Shotgun Proteomics and Quantitative Pathway Analysis of the Mechanisms of Action of Dehydroeffusol, a Bioactive Phytochemical with Anticancer Activity from Juncus effusus.

Dehydroeffusol (DHE) is a phenanthrene isolated from the Chinese medicinal plant Juncus effusus. Biological evaluation of DHE reveals in vitro and in vivo anticancer effects. We performed a shotgun proteomic analysis using liquid chromatography-tandem mass spectrometry to investigate the changes in the protein profiles in cancer cells upon DHE treatment. DHE affected cancer-associated signaling pathways, including NF-κB, ß-catenin, and endoplasmic reticulum stress. Through quantitative pathway and key node analysis of the proteomics data, activating transcription factor 2 (ATF-2) and c-Jun kinase (JNK) were found to be the key components in DHE's modulated biological pathways. Based on the pathway analysis as well as chemical similarity to estradiol, DHE is proposed to be a phytoestrogen. The proteomic, bioinformatic, and chemoinformatic analyses were further verified with individual cell-based experiments. Our study demonstrates a workflow for identifying the mechanisms of action of DHE through shotgun proteomic analysis.

Chemical biology of anticancer gold(III) and gold(I) complexes.

Gold complexes have recently gained increasing attention in the design of new metal-based anticancer therapeutics. Gold(III) complexes are generally reactive/unstable under physiological conditions via intracellular redox reactions, and the intracellular Au(III) to Au(I) reduction reaction has recently been "traced" by the introduction of appropriate fluorescent ligands. Similar to most Au(I) complexes, Au(III) complexes can inhibit the activities of thiol-containing enzymes, including thioredoxin reductase, via ligand exchange reactions to form Au-S(Se) bonds. Nonetheless, there are examples of physiologically stable Au(III) and Au(I) complexes, such as [Au(TPP)]Cl (H2TPP = 5,10,15,20-tetraphenylporphyrin) and [Au(dppe)2]Cl (dppe = 1,2-bis(diphenylphosphanyl)ethane), which are known to display highly potent in vitro and in vivo anticancer activities. In this review, we summarize our current understanding of anticancer gold complexes, including their mechanisms of action and the approaches adopted to improve their anticancer efficiency. Some recent examples of gold anticancer chemotherapeutics are highlighted.

A binuclear gold(I) complex with mixed bridging diphosphine and bis(N-heterocyclic carbene) ligands shows favorable thiol reactivity and inhibits tumor growth and angiogenesis in vivo.

In the design of anticancer gold(I) complexes with high in vivo efficacy, tuning the thiol reactivity to achieve stability towards blood thiols yet maintaining the thiol reactivity to target cellular thioredoxin reductase (TrxR) is of pivotal importance. Herein we describe a dinuclear gold(I) complex (1-PF6) utilizing a bridging bis(N-heterocyclic carbene) ligand to attain thiol stability and a diphosphine ligand to keep appropriate thiol reactivity. Complex 1-PF6 displays a favorable stability that allows it to inhibit TrxR activity without being attacked by blood thiols. In vivo studies reveal that 1-PF6 significantly inhibits tumor growth in mice bearing HeLa xenograft and mice bearing highly aggressive mouse B16-F10 melanoma. It inhibits angiogenesis in tumor models and inhibits sphere formation of cancer stem cells in vitro. Toxicology studies indicate that 1-PF6 does not show systemic anaphylaxis on guinea pigs and localized irritation on rabbits.

Luminescent cyclometalated platinum(II) complex forms emissive intercalating adducts with double-stranded DNA and RNA: differential emissions and anticancer activities.

Luminescent metallo-intercalators are potent biosensors of nucleic acid structure and anticancer agents targeting DNAs. There are few examples of luminescent metallo-intercalators which can simultaneously act as emission probes of nucleic acid structure and display promising anticancer activities. Herein, we describe a luminescent platinum(II) complex, [Pt(C^N^N)(C≡NtBu)]ClO4 (1 a, HC^N^N= 6-phenyl-2,2'-bipyridyl), that intercalates between the nucleobases of nucleic acids, accompanied by an increase in emission intensity and/or a significant change in the maximum emission wavelength. The changes in emission properties measured with double-stranded RNA (dsRNA) are different from those with dsDNA used in the binding reactions. Complex 1 a exhibited potent anticancer activity towards cancer cells in vitro and inhibited tumor growth in a mouse model. The stabilization of the topoisomerase I-DNA complex with resulting DNA damage by 1 a is suggested to contribute to its anticancer activity.

