Reduced Expression of Metallothionein-I/II in Renal Proximal Tubules Is Associated with Advanced Chronic Kidney Disease.

Chronic kidney disease (CKD) is a commonly occurring complex renal syndrome that causes overall mortality in many diseases. The clinical manifestations of CKD include renal tubulointerstitial fibrosis and loss of renal function. Metallothionein-I/II (MT-I/II) is potentially expressed in the liver and kidney, and possesses antioxidant and metal detoxification properties. However, whether MT-I/II expression is associated with the prognosis of nephropathy remains unknown. In this study, we investigated the MT-I/II level in human CKD, using immunohistochemistry. MT-I/II is located on the proximal tubules and is notably reduced in patients with CKD. MT-I/II expression was significantly correlated with the functional and histological grades of CKD. In an aristolochic acid (AAI)-induced nephropathy mouse model, MT-I/II was abundantly increased after AAI injection for 7 days, but decreased subsequently compared to that induced in the acute phase when injected with AAI for 28 days. Furthermore, we found that ammonium pyrrolidinedithiocarbamate (PDTC) restored AAI-induced MT-I/II reduction in HK2 cells. The injection of PDTC ameliorated AAI-induced renal tubulointerstitial fibrosis and reduced the concentrations of blood urea nitrogen and creatinine in mouse sera. Taken together, our results indicate that MT-I/II reduction is associated with advanced CKD, and the retention of renal MT-I/II is a potential therapeutic strategy for CKD.

Expression of Nik-related kinase in smooth muscle cells attenuates vascular inflammation and intimal hyperplasia.

Inflammation of the vascular microenvironment modulates distinct types of vascular cells, and plays important roles in promoting atherosclerosis, stenosis/restenosis, and vascular-related diseases. Nik-related kinase (Nrk), a member of the Ste20-type kinase family, has been reported to be selectively expressed in embryonic skeletal muscle. However, whether Nrk is expressed in adult vascular smooth muscle, and if it influences intimal hyperplasia is unclear. Here, we found that Nrk is abundantly expressed in cultured vascular smooth muscle cells (VSMC) and mouse arterial intima. Treatment of mouse VSMCs with lipopolysaccharide (LPS) or platelet-derived growth factor significantly reduced Nrk expression. In addition, expression of Nrk was significantly reduced in regions of neointimal formation caused by guide-wire carotid artery injuries in mice, as well as in human atherosclerotic tissues, when compared to normal vessels. We identified that expression of matrix metalloproteinases (MMP3, MMP8 and MMP12) and inflammatory cytokines/chemokines (CCL6, CCL8, CCL11, CXCL1, CXCL3, CXCL5 and CXCL9) are synergistically induced by Nrk siRNA in LPS-treated mouse VSMCs. Moreover, we found that resveratrol significantly impaired LPS- and Nrk siRNA-induced expression of MMP3, CCL8, CCL11, CXCL3 and CXCL5. These results suggested that Nrk may play important roles in regulating pathological progression of atherosclerosis or neointimal- hyperplasia-related vascular diseases.

ZNF479 downregulates metallothionein-1 expression by regulating ASH2L and DNMT1 in hepatocellular carcinoma.

Decreased expression of metallothionein-1 (MT-1) is associated with a poor prognosis in hepatocellular carcinoma (HCC). Here, we found that MT-1 expression was suppressed by 14-3-3ε, and MT-1 overexpression abolished 14-3-3ε-induced cell proliferation and tumor growth. We identified that 14-3-3ε induced expression of ZNF479, a novel potential transcriptional regulator by gene expression profiling and ZNF479 contributed to 14-3-3ε-suppressed MT-1 expression. ZNF479 induced the expression of DNMT1, UHRF1, and mixed-lineage leukemia (MLL) complex proteins (ASH2L and Menin), and increased tri-methylated histone H3 (H3K4me3) levels, but suppressed H3K4 (H3K4me2) di-methylation. ZNF479-suppressed MT-1 expression was restored by silencing of ASH2L and DNMT1. Furthermore, ZNF479 expression was higher in HCC tissues than that in the non-cancerous tissues. Expression analyses revealed a positive correlation between the expression of ZNF479 and DNMT1, UHRF1, ASH2L, and Menin, and an inverse correlation with that of ZNF479, ASH2L, Menin, and MT-1 isoforms. Moreover, correlations between the expression of ZNF479 and its downstream factors were more pronounced in HCC patients with hepatitis B. Here, we found that ZNF479 regulates MT-1 expression by modulating ASH2L in HCC. Approaches that target ZNF479/MLL complex/MT-1 or related epigenetic regulatory factors are potential therapeutic strategies for HCC.

Cordycepin Suppresses Endothelial Cell Proliferation, Migration, Angiogenesis, and Tumor Growth by Regulating Focal Adhesion Kinase and p53.

