PTEN, a general negative regulator of cyclin D expression

The tumor-suppressor phosphatase with tensin homology （PTEN） is frequently mutated in many malignancies and is one of the most well studied tumor suppressor genes [ 1, 2]. PTEN, a lipid and protein dual phosphatase, plays a vital role in embryonic development, cell growth, apoptosis and cell migration. The well-known function of PTEN is phosphatidylinositol-3 （PI3）-phosphatase, which functions as a negative regulator of the PI3 kinase （PI3K） pathway. It is well established that PTEN regulates the G I-S transition by modulating the expression of cyclin D 1 and p27gipl.     

Fucoxanthin induces growth arrest and apoptosis in human bladder cancer T24 cells by up-regulation of p21 and down-regulation of mortalin

Fucoxanthin, a natural carotenoid, has been reported to have anti-cancer activity in human colon cancer cells, human prostate cancer cells, human leukemia cells, and human epithelial cervical cancer cells. This study was undertaken to evaluate the molecular mechanisms of fuco- xanthin against human bladder cancer T24 cell line. MTT analysis results showed that 5 and 10 ixM fucoxanthin inhibited the proliferation of T24 cells in a dose- and time- dependent manner accompanied by the growth arrest at Go/G1 phase of cell cycle, which is mediated by the up-regu- lation of p21, a cyclin-dependent kinase （CDK）-inhibitory protein and the down-regulation of CDK-2, CDK-4, cyclin D1, and cyclin E. In addition, 20 and 40 μM fucoxanthin induced apoptosis of T24 cells by the abrogation of morta- lin-p53 complex and the reactivation of nuclear mutant- type p53, which also had tumor suppressor function as wild-type p53. All these results demonstrated that the anti- cancer activity of fucoxanthin on T24 cells was associated with cell cycle arrest at Go/G1 phase by up-regulation of p21 at low doses and apoptosis via decrease in the expres- sion level of mortalin, which is a stress regulator and a mem- ber of heat shock protein 70, followed by up-regulation of cleaved caspase-3 at high doses.     

The modulation of radiation-induced cell death by genistein in K562 cells：Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression ofTK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G I/S progression and G2-arrest, and their relationship with TKI in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.     

Silencing RhoA inhibits migration and invasion through Wnt/β-catenin pathway and growth through cell cycle regulation in human tongue cancer

Ras homolog gene family member A （RhoA） has been iden- tified as a critical regulator of tumor aggressive behavior. In this study, we assessed the role of RhoA in the mechan- isms underlying growth, migration, and invasion of squa- mous cell carcinoma of tongue （TSCC）. Stable RhoA knockdown of TSCC cell lines SCC-4 and CAL27 were achieved using Lentiviral transfection. The effects of RhoA depletion on cell migration, invasion, and cell proliferation were determined. The possible underlying mechanism of RhoA depletion on TSCC cell line was also evaluated by determining the expression of Galectin-3 （Gal-3）, β-catenin, and matrix metalloproteinase-9 （MMP-9） in vivo. Meanwhile, the underlying mechanism of TSCC growth was studied by analysis of cyclin D1/2, p21clel/WArl, and p27 kiap 1 protein levels. Immunohistochemical assess- ments were performed to further prove the alteration of Gal-3 and β-catenin expression. We found that, in mice injected with human TSCC cells in the tongue, RhoA levels were higher in primary tumors and metastasized lymph nodes compared with those in the normal tissues. Silencing of RhoA significantly reduced the tumor growth, decreased the levels of Gai-3, β-catenin, MMP-9, and cyclin D1/2, and increased the levels of p21 CIPI/WAFI and p27Kiap 1. In vitro, RhoA knockdown also led to inhibition of cell migration, in- vasion, and proliferation. Our data suggest that RhoA plays a significant role in TSCC progression by regulating cell migra- tion and invasion through Wnt/β-catenin signaling pathway and cell proliferation through cell cycle regulation, respecti- vely. RhoA might be a novel therapeutic target of TSCC.     

mad—overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Mad protein has been shown as an antagonist of cMyc protein in some cell lines.The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally.An eukarryotic vector pCDNA Ⅲ containing full ORF fragment of mad cDNA was transfected into targeted cells.Under G418 selection,stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells.DNA synthesis,cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cells were partially inhibited in comparison to control cells.Flos cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase,resulting in the retardation of cell proliferation.RT-PCR detected a marked inhibition of the expression of cdc25A,an important regulator gene of G0/G1 to S phase in cell cycle.It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-74040M1 cells in the absence of serume.Thus,Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.     

