Synthesis of 1-substituted carbazolyl-1,2,3,4-tetrahydro- and carbazolyl-3,4-dihydro-ß-carboline analogs as potential antitumor agents.

A series of 1-substituted carbazolyl-1,2,3,4-tetrahydro- and carbazolyl-3,4-dihydro-ß-carboline analogs have been synthesized and evaluated for antitumor activity against human tumor cells including KB, DLD, NCI-H661, Hepa, and HepG2/A2 cell lines. Among these, compounds 2, 6, 7, and 9 exhibited the most potent and selective activity against the tested tumor cells. As for inhibition of topoisomerase II, compounds 1-14 and 18 showed better activity than etoposide. Among them, compounds 3, 4, 7, 9, and 10 exhibited potent activity. The structure and activity relationship (SAR) study revealed correlation between carbon numbers of the side chain and biological activities. The molecular complex with DNA for compound 2 was proposed.

Structure-activity relationship and mechanism of action studies of manzamine analogues for the control of neuroinflammation and cerebral infections.

Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3beta (GSK-3beta), which is a putative target of manzamines. On the basis of the results presented here, it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.

Targeting stem cells-clinical implications for cancer therapy.

Cancer stem cells (CSC), also called tumor initiating cells (TIC), are considered to be the origin of replicating malignant tumor cells in a variety of human cancers. Their presence in the tumor may herald malignancy potential, mediate resistance to conventional chemotherapy or radiotherapy, and confer poor survival outcomes. Thus, CSC may serve as critical cellular targets for treatment. The ability to therapeutically target CSC hinges upon identifying their unique cell surface markers and the underlying survival signaling pathways. While accumulating evidence suggests cell-surface antigens (such as CD44, CD133) as CSC markers for several tumor tissues, emerging clinical needs exist for the identification of new markers to completely separate CSC from normal stem cells. Recent studies have demonstrated the critical role of the tumor suppressor PTEN/PI3 kinase pathway in regulating TIC in leukemia, brain, and intestinal tissues. The successful eradication of tumors by therapies targeting CSC will require an in-depth understanding of the molecular mechanisms governing CSC self renewal, differentiation, and escape from conventional therapy. Here we review recent progress from brain tumor and intestinal stem cell research with a focus on the PTEN-Akt-Wnt pathway, and how the components of CSC pathways may serve as biomarkers for diagnosis, prognosis, and therapeutics.

Proteomics cataloging analysis of human expressed prostatic secretions reveals rich source of biomarker candidates.

Expressed prostatic secretions (EPS) contain proteins of prostate origin that may reflect the health status of the prostate and be used as diagnostic markers for prostate diseases including prostatitis, benign prostatic hyperplasia, and prostate cancer. Despite their importance and potential applications, a complete catalog of EPS proteins is not yet available. We, therefore, undertook a comprehensive analysis of the EPS proteome using 2-D micro-LC combined with MS/MS. Using stringent filtering criteria, we identified a list of 114 proteins with at least two unique-peptide hits and an additional 75 proteins with only a single unique-peptide hit. The proteins identified include kallikrein 2 (KLK2), KLK3 (prostate-specific antigen), KLK11, and nine cluster of differentiation (CD) molecules including CD10, CD13, CD14, CD26, CD66a, CD66c, CD 143, CD177, and CD224. To our knowledge, this list represents the first comprehensive characterization of the EPS proteome, and it provides a candidate biomarker list for targeted quantitative proteomics analysis using a multiple reaction monitoring (MRM) approach. To help prioritize candidate biomarkers, we constructed a protein-protein interaction network of the EPS proteins using Cytoscape (www.cytoscape.org), and overlaid the expression level changes from the Oncomine database onto the network.

Proteomics analysis of the interactome of N-myc downstream regulated gene 1 and its interactions with the androgen response program in prostate cancer cells.

NDRG1 is known to play important roles in both androgen-induced cell differentiation and inhibition of prostate cancer metastasis. However, the proteins associated with NDRG1 function are not fully enumerated. Using coimmunoprecipitation and mass spectrometry analysis, we identified 58 proteins that interact with NDRG1 in prostate cancer cells. These proteins include nuclear proteins, adhesion molecules, endoplasmic reticulum (ER) chaperons, proteasome subunits, and signaling proteins. Integration of our data with protein-protein interaction data from the Human Proteome Reference Database allowed us to build a comprehensive interactome map of NDRG1. This interactome map consists of several modules such as a nuclear module and a cell membrane module; these modules explain the reported versatile functions of NDRG1. We also determined that serine 330 and threonine 366 of NDRG1 were phosphorylated and demonstrated that the phosphorylation of NDRG1 was prominently mediated by protein kinase A (PKA). Further, we showed that NDRG1 directly binds to beta-catenin and E-cadherin. However, the phosphorylation of NDRG1 did not interrupt the binding of NDRG1 to E-cadherin and beta-catenin. Finally, we showed that the inhibition of NDRG1 expression by RNA interference decreased the ER inducible chaperon GRP94 expression, directly proving that NDRG1 is involved in the ER stress response. Intriguingly, we observed that many members of the NDRG1 interactome are androgen-regulated and that the NDRG1 interactome links to the androgen response network through common interactions with beta-catenin and heat shock protein 90. Therefore we overlaid the transcriptomic expression changes in the NDRG1 interactome in response to androgen treatment and built a dual dynamic picture of the NDRG1 interactome in response to androgen. This interactome map provides the first road map for understanding the functions of NDRG1 in cells and its roles in human diseases, such as prostate cancer, which can progress from androgen-dependent curable stages to androgen-independent incurable stages.