Gold(III) porphyrin 1a prolongs the survival of melanoma-bearing mice and inhibits angiogenesis.

BACKGROUND: Gold(III) meso-tetraphenylporphyrin (gold-1a) has previously been shown to prolong the survival of hepatocellular carcinoma (HCC)-bearing rats and nasopharyngeal carcinoma (NPC) metastasis-bearing mice. It has also been proved to inhibit the tumor growth of mice bearing NPC, neuroblastoma and colon carcinoma. Mechanistically, gold-1a induces apoptosis, inhibits cell migration and invasion. In this study the efficacies of gold-1a in inhibiting melanoma and angiogenesis were investigated. MATERIAL AND METHODS: A mouse melanoma model was used to investigate the efficacy of gold-1a in inhibiting angiogenesis, tumor growth and prolonging the survival of the tumor-bearing animals. The model was established by inoculation of 2 × 10(5) B16-F1 mouse melanoma cells into the right back flanks of the mice by subcutaneous inoculation. When the tumors grew to 0.2-0.4 cm in diameters, the mice were treated with gold-1a, solvent control or dacarbazine (DTIC) for comparison. Tumor sizes and animal survivals were monitored throughout the experiment. Tumor tissues were collected and immunohistochemically stained with CD31 antibodies for evaluation of intra-tumoral microvessel density (iMVD). RESULTS AND CONCLUSION: Gold-1a significantly prolonged the survivals, reduced angiogenesis and tumor growth rates of melanoma-bearing mice. The compound induced necrosis and apoptosis in the mouse melanoma tissues. Gold-1a also downregulated the expression of genes playing roles in angiogenesis. Gold-1a may potentially be used to treat melanoma in patients.

Specific inhibitors of the protein tyrosine phosphatase Shp2 identified by high-throughput docking.

The protein tyrosine phosphatase Shp2 is a positive regulator of growth factor signaling. Gain-of-function mutations in several types of leukemia define Shp2 as a bona fide oncogene. We performed a high-throughput in silico screen for small-molecular-weight compounds that bind the catalytic site of Shp2. We have identified the phenylhydrazonopyrazolone sulfonate PHPS1 as a potent and cell-permeable inhibitor, which is specific for Shp2 over the closely related tyrosine phosphatases Shp1 and PTP1B. PHPS1 inhibits Shp2-dependent cellular events such as hepatocyte growth factor/scatter factor (HGF/SF)-induced epithelial cell scattering and branching morphogenesis. PHPS1 also blocks Shp2-dependent downstream signaling, namely HGF/SF-induced sustained phosphorylation of the Erk1/2 MAP kinases and dephosphorylation of paxillin. Furthermore, PHPS1 efficiently inhibits activation of Erk1/2 by the leukemia-associated Shp2 mutant, Shp2-E76K, and blocks the anchorage-independent growth of a variety of human tumor cell lines. The PHPS compound class is therefore suitable for further development of therapeutics for the treatment of Shp2-dependent diseases.

Restoration of XAF1 expression induces apoptosis and inhibits tumor growth in gastric cancer.

XAF1 (XIAP-associated factor 1) is a novel XIAP binding protein that can antagonize XIAP and sensitize cells to other cell death triggers. Our previous results have shown that aberrant hypermethylation of the CpG sites in XAF1 promoter is strongly associated with lower expression of XAF1 in gastric cancers. In our study, we investigated the effect of restoration of XAF1 expression on growth of gastric cancers. We found that the restoration of XAF1 expression suppressed anchorage-dependent and -independent growth and increased sensitivity to TRAIL and drug-induced apoptosis. Stable cell clones expressing XAF1 exhibited delayed tumor initiation in nude mice. Restoration of XAF1 expression mediated by adenovirus vector greatly increased apoptosis in gastric cancer cell lines in a time- and dose-dependent manner and sensitized cancer cells to TRAIL and drugs-induced apoptosis. Adeno-XAF1 transduction induced cell cycle G2/M arrest and upregulated the expression of p21 and downregulated the expression of cyclin B1 and cdc2. Notably, adeno-XAF1 treatment significantly inhibited tumor growth, strongly enhanced the antitumor activity of TRAIL in a gastric cancer xenograft model in vivo, and significantly prolonged the survival time of animals bearing tumor xenografts. Complete eradication of established tumors was achieved on combined treatment with adeno-XAF1 and TRAIL. Our results document that the restoration of XAF1 inhibits gastric tumorigenesis and tumor growth and that XAF1 is a promising candidate for cancer gene therapy.