Focal adhesion kinase (FAK) plays an important role in vascular development, including the regulation of endothelial cell (EC) adhesion, migration, proliferation, and survival. 3'-deoxyadenosine (cordycepin) is known to suppress FAK expression, cell migration, and the epithelial⁻mesenchymal transition in hepatocellular carcinoma (HCC). However, whether cordycepin affects FAK expression and cellular functions in ECs and the specific molecular mechanism remain unclear. In this study, we found that cordycepin suppressed FAK expression and the phosphorylation of FAK (p-FAK) at Tyr397 in ECs. Cordycepin inhibited the proliferation, wound healing, transwell migration, and tube formation of ECs. Confocal microscopy revealed that cordycepin significantly reduced FAK expression and decreased focal adhesion number of ECs. The suppressed expression of FAK was accompanied by induced p53 and p21 expression in ECs. Finally, we demonstrated that cordycepin suppressed angiogenesis in an in vivo angiogenesis assay and reduced HCC tumor growth in a xenograft nude mice model. Our study indicated that cordycepin could attenuate cell proliferation and migration and may result in the impairment of the angiogenesis process and tumor growth via downregulation of FAK and induction of p53 and p21 in ECs. Therefore, cordycepin may be used as a potential adjuvant for cancer therapy.

Correction: Regulation of Aldo-keto-reductase family 1 B10 by 14-3-3ε and their prognostic impact of hepatocellular carcinoma.

[This corrects the article DOI: 10.18632/oncotarget.5734.].

Cordycepin disrupts leukemia association with mesenchymal stromal cells and eliminates leukemia stem cell activity.

Maintaining stemness of leukemic stem cells (LSCs) and reciprocal interactions between leukemia and stromal cells support leukemic progression and resistance to chemotherapy. Targeting the niche-based microenvironment is thus a new approach for leukemia therapy. Cordycepin is an analogue of adenosine and has been suggested to possess anti-leukemia properties. However, whether cordycepin influences association of leukemia and mesenchymal stromal cells has never been investigated. Here we show that cordycepin reduces CD34+CD38- cells in U937 and K562 cells and induces Dkk1 expression via autocrine and paracrine regulation in leukemia and mesenchymal stromal/stem cells (MSCs). Cordycepin suppresses cell attachment of leukemia with MSCs and downregulates N-cadherin in leukemia and VCAM-1 in MSCs. Moreover, incubation with leukemic conditioned media (CM) significantly induces IL-8 and IL-6 expression in MSCs, which is abrogated by cordycepin. Suppression of leukemic CM-induced VCAM-1 and IL-8 by cordycepin in MSCs is mediated by impairing NFκB signaling. Finally, cordycepin combined with an adenosine deaminase inhibitor prolongs survival in a leukemic mouse model. Our results indicate that cordycepin is a potential anti-leukemia therapeutic adjuvant via eliminating LSCs and disrupting leukemia-stromal association.

Regulation of aldo-keto-reductase family 1 B10 by 14-3-3ε and their prognostic impact of hepatocellular carcinoma.

14-3-3ε is overexpressed in hepatocellular carcinoma (HCC) and its expression significantly associates with a poor prognostic outcome. To uncover how 14-3-3ε contributes to the tumor progression of HCC, we investigated the potential downstream targets regulated by 14-3-3ε. We found that 14-3-3ε increases expression and nuclear translocation of ß-catenin and that 14-3-3ε-induced cell proliferation is attenuated by ß-catenin silencing in HCC cells. Moreover, 14-3-3ε induces aldo-keto reductase family 1 member B10 (AKR1B10) expression through the activation of ß-catenin signaling. Knockdown of AKR1B10 by siRNAs abolished 14-3-3ε-induced in vitro cell proliferation, anchorage-independent growth as well as in vivo tumor growth. Furthermore, AKR1B10 silencing increased retinoic acid (RA) levels in the serum of tumor-bearing mice and RA treatment attenuated 14-3-3ε-induced HCC cell proliferation. We further examined 14-3-3ε and AKR1B10 expression and clinicopathological characteristics of HCC tumors. Although the expression of AKR1B10 was significantly correlated with 14-3-3ε, an increase of AKR1B10 expression in 14-3-3ε positive patients paradoxically had better overall survival and disease-free survival rates as well as lower metastatic incidence than those without an AKR1B10 increase. Finally, we found a loss of AKR1B10 expression in cells exhibiting a high capacity of invasiveness. Silencing of AKR1B10 resulted in inducing snail and vimentin expression in HCC cells. These results indicate that AKR1B10 may play a dual role during HCC tumor progression. Our results also indicate that 14-3-3ε regulates AKR1B10 expression by activating ß-catenin signaling. A combination of 14-3-3ε with AKR1B10 is a potential therapeutic target and novel prognostic biomarker of HCC.

The essential role of endothelial nitric oxide synthase activation in insulin-mediated neuroprotection against ischemic stroke in diabetes.