Taiwanin A induced cell cycle arrest and p53-dependent apoptosis in human hepatocellular carcinoma HepG2 cells.

Taiwanin A, a lignan isolated from Taiwania cryptomerioides Hayata, has previously been reported to have cytotoxicity against human tumor cells, but the mechanisms are unclear. In this study, we examined the molecular mechanism of cell death of human hepatocellular carcinoma HepG2 cells induced by Taiwanin A. Taiwanin A has been found to induce cell cycle arrest at G2/M phase as well as caspase-3-dependent apoptosis within 24 h. We performed both in vitro turbidity assay and immunofluorescence staining of tubulin to show that Taiwanin A can inhibit microtubule assembly. Moreover, the tumor suppressor protein p53 in HepG2 cells was activated by Taiwanin A within 12 h. Inhibition of p53 by either pifithrin-alpha or by short hairpin RNA which blocks p53 expression attenuates Taiwanin A cytotoxicity. Our results demonstrate that Taiwanin A can act as a new class of microtubule damaging agent, arresting cell cycle progression at mitotic phase and inducing apoptosis through the activation of p53.

The beta-carboline analog Mana-Hox causes mitotic aberration by interacting with DNA.

Mana-Hox, an analog of beta-carbolines with anticancer activity, induces aberrant mitosis and delays mitotic exit. However, the cellular target is not known. In this study, we visualized the intracellular localization of Mana-Hox. Mana-Hox rapidly penetrated into cells (within 1 min) and concentrated on disorganized metaphase chromosomes after 13 hr of exposure. We demonstrated that Mana-Hox is a noncovalent DNA binder that can interact with DNA through intercalation and/or through minor groove binding. Furthermore, Mana-Hox also inhibits topoisomerase II relaxation activity in vitro, suggesting that Mana-Hox could perturb mitotic chromosome decatenation. Overall, Mana-Hox binding to DNA plays a critical role in the induction of aberrant mitosis and contributes to its anticancer activity.

Hedamycin, a DNA alkylator, induces (gamma)H2AX and chromosome aberrations: involvement of phosphatidylinositol 3-kinase-related kinases and DNA replication fork movement.

Genotoxic treatments, such as UV light, camptothecin, and adozelesin, stall DNA replication and subsequently generate DNA strand breaks. Typically, DNA breaks are reflected by an increase in ataxia and Rad-related kinase (ATR)-regulated phosphorylation of H2AX (gammaH2AX) and require replication fork movement. This study examined the potential of the monofunctional DNA alkylating agent hedamycin, a powerful inhibitor of DNA replication, to induce DNA strand breaks, phosphorylated H2AX (gammaH2AX) foci, and chromosome aberrations. Hedamycin treatment of HCT116 carcinoma cells resulted in a rapid induction of DNA strand breaks accompanied by increasing H2AX phosphorylation and focalization. Unlike many other treatments that also stall replication, such as UV, camptothecin, and adozelesin, gammaH2AX formation was not suppressed in ATR-compromised cells but actually increased. Similarly, hedamycin induction of gammaH2AX is not dependent on ataxia telangiectasia mutated or DNA-protein kinase, and pretreatment of cells with the phosphatidylinositol 3-kinase-related kinase inhibitor caffeine did not substantially reduce induction of H2AX phosphorylation by hedamycin. Furthermore, the DNA replication inhibitor aphidicolin only modestly depressed hedamycin-induced gammaH2AX formation, indicating that hedamycin-induced DNA double-strand breaks are not dependent on fork progression. In contrast, camptothecin- and adozelesin-induced gammaH2AX was strongly suppressed by aphidicolin. Moreover, after 24 hours following a short-term hedamycin treatment, cells displayed high levels of breaks in interphase nuclear DNA and misjoined chromosomes in metaphase cells. Finally, focalization of a tightly bound form of Ku80 was observed in interphase cells, consistent with the subsequent appearance of chromosomal aberrations via abnormal nonhomologous end joining. Overall, this study has revealed a disparate type of DNA damage response to stalled replication induced by a bulky DNA adduct inducer, hedamycin, that seems not to be highly dependent on ATR or DNA replication.

Characterization of the cytotoxic mechanism of Mana-Hox, an analog of manzamine alkaloids.