Crystal structure of the MAP3K TAO2 kinase domain bound by an inhibitor staurosporine

Mitogen-activated protein kinase (MAPK) signal transduction pathways are ubiquitous ineukaryotic cells,which transfer signals from the cell surface to the nucleus,controlling multiple cellularprograms.MAPKs are activated by MAPK kinases [MAP2Ks or MAP/extracellular signal-regulated kinase(ERK) kinases (MEK)],which in turn are activated by MAPK kinase kinases (MAP3Ks).TAO2 is a MAP3Klevel kinase that activates the MAP2Ks MEK3 and MEK6 to activate p38 MAPKs.Because p38 MAPKs arekey regulators of expression of inflammatory cytokines,they appear to be involved in human diseases suchas asthma and autoimmunity.As an upstream activator of p38s,TAO2 represents a potential drug target.Here we report the crystal structure of active TAO2 kinase domain in complex with staurosporine,a broad-range protein kinase inhibitor that inhibits TAO2 with an IC_(50) of 3 μM.The structure reveals that staurosporineoccupies the position where the adenosine of ATP binds in TAO2,and the binding of the inhibitor mimicsmany features of ATP binding.Both polar and nonpolar interactions contribute to the enzyme-inhibitorrecognition.Staurosporine induces conformational changes in TAO2 residues that surround the inhibitormolecule,but causes very limited global changes in the kinase.The structure provides atomic details forTAO2-staurosporine interactions,and explains the relatively low potency of staurosporine against TAO2.The structure presented here should aid in the design of inhibitors specific to TAO2 and related kinases.     

The molecular mechanisms that underlie the tumor suppressor function of LKB1

Germline mutations of the LKB1 tumor suppressor gene result in Peutz-Jeghers syndrome （PJS） characterized by intestinal hamartomas and increased incidence of epithelial cancers. Inactivating mutations in LKB1 have also been found in certain sporadic human cancers and with particularly high frequency in lung cancer. LKB1 has now been demonstrated to play a crucial role in pulmonary tumorigenesis, controlling initiation, differentiation, and metastasis. Recent evidences showed that LKB1 is a multitasking kinase, with great potential in orchestrating cell activity. Thus far, LKB1 has been found to play a role in cell polarity, energy metabolism, apoptosis, cell cycle arrest, and cell proliferation, all of which may require the tumor suppressor function of this kinase and/or its catalytic activity. This review focuses on remarkable recent findings concerning the molecular mechanism by which the LKB1 protein kinase operates as a tumor suppressor and discusses the rational treatment strategies to individuals suffering from PJS and other common disorders related to LKB1 signaling.     

Therapeutic silencing miR-146b-5p improves cardiac remodeling in a porcine model of myocardial infarction by modulating the wound reparative phenotype

Fibrotic remodeling is an adverse consequence of immune response-driven phenotypic modulation of cardiac cells following myocardial infarction(Ml).MicroRNA-146b(miR-146b)is an active regulator of immunomodulation,but its function in the cardiac inflammatory cascade and its clinical implication in fibrotic remodeling following Ml remain largely unknown.Herein,miR-146b-5p was found to be upregulated in the infarcted myocardium of mice and the serum of myocardial ischemia patients.Gain-and loss-of-function experiments demonstrated that miR-146b-5p was a hypoxia-induced regulator that governed the pro-fibrotic phenotype transition of cardiac cells.Overexpression of miR-146b-5p activated fibroblast proliferation,migration,and fibroblast-to-myofibroblast transition,impaired endothelial cell function and stress survival,and disturbed macrophage paracrine signaling.Interestingly,the opposite effects were observed when miR-146b-5p expression was inhibited.Luciferase assays and rescue studies demonstrated that the miR-146b-5p target genes mediating the above phenotypic modulations included interleukin 1 receptor associated kinase 1(IRAKI)and carcinoembryonic antigen related cell adhesion molecule 1(CEACAM1).Local delivery of a miR-146b-5p antagomir significantly reduced fibrosis and cell death,and upregulated capillary and reparative macrophages in the infarcted myocardium to restore cardiac remodeling and function in both mouse and porcine Ml models.Local inhibition of miR-146b-5p may represent a novel therapeutic approach to treat cardiac fibrotic remodeling and dysfunction following Ml.     

