Nuclear matrix protein,prohibitin, was down‐regulated and translocated from nucleus to cytoplasm during the differentiation of osteosarcoma MG‐63 cells induced by ginsenoside Rg1, cinnamic acid,and tanshinone IIA (RCT)

Ginsenoside Rg1, cinnamic acid, and tanshinone IIA (RCT) are effective anticancer and antioxidant constituents of traditional Chinese herbal medicines of Ginseng, Xuanseng, and Danseng. The molecular mechanisms of anticancer effects of those constituents and their targets are unknown. Prohibitin, an inner membrane‐bound chaperone in mitochondrion involved in the regulation of cell growth, proliferation, differentiation, aging, and apoptosis, was chosen as a candidate molecular target because of its frequent up‐regulation in various cancer cells. We demonstrated that prohibitin existed in the filaments of the nuclear matrix of the MG‐63 cell and its expression was down‐regulated by the treatment of RCT using proteomic methodologies and Western blot analysis. Immunogold electro‐microscopy also found that prohibitin was localized on nuclear matrix intermediate filaments (NM‐IF) that had undergone restorational changes after RCT treatment. Prohibitin may function as a molecular chaperone that might interact with multiple oncogenes and tumor suppressor genes. We found that oncogenes c‐myc and c‐fos and tumor suppressor genes P53 and Rb were regulated by RCT as well and that these gene products co‐localized with prohibitin. Our study identified prohibitin as a molecular target of the effective anticancer constituents of Ginseng, Xuanseng, and Danseng that down‐regulated prohibitin in nuclear matrix, changed prohibtin trafficking from nucleolus to cytoplasm, and regulated several oncogenes and tumor suppressor genes. Prohibitin downregulation and cellular trafficking from nucleolus to cytoplasm indicated RCT protective roles in cancer prevention and treatment. J. Cell. Biochem. 108: 926–934, 2009. © 2009 Wiley‐Liss, Inc.     

人参皂苷Rg1组合诱导人成骨肉瘤MG-63细胞分化过程中Prohibitin的表达与定位变化

选择性抽提经人参皂苷Rg1组合（RCT）诱导处理前后的人成骨肉瘤MG-63细胞核基质,对prohibitin在核基质中的存在、分布及其与相关基因产物在RCT处理前后MG-63细胞中的共定位关系进行观察研究.蛋白质组学分析结果显示,prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在RCT处理后细胞核基质中表达下调;蛋白质印迹杂交确证了prohibitin在MG-63细胞核基质中的存在及其在RCT处理后下调变化;免疫荧光显微镜观察进一步证实prohibitin定位在核基质上,经RCT处理后出现分布位置与表达水平变化;激光共聚焦显微镜观察可见prohibitin与c-Fos、c-Myc、p53和Rb基因产物均存在共定位关系,并在RCT处理后共定位分布区域出现变化.本研究证实了prohibitin是一种新发现的核基质蛋白,其在核基质上的定位与表达在RCT诱导分化前后发生显著变化,并与相关癌基因、抑癌基因产物存在共定位关系.实验表明RCT处理引起的prohibitin的变化与人成骨肉瘤MG-63细胞的诱导分化与调控具有密切关系,为深入揭示RCT等中药有效成分诱导肿瘤细胞分化的机理提供了重要科学依据和深入探索的新方向.     

