Thioacetamide-Induced Norepinephrine Production by Hepatocytes is Associated with Hepatic Stellate Cell Activation and Liver Fibrosis.

BACKGROUND: Collagen production by activated hepatic stellate cells (HSCs) to encapsulate injury is part of the natural wound-healing response in injured liver. However, persistent activation of HSCs can lead to pathological fibrogenesis. Such persistent HSC activation could be mediated by norepinephrine (NE), a reaction product of dopamine beta-hydroxylase (DBH). OBJECTIVE: To investigate the potential paracrine role of NE in hepatotoxin thioacetamide (TAA)-induced liver fibrosis. METHODS: In TAA-treated mice, fibrotic liver tissue showed significant increases in the mRNA expression of DBH up to 14-fold and collagen up to 7-fold. Immunohistochemical staining showed increased DBH protein expression in fibrotic liver tissue. Parenchymal hepatocyte cell line HepG2 expressed DBH and secreted NE, and the conditioned medium of HepG2 cells promoted collagenesis in nonparenchymal HSC cell line LX-2. TAA treatment increased DBH expression by 170% in HepG2 cells, as well as increased NE by 120% in the conditioned medium of HepG2 cells. The conditioned medium of TAA-treated HepG2 cells was used to culture LX-2 cells, and was found to increase collagen expression by 80% in LX-2 cells. Collagen expression was reduced by pre-treating HepG2 cells with siRNA targeting DBH or by adding NE antagonists to the conditioned medium. RESULTS: Finally, TAA-induced oxidative stress in HepG2 cells was associated with induction of DBH expression. Collectively, our results suggest a potential role for DBH/NE-mediated crosstalk between hepatocytes and HSCs in fibrogenesis. CONCLUSION: From a therapeutic standpoint, antagonism of DBH/NE induction in hepatocytes might be a useful strategy to suppress pathological fibrogenesis.

Colossolactone H, a new Ganoderma triterpenoid exhibits cytotoxicity and potentiates drug efficacy of gefitinib in lung cancer.

Three pentacyclic triterpene dilactones were isolated from the fruiting bodies of Ganoderma colossum, a medicinal mushroom. Colossolactone H (colo H) as a new compound and the most cytotoxic among the isolates was studied for its anticancer mechanism and the potential use in cancer therapy. Gene expression profiling analysis indicated that treatment of lung cancer cells with colo H caused upregulation of 252 genes and downregulation of 398 genes. Gene ontology enrichment analysis indicated that the downregulated genes were the most significantly enriched in cell cycle progression, and the upregulated genes were significantly enriched in metabolic process, cellular response to stimulus, and oxidation reduction. Accordingly, colo H was found to halt cell growth and induce cell apoptosis via the elevation of cellular reactive oxygen species to cause DNA damage and the increase of tumor suppressor p53 protein. These events facilitate additive cytotoxicity of colo H and gefitinib for gefitinib-resistant H1650 lung cancer cells. Furthermore, combination of colo H and gefitinib effectively inhibited the growth of tumor xenografts in athymic mice. In addition to the efficacy in adjunctive cancer therapy, we have also demonstrated the isolation of colo H from cultivated G. colossum. Thus it is feasible to use colo H or Ganoderma colossum for cancer therapy.

GPKOW is essential for pre-mRNA splicing in vitro and suppresses splicing defect caused by dominant-negative DHX16 mutation in vivo.