iASPP protects the heart from ischemia injury by inhibiting p53 expression and cardiomyocyte apoptosis

Currently, there remains a great need to elucidate the molecular mechanism of acute myocardial infarction in order to facilitate the development of novel therapy. Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is a member of the ASPP family proteins and an evolutionarily preserved inhibitor of p53 that is involved in many cellular processes, including apoptosis of cancer cells. The purpose of this study was to investigate the possible role of iASPP in acute myocardial infarction. The protein level of iASPP was markedly reduced in the ischemic hearts in vivo and hydrogen peroxide-exposed cardiomyocytes in vitro. Overexpression of iASPP reduced the infarct size and cardiomyocyte apoptosis of mice subjected to 24 h of coronary artery ligation. Echocardiography showed that cardiac function was improved as indicated by the increase in ejection fraction and fractional shortening. In contrast, knockdown of iASPP exacerbated cardiac injury as manifested by impaired cardiac function, increased infarct size, and apoptosis rate. Mechanistically, overexpression of iASPP inhibited, while knockdown of iASPP increased the expressions of p53 and Bax, the key regulators of apoptosis. Taken together, our results suggested that iASPP is an important regulator of cardiomyocyte apoptosis, which represents a potential target in the therapy of myocardial infarction.     

Activation and signaling of the p38 MAP kinase pathway

The family members of the mitogen-activated protein (MAP) kinases mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the four main sub-groups, the p38 group of MAP kinases, serve as a nexus for signal transduction and play a vital role in numerous biological processes. In this review, we highlight the known characteristics and components of the p38 pathway along with the mechanism and consequences of p38 activation. We focus on the role of p38 as a signal transduction mediator and examine the evidence linking p38 to inflammation, cell cycle, cell death, development, cell differentiation, senescence and tumorigenesis in specific cell types. Upstream and downstream components of p38 are described and questions remaining to be answered are posed. Finally, we propose several directions for future research on p38.     

HBx transfection limits proliferative capacity of podocytes through cell cycle regulation

Our previous studies have shown that podocyte number is significantly decreased in glomeruli of children with hepa- titis B virus （HBV）-associated glomerulonephritis. In this study, we aimed to explore whether exogenous expression of HBx protein could directly inhibit podocyte proliferation in vitro, and to investigate its role in cell cycle regulation. HBx gene was delivered into cultured mouse podocytes through an adenovirus-based vector. Cell morphology was evaluated with Wright-Giemsa staining. Cell growth and proliferation were measured by 3-（4,5-dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide （MTT） and 5,6-carboxy- fluorescein diacetate, succinimidyl ester （CFSE）-based pro- liferation assays. Cell cycle phase was analyzed by flow cytometry, and the expression of cell cycle regulatory pro- teins was examined by western blot analysis. It was found that the aberrant nuclear changes like double and multiple micronuclei, which reflect mitotic catastrophe, accumulated in podocytes after 5 days post-infection. MTT assay showed that Ad.HBx-infected podocytes grew much more slowly than controls at day 4 post-infection and thereafter. Furthermore, CFSE-based proliferation assay also showed that the prolifer- ation of HBx-expressing podocytes was significantly inhibited than that of controls at 3-day post-infection, and that the dif- ference became much more obvious at day 5 post-infection. Cell cycle analysis showed that the transfection of HBx resulted in significant up-regulation of both cyclin B1 and CDK-inhibitor p21 expression and G2/M phase arrest, and slight down-regulation of cyclin A expression. These results demonstrated that exogenous expression of HBx might limit the proliferative capacity of podocytes through cell cycle regu- lation, thus suggesting that HBx may play a role in podocyte injuries in HBV-associated glomerulonephritis.     