HMBA诱导人肝癌SMMC-7721 细胞分化过程中 Nucleophosmin 的表达与定位变化

通过选择性抽提经环六亚甲基双乙酰胺（hexamethylene bisacetamide,HMBA）诱导处理前后的人肝癌SMMC-7721细胞核基质,并运用亚细胞蛋白质组学等分析技术,研究nucleophosmin (NPM)在核基质上的表达和定位变化,及其与相关基因产物的共定位关系,观察研究了nucleophosmin 在诱导分化前后人肝癌SMMC-7721细胞核基质中的存在、分布及其与相关基因产物的共定位关系.双向凝胶电泳和质谱鉴定结果显示,nucleophosmin 存在于 SMMC-7721 细胞核基质蛋白组分中,在 HMBA 处理后细胞核基质中表达下调.蛋白质印迹杂交实验结果确证了 nucleophosmin 在核基质中的存在及其在诱导处理后细胞核基质中表达下调的变化.免疫荧光显微镜观察显示,nucleophosmin 定位在 SMMC-7721细胞核基质上,经 HMBA处理后出现分布位置与表达水平的变化.激光扫描共聚焦显微镜观察结果显示,SMMC-7721细胞中,nucleophosmin与 c-fos、c-myc、rb、p53 等基因产物具有共定位关系,但在诱导处理后细胞内的共定位区域发生了改变.研究结果证实,nucleophosmin 是一种核基质蛋白,定位于核基质纤维上,nucleophosmin 在人肝癌 SMMC-7721 细胞诱导分化过程中的表达分布,及其与相关癌基因、抑癌基因产物的关系对 SMMC-7721 细胞分化具有重要影响.     

PXR-mediated transcriptional activation of CYP3A4 by cryptotanshinone and tanshinone IIA

Danshen (Radix Salvia miltiorrhiza) is a famous Traditional Chinese Medicine used widely for the treatment of coronary heart disease and cerebrovascular disease. Diterpenoid tanshinones including tanshinone I, tanshinone IIA and cryptotanshinone are the major bioactive components from Danshen herb. Previous reports have demonstrated that Danshen extracts could induce the expression of CYP3A in rodents, however, the constituents responsible for Danshen-mediated CYP3A induction and the underlying molecular mechanisms remain unknown. The discovery of a family of nuclear receptors such as pregnane X receptor (PXR), constitutive androstane receptor (CAR) and glucocorticoid receptor (GR) gives insight into the molecular explanation of CYP3A induction by xenobiotics. In the present study, interactions between Danshen constituents and human PXR were evaluated using a reporter gene assay. Our observations showed that Danshen ethanol extract could activate human PXR and induce the CYP3A4 reporter construct in HepG2 cells. Tanshinone IIA and cryptotanshinone were identified as efficacious PXR agonists, and cryptotanshinone activated the CYP3A4 promoter more strongly than tanshinone IIA. Furthermore, CAR and GR were also involved in the induction of CYP3A4 expression by tanshinones, though their roles seemed not as important as PXR. Treatment of LS174T cells with cryptotanshinone or tanshinone IIA resulted in a significant increase of CYP3A4 mRNA, which was consistent with the results from the reporter gene assay. Collectively, activation of PXR and the resultant CYP3A4 induction mediated by cryptotanshinone and tanshinone IIA provide a molecular mechanism for previously observed CYP3A induction by Danshen extracts, and our findings also suggest that caution should be taken when Danshen products are used in combination with therapeutic drugs metabolized by CYP3A4.     

Nucleophosmin is a binding partner of nucleostemin in human osteosarcoma cells

Nucleostemin (NS) is expressed in the nucleoli of adult and embryonic stem cells and in many tumors and tumor-derived cell lines. In coimmunoprecipitation experiments, nucleostemin is recovered with the tumor suppressor p53, and more recently we have demonstrated that nucleostemin exerts its role in cell cycle progression via a p53-dependent pathway. Here, we report that in human osteosarcoma cells, nucleostemin interacts with nucleophosmin, a nucleolar protein believed to possess oncogenic potential. Nucleostemin (NS) and nucleophosmin (NPM) displayed an extremely high degree of colocalization in the granular component of the nucleolus during interphase, and both proteins associated with prenucleolar bodies in late mitosis before the reformation of nucleoli. Coimmunoprecipitation experiments revealed that NS and NPM co-reside in complexes, and yeast two-hybrid experiments confirmed that they are interactive proteins, revealing the NPM-interactive region to be the 46-amino acid N-terminal domain of NS. In bimolecular fluorescence complementation studies, bright nucleolar signals were observed, indicating that these two proteins directly interact in the nucleolus in vivo. These results support the notion that cell cycle regulatory proteins congress and interact in the nucleolus, adding to the emerging concept that this nuclear domain has functions beyond ribosome production.     