Human GPKOW [G-patch (glycine-rich) domain and KOW (Kyrpides, Ouzounis and Woese) domain] protein contains a G-patch domain and two KOW domains, and is a homologue of Arabidopsis MOS2 and Saccharomyces Spp2 protein. GPKOW is found in the human spliceosome, but its role in pre-mRNA splicing remains to be elucidated. In this report, we showed that GPKOW interacted directly with the DHX16/hPRP2 and with RNA. Immuno-depletion of GPKOW from HeLa nuclear extracts resulted in an inactive spliceosome that still bound DHX16. Adding back recombinant GPKOW restored splicing to the depleted extract. In vivo, overexpression of GPKOW partially suppressed the splicing defect observed in dominant-negative DHX16 mutant expressing cells. Mutations at the G-patch domain greatly diminished the GPKOW-DHX16 interaction; however, the mutant was active in splicing and was able to suppress splicing defect. Mutations at the KOW1 domain slightly altered the GPKOW-RNA interaction, but the mutant was less functional in vitro and in vivo. Our results indicated that GPKOW can functionally impact DHX16 but that interaction between the proteins is not required for this activity.

The antileukemia activity of natural product HQ17(3) is possibly associated with downregulation of miR-17-92 cluster.

The compound 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)] was purified from the sap of the lacquer tree Rhus succedanea. HQ17(3) has cytotoxic effect on cancer cells and can inhibit topoisomerase (topo) IIα activity. We treated various cancer cells with different doses of HQ17(3) and found that leukemia cells were most sensitive to HQ17(3). After analysis of microRNA (miRNA) profiling, we found that treatment with HQ17(3) caused downregulation of miR-17-92 cluster in some leukemia cells. These changes partially restored the normal levels from leukemia-specific miRNA expression signature. Messenger RNAs of tumor suppressor proteins, such as pRB, PTEN, and Dicer, are targets of miR-17-92 cluster. Their protein levels were increased after the treatment. c-Myc is a regulatory protein for miR-17-92 gene. Similar to topo IIα, we found that c-Myc decreased its activity after the HQ17(3) treatment, which may explain the downregulation of miR-17-92 cluster. Combined with 5-fluorouracil, NaAsO2, or ABT-737, HQ17(3) elicited additive inhibitory effects on leukemia cells. In conclusion, the high sensitivity of leukemia cells to HQ17(3) may be associated with the reduction of topo IIα and c-Myc activities, as well as with the downregulation of the miR-17-92 cluster expression.

ALDH-positive lung cancer stem cells confer resistance to epidermal growth factor receptor tyrosine kinase inhibitors.

Molecular targeting therapeutics, such as EGFR tyrosine kinase inhibitors (TKIs), are important treatment strategies for lung cancer. Currently, the major challenge confronting targeted cancer therapies is the development of resistance. Cancer stem cells (CSCs) represent a rare population of undifferentiated tumorigenic cells responsible for tumor initiation, maintenance and spreading. Resistance to conventional chemotherapeutic drugs is a common characteristic of CSCs. However, the issue of whether CSCs contribute to EGFR TKI resistance in lung cancer is yet to be established. In the current study, we explored the association of ALDH1A1 expression with EGFR TKI resistance in lung cancer stem cells. ALDH1A1-positive lung cancer cells displayed resistance to gefitinib, compared to ALDH1A1-negative lung cancer cells. Moreover, PC9/gef cells (gefitinib-resistant lung cancer cells) presented a higher proportion of ALDH1A1-positive cells, compared to PC9 cells (gefitinib-sensitive lung cancer cells). Clinical sample studies were consistent with results from cell culture model systems showing that lung cancer cells with resistance to EGFR TKI and chemotherapy drugs contain significantly increased proportions of ALDH1A1-positive cells. These findings collectively suggest that ALDH1A1 positivity in cancer stem cells confers resistance to EGFR TKI in lung cancer.

10'(Z),13'(E)-heptadecadienylhydroquinone inhibits swarming and virulence factors and increases polymyxin B susceptibility in Proteus mirabilis.

