞浆中α-SMA蛋白质含量。结果:SB203580呈剂量依赖性地抑制IL-18诱导的HK-2细胞α-SMA基因表达(P0.05)。结论:p38MAPK通路是调控IL-18诱导肾小管上皮细胞转分化的主要信号通路之一。     

Flavonoids from tartary buckwheat induce G2/M cell cycle arrest and apoptosis in human hepatoma HepG2 cells

The cytotoxicity and antioxidant activity on human hepa toma cell line HepG2 of three flavonoids homogenous com pounds from tartary buckwheat seeds and bran, namely quercetin, isoquercetin, and rutin, were investigated. The total antioxidant competency detection results indicated that the antioxidant capacity of quercetin was the strongest in a biological response system. A [3-（4,5-dimethylthiazol-2-yl）-2,5diphenyltetrazolium bromide] assay showed that quercetin exhibited the strongest cytotoxic effects against the HepG2 cell line. Flow cytometric analysis indicated that quercetin significantly increased the production of reactive oxygen species, and led to the G2/M phase arrest accom panied by an increase of apoptotic cell death after 48 h of incubation. Quercetininduced cell apoptosis was shown to involve p53 and p21 upregulation, Cyclin D1, Cdk2, and Cdk7 downregulation. These results suggested that the in duction of G2/M arrest, apoptosis, and cell death by quer cetin may associate with increased expression of p53 and p21, decrease of Cyclin D1, Cdk2, and Cdk7 levels, and generation of reactive oxygen species in cells. This study will help to better understand and fully utilize medicinal resources of plant flavonoids.