Nucleolar Disruption Ensures Nuclear Accumulation of p21 upon DNA Damage

p21^cip1 is a protein with a dual function in oncogenesis depending mainly on its intracellular localization: tumor suppressor in the nucleus and oncogenic in the cytoplasm. After DNA damage, p21^cip1 increases and accumulates in the nucleus to ensure cell cycle arrest. We show here that the nuclear accumulation of p21^cip1 is not only a consequence of its increased levels but to a DNA damage cellular response, which is ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) and p53 independent. Furthermore, after DNA damage, p21^cip1 not only accumulates in the nucleoplasm but also in the disrupted nucleolus. Inside the nucleolus, it is found in spherical structures, which are not a protrusion of the nucleoplasm. The steady‐state distribution of p21^cip1 in the nucleolus resulted from a highly dynamic equilibrium between nucleoplasmic and nucleolar p21^cip1 and correlated with the inhibition of p21^cip1 nuclear export. Most interestingly, inhibition of ribosomal export after expressing a dominant‐negative mutant of nucleophosmin induced p21^cip1 accumulation in the nucleus and the nucleolus in the absence of DNA damage. This proved the existence of a nucleolar export route to the cytoplasm for p21^cip1 in control conditions that would be inhibited upon DNA damage leading to nuclear and nucleolar accumulation of p21^cip1.     

The localization of hnRNP A2/B1 in nuclear matrix and the aberrant expression during the RA-induced differentiation of human neuroblastoma SK-N-SH cells

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in the synthesis of RNA. Its expression is up-regulated in many tumor cell lines. In this study, we investigated the distribution of hnRNP A2/B1 in the nuclear matrix, including its co-localization with expression products of related genes. Results from 2-DE PAGE and MS showed that hnRNP A2/B1 is involved with components of nuclear matrix proteins of SK-N-SH cells, and that its expression level is down-regulated after retinoic acid (RA) treatment. Protein immunoblotting results further confirm the existence of hnRNP A2/B1 in the nuclear matrix, as well as its down-regulation after RA treatment. Immunofluorescence microscopy observation showed that hnRNP A2/B1 localized in nuclear matrix of SK-N-SH cells and its distribution regions were altered after RA treatment. Laser scanning confocal microscopy observation showed that hnRNP A2/B1 co-localized with c-Myc, c-Fos, P53, and Rb in SK-N-SH cells. The co-localized region was altered as a result of RA treatment. Our data proved that hnRNP A2/B1 is a nuclear matrix protein and can be up-regulated in human neuroblastoma. The expression and distribution of hnRNP A2/B1 can affect the differentiation of SK-N-SH cells, as well as its co-localization with related oncogenes and tumor suppressor genes.     

Genes,dreams, and cancer.

There have been tremendous advances in our understanding of cancer from the application of molecular biology over the past decade. The disease is caused by a series of defects in the genes that accelerate growth--oncogenes--and those that slow down cellular turnover--tumour suppressor genes. The proteins they encode provide a promising hunting ground in which to design and test new anticancer drugs. Several treatment strategies are now under clinical trial entailing direct gene transfer. These include the use of gene marking to detect minimal residual disease, the production of novel cancer vaccines by the insertion of genes which uncloak cancer cells so making them visible to the host''s immune system, the isolation and coupling of cancer specific molecular switches upstream of drug activating genes, and the correction of aberrant oncogenes or tumour suppressor genes. The issues in these approaches are likely to have a profound impact on the management of cancer patients as we enter the next century.     