In this study, we demonstrated that 10'(Z), 13'(E)-heptadecadienylhydroquinone (HQ17-2), isolated from the lacquer tree, could decrease swarming motility and hemolysin activity but increase polymyxin B (PB) susceptibilityof Proteus mirabilis which is intrinsically highly-resistant to PB. The increased PB susceptibility induced by HQ17-2 was also observed in clinical isolates and biofilm-grown cells. HQ17-2 could inhibit swarming in the wild-type and rppA mutant but not in the rcsB mutant, indicating that HQ17-2 inhibits swarming through the RcsB-dependent pathway, a two-component signaling pathway negatively regulating swarming and virulence factor expression. The inhibition of hemolysin activity by HQ17-2 is also mediated through the RcsB-dependent pathway, because HQ17-2 could not inhibit hemolysin activity in the rcsB mutant. Moreover, the finding that HQ17-2 inhibits the expression of flhDC gene in the wild-type and rcsB-complemented strain but not in the rcsB mutant supports the notion. By contrast, HQ17-2 could increase PB susceptibility in the wild-type and rcsB mutant but not in the rppA mutant, indicating that HQ17-2 increases PB susceptibility through the RppA-dependent pathway, a signaling pathway positively regulating PB resistance. In addition, HQ17-2 could inhibit the promoter activities of rppA and pmrI, a gene positively regulated by RppA and involved in PB resistance, in the wild-type but not in the rppA mutant. The inhibition of rppA and pmrI expression caused lipopolysaccharide purified from HQ17-2-treated cells to have higher affinity for PB. Altogether, this study uncovers new biological effects of HQ17-2 and provides evidence for the potential of HQ17-2 in clinical applications.

A cysteine-reactive alkyl hydroquinone modifies topoisomerase IIα, enhances DNA breakage, and induces apoptosis in cancer cells.

We previously reported that the anticancer activity of a botanical compound 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)] was attributed to topoisomerase (Topo) IIα poisoning and the induction of oxidative damage. HQ17(3) irreversibly inhibits Topo IIα activity in vitro and is more cytotoxic in leukemia HL-60 cells than in Topo IIα-deficient variant HL-60/MX2 cells, which suggests that Topo IIα is a cellular target of HQ17(3). This study further characterizes the molecular mechanisms of the anticancer activity of HQ17(3). Proteomic analyses indicated that HQ17(3) reacted with Cys-427, Cys-733, and Cys-997 of recombinant Topo IIα in vitro, whereas it reacted with Cys-427 of cellular Topo IIα in Huh7 hepatoma cells. The modification of HQ17(3) inhibited Topo IIα catalytic activity, increased the Topo IIα-DNA cleavage complex, and caused the accumulation of DNA breakage. In Huh7 cells, HQ17(3) treatment caused prompt inhibition of DNA synthesis and consequently induced the expression of DNA damage-related genes DDIT3, GADD45A, and GADD45G. Topo IIα inhibition, apoptosis, and oxidative stress were found to account for cytotoxicity caused by HQ17(3). Pretreatment of Huh7 cells with N-acetylcysteine (NAC) partially attenuated mitochondrial membrane damage, DNA breakage, and caspase activation. However, NAC pretreatment did not diminish HQ17(3)-induced cell death. These results suggest that the anticancer activity of HQ17(3) is attributed significantly to Topo IIα poisoning. The structural feature of HQ17(3) can be used as a model for the design of Topo IIα inhibitors and anticancer drugs.

Slug confers resistance to the epidermal growth factor receptor tyrosine kinase inhibitor.