BACKGROUND: Stroke patients with diabetes have a higher mortality rate, worse neurologic outcome, and more severe disability than those without diabetes. Results from clinical trials comparing the outcomes of stroke seen with intensive glycemic control in diabetic individuals are conflicting. Therefore, the present study was aimed to identify the key factor involved in the neuroprotective action of insulin beyond its hypoglycemic effects in streptozotocin-diabetic rats with ischemic stroke. METHODS: Long-Evans male rats were divided into three groups (control, diabetes, and diabetes treated with insulin) and subjected to focal cerebral ischemia-reperfusion (FC I/R) injury. RESULTS: Hyperglycemia aggravated FC I/R injuries with an increase in cerebral infarction and neurologic deficits, inhibition of glucose uptake and membrane-trafficking activity of glucose transporter 1, and reduction of Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in the cerebrum. Insulin treatment alleviated hyperglycemia and the symptoms of diabetes in streptozotocin-diabetic rats. Insulin administration also significantly decreased cerebral infarction and neurologic deficits and increased phosphorylation of Akt and eNOS protein in the cerebrum of FC I/R-injured diabetic rats. However, the glucose uptake and membrane trafficking activity of glucose transporter 1 in the cerebrum were not restored by insulin treatment. Coadministration of the eNOS inhibitor, N-iminoethyl-L-ornithine, with insulin abrogated beneficial effects of insulin on cerebral infarct volume and neurologic deficits in FC I/R-injured diabetic rats without affecting the hypoglycemic action of insulin. CONCLUSIONS: These results suggest that eNOS activation is required for the neuroprotection of insulin against ischemic stroke in patients with diabetes.

Cordycepin suppresses integrin/FAK signaling and epithelial-mesenchymal transition in hepatocellular carcinoma.

Cordycepin, also known as 3-deoxyadenosine, is an analogue of adenosine extracted from the traditional Chinese medicine "Dong Chong Xia Cao". Cordycepin is an active small molecular weight compound and is implicated in modulating multiple physiological functions including immune activation, anti-aging and anti-tumor effects. Several studies have indicated that cordycepin suppresses tumor progression. However, the signaling pathways involved in cordycepin regulating cancer cell motility, invasiveness and epithelial-mesenchymal transition (EMT) remain unclear. In this study, we found that cordycepin inhibits hepatocellular carcinoma (HCC) cell proliferation and migration/invasion. Treatment of cordycepin results in the increasing expression of epithelial marker, Ecadherin while no significant effect was found on N-cadherin α-catenin and ß-catenin. Furthermore, although the expression of focal adhesion kinase (FAK) was slightly reduced, the level of phosphorylated FAK was significantly reduced by the treatment of cordycepin. In addition, cordycepin significantly suppresses the expression of integrin α3, integrin α6 and integrin ß1 which are crucial interacting partners of FAK in regulating the focal adhesion complex. These results suggest cordycepin may contribute to EMT, antimigration/ invasion and growth inhibitory effects of HCC by suppressing E-cadherin and integrin/FAK signaling. Thus, cordycepin is a potential therapeutic or supplementary agent for preventing HCC tumor progression.

Cordycepin regulates GSK-3ß/ß-catenin signaling in human leukemia cells.

BACKGROUND: Leukemia stem cells (LSCs) are a limitless cell source for the initiation and maintenance of leukemia. Activation of the Wnt/ß-catenin pathway is required for the survival and development of LSCs. Therefore, targeting ß-catenin is considered a therapeutic strategy for the treatment of leukemia. The goal of this study was to explore whether cordycepin, an active component of the traditional medicine Cordyceps sinensis, regulates ß-catenin expression in leukemia cells. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we found that cordycepin significantly suppressed cell proliferation in all malignant cancer cells, including U937, K562, A549, HepG2, SK-Hep1 and MCF7 in a dose-dependent manner. However, cordycepin reduced ß-catenin levels in U937, K562 and THP1 leukemia cells and had no effect on other solid cancer cells. In addition, treatment with cordycepin significantly suppressed leukemia colony formation in soft agar assay. Cordycepin enhanced proteasome-dependent degradation and inhibited nuclear translocation of ß-catenin in leukemia cells. Cordycepin-reduced ß-catenin stability was restored by the addition of a pharmacological inhibitor of GSK-3ß, indicating that cordycepin-suppressed ß-catenin stability is mediated by the activation of GSK-3ß. Furthermore, cordycepin abolished the effect of Wnt3a-induced ß-catenin in leukemia cells. In addition, cordycepin-impaired ß-catenin is regulated by Akt activation but is not significantly influenced by AMPK or mTOR signal pathways. SIGNIFICANCE: Our findings show for the first time that codycepin selectively reduces ß-catenin stability in leukemia but not in other solid tumor cells. This suppressive effect is mediated by regulating GSK-3ß. A synergistic combination of cordycepin with other treatments should be used as a novel strategy to eradicate leukemia via elimination of LSCs.