Molecular modeling reveals binding interface of γ-tubulin with GCP4 and interactions with noscapinoids.

The initiation of microtubule assembly within cells is guided by a cone shaped multi-protein complex, γ-tubulin ring complex (γTuRC) containing γ-tubulin and atleast five other γ-tubulin-complex proteins (GCPs), i.e., GCP2, GCP3, GCP4, GCP5, and GCP6. The rim of γTuRC is a ring of γ-tubulin molecules that interacts, via one of its longitudinal interfaces, with GCP2, GCP3, or GCP4 and, via other interface, with α/ß-tubulin dimers recruited for the microtubule lattice formation. These interactions however, are not well understood in the absence of crystal structure of functional reconstitution of γTuRC subunits. In this study, we elucidate the atomic interactions between γ-tubulin and GCP4 through computational techniques. We simulated two complexes of γ-tubulin-GCP4 complex (we called dimer1 and dimer2) for 25 ns to obtain a stable complex and calculated the ensemble average of binding free energies of -158.82 and -170.19 kcal/mol for dimer1 and -79.53 and -101.50 kcal/mol for dimer2 using MM-PBSA and MM-GBSA methods, respectively. These highly favourable binding free energy values points to very robust interactions between GCP4 and γ-tubulin. From the results of the free-energy decomposition and the computational alanine scanning calculation, we identified the amino acids crucial for the interaction of γ-tubulin with GCP4, called hotspots. Furthermore, in the endeavour to identify chemical leads that might interact at the interface of γ-tubulin-GCP4 complex; we found a class of compounds based on the plant alkaloid, noscapine that binds with high affinity in a cavity close to γ-tubulin-GCP4 interface compared with previously reported compounds. All noscapinoids displayed stable interaction throughout the simulation, however, most robust interaction was observed for bromo-noscapine followed by noscapine and amino-noscapine. This offers a novel chemical scaffold for γ-tubulin binding drugs near γ-tubulin-GCP4 interface.

Molecular insight into γ-γ tubulin lateral interactions within the γ-tubulin ring complex (γ-TuRC).

γ-Tubulin is essential for the nucleation and organization of mitotic microtubules in dividing cells. It is localized at the microtubule organizing centers and mitotic spindle fibres. The most well accepted hypothesis for the initiation of microtubule polymerization is that α/ß-tubulin dimers add onto a γ-tubulin ring complex (γTuRC), in which adjacent γ-tubulin subunits bind to the underlying non-tubulin components of the γTuRC. This template thus determines the resulting microtubule lattice. In this study we use molecular modelling and molecular dynamics simulations, combined with computational MM-PBSA/MM-GBSA methods, to determine the extent of the lateral atomic interaction between two adjacent γ-tubulins within the γTuRC. To do this we simulated a γ-γ homodimer for 10 ns and calculated the ensemble average of binding free energies of -107.76 kcal/mol by the MM-PBSA method and of -87.12 kcal/mol by the MM-GBSA method. These highly favourable binding free energy values imply robust lateral interactions between adjacent γ-tubulin subunits in addition to their end-interactions longitudinally with other proteins of γTuRC. Although the functional reconstitution of γ-TuRC subunits and their stepwise in vitro assembly from purified components is not yet feasible, we nevertheless wanted to recognize hotspot amino acids responsible for key γ-γ interactions. Our free energy decomposition data from converting a compendium of amino acid residues identified an array of hotspot amino acids. A subset of such mutants can be expressed in vivo in living yeast. Because γTuRC is important for the growth of yeast, we could test whether this subset of the hotspot mutations support growth of yeast. Consistent with our model, γ-tubulin mutants that fall into our identified hotspot do not support yeast growth.

Synergistic suppression of noscapine and conventional chemotherapeutics on human glioblastoma cell growth.

