Aldosterone Suppresses Endothelial Mitochondria through Mineralocorticoid Receptor/Mitochondrial Reactive Oxygen Species Pathway.

Excessive aldosterone secretion causes endothelial dysfunction, vascular inflammation, and vascular fibrosis in patients with primary aldosteronism (PA). Endothelial function is closely related to endothelial mitochondria. However, the effects of elevated aldosterone levels on endothelial mitochondria remain unclear. In this study, we used primary cultured human umbilical vein endothelial cells (HUVECs) to investigate the effects of aldosterone on endothelial mitochondria. Mineralocorticoid receptor (MR) small interfering (si)RNA or glucocorticoid receptor (GR) siRNA were used to confirm the pathway by which aldosterone exerts its effects on the mitochondria of HUVECs. The results showed that excess aldosterone suppressed mitochondrial DNA copy numbers, anti-mitochondrial protein, and SOD2 protein expression in a dose- and time-dependent manner. These effects were attenuated by treatment with MR siRNA, but not with GR siRNA. Furthermore, it was attenuated by treatment with a mitochondria-targeted antioxidant (Mito-TEMPO, associated with mitochondrial reactive oxygen species (ROS) production), but not N-acetyl-L-cysteine (associated with cytosolic ROS production), which suggests that the process was through the mitochondrial ROS pathway, but not the cytosolic ROS pathway. In conclusion, aldosterone excess suppressed endothelial mitochondria through the MR/mitochondrial ROS pathway.

Sirtuin 3 Protects against Urban Particulate Matter-Induced Autophagy in Human Bronchial Epithelial Cells.

Urban particulate matter (urban PM) is a heterogeneous mixture of various types of particles originating from different sources. Exposure to high concentrations of urban PM leading to adverse health effects is evaluated by using in vitro cultures of human lung epithelial cells. However, the mechanism underlying the correlation between high concentrations of urban PM exposure and adverse health effects has not been fully elucidated; urban PM-induced oxidative stress is considered as an important mechanism of urban PM-mediated cytotoxicity. Sirtuin 3 (SIRT3), a primary mitrochondrial deacetylase, controls cellular reactive oxygen species (ROS) production, and expression of antioxidant enzymes. In this study, we examined the role of SIRT3 in the regulation of urban PM-induced oxidative stress in normal primary human bronchial epithelial cells (HBEpiCs). Cell viability showed a time- and concentration-dependent decrease when exposed to urban PM, which could indicate that the amount of lactate dehydrogenase released from the cell in response to urban PM is related to cell viability in HBEpiC. The effects of urban PM on morphological and biochemical markers of autophagy in HBEpiC were analyzed by electron microscopy and Western blotting. Overexpression of SIRT3 inhibited urban PM-induced ROS generation, while concomitantly increasing the expression of antioxidant enzymes, and decreasing NF-κB activation and release of inflammation factors. Up-regulation of SIRT3 significantly inhibited the expression of autophagy markers and autophagic vacuole formation. Our findings provide a valuable insight into the potential role of the SIRT3 enzyme in regulating urban PM-induced autophagy by mediating urban PM-induced oxidative stress, which may contribute to urban PM-induced impairment of airway epithelial cell function.

Role of SIRT1 in regulation of epithelial-to-mesenchymal transition in oral squamous cell carcinoma metastasis.

BACKGROUND: The epithelial-to-mesenchymal transition (EMT) process results in a loss of cell-cell adhesion, increased cell mobility, and is crucial for enabling the metastasis of cancer cells. Recently, the enzyme SIRT1 has been implicated in a variety of physiological processes; however, its role in regulating oral cancer metastasis and EMT is not fully elucidated. Here, we propose a mechanism by which the enzyme sirtuin1 (SIRT1) regulates the EMT process in oral cancer by deacetylating Smad4 and repressing the effect of TGF-ß signaling on matrix metalloproteinase-7 (MMP7). METHODS: The roles of SIRT1 in tumor cell migration/invasion and metastasis to the lungs were investigated using the Boyden chamber assay and orthotopic injections, respectively. RNA interference was used to knockdown either SIRT1 or Smad4 expression in oral squamous cell carcinoma (OSCC) cell lines. Immunoblotting, zymographic assays, and co-immunoprecipitation were used to examine the effects of SIRT1 overexpression on MMP7 expression and activity, as well as on SIRT1/ Smad4 interaction. RESULTS: We found that compared with normal human oral keratinocytes (HOKs), SIRT1 was underexpressed in OSCC cells, and also in oral cancer tissues obtained from 14 of 21 OSCC patients compared with expression in their matched normal tissues. Overexpression of SIRT1 inhibited migration of OSCC cells in vitro, as well as their metastasis to the lung in vivo. Furthermore, up-regulation of SIRT1 in metastatic OSCCs significantly inhibited the migration and invasion abilities of OSCC cells, while concomitantly increasing the expression of E-cadherin, and decreasing the expressions of mesenchymal markers. We also identified Smad4, a TGF-ß-activated transcription factor, as a direct target protein for SIRT1. Overexpression of SIRT1 in OSCC cells led to decreased levels of acetylated Smad4, and inhibition of TGF-ß-induced signaling. By associating and deacetylating Smad4, SIRT1 enzyme can influence MMP7 expression, MMP enzyme activity, and consequently, cell migration, invasion, and tumor metastasis in OSCCs. CONCLUSIONS: These findings provide a valuable insight into the potential role of the SIRT1 enzyme in regulating cell migration and invasion in oral squamous cell carcinoma. Our findings suggest the SIRT1/Smad4/MMP7 pathway as a target for oral cancer driven by EMT.