RATIONALE: Non-small cell lung cancers carrying epidermal growth factor receptor (EGFR) mutations respond well to EGFR tyrosine kinase inhibitors (TKIs), but patients ultimately develop drug resistance and relapse. Although epithelial-mesenchymal transition (EMT) can predict resistance to EGFR TKIs, the molecular mechanisms are still unknown. OBJECTIVES: To examine the role of EMT regulators in resistance to gefitinib. METHODS: The expression level of EMT regulators in gefitinib-sensitive cells (PC9) and gefitinib-resistant cells (PC9/gef) was determined using quantitative real-time reverse transcription-polymerase chain reaction and Western blot analysis. Molecular manipulations (silencing or overexpression) were performed to investigate the effects of EMT regulators on gefitinib resistance in vitro, and a xenograft mouse model was used for in vivo confirmation. In addition, cancer cells from 44 patients with malignant pleural effusions of lung adenocarcinoma were collected for analysis of EMT regulator mRNA by quantitative real-time reverse transcription-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Slug expression, but not that of snail, twist, or zeb-1, was significantly increased in PC9/gef compared with PC9 cells. Slug knockdown in PC9/gef cells reversed resistance to gefitinib, and overexpression of Slug in PC9 cells protected cells from gefitinib-induced apoptosis. Silencing of Slug in gefitinib-resistant cells restored gefitinib-induced apoptosis primarily through Bim up-regulation and activation of caspase-9. Slug enhanced tumor growth in a xenograft mouse model, even with gefitinib treatment. In clinical samples, Slug expression was significantly higher in cancer cells with resistance to EGFR TKIs than in treatment-naive cancer cells. CONCLUSIONS: Slug contributes to the resistance to gefitinib and may be a potential therapeutic target for treating resistance to EGFR TKIs.

Arachidin-1, a peanut stilbenoid, induces programmed cell death in human leukemia HL-60 cells.

The stilbenoids, arachidin-1 (Ara-1), arachidin-3, isopentadienylresveratrol, and resveratrol, have been isolated from germinating peanut kernels and characterized as antioxidant and anti-inflammatory agents. Resveratrol possesses anticancer activity, and studies have indicated that it induces programmed cell death (PCD) in human leukemia HL-60 cells. In this study, the anticancer activity of these stilbenoids was determined in HL-60 cells. Ara-1 had the highest efficacy in inducing PCD in HL-60 cells, with an approximately 4-fold lower EC50 than resveratrol. Ara-1 treatment caused mitochondrial membrane damage, activation of caspases, and nuclear translocation of apoptosis-inducing factor, resulting in chromosome degradation and cell death. Therefore, Ara-1 induces PCD in HL-60 cells through caspase-dependent and caspase-independent pathways. Ara-1 demonstrates its efficacy as an anticancer agent by inducing caspase-independent cell death, which is an alternative death pathway of cancer cells with mutations in key apoptotic genes. These findings indicate the merits of screening other peanut stilbenoids for anticancer activity.

Nuclear retention of unspliced pre-mRNAs by mutant DHX16/hPRP2, a spliceosomal DEAH-box protein.

Defective or imbalanced expression of spliceosomal factors has been linked to human disease; however, how a defective spliceosome affects intron-containing gene transcripts in human cells is largely unknown. DEAH-box protein DHX16 is a human orthologue of Saccharomyces cerevisiae spliceosomal protein Prp2, an RNA-dependent ATPase that activates the spliceosome before the first catalytic step of splicing. Yeast prp2 mutants accumulate unspliced RNAs from the vast majority of intron-containing genes. Here we used a genomic tiling microarray to screen transcripts from four chromosomes in human cells expressing a dominant negative DHX16 mutant and identified a number of gene transcripts that retained their introns. The mutant protein also affected gene transcripts that are sensitive to pladienolide, an SF3b inhibitor. The unspliced RNAs were retained in the nucleus, and block of nonsense-mediated decay did not affect their accumulation. Thus, a perturbation of human PRP2/DHX16 results in accumulation of unspliced transcripts, similar to the outcome in yeast prp2 mutants. The results further suggest that mutant DHX16/hPRP2 causes a defective spliceosome to retain unspliced gene transcripts in the nuclei of human cells.

Endonuclease V-mediated deoxyinosine excision repair in vitro.