AIM: Noscapine (NOS) is a non-narcotic opium alkaloid with anti-tumor activity. The aim of this study was to investigate the effects of the combination of NOS with conventional chemotherapeutics temozolamide (TMZ), bis-chloroethylnitrosourea (BCNU), or cisplatin (CIS)on human glioblastoma cells. METHODS: U87MG human glioblastoma cells were examined. Cell proliferation was quantified using MTT assay. Western blotting and flow cytometry were used to examine apoptosis and the expression of active caspase-3 and cleaved PARP. Mouse tumor xenograft model bearing U87MG cells was treated with TMZ (2 mg·kg(-1)·d(-1), ip) or CIS (2 mg/kg, ip 3 times a week) alone or in combination with NOS (200 mg·kg(-1)·d(-1), ig) for 3 weeks. Immunohistochemistry was used to investigate the expression of active caspase-3 and Ki67 following treatment in vivo. The safety of the combined treatments was evaluated based on the body weight and histological studies of the animal's organs. RESULTS: NOS (10 or 20 mol/L) markedly increased the anti-proliferation effects of TMZ, BCNU, and CIS on U87MG cells in vitro. The calculated combination index (CI) values of NOS-CIS, NOS-TMZ, and NOS-BCNU (20 µmol/L) were 0.45, 0.51, and 0.57, respectively, demonstrating synergistic inhibition of the drug combinations. In tumor xenograft models, combined treatment with NOS robustly augmented the anti-cancer actions of TMZ and CIS, and showed no detectable toxicity. The combined treatments significantly enhanced the apoptosis, the activated caspase-3 and PARP levels in U87MG cells in vitro, and reduced Ki67 staining and increased the activated caspase-3 level in the shrinking xenografts in vivo. CONCLUSION: NOS synergistically potentiated the efficacy of FDA-approved anti-cancer drugs against human glioblastoma cells, thereby allowing them to be used at lower doses and hence minimizing their toxic side effects.

Chemoprevention of familial adenomatous polyposis by bromo-noscapine (EM011) in the Apc(Min/+) mouse model.

Germline mutation of the tumor suppressor gene, adenomatous polyposis coli (APC), is responsible for familial adenomatous polyposis (FAP) with nearly 100% risk for colon cancer at an early age. Although FAP is involved in only 1% of all colon cancer cases, over 80% of sporadic cancers harbor somatic mutations of APC. We show here that bromo-noscapine (EM011), a rationally designed synthetic derivative of a natural nontoxic tubulin-binding alkaloid-noscapine, that reduces the dynamics of microtubules, causes a reversible G(2) /M arrest in wild type murine embryonic fibroblasts (MEFs), but an aberrant exit from a brief mitotic block, followed by apoptosis in MEFs after APC deletion with small interfering RNA. Furthermore, both ß-catenin levels and activity fell to half the original levels with a concomitant reduction of cell proliferation-inducing cyclin D1, c-Myc, and induction of cytostatic protein p21 before caspase-3 activation. Additionally, we show a statistically significant reduction in the number of newly emerging intestinal polyps (to 35% compared with untreated mice) as well as the mean size of polyps (to 42% compared with untreated mice) in EM011-treated Apc(Min/+) mice as compared to their sham-treated control littermates. The remaining polyps in the EM011 treated group of Apc(Min/+) mice showed evidence of elevated apoptosis as revealed by immunohistochemistry. We failed to detect any evidence of histopathological and hematological toxicities following EM011 treatment. Taken together, our data are persuasive that a clinical trial of EM011 is possible for the prevention/amelioration of polyposis in FAP patients.

Copper(I) mediated facile synthesis of potent tubulin polymerization inhibitor, 9-amino-α-noscapine from natural α-noscapine.

Facile synthesis of natural α-noscapine analogue, 9-amino-α-noscapine, a potent inhibitor of tubulin polymerization for cancer therapy, is achieved via copper(I) iodide mediated in situ aromatic azidation and reduction of 9-bromo-α-noscapine (obtained by bromination of natural α-noscapine) with NaN(3) in DMSO at 130°C in the presence of L-proline as an amino acid promoter. The protocol developed here avoided isolation of 9-azido-α-noscapine and did not cleave the sensitive C-C bond between two heterocyclic phthalide and isoquinoline units.

In silico inspired design and synthesis of a novel tubulin-binding anti-cancer drug: folate conjugated noscapine (Targetin).