Induction of apoptosis and inhibition of cell adhesive and invasive effects by tanshinone IIA in acute promyelocytic leukemia cells in vitro

Tanshinone IIA, a diterpene quinone extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, is used widely and successfully in clinics in China for treating inflammatory diseases. Recently tanshinone IIA has been reported to have apoptosis inducing effects on a large variety of cancer cells. In this study, the anti-proliferation and apoptosis inducing effects of tanshinone IIA as well as its influence on cell adhesion to and invasion through the extracellular matrix (ECM) on acute promyelocytic leukemia (APL) NB4 cells in vitro were studied. Cell proliferation was assessed by MTT assay, cell apoptosis was observed by Hoechst 33258 staining and flow cytometry (FCM); The variation of caspase-3 and apoptotic related genes were assayed by Western blotting, cell mitochondrial membrane potential as well as cell adhesive and invasive effects were also investigated by using standard methods. The results showed that tanshinone IIA exhibited induction of apoptosis by activation of caspase-3, downregulation of anti-apoptotic protein bcl-2 and bcl-xl and upregulation of pro-apoptotic protein bax, as well as disruption of the mitochondrial membrane potential. Furthermore, treatment by tanshinone IIA could reduce cell adhesion to and invasion through ECM in leukemia NB4 cells. These data provide a potential mechanism for tanshinone IIA-induced apoptosis and cell growth inhibition in leukemia NB4 cells, suggesting that tanshinone IIA may serve as an effective adjunctive reagent for the treatment of APL.Contributed equally to this study.     

ARF impedes NPM/B23 shuttling in an Mdm2-sensitive tumor suppressor pathway

The ARF tumor suppressor is widely regarded as an upstream activator of p53-dependent growth arrest and apoptosis. However, recent findings indicate that ARF can also regulate the cell cycle in the absence of p53. In search of p53-independent ARF targets, we isolated nucleophosmin (NPM/B23), a protein we show is required for proliferation, as a novel ARF binding protein. In response to hyperproliferative signals, ARF is upregulated, resulting in the nucleolar retention of NPM and concomitant cell cycle arrest. The Mdm2 oncogene outcompetes NPM/B23 for ARF binding, and introduction of Mdm2 reverses ARF's p53-independent properties: in vitro, NPM is released from ARF-containing protein complexes, and in vivo S phase progression ensues. ARF induction by oncogenes or replicative senescence does not alter NPM/B23 protein levels but rather prevents its nucleocytoplasmic shuttling without inhibiting rRNA processing. By actively sequestering NPM in the nucleolus, ARF utilizes an additional mechanism of tumor suppression, one that is readily antagonized by Mdm2.     

Localization of nucleophosmin in nuclear matrix and changes in its expression during the differentiation of human neuroblastoma induced by retinoic acid

In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK‐N‐SH cells pre‐ and post‐treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two‐dimensional gel electrophoresis and MALDI‐TOF showed that NPM was a component of the nuclear matrix and its expression in SK‐N‐SH cells post‐treated with RA was down‐regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK‐N‐SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c‐myc, c‐fos, p53, and Rb in SK‐N‐SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK‐N‐SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation. J. Cell. Biochem. 111: 67–74, 2010. © 2010 Wiley‐Liss, Inc.     

Tanshinone IIA suppress the proliferation of HNE-1 nasopharyngeal carcinoma an in vitro study

Nasopharyngeal carcinoma (NPC) at present is considered to be one of the fatal diseases detected commonly in the people belonging to Southeast Asia and southern China. According to the WHO reports among the detected cases of NPC worldwide, 80% are from China. The present study investigates the effect of tanshinone IIA on the migration and invasion potential of HNE-1NPC cells and studied the detailed mechanism involved. Effect of the tanshinone IIA on viability of the HNE-1NPC cells was analyzed by MTS assay. Cell matrigel invasion and wound-healing motility assays, respectively were used for the analysis of invasion and migration potential of HNE-1 cells. Tanshinone IIA inhibited the viability of HNE-1cells in a dose dependent manner. Migration and invasion potential of the tanshinone IIA treated cells was reduced significantly (P < 0.05) compared to the control cells after 48 h. Analysis of the proteins involved in migration and invasion revealed a significant decrease in the expression of matrix metalloproteinase (MMP)-2 and MMP-9 on treatment with tanshinone IIA. It also inhibited the p65 and p50 expression in the nuclear fractions of HNE-1 cells after 48 h. Thus, tanshinone IIA inhibits migration and invasion potential of the HNE-1NPC cells through reduction in the expression of matrix metalloproteinases. Therefore, tanshinone IIA can be used for the treatment of NPC.     