Deoxyinosine (dI) in DNA can arise from hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the nfi gene product, in Escherichia coli. Repair was studied in vitro using M13mp18 derived heteroduplexes containing a site-specific deoxyinosine. Unpaired dI/G mismatch resides within the recognition site for XhoI restriction endonucleases, permitting evaluation of repair occurring on deoxyinosine-containing DNA strand. Our results show that dI lesions were efficiently repaired in nfi(+)E. coli extracts but the repair level was much reduced in nfi mutant extracts. We subjected the deoxyinosine-containing heteroduplex to a purified system consisting of soluble endonuclease V fusion protein, DNA polymerase I, and DNA ligase, along with the four deoxynucleoside triphosphates. Interestingly we found these three proteins alone are sufficient to process the dI lesion efficiently. We also found that the 3'-exonuclease activity of DNA polymerase I is sufficient to remove the dI lesion in this minimum reconstituted assay.

Identification of an Alkylhydroquinone from Rhus succedanea as an Inhibitor of Tyrosinase and Melanogenesis.

The alkylhydroquinone 10'(Z)-heptadecenylhydroquinone [HQ17(1)], isolated from the sap of the lacquer tree Rhus succedanea, was found to inhibit the activity of tyrosinase and to suppress melanin production in animal cells. The IC50 of HQ17(1) as a tyrosinase inhibitor was 37 microM versus 70 microM for hydroquinone (HQ), a known inhibitor of tyrosinase and melanogenesis. For the inhibition of melanin production in mouse B16 melanoma cells, the EC50 of HQ17(1) was 40 microM versus 124 microM for HQ. HQ17(1) induced much less oxidative stress than did HQ. The effectiveness in inhibiting melanin production could be mimicked by intermittent exposure of cells to HQ17(1). The potent inhibitory effects of HQ17(1) on tyrosinase activity and melanin production are likely due to its heptadecenyl chain, which facilitates retention of the compound in cell membrane compartments and may impede oxidation of the hydroquinone ring. As tyrosinase activity accounts for postharvest browning of botanical products and animal skin melanogenesis, HQ17(1) could be useful for the preservation of these products or as a skin-whitening cosmetic.

Anticancer activity of botanical alkyl hydroquinones attributed to topoisomerase II poisoning.

Cytotoxic alkyl hydroquinone compounds have been isolated from many plants. We previously isolated 3 structurally similar cytotoxic alkyl hydroquinone compounds from the sap of the lacquer tree Rhus succedanea L. belonging to the sumac family, which have a long history of medicinal use in Asia. Each has an unsaturated alkyl chain attached to the 2-position of a hydroquinone ring. One of these isolates, 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)], being the most cytotoxic, was chosen for studying the anticancer mechanism of these compounds. We found that HQ17(3) was a topoisomerase (Topo) II poison. It irreversibly inhibited Topo IIalpha activity through the accumulation of Topo II-DNA cleavable complexes. A cell-based assay showed that HQ17(3) inhibited the growth of leukemia HL-60 cells with an EC50 of 0.9 microM, inhibited the topoisomerase-II-deficient cells HL-60/MX2 with an EC50 of 9.6 microM, and exerted no effect on peripheral blood mononuclear cells at concentrations up to 50 microM. These results suggest that Topo II is the cellular drug target. In HL-60 cells, HQ17(3) promptly inhibited DNA synthesis, induced chromosomal breakage, and led to cell death with an EC50 about one-tenth that of hydroquinone. Pretreatment of the cells with N-acetylcysteine could not attenuate the cytotoxicity and DNA damage induced by HQ17(3). However, N-acetylcysteine did significantly reduce the cytotoxicity of hydroquinone. In F344 rats, intraperitoneal injection of HQ17(3) for 28 days induced no clinical signs of toxicity. These results indicated that HQ17(3) is a potential anticancer agent, and its structural features could be a model for anticancer drug design.

A novel immunogenic spore coat-associated protein in Bacillus anthracis: characterization via proteomics approaches and a vector-based vaccine system.