Our screen for tubulin-binding small molecules that do not depolymerize bulk cellular microtubules, but based upon structural features of well known microtubule-depolymerizing colchicine and podophyllotoxin, revealed tubulin binding anti-cancer property of noscapine (Ye et al. in Proc Natl Acad Sci USA 95:2280-2286, 1998). Guided by molecular modelling calculations and structure-activity relationships we conjugated at C9 of noscapine, a folate group-a ligand for cellular folate receptor alpha (FRα). FRα is over-expressed on some solid tumours such as ovarian epithelial cancers. Molecular docking experiments predicted that a folate conjugated noscapine (Targetin) accommodated well inside the binding cavity (docking score -11.295 kcal/mol) at the interface between α- and ß-tubulin. The bulky folate moiety of Targetin is extended toward lumen of microtubules. The binding free energy (ΔG (bind)) computed based on molecular mechanics energy minimization was -221.01 kcal/mol that revealed favourable interaction of Targetin with the receptor. Chemical synthesis, tubulin-binding experiments, and anti-cancer activity in vitro corroborate fully well with the molecular modelling experiments. Targetin binds tubulin with a dissociation constant (K (d) value) of 149 ± 3.0 µM and decreases the transition frequencies between growth and shortening phases of microtubule assembly dynamics at concentrations that do not alter the total polymer mass. Cancer cells in general were more sensitive to Targetin compared with the founding compound noscapine (IC(50) in the range of 15-40 µM). Quite strikingly, ovarian cancer cells (SKOV3 and A2780), known to overexpress FRα, were much more sensitive to targetin (IC(50) in the range of 0.3-1.5 µM).

Rational design, synthesis and biological evaluations of amino-noscapine: a high affinity tubulin-binding noscapinoid.

Noscapine and its derivatives are important microtubule-interfering agents shown to have potent anti-tumor activity. The binding free energies (ΔG (bind)) of noscapinoids computed using linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model were in agreement with the experimental ΔG (bind) with average root mean square error of 0.082 kcal/mol. This LIE-SGB model guided us in designing a novel derivative of noscapine, amino-noscapine [(S)-3-((R)-9-amino-4-methoxy-6-methyl-5,6,7,8-tetrahydro [1, 3] dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxy isobenzo-furan-1(3H)-one] that has higher tubulin binding activity (predicted ΔG (bind) = -6.438 kcal/mol and experimental ΔG (bind) = -6.628 kcal/mol) than noscapine, but does not significantly change the total extent of the tubulin subunit/polymer ratio. The modes of interaction of amino-noscapine with the binding pocket of tubulin involved three hydrogen bonds and are distinct compared to noscapine which involved only one hydrogen bond. Also the patterns of non-bonded interactions are albeit different between both the lignads. The 'blind docking' approach (docking of ligand with different binding sites of a protein and their evaluations) as well as the reasonable accuracy of calculating ΔG (bind) using LIE-SGB model constitutes the first evidence that this class of compounds binds to tubulin at a site overlapping with colchicine-binding site or close to it. Our results revealed that amino-noscapine has better anti-tumor activity than noscapine.

Non-toxic melanoma therapy by a novel tubulin-binding agent.

(S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquino-lin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (EM011) is a tubulin-binding agent with significant anticancer activity. Here we show that EM011 modulates microtubule dynamics at concentrations that do not alter the total polymer mass of tubulin. In particular, EM011 decreases the transition frequencies between growth and shortening phases and increases the duration microtubules spend in an idle 'pause' state. Using B16LS9 murine melanoma cells, we show that EM011 briefly arrests cell-cycle progression at the G2/M phase by formation of multiple aster spindles. An aberrant mitotic exit without cytokinesis then occurs, leading to the accumulation of abnormal multinucleated cells prior to apoptosis. Our pharmacokinetic studies conformed to a linear dose-response relationship upto 150 mg/kg. However, non-linearity was observed at 300 mg/kg. In a syngeneic murine model of subcutaneous melanoma, better antitumor responses were seen at 150 mg/kg compared to 300 mg/kg of EM011. Unlike currently available chemotherapeutics, EM011 is non-toxic to normal tissues and most importantly, does not cause any immunosuppression and neurotoxicity. Our data thus warrant a clinical evaluation of EM011 for melanoma therapy.