Identification of brain-derived neurotrophic factor as a novel functional protein in hepatocellular carcinoma.

This study aims to identify a novel molecule that may contribute to hepatocarcinogenesis in a rat orthotopic hepatocellular carcinoma model. The hepatocellular carcinoma model was generated by injection of tumor cells into the left lobe of the liver. Proteomic approaches, including ProteinChip and two-dimensional electrophoresis, were used to identify proteins from serially collected rat serum samples. By both ProteinChip and two-dimensional electrophoresis techniques, the level of a 27-kDa protein was found to be augmented in serum samples during tumor development, decreased after left lobectomy, and reincreased at the time of tumor recurrence. The protein was identified to be brain-derived neurotrophic factor (BDNF). By using specific primers and monoclonal antibody, the expression pattern of BDNF was confirmed in tumor tissue but not in the adjacent nontumorous liver tissue. In addition, the truncated isoform of BDNF receptor-tyrosine protein kinase receptor B was only found in tumor tissue. An in vitro study showed that exogenous BDNF could induce tumor cell proliferation predominantly in relatively small numbers of inoculated cells. Administration of BDNF to tumor cell lines induced significantly increased expression of heat shock protein 90 (Hsp90) and cyclin D1, and blocking the activity of Hsp90 could reverse the up-regulation of cyclin D1 induced by BDNF. The present study revealed that BDNF and its receptor were uniquely expressed in tumor tissue and cell lines of hepatocellular carcimona but not in nontumorous liver tissue and normal cell line. BDNF could stimulate tumor cell proliferation in a Hsp90-dependent manner.

Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer.

Survivin plays an important role in cancer development. We aim to show here that suppression of survivin expression or function by antisense and dominant-negative (DN) mutant can inhibit gastric cancer carcinogenesis and angiogenesis in vivo. Plasmid constructs expressing survivin antisense and DN mutant replacing the cysteine residue at amino acid 84 with alanine (Cys84Ala) were prepared and introduced into BCG-823 and MKN-45 gastric cancer cells to establish stable transfectants. We showed that both antisense and DN mutant stable transfectants exhibited abnormal morphology, with decreased cell growth and increased rate of spontaneous apoptosis and mitotic catastrophe. Furthermore, in nude mice xenografts, these cells exhibited decreased de novo gastric tumor formation and reduced development of angiogenesis. Results from these studies strongly suggest that survivin is a promising target for gastric cancer treatment.

Alkynyl gold(I) phosphane complexes: Evaluation of structure-activity-relationships for the phosphane ligands, effects on key signaling proteins and preliminary in-vivo studies with a nanoformulated complex.

Gold alkynyl complexes with phosphane ligands of the type (alkynyl)Au(I)(phosphane) represent a group of bioorganometallics, which has only recently been evaluated biologically in more detail. Structure-activity-relationship studies regarding the residues of the phosphane ligand (P(Ph)3, P(2-furyl)3, P(DAPTA)3, P(PTA)3, P(Et)3, P(Me)3) of complexes with an 4-ethynylanisole alkyne ligand revealed no strong differences concerning cytotoxicity. However, a relevant preference for the heteroatom free alkyl/aryl residues concerning inhibition of the target enzyme thioredoxin reductase was evident. Complex 1 with the triphenylphosphane ligand was selected for further studies, in which clear effects on cell morphology were monitored by time-lapse microscopy. Effects on cellular signaling were determined by ELISA microarrays and showed a significant induction of the phosphorylation of ERK1 (extracellular signal related kinase 1), ERK2 and HSP27 (heat shock protein 27) in HT-29 cells. Application of 1 in-vivo in a mouse xenograft model was found to be challenging due to the low solubility of the complex and required a formulation strategy based on a peanut oil nanoemulsion.