丹参酮ⅡA对人乳腺癌细胞株恶性表型逆转的研究

目的：研究丹参酮ⅡA体外诱导人乳腺癌细胞分化,逆转其恶性表型作用。方法：在体外培养的基础上,观察细胞形态学、细胞增殖动力学的变化。采用流式细胞仪的方法,定量检测了ER、nm23-1蛋白、抑癌基因c—fos、癌基因c—myc。结果：经无毒剂量的丹参酮ⅡA处理后,人乳腺癌细胞株MDA—MB-231形态趋向良性分化,细胞增殖指数明显降低,ER、nm23-1蛋白、抑癌基因c-fos明显升高,癌基因c-myc明显降低。结论：丹参酮ⅡA对人乳腺癌细胞株恶性表型有逆转作用,可能是通过抑制癌基因的表达,增加抑癌基因的表达,改变细胞形态结构和生物学特性。     

Effect of roscovitine,a selective cyclin B-dependent kinase 1 inhibitor,on assembly of the nucleolus in mitosis

It is well known that at the beginning of mitosis the nucleolus disassembles but then reassembles at the end of mitosis. However, the mechanisms of these processes are still unclear. In the present work, we show for the first time that selective inhibition of cyclin B-dependent kinase 1 (CDK1) by roscovitine induces premature assembly of the nucleolus in mammalian cells in metaphase. Treatment of metaphase cells with roscovitine induces formation of structures in their cytoplasm that contain major proteins of the mature nucleolus participating in rRNA processing, such as B23/nucleophosmin, C23/nucleolin, fibrillarin, Nop52, as well as partially processed (immature) 46-45S pre-rRNA. This effect is reproducible in cells of various types; this indicates that general mechanisms regulate early stages of the nucleolus reassembly with CDK1 participation in mammalian cells. Based on our and literature data, we suggest that inactivation of the CDK1-cyclin B complex at the end of mitosis results in dephosphorylation of B23/nucleophosmin and C23/nucleolin; this facilitates their interaction with pre-rRNA and leads to formation of insoluble supramolecular complexes--nucleolus-derived foci.     

Dysregulation of glucose transport, glycolysis, TCA cycle and glutaminolysis by oncogenes and tumor suppressors in cancer cells

A common set of functional characteristics of cancer cells is that cancer cells consume a large amount of glucose, maintain high rate of glycolysis and convert a majority of glucose into lactic acid even in the presence of oxygen compared to that of normal cells (Warburg's Effects). In addition, cancer cells exhibit substantial alterations in several energy metabolism pathways including glucose transport, tricarboxylic acid (TCA) cycle, glutaminolysis, mitochondrial respiratory chain oxidative phosphorylation and pentose phosphate pathway (PPP). In the present work, we focused on reviewing the current knowledge about the dysregulation of the proteins/enzymes involved in the key regulatory steps of glucose transport, glycolysis, TCA cycle and glutaminolysis by several oncogenes including c-Myc and hypoxia inducible factor-1 (HIF-1) and tumor suppressor, p53, in cancer cells. The dysregulation of glucose transport and energy metabolism pathways by oncogenes and lost functions of the tumor suppressors have been implicated as important biomarkers for cancer detection and as valuable targets for the development of new anticancer therapies.     

Derangement of growth and differentiation control in oncogenesis.

Human neoplasms develop following the progressive accumulation of genetic and epigenetic alterations to oncogenes and tumor suppressor genes. These alterations confer a growth advantage to the cancer cell, leading to its clonal proliferation, invasion into surrounding tissues, and spread to distant organs. Genes that are altered in neoplasia affect three major biologic pathways that normally regulate cell growth and tissue homeostasis: the cell cycle, apoptosis, and differentiation. While each of these pathways can be defined by a unique set of molecular events, they are not biologically separate. Rather, they function more as an integrated molecular network, and perturbations in one pathway can have profound consequences on another. Insights into what distinguishes the regulation of growth and differentiation in a normal cell versus a cancer cell have led to the development of novel anticancer therapies.     