Runt-related transcription factor 1 promotes apoptosis and inhibits neuroblastoma progression in vitro and in vivo.

BACKGROUND: Runt-related transcription factor 1 (RUNX1) is a heterodimeric transcription factor that binds to the core element of many enhancers and promoters and can accelerate apoptosis in various tumors. However, the regulatory mechanisms underlying RUNX1 expression in neuroblastoma (NB), a highly malignant tumor in childhood, remain largely unclear. In this study, we aimed to assess the role of RUNX1 in NB and to reveal the underlying mechanisms that may contribute to finding a potential therapeutics strategy against NB. METHODS: Growth, invasion, metastasis and angiogenesis were assessed using Cell Counting Kit-8 (CCK-8) immunocytochemistry, and studies involving soft agar, cell invasion, tube formation and whole animals. The levels of expression were measured using real-time quantitative PCR for RNA, Western blot and immunostaining analyses for proteins. Luciferase reporter and chromatin immunoprecipitation assays indicated that RUNX1 directly binds within the BIRC5, CSF2RB and NFKBIA promoter regions to facilitate transcription. The level of apoptosis was assessed by determining mitochondrial membrane potential and flow cytometry. RESULTS: RUNX1 was highly expressed in ganglioneuroma (GN) and well-differentiated (WD) tissues relative to the poorly differentiated (PD) and undifferentiated (UD) ones. Moreover, RUNX1 effectively reduced cell viability, invasion, metastasis, angiogenesis, and promoted apoptosis in vitro and in vivo. RUNX1 reduced BIRC5 transcription and increased CSF2RB and NFKBIA transcription by directly binding BIRC5, CSF2RB and NFKBIA promoters. In addition, cytotoxic drugs, especially cisplatin, significantly increased RUNX1 expression in NB cells and promoted apoptosis. CONCLUSIONS: These data show that RUNX1 is an independent surrogate marker for the progression of NB and it can be used for monitoring NB prognosis during therapy.

9-bromonoscapine-induced mitotic arrest of cigarette smoke condensate-transformed breast epithelial cells.

In the present investigation, we determined the chemotherapeutic efficacy of 9-bromonoscapine (Br-Nos), a more potent noscapine analog, on MCF10A, spontaneously immortalized human normal breast epithelial cells and MCF10A-CSC3, cigarette smoke condensate (CSC)-transformed cells. The results from cytogenetic analysis showed that Br-Nos induced polyploidy and telomeric association in MCF10A-CSC3 cells, while MCF10A cells remained unaffected. Our immunofluorescence data further demonstrated that MCF10A-CSC3 cells were susceptible to mitotic catastrophe on exposure to Br-Nos and failed to recover after drug withdrawal. MCF10A-CSC3 cells exhibited Br-Nos-induced aberrant multipolar spindle formation, which irreversibly impaired the alignment of replicated chromosome to the equatorial plane and finally culminated in cell death. Although MCF10A cells upon Br-Nos treatment showed bipolar spindles with some uncongressed chromosomes, these cells recovered fairly well after drug withdrawal. Our flow-cytometry analysis data reconfirmed that MCF10A-CSC3 cells were more susceptible to cell death compared to MCF10A cells. Furthermore, our results suggest that decreased levels of cdc2/cyclin B1 and cdc2 kinase activity are responsible for Br-Nos-induced mitotic cell arrest leading to cell death in MCF10A-CSC3 cells. This study thus explores the underlying mechanism of Br-Nos-induced mitotic catastrophe in CSC-transformed MCF10A-CSC3 cells and its potential usefulness as a chemotherapeutic agent for prevention of cigarette smoke-induced breast cancer growth.

p53 and p21 determine the sensitivity of noscapine-induced apoptosis in colon cancer cells.