New generation anthrax vaccines have been actively explored with the aim of enhancing efficacies and decreasing undesirable side effects that could be caused by licensed vaccines. Targeting novel antigens and/or eliminating the requirements for multiple needle injections and adjuvants are major objectives in the development of new anthrax vaccines. Using proteomics approaches, we identified a spore coat-associated protein (SCAP) in Bacillus anthracis. An Escherichia coli vector-based vaccine system was used to determine the immunogenicity of SCAP. Mice generated detectable SCAP antibodies three weeks after intranasal immunization with an intact particle of ultraviolet (UV)-irradiated E. coli vector overproducing SCAP. The production of SCAP antibodies was detected via western blotting and SCAP-spotted antigen-arrays. The adjuvant effect of a UV-irradiated E. coli vector eliminates the necessity of boosting and the use of other immunomodulators which will foster the screening and manufacturing of new generation anthrax vaccines. More importantly, the immunogenic SCAP may potentially be a new candidate for the development of anthrax vaccines.

Proteomics integrated with Escherichia coli vector-based vaccines and antigen microarrays reveals the immunogenicity of a surface sialidase-like protein of Propionibacterium acnes.

Proteomics is a powerful tool for the identification of proteins, which provides a basis for rational vaccine design. However, it is still a highly technical and time-consuming task to examine a protein's immunogenicity utilizing traditional approaches. Here, we present a platform for effectively evaluating protein immunogenicity and antibody detection. A tetanus toxin C fragment (Tet-c) was used as a representative antigen to establish this platform. A cell wall-anchoring sialidase-like protein (SLP) of Propionibacterium acnes was utilized to assess the efficacy of this platform. We constructed an Escherichia coli vector-based vaccine by overexpressing Tet-c or SLP in E. coli and utilized an intact particle of E. coli itself as a vaccine (E. coli Tet-c or SLP vector). After ultraviolet (UV) irradiation, the E. coli vector-based vaccines were administered intranasally into imprinting control region mice without adding exogenous adjuvants. For antibody detection, we fabricated antigen microarrays by printing with purified recombinant proteins including Tet-c and SLP. Our results demonstrated that detectable antibodies were elicited in mice 6 weeks after intranasal administration of UV-irradiated E. coli vector-based vaccines. The antibody production of Tet-c and SLP was significantly elevated after boosting. Notably, the platform with main benefits of using E. coli itself as a vaccine carrier provides a critical template for applied proteomics aimed at screening novel vaccine targets. In addition, the novel immunogenic SLP potentially serves as an antigen candidate for the development of vaccines targeting P. acnes-associated diseases.

Specific stabilization of DNA triple helices by indolo[2,1-b]quinazolin-6,12-dione derivatives.

Derivatives of indolo[2,1-b]quinazolinone containing aminoalkylamino side chains were synthesized as specific DNA triplex stabilizing agents. The aminoalkylamino side chains are essential for triplex stabilization. The position-8 fluorine atom or a methyl group to the nitrogen adjacent to the planar core can enhance triplex stability by 6 degrees C and the effect is additive. Conformational analysis reveals that the orientation of the side chain underlies the ability of this compound to stabilize a DNA triplex.

Inhibition of swarming and virulence factor expression in Proteus mirabilis by resveratrol.

Resveratrol (3,5,4-trihydroxy-trans-stilbene) is a phytoalexin compound with anti-inflammatory and antioxidant activities. The effect of resveratrol on swarming and virulence factor expression of Proteus mirabilis, an important pathogen infecting the urinary tract, was determined on swarming agar plates with and without the compound. Bacteria harvested at different times were assayed for cell length and the production of flagella, haemolysin and urease. Resveratrol inhibited P. mirabilis swarming and virulence factor expression in a dose-dependent manner. Resveratrol significantly inhibited swarming at 15 microg ml(-1), and completely inhibited swarming at 60 microg ml(-1). Inhibition of swarming and virulence factor expression was mediated through RsbA, a His-containing phosphotransmitter of the bacterial two-component signalling system possibly involved in quorum sensing. Complementation of an rsbA-defective mutant with the rsbA gene restored its responsiveness to resveratrol. The compound also inhibited the ability of P. mirabilis to invade human urothelial cells. These findings suggest that resveratrol has potential to be developed as an antimicrobial agent against P. mirabilis infection.