Protection of Xenopus laevis embryos against alcohol-induced delayed gut maturation and growth retardation by peroxiredoxin 5 and catalase.

Accumulated evidence indicates that maternal alcohol consumption causes fetal enteric damage and growth retardation. In this study, we investigated the underlying molecular mechanisms in a Xenopus model of fetal alcohol exposure. We established a condition of transient alcohol exposure that produces tadpoles with delayed gut maturation and decreased body length. We then investigated the roles of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by microinjecting plasmids expressing catalase and peroxiredoxin 5 (PRDX5) into two-cell stage embryos. Finally, the effects of these enzymes on the expression of key gut developmental genes were determined by animal cap explant assay. We showed that exposure of Xenopus embryos to 0.5% alcohol from stage 13 to stage 22 produced tadpoles with delayed gut maturation, reduced growth, and down-regulation in several gut developmental genes, with VegT, Pax6 and Sox17 most vulnerable. We further demonstrated that microinjection of catalase attenuated alcohol-induced ROS production and restored the expression of VegT and Pax6, but protected the embryos from delayed gut development and retarded growth only partially. By contrast, microinjection of PRDX5 reduced both ROS and RNS production, and prevented the gut and growth defects, and restored VegT, Pax6 and Sox17 gene expression. A positive correlation was found between delayed gut maturation and reduced body length. These results indicate the crucial roles of both the ROS-Pax6 and RNS-Sox17 signaling axes in alcohol-induced fetal gut defects and growth retardation. In addition, they suggest strongly a cause-and-effect relationship between alcohol-induced delayed gut maturation and growth retardation.

IFITM1 promotes the metastasis of human colorectal cancer via CAV-1.

Interferon-induced transmembrane protein 1 (IFITM1) is one of the interferon-induced transmembrane protein family members. In this study, we reported that the elevated IFITM1 expression in human colorectal cancer (CRC) significantly correlated with CRC lymph node and distance metastasis as well as a more advanced clinical stage. Importantly, elevated IFITM1 expression is an independent prognostic factor for poor survival. To investigate the molecular mechanisms, we showed that over-expression of IFITM1 in CRC cells promoted, whereas knockdown of IFITM1 expression inhibited, cell migration/invasion and tumorigenicity in vitro. Furthermore, we identified Caveolin-1 (CAV1) as a downstream target of IFITM1-induced cell invasion, as knockdown of CAV1 abrogated siIFITM1 mediated inhibition of cell invasion in CRC cells. In addition, in a CRC cohort of 229 patients, the expression of IFITM1 inversely correlated with the expression of CAV1. These results suggested that IFITM1 promotes the aggressiveness of CRC cells, and it is a potential prognostic marker and therapeutic target for CRC.

Potent inhibition of SARS-associated coronavirus (SCOV) infection and replication by type I interferons (IFN-alpha/beta) but not by type II interferon (IFN-gamma).

We sought to investigate the anti-severe acute respiratory syndrome (SARS)-associated coronavirus (SCoV) activities of type I (alpha and beta) and type II (gamma) interferons (IFN) in vitro. Type I IFNs protected cells from cytopathic effects (CPE) induced by SCoV, and inhibited viral genomic RNA replication in FRhk-4 cells (measured by quantitative RT-PCR) in a dose-dependent manner. Intracellular viral RNA copies were reduced 50% by IFN-alpha at a concentration of 25 U/ml and by IFN-beta at a concentration of 14 U/ml. IFN-gamma had fewer effects on inhibition of viral infection and replication. The type I IFN receptor signaling pathway in host cells is mainly involved in the inhibition of SCoV infection and replication. Type I IFNs could be used as potential agents for anti-SARS treatment.

A gold(III) porphyrin complex as an anti-cancer candidate to inhibit growth of cancer-stem cells.

A cytotoxic gold(III) complex of meso-tetraphenylporphyrin ([Au(TPP)]Cl, denoted gold-1a) blocks the self-renewal ability of cancer stem-like cells and shows appealing safety pharmacological profiles in rodents.

Gold(III) porphyrin 1a inhibited nasopharyngeal carcinoma metastasis in vivo and inhibited cell migration and invasion in vitro.