We have previously discovered the naturally occurring antitussive alkaloid noscapine as a tubulin-binding agent that attenuates microtubule dynamics and arrests mammalian cells at mitosis via activation of the c-Jun NH(2)-terminal kinase pathway. It is well established that the p53 protein plays a crucial role in the control of tumor cell response to chemotherapeutic agents and DNA-damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. In this study, we compared chemosensitivity, cell cycle distribution, and apoptosis on noscapine treatment in four cell lines derived from the colorectal carcinoma HCT116 cells: p53(+/+) (p53-wt), p53(-/-) (p53-null), p21(-/-) (p21-null), and BAX(-/-) (BAX-null). Using these isogenic variants, we investigated the roles of p53, BAX, and p21 in the cellular response to treatment with noscapine. Our results show that noscapine treatment increases the expression of p53 over time in cells with wild-type p53 status. This increase in p53 is associated with an increased apoptotic BAX/Bcl-2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. Conversely, loss of p53 and p21 alleles had a counter effect on both BAX and Bcl-2 expression and the p53-null and p21-null cells were significantly resistant to the antiproliferative and apoptotic effects of noscapine. All but the p53-null cells displayed p53 protein accumulation in a time-dependent manner on noscapine treatment. Interestingly, despite increased levels of p53, p21-null cells were resistant to apoptosis, suggesting a proapoptotic role of p21 and implying that p53 is a necessary but not sufficient condition for noscapine-mediated apoptosis.

Reversal of P-glycoprotein-mediated multidrug resistance in cancer cells by the c-Jun NH2-terminal kinase.

A significant impediment to the success of cancer chemotherapy is multidrug resistance (MDR). A typical form of MDR is attributable to the overexpression of membrane transport proteins, such as P-glycoprotein, resulting in an increased drug efflux. In this study, we show that adenovirus-mediated enhancement of the c-Jun NH2-terminal kinase (JNK) reduces the level of P-glycoprotein in a dose- and time-dependent manner. Protein turnover assay shows that the decrease of P-glycoprotein is independent of its protein stability. Instead, this occurs primarily at the mRNA level, as revealed by reverse transcription-PCR analysis. We find that P-glycoprotein down-regulation requires the catalytic activity of JNK and is mediated by the c-Jun transcription factor, as either pharmacologic inhibition of JNK activity or dominant-negative suppression of c-Jun remarkably abolishes the ability of JNK to down-regulate P-glycoprotein. In addition, electrophoretic mobility shift assay reveals that adenoviral JNK increases the activator protein binding activity of the mdr1 gene in the MDR cells. We further show that the decrease of P-glycoprotein level is associated with a significant increase in intracellular drug accumulation and dramatically enhances the sensitivity of MDR cancer cells to chemotherapeutic agents. Our study provides the first direct evidence that enhancement of the JNK pathway down-regulates P-glycoprotein and reverses P-glycoprotein-mediated MDR in cancer cells.

Rational design of the microtubule-targeting anti-breast cancer drug EM015.

We studied in silico docking of noscapine onto tubulin, combined with calculations of surface charge, pi-pi, van der Waals, and hydrogen bonding interactions, to rationally design a new compound, EM015. This tubulin-binding semisynthetic compound is a selective and potent anti-breast cancer agent and displays a 20-fold lower IC(50) against many tumor cells compared with our founding compound, (S)-6,7-dimethoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro[1,3]-dioxolo-[4,5-g]isoquinolin-5-yl)isobenzo-furan-1(3H)-one (noscapine). Furthermore, EM015 is also effective against a variety of drug-resistant cells. Surprisingly, the cell cycle profile of nontumorigenic normal cells is not affected. Many antimicrotubule cancer drugs in clinic today, particularly taxanes and Vincas, face challenges including frequent visits to the hospital for prolonged i.v. infusions, toxicities, and tumor recurrences due to drug resistance. EM015, on the other hand, is orally available, regresses breast tumor xenografts in nude mice models, and increases longevity. Furthermore, we have failed to observe any detectable toxicity in tissues, such as liver, kidney, spleen, lung, heart, and brain, as well as neurons, which are common targets of antimicrotubule drug therapy. Thus, EM015 has a great promise in the clinic.

Nonimmunosuppressive chemotherapy: EM011-treated mice mount normal T-cell responses to an acute lymphocytic choriomeningitis virus infection.