Ganoderiol F, a ganoderma triterpene, induces senescence in hepatoma HepG2 cells.

Ganoderiol F (GolF), a tetracyclic triterpene, was isolated from Ganoderma amboinense and found to induce senescence of cancer cell lines. GolF induced growth arrest of cancer cell lines HepG2, Huh7 and K562, but exerted much less effect in hepatoma Hep3B cells and normal lung fibroblast MRC5 cells, and no effect on peripheral blood mononuclear cells. GolF treatment of the cancer cells, with the exception of Hep3B, resulted in prompt inhibition of DNA synthesis and arrest of cell progression cycle in G1 phase. Short-term exposure of HepG2 cells to GolF temporarily arrested progression of the cell cycle; cell growth was recovered if the drug was withdrawn from the medium after a 24-h exposure. After 18 days of continuous treatment of HepG2 cells with 30 muM GolF, over 50% of cells were found to be enlarged and flattened, and were beta-galactosidase positive phenotypes of senescent cells. GolF was found to inhibit activity of topoisomerases in vitro, which may contribute to the inhibition of cellular DNA synthesis. Activation of the mitogen-activated protein kinase EKR and up-regulation of cyclin-dependent kinase inhibitor p16 were found in early stages of GolF treatment and were presumed to cause cell-cycle arrest and trigger premature senescence of HepG2 cells. The growth-arrest and senescence induction capability on cancer cells suggest anticancer potential of GolF.

Ganoderic acid X, a lanostanoid triterpene, inhibits topoisomerases and induces apoptosis of cancer cells.

Lanostanoid triterpenes isolated from Ganoderma amboinense were found to inhibit the growth of numerous cancer cell lines, and some of them inhibited the activities of topoisomerases I and IIalpha in vitro. Among the bioactive isolates, one of the most potent triterpene was identified to be 3 alpha-hydroxy-15 alpha-acetoxy-lanosta-7,9(11),24-trien-26-oic acid, ganoderic acid X (GAX). Treatment of human hepatoma HuH-7 cells with GAX caused immediate inhibition of DNA synthesis as well as activation of ERK and JNK mitogen-activated protein kinases, and cell apoptosis. Molecular events of apoptosis including degradation of chromosomal DNA, decrease in the level of Bcl-xL, the disruption of mitochondrial membrane, cytosolic release of cytochrome c and activation of caspase-3 were elucidated. The ability of GAX to inhibit topoisomerases and to sensitize the cancer cells toward apoptosis fulfills the feature of a potential anticancer drug.

A new N-acetylgalactosamine containing peptide as a targeting vehicle for mammalian hepatocytes via asialoglycoprotein receptor endocytosis.

Galactoside-containing cluster ligands have high affinity for asialoglycoprotein receptors (ASGP-r), which are found in abundance in mammalian parenchymal liver cells. These ligands may be conjugated with a therapeutic drug to improve the efficiency of delivery to diseased liver cells. This report describes a new synthetic route towards clustering glycopeptides containing N-acetyl-D-galactosamine (GalNAc). The building block Fmoc-alpha-(ah-Ac3GalNAc)-L-glutamate allowed access to the target compound YEEE(alpha-ah-GalNAc)(3), a structural mimic of YEE(ah-GalNAc)(3), via solid phase peptide synthesis (SPPS). Fatty acid, poly-lysine, fluorescein and biotin conjugates further demonstrate the facility of the described method. Using fluorescein labeling and 131I labeling, in vitro and in vivo assays confirmed that YEEE(alpha-ah-GalNAc)(3) possesses both specificity and affinity to the liver, similar to the agent YEE(ah-GalNAc)(3), which targets liver lesions. The synthesis described in this report represents a considerable improvement in synthesizing a ligand for ASGP-r by simplifying both the preparation of the starting material and the procedure for conjugating the galactosidase cluster to drugs.