A physiologically stable gold compound, gold(III) meso-tetraphenylporphyrin (gold-1a), has been shown to be effective in inducing apoptosis and prolonging the survival of hepatocellular carcinoma (HCC)-bearing rats as well as inhibiting the tumor growth of mice bearing nasopharyngeal carcinoma (NPC), neuroblastoma and colon carcinoma. In this study, we showed that gold-1a prolonged the survival of NPC metastasis-bearing mice and inhibited intrahepatic and lung metastasis. Histologically, gold-1a markedly reduced tumor microvessel formation. Consistently, in in vitro studies, gold-1a inhibited migration and invasion of C666-1 human NPC cells. The data strongly support the use of gold(III) compounds to treat cancer metastasis.

Gold(III) compound is a novel chemocytotoxic agent for hepatocellular carcinoma.

Recently, a series of gold(III) meso-tetraarylporphyrins that are stable against demetallation in physiological conditions have been synthesized. In the present study, the antitumor effects of one of these compounds, gold(III) meso-tetraarylporphyrin 1a (gold-1a) was investigated in an orthotopic rat hepatocellular carcinoma (HCC) model as well as using a HCC cell line. The rat HCC model was induced by injection of rat hepatoma cells, McA-RH7777, into the left lobe of the liver. Seven days after tumor cell inoculation, gold-1a was injected directly into the tumor nodule at different doses, followed by the same doses via intraperitoneal injection twice a week. Gold-1a administration significantly prolonged the survival of HCC-bearing rats. Importantly, gold-1a induced necrosis as well as apoptosis in the tumor tissues, but not in the normal liver tissues. Furthermore, gold-1a treatment neither caused significant drop in body weight of the rats nor affected plasma aspartate aminotransferase level. In the in vitro studies, we observed that gold-1a treatment inhibited the proliferation of McA-RH7777 cells. Gold-1a upregulated genes that increase apoptosis, stabilize p53, decrease proliferation and downregulated genes playing roles in angiogenesis, invasion, and metabolism, as demonstrated by microarray. In particular, the compound upregulated 2 members of the growth arrest and DNA damage (Gadd) inducible gene family, Gadd34 and Gadd153. Suppression of Gadd34 and Gadd153 in McA-RH7777 cells by small hairpin RNA reduced the gold-1a-induced apoptosis and growth inhibition, indicating that gold-1a mediated its effects via upregulation of Gadd34 and Gadd153. Results from our study demonstrated that gold-1a might be a novel promising chemocytotoxic agent for treating HCC.

Allograft inflammatory factor-1 (AIF-1) is crucial for the survival and pro-inflammatory activity of macrophages.

Our previous studies revealed that macrophages played an important role in linking injury, inflammatory and immune response in small-for-size liver transplantation. However, the molecular basis that promoted macrophage activation was not clear. In the present study, we explored the potential role of allograft inflammatory factor-1 (AIF-1) in mediating the survival and pro-inflammatory activity of macrophages in a macrophage cell line. First, the expression of AIF-1 was investigated with the stimulation of pro-inflammatory cytokines and anti-inflammatory treatment. Second, the level of inducible nitric oxide synthase (iNOS) and the survival and migration activity of macrophages were determined with the alterations of AIF-1 expression. Finally, a potential molecule that regulated AIF-1 expression was identified by the proteomic approach. The macrophage cell line expressed a certain level of endogenous AIF-1, which could be enhanced by pro-inflammatory cytokines IL-1beta or tumor necrosis factor-alpha and suppressed by anti-inflammatory drug sodium salicylate. AIF-1 augmentation induced by AIF-1/PCDNA3.1(+) transfection enhanced the levels of iNOS and monocyte chemoattractant protein-1, and promoted the cell migration. On the other hand, suppression of AIF-1 expression by AIF-1/short interference RNA (siRNA) inhibited iNOS production, induced macrophage cell apoptosis and blocked the cell migration. Using two-dimensional electrophoresis, a disintegrin and metalloproteinase domain 3 (ADAM3) was identified after AIF-1/siRNA transfection. Transfection of ADAM3/PCDNA3.1(+) up-regulated the expression of AIF-1 and iNOS, whereas suppression of ADAM3 expression down-regulated AIF-1 and iNOS expression. In conclusion, AIF-1 played an important role in the survival and pro-inflammatory activity of macrophages, and ADAM3 might be an upstream molecule that regulated AIF-1 expression.