Myelosuppression and associated immunosuppression are major problems in cancer chemotherapy. Thus, infection remains a significant source of morbidity and mortality during chemotherapy of cancer patients. Viral infections, particularly herpes simplex virus, varicella zoster virus, and cytomegalovirus, result either due to reactivation of latent viruses or new infections as sequelae of chemotherapy and debilitated cell-mediated immunity. Ultimately, the resolution of these infections can only be achieved after the control of malignancy and regaining the patient's ability to mount adequate immune responses. We show here that EM011, a tubulin-binding, nontoxic, orally available anticancer agent, does not alter absolute CD4(+), CD8(+), B220(+), and NK1.1(+) cell counts in immunocompetent mice. More importantly, EM011 treatment at tumor-suppressive dosages (300 mg/kg) does not suppress cell-mediated immune responses in mice experimentally challenged with acute lymphocytic choriomeningitis virus infection, in that mice mount robust virus-specific CD8(+) and CD4(+) T-cell immune responses while maintained on daily drug treatment. Thus, CD8(+) and CD4(+) T-cell expansion and acquisition of effector functions is not perturbed by EM011 treatment. These data provide compelling evidence to support the nonimmunosuppressive nature of EM011 therapy and provide strong impetus for combining chemotherapy with immunotherapy as a novel anticancer strategy.

Preclinical pharmacokinetics and bioavailability of noscapine, a tubulin-binding anticancer agent.

BACKGROUND: Noscapine, a naturally occurring antitussive phthalideisoquinoline alkaloid, is a tubulin-binding agent currently in Phase I/II clinical trials for anticancer therapy. Unlike currently available antimitotics such as taxanes and vincas, noscapine is water-soluble, well tolerated, and shows no detectable toxicity. OBJECTIVE: The goal was to develop a simple, sensitive, quantitative, selective, and less time-consuming high-performance liquid chromatography (HPLC) method for determination of noscapine and to study its pharmacokinetics in mice models. METHOD: Noscapine was extracted from mice plasma using the protein-precipitation method and detected using a reversed-phase C8 column with mobile phase consisting of 35% acetonitrile and 65% ammonium acetate buffer (pH 4.5) at 232 nm wavelength. Pharmacokinetic studies of noscapine were performed in mice following intravenous bolus at 10 mg/kg and oral administrations at 75, 150, and 300 mg/kg. RESULTS: The standard curves for noscapine estimation were linear between 390 and 50,000 ng/ml (lower limit of quantification was 390 ng/ml) and the recovery was approximately 80%. Following 10 mg/kg intravenous dose, mean plasma concentrations of 7.88 microg/ml were achieved at 5 min in mice and declined with undetectable levels at 4 h. The mean total body clearance was 4.78 l/h. The mean volume of distribution (V (d)) was 5.05 l. Non-compartmental analysis yielded the mean area under the plasma concentration-time curve (AUC) for noscapine as 53.42, 64.08, and 198.35 h microg/ml reaching maximum plasma concentrations (C (max)) of 12.74, 23.24, and 46.73 microg/ml at a t (max) of 1.12, 1.50, and 0.46 h at the linearly increasing dose levels. CONCLUSION: A rapid and simple HPLC/UV method for the quantification of noscapine in plasma has been developed to study pharmacokinetics of noscapine at tumor-suppressive doses in the mouse. Since orally available anticancer drugs are rare, therefore, noscapine, an innocuous agent, having a mean oral bioavailability of 31.5% over the studied dose range merits its further advancement in humans for anticancer therapy.

Treatment of hormone-refractory breast cancer: apoptosis and regression of human tumors implanted in mice.