Mana-Hox is a synthetic analog of manzamines, which are beta-carboline alkaloids isolated from marine sponges. Mana-Hox exhibited cytotoxicity against various tumor cell lines with the IC(50) range from 1 to 5 microM. Cell cycle synchronization and flow cytometric analysis showed that Mana-Hox delayed cell cycle progression at mitosis. At the concentration that delayed mitotic progression, bipolar spindle with lagged chromosomes and multipolar spindle with disorganized chromosomes were detected. The presence of such aberrant mitotic cells accompanied by the activation of spindle checkpoint that delayed cells exit from mitosis. However, after a short delay, lagged chromosomes were able to display in the abnormal metaphase plates, and subsequent cell division resulting in chromosome missegregation. Furthermore, the aberrant mitotic cells showed lower viability, indicating that Mana-Hox-induced cell death resulting from chromosome missegregation. This study is the first to explore cytotoxic mechanism of a manzamine-related compound and understand its potential as a lead compound for the development of future anticancer agents.

DNA damage responses triggered by a highly cytotoxic monofunctional DNA alkylator, hedamycin, a pluramycin antitumor antibiotic.

Long-term exposure (72 h) to hedamycin, a monofunctional DNA alkylator of the pluramycin class of antitumor antibiotics, decreased growth of mammalian cells by 50% at subnanomolar concentrations. Short-term treatment (4 h) rapidly reduced DNA synthesis by 50% also at subnanomolar concentrations, but substantially higher levels were needed to block RNA synthesis while protein synthesis even at very high hedamycin concentrations remained unaffected. Hedamycin treatment at concentrations below its growth IC(50) induced only a transient and temporary accumulation of cells in G(2). Somewhat higher concentrations resulted in substantial S-phase arrest, and at increasing concentrations, complete cell cycle arrest in G(1) was observed without the appearance of a sub-G(1) cell population. Neither inhibition of cell growth nor cell cycle arrest appeared to be dependent on ataxia and Rad-related kinase expression. DNA damage checkpoint proteins including p53, chk1, and chk2 were differentially activated by hedamycin depending on the concentration and duration of treatment. The level of downstream cell cycle regulators such as cdc25A, E2F1, cyclin E, and p21 were also altered under conditions that induced cell cycle arrest, but atypically, p21 overexpression was observed only in S-phase-arrested cells. Apoptotic indicators were only observed at moderate hedamycin concentrations associated with S-phase arrest, while increasing concentrations, when cells were arrested in G(1), resulted in a reduction of these signals. Taken together, the responses of cells to hedamycin are distinct with regard to its effect on cell cycle but also in the unusual concentration-dependent manner of activation of DNA damage and cell cycle checkpoint proteins as well as the induction of apoptotic-associated events.

RNA polymerase I transcribes procyclin genes and variant surface glycoprotein gene expression sites in Trypanosoma brucei.

In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to alpha-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, alphabeta-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

The 5' end structure of transcripts derived from the rRNA gene and the RNA polymerase I transcribed protein coding genes in Trypanosoma brucei.

Due to trans-splicing and polycistronic transcription, the 5' end structure of precursor RNAs of protein coding genes in Trypanosoma brucei has not yet been characterized. In eukaryotes, in general, the 5' ends of transcripts generated by RNA polymerase (pol) I and pol II are different. Pol I derived precursor RNAs contain an unmodified tri- or diphosphate group at their 5' ends. In contrast, pol II primary transcripts, the 5' triphosphate (initially also part of the pre-mRNA) is rapidly modified by the addition of methylated guanosine triphosphate, immediately after transcription initiation. We determined the 5' end structure of precursor RNAs of the rRNA gene and the RNA pol I transcribed protein coding gene by the differential display of RNA ligase mediated amplification of cDNA ends (DDRLACE) method. Comparing the ability of the 5' end of RNA transcripts to ligate with an RNA primer following different pre-treatments, the structure of the 5' end of RNA transcripts was characterized. We found that: (1). the 5' end of putative precursor RNAs from a pol I transcribed protein coding gene and the rRNA gene was uncapped; (2). approximately 20% of the putative rRNA precursor contained a 5' tri- or diphosphate group, representing the primary transcript and approximately 80% of the putative rRNA precursor were dephosphorylated and contained a 5' hydroxyl group; (3). the majority of putative neomycin resistance gene precursor RNAs, driven by the procyclin gene promoter (a pol I promoter), contained a 5' hydroxyl group. The procyclin-neo primary transcript, as being those containing a 5' tri- or diphosphate, was below a detectable level in the steady state RNA; and (4). we did not detect pol I transcribed precursor RNAs that contained a 5' monophosphate group. The observation that the putative pre-RNAs derived from the procyclin gene promoter, similar to those of rRNA do not have a 5' capped structure, is consistent with the notion that transcription of pol I transcribed protein coding genes is crucially dependent on trans-splicing for the cap addition.