Src-homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis.

BACKGROUND: Tumor invasion and metastasis represent a major unsolved problem in cancer pathogenesis. Recent studies have indicated the involvement of Src-homology 2 domain-containing tyrosine phosphatase 2 (SHP2) in multiple malignancies; however, the role of SHP2 in oral cancer progression has yet to be elucidated. We propose that SHP2 is involved in the progression of oral cancer toward metastasis. METHODS: SHP2 expression was evaluated in paired oral cancer tissues by using immunohistochemical staining and real-time reverse transcription polymerase chain reaction. Isogenic highly invasive oral cancer cell lines from their respective low invasive parental lines were established using a Boyden chamber assay, and changes in the hallmarks of the epithelial-mesenchymal transition (EMT) were assessed to evaluate SHP2 function. SHP2 activity in oral cancer cells was reduced using si-RNA knockdown or enforced expression of a catalytically deficient mutant to analyze migratory and invasive ability in vitro and metastasis toward the lung in mice in vivo. RESULTS: We observed the significant upregulation of SHP2 in oral cancer tissues and cell lines. Following SHP2 knockdown, the oral cancer cells markedly attenuated migratory and invasion ability. We observed similar results in phosphatase-dead SHP2 C459S mutant expressing cells. Enhanced invasiveness was associated with significant upregulation of E-cadherin, vimentin, Snail/Twist1, and matrix metalloproteinase-2 in the highly invasive clones. In addition, we determined that SHP2 activity is required for the downregulation of phosphorylated ERK1/2, which modulates the downstream effectors, Snail and Twist1 at a transcript level. In lung tissue sections of mice, we observed that HSC3 tumors with SHP2 deletion exhibited significantly reduced metastatic capacity, compared with tumors administered control si-RNA. CONCLUSIONS: Our data suggest that SHP2 promotes the invasion and metastasis of oral cancer cells. These results provide a rationale for further investigating the effects of small-molecule SHP2 inhibitors on the progression of oral cancer, and indicate a previously unrecognized SHP2-ERK1/2-Snail/Twist1 pathway that is likely to play a crucial role in oral cancer invasion and metastasis.

Promoter hypermethylation of the gene encoding heat shock protein B1 in oral squamous carcinoma cells.

OBJECTIVE: The mechanism responsible for the regulation of HSPB1 expression in oral squamous cell carcinoma was explored in this study. STUDY DESIGN: The expression and the methylation status of HSPB1 in oral squamous carcinoma cells were examined using real-time reverse transcription-PCR, methylation-specific PCR and pyrosequencing. RESULTS: HSPB1 expression was weakly expressed in oral squamous carcinoma cell lines (N = 4) as compared to that of normal human oral keratinocytes. The lower expressed HSPB1 was associated with promoter hypermethylation of the HSPB1 gene, and the expression of HSPB1 could be induced by treating the cells with a DNA methyltransferase inhibitor, RG108. Promoter hypermethylation of the HSPB1 gene was also noted in primary oral squamous carcinomas, concomitant with reduced levels of HSPB1 gene expression, as compared to those of the paired neighboring normal tissues. CONCLUSION: Aberrant promoter hypermethylation of the HSPB1 gene may explain the reduced expression of HSPB1 noted in oral cancer cells.

Isocostunolide, a sesquiterpene lactone, induces mitochondrial membrane depolarization and caspase-dependent apoptosis in human melanoma cells.

Isocostunolide is a sesquiterpene lactone isolated from the roots of Inula helenium. Its chemical structure was determined by NMR and FAB-MS spectra. No biological activities of this compound have yet been reported. In this study, we found isocostunolide could effectively induce cytotoxicity in three cancer cell lines (A2058, HT-29, and HepG2), with an IC(50) of 3.2, 5.0, and 2.0 micro g/mL, respectively. DNA flow cytometric analysis indicated that isocostunolide actively induced apoptosis of cancer cells accompanied by a marked loss of G0/G1 phase cells. To address the mechanism of the apoptotic effect of isocostunolide, we analyzed the induction of apoptosis-related proteins in A2058. The levels of pro-caspase-8, Bid, pro-caspase-3, and poly(ADP-ribose) polymerase (PARP) decreased. However, the level of Fas was increased markedly in a dose-dependent manner. Furthermore, this compound markedly induced a depolarization of mitochondrial membranes to facilitate cytochrome c release into cytosol. The findings suggest that isocostunolide may activate a mitochondria-mediated apoptosis pathway. To address this, we found that isocostunolide-induced loss of mitochondrial membrane potential occurred via modulation of the Bcl-2 family proteins. The production of intracellular reactive oxygen species (ROS) in A2058 was not elicited. In summary, for the first time, we have isolated and characterized isocostunolide from I. helenium. This compound induces apoptosis through a mitochondria-dependent pathway in A2058 cells.