Following surgery, the hormone dependence of breast tumors is exploited for therapy using antagonists such as tamoxifen, although occasional hormone-resistant clones do appear. Another chemotherapeutic strategy uses microtubule inhibitors such as taxanes. Unfortunately, these agents elicit toxicities such as leukocytopenia, diarrhea, alopecia, and peripheral neuropathies and are also associated with the emergence of drug resistance. We have previously described a tubulin-binding, natural compound, noscapine, that was nontoxic and triggered apoptosis in many cancer types albeit at 10 mumol/L or higher concentrations depending on the cell type. We now show that a synthetic analogue of noscapine, 9-bromonoscapine, is approximately 10-fold to 15-fold more potent than noscapine in inhibiting cell proliferation and induces apoptosis following G2-M arrest in hormone-insensitive human breast cancers (MDA-MB-231). Furthermore, a clear loss of mitochondrial membrane potential, release of cytochrome c, activation of the terminal caspase-3, and the cleavage of its substrates such as poly(ADP-ribose) polymerase, suggest an intrinsic apoptotic mechanism. Taken together, these data point to a mitochondrially mediated apoptosis of hormone-insensitive breast cancer cells. Human tumor xenografts in nude mice showed significant tumor volume reduction and a surprising increase in longevity without signs of obvious toxicity. Thus, our data provide compelling evidence that 9-bromonoscapine can be useful for the therapy of hormone-refractory breast cancer.

Synthesis of microtubule-interfering halogenated noscapine analogs that perturb mitosis in cancer cells followed by cell death.

We have previously identified the naturally occurring non-toxic antitussive phthalideisoquinoline alkaloid, noscapine as a tubulin-binding agent that arrests mitosis and induces apoptosis. Here we present high-yield efficient synthetic methods and an evaluation of anticancer activity of halogenated noscapine analogs. Our results show that all analogs display higher tubulin-binding activity than noscapine and inhibit proliferation of human cancer cells (MCF-7, MDA-MB-231 and CEM). Surprisingly, the bromo-analog is approximately 40-fold more potent than noscapine in inhibiting cellular proliferation of MCF-7 cells. The ability of these analogs to inhibit cellular proliferation is mediated by cell cycle arrest at the G2/M phase, in that all analogs except 9-iodonoscapine, caused selective mitotic arrest with a higher efficiency than noscapine followed by apoptotic cell death as shown by immunofluorescence and quantitative FACS analyses. Furthermore, our results reveal the appearance of numerous fragmented nuclei as evidenced by DAPI staining. Thus, our data indicate a great potential of these compounds for studying microtubule-mediated processes and as chemotherapeutic agents for the management of human cancers.

Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma.

Cellular microtubules, polymers of tubulin, alternate relentlessly between phases of growth and shortening. We now show that noscapine, a tubulin-binding agent, increases the time that cellular microtubules spend idle in a paused state. As a result, most mammalian cell types observed arrest in mitosis in the presence of noscapine. We demonstrate that noscapine-treated murine melanoma B16LS9 cells do not arrest in mitosis but rather become polyploid followed by cell death, whereas primary melanocytes reversibly arrest in mitosis and resume a normal cell cycle after noscapine removal. Furthermore, in a syngeneic murine model of established s.c. melanoma, noscapine treatment resulted in an 85% inhibition of tumor volume on day 17 when delivered by gavage compared with untreated animals (P

Aqueous phase adsorption of different sized molecules on activated carbon fibers: Effect of textural properties.

The effect that the textural properties of rayon-based activated carbon fibers (ACFs), such as the BET surface area and pore size distribution (PSD), have on the adsorption of differently sized molecules, namely, brilliant yellow (BY), methyl orange (MO) and phenol (PH), was investigated in the aqueous phase. ACF samples with different BET areas and PSDs were produced by steam-activating carbonized fibers for different activation times (0.25, 0.5, and 1 h). The samples activated for 0.25 h were predominantly microporous, whereas those activated for relatively longer times contained hierarchical micro-mesopores. The adsorption capacities of the ACFs for the adsorbate increased with increasing BET surface area and pore volume, and ranged from 51 to 1306 mg/g depending on the textural properties of the ACFs and adsorbate size. The adsorption capacities of the hierarchical ACF samples followed the order BY > MO > PH. Interestingly, the number of molecules adsorbed by the ACFs followed the reverse order: PH > MO > BY. This anomaly was attributed to the increasing molecular weight of the PH, MO and BY molecules. The equilibrium adsorption data were described using the Langmuir isotherm. This study shows that suitable textural modifications to ACFs are required for the efficient aqueous phase removal of an adsorbate.