MicroRNAs Contribute to the Anticancer Effect of 1'-Acetoxychavicol Acetate in Human Head and Neck Squamous Cell Carcinoma Cell Line HN4

1'-Acetoxychavicol acetate (ACA), extracted from rhizomes of tropical ginger, possesses antitumor properties against a wide variety of malignancies. MicroRNAs have been found to act as oncogenes and as tumor suppressor genes in the development of cancer. The purpose of this study was to investigate the miRNA involved in the molecular mechanisms of ACA action on tumor inhibition. It was found that ACA significantly inhibited the growth of human head and neck squamous cell carcinoma cell line HN4 and induced cell apoptosis. Further studies indicated that ACA downregulated the expression of miR-23a in HN4 cells. Transfection with anti-miR-23a inhibited the proliferation of HN4 cells and induced cell apoptosis. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was confirmed to be the target of miR-23a. Taken together, our findings suggest that ACA might have anticancer effects against human head and neck cancer through downregulation of miR-23a, which can repress tumor suppressor PTEN.     

Transformation effector and suppressor genes.

Much has been learned about the molecular basis of cancer from the study of the dominantly acting viral and cellular oncogenes and their normal progenitors, the proto-oncogenes. More recent studies have resulted in the isolation and characterization of several genes prototypic of a second class of cancer genes. Whereas oncogenes act to promote the growth of cells, members of this latter class of genes act to inhibit cellular growth and are believed to contribute to the tumorigenic phenotype only when their activities are absent. This new class of cancer genes is referred to by a number of different names including; anti-oncogenes, recessive oncogenes, growth suppressor genes, tumor suppressor genes and emerogenes. Although only a few of these cancer genes have been identified, to date, it is likely that many additional genes of this class await identification. A third class of genes, necessary for the development of the cancer phenotype, is comprised of the transformation effector genes. These are normal cellular genes that encode proteins that cooperate with or activate oncogene functions and thereby induce the development of the neoplastic phenotype. The inactivation of transformation effector functions would therefore inhibit the ability of certain dominantly acting oncogenes to transform cells. The approaches outlined here describe functional assays for the isolation and molecular characterization of transformation effector and suppressor genes.     

The Relative Concentration of Solids in the Nucleolus, Nucleus, and Cytoplasm of the Developing Nerve Cell of the Chick

Growing and differentiating nerve cells of the fifth cranial ganglion of the chick embryo were studied by several means. During the period of 70 hours to 11 days of incubation (Hamburger-Hamilton stages 19 to 37) average cell mass increased more than 4.5 times while cells changed from relatively undifferentiated neuroblasts to morphologically characteristic nerve cells with long processes. By making simplifying assumptions about thickness of nucleus and nucleolus, relative to cytoplasmic thickness, it was possible to calculate solute concentration of nucleus and nucleolus relative to that of the cytoplasm from measurements of optical retardations through living cells. Differences in relative solute concentration were observed in nucleolus, cytoplasm, and nucleoplasm in the approximate ratio 1.2:1.0:0.8, respectively. The ratio remained essentially constant during the growth period examined despite the fact that the cell components grow at markedly different rates. This suggests that solid concentrations are physical characteristics of nucleus, nucleolus, and cytoplasm which are maintained even during rapid growth and differentiation. By cytochemical means it was demonstrated that mass increase in the nucleus is not associated with increase in deoxyribonucleic acid. Both ribonucleic acid and protein are in greater concentration in nucleolus and cytoplasm than in the nucleoplasm. Electron microscopy shows interruptions in the nuclear envelope as well as an approximately even distribution of electron density in nucleus and cytoplasm. It is pointed out that consistent differences in solid concentration can exist on either side of the nuclear envelope even though it contains "pores." Implications of these data are discussed.