Localization and secretory pathways of a 58K-like protein in multi-vesicular bodies in callus of Arabidopsis thaliana

Multi-vesicular bodies in endocytosis and protoplasts are special cellular structures that are consid-ered to be originated from invagination of plasma membranes. However, the genesis and function of multi-vesicular bodies, the relationship with Golgi bodies and cell walls, and their secretory pathways remain controversial and ambiguous. Using a monoclonal antibody against an animal 58K protein, we have detected, by Western blotting and confocal microscopy, that a 58K-like protein is present in the calli of Arabidopsis thaliana and Hypericum perforatum. The results of immuno-electron microscopy showed that the 58K-like protein was located in the cisternae of Golgi bodies, secretory vesicles, multi-vesicular bodies, cell walls and vacuoles in callus of Arabidopsis thaliana, suggesting that the multi-vesicular bodies may be originated from Golgi bodies and function as a transporter carrying substances synthesized in Golgi bodies to cell walls and vacuoles. It seems that multi-vesicular bodies have a close relationship with the development of the cell wall and vacuole. The possible secretory pathways of multi-vesicular bodies might be in exocytosis, in which multi-vesicular bodies carry sub-stances to the cell wall for its construction, and in endocytosis, in which multi-vesicular bodies carry substances to the vacuole for its development, depending on what they carry and where the materials are transported. We hence propose that there is more than one pathway for the secretion of multi-vesicular bodies. In addition, our results provided a paradigm that a plant molecule, such as the 58k-like protein in callus of Arabidopsis thaliana, can be detected using a cross-reactive monoclonal antibody induced by an animal protein, and illustrate the existence of analog molecules in both animal and plant kingdoms.     

Mitogen-activated protein kinases mediate the oxidative burst and saponin synthesis induced by chitosan in cell cultures of Panax ginseng

The mitogen-activated protein kinase (MAPK) cascade is a key signaling pathway responsible for the transduction of signals from the cell surface to the cell interior and the nucleus. MAPKs are involved in vari-ety of physiological process including cell growth, development, meiosis, cell death and cell differentia-tion[1—3]. Typically, the components of MAPK cas-cades include the MAPK, a mitogen-activated protein kinase kinase (MAPKK) and a mitogen-activated pro-tein kinase kinase kin…     

miR-199b-5p enhances the proliferation of medullary thymic epithelial cells via regulating Wnt signaling by targeting Fzd6

Thymic epithelial cells (TECs) are essential regulators of T-cell development and selection. miRNAs play critical roles in regulating TEC proliferation during the process of thymic aging. Our previous studies revealed that miR-199b-5p was upregulated in TECs from 1- to 3-month-old mice. But its function and potential mechanism are not clear. We hypothesized that miR-199b-5p may play an important role in age-related thymus involution via targeting some genes. To confirm it, the murine thymic epithelial cell line 1 (MTEC1) cells were used. Our results showed that overexpression of miR-199b-5p can enhance MTEC1 cell proliferation. On the contrary, repression of miR-199b-5p can inhibit MTEC1 cell proliferation. Meanwhile, it was confirmed that frizzled receptor 6 (Fzd6) is the direct target gene of miR-199b-5p. Furthermore, overexpression of miR-199b-5p can upregulate the expressions of β-catenin, Tcf7, Wnt4, and C-myc to activate Wnt signaling and cell cycle signaling. Silence of Fzd6 and co-transfection with siFzd6 and miR-199b-5p mimic/inhibitor confirmed that the biological function of miR-199b-5p is indeed by targeting Fzd6 in medullary TECs. Overall, miR-199b-5p is an important regulator in medullary TEC proliferation through targeting Fzd6 to activate Wnt signaling and cell cycle signaling. Our data indicate that miR-199b-5p may block the process of thymic aging and be a potential therapeutic target for thymus involution.