Mary Ann Jordan

Publications of Mary Ann Jordan

• Modeling the effects of drug binding on the dynamic instability of microtubules.

  Authors: Peter Hinow, Vahid Rezania, Manu Lopus, Mary Ann Jordan, Jack A Tuszyński

  Physical biology. 08/2011; 8(5):056004.

  We propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady-state microtubules assembled from MAP-free tubulin in the possible presence of aWe propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady-state microtubules assembled from MAP-free tubulin in the possible presence of a microtubule-associated drug. As an example of the latter, we both experimentally and theoretically study the perturbation of microtubule dynamic instability by S-methyl-D-DM1, a synthetic derivative of the microtubule-targeted agent maytansine and a potential anticancer agent. Our model predicts that among the drugs that act locally at the microtubule tip, primary inhibition of the loss of GDP tubulin results in stronger damping of microtubule dynamics than inhibition of GTP tubulin addition. On the other hand, drugs whose action occurs in the interior of the microtubule need to be present in much higher concentrations to have visible effects.

• Characterization and detection of cellular and proteomic alterations in stable stathmin-overexpressing, taxol-resistant BT549 breast cancer cells using offgel IEF/PAGE difference gel electrophoresis.

  Authors: Manimalha Balasubramani, Chitose Nakao, Guy T Uechi, John Cardamone, Kathy Kamath, Kristen L Leslie, Raghavan Balachandran, Leslie Wilson, Billy W Day, Mary Ann Jordan

  Mutation research. 06/2011; 722(2):154-64.

  Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers.Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.

• Combinatorial Tau pseudophosphorylation: markedly different regulatory effects on microtubule assembly and dynamic instability than the sum of the individual parts.

  Authors: Erkan Kiris, Donovan Ventimiglia, Mehmet E Sargin, Michelle R Gaylord, Alphan Altinok, Kenneth Rose, B S Manjunath, Mary Ann Jordan, Leslie Wilson, Stuart C Feinstein

  The Journal of biological chemistry. 02/2011; 286(16):14257-70.

  Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias.Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias. A huge literature has led to the widely held notion that aberrant Tau hyperphosphorylation is central to these disorders. Unfortunately, our mechanistic understanding of the functional effects of combinatorial Tau phosphorylation remains minimal. Here, we generated four singly pseudophosphorylated Tau proteins (at Thr(231), Ser(262), Ser(396), and Ser(404)) and four doubly pseudophosphorylated Tau proteins using the same sites. Each Tau preparation was assayed for its abilities to promote microtubule assembly and to regulate microtubule dynamic instability in vitro. All four singly pseudophosphorylated Tau proteins exhibited loss-of-function effects. In marked contrast to the expectation that doubly pseudophosphorylated Tau would be less functional than either of its corresponding singly pseudophosphorylated forms, all of the doubly pseudophosphorylated Tau proteins possessed enhanced microtubule assembly activity and were more potent at regulating dynamic instability than their compromised singly pseudophosphorylated counterparts. Thus, the effects of multiple pseudophosphorylations were not simply the sum of the effects of the constituent single pseudophosphorylations; rather, they were generally opposite to the effects of singly pseudophosphorylated Tau. Further, despite being pseudophosphorylated at different sites, the four singly pseduophosphorylated Tau proteins often functioned similarly, as did the four doubly pseudophosphorylated proteins. These data lead us to reassess the conventional view of combinatorial phosphorylation in normal and pathological Tau action. They may also be relevant to the issue of combinatorial phosphorylation as a general regulatory mechanism.

• Microtubule-binding agents: a dynamic field of cancer therapeutics.

  Authors: Charles Dumontet, Mary Ann Jordan

  Nature reviews. Drug discovery. 10/2010; 9(10):790-803.

  Microtubules are dynamic filamentous cytoskeletal proteins composed of tubulin and are an important therapeutic target in tumour cells. Agents that bind to microtubules have been part of theMicrotubules are dynamic filamentous cytoskeletal proteins composed of tubulin and are an important therapeutic target in tumour cells. Agents that bind to microtubules have been part of the pharmacopoeia of anticancer therapy for decades and until the advent of targeted therapy, microtubules were the only alternative to DNA as a therapeutic target in cancer. The screening of a range of botanical species and marine organisms has yielded promising new antitubulin agents with novel properties. In the current search for novel microtubule-binding agents, enhanced tumour specificity, reduced neurotoxicity and insensitivity to chemoresistance mechanisms are the three main objectives.

• Maytansine and cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule dynamics by binding to microtubules.

  Authors: Manu Lopus, Emin Oroudjev, Leslie Wilson, Sharon Wilhelm, Wayne Widdison, Ravi Chari, Mary Ann Jordan

  Molecular cancer therapeutics. 10/2010; 9(10):2689-99.

  Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity haveMaytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. Recently, antibody-conjugated maytansine derivatives have been developed to overcome these drawbacks. Several conjugates show promising early clinical results. We evaluated the effects on microtubule polymerization and dynamic instability of maytansine and two cellular metabolites (S-methyl-DM1 and S-methyl-DM4) of antibody-maytansinoid conjugates that are potent in cells at picomolar levels and that are active in tumor-bearing mice. Although S-methyl-DM1 and S-methyl-DM4 inhibited polymerization more weakly than maytansine, at 100 nmol/L they suppressed dynamic instability more strongly than maytansine (by 84% and 73%, respectively, compared with 45% for maytansine). However, unlike maytansine, S-methyl-DM1 and S-methyl-DM4 induced tubulin aggregates detectable by electron microscopy at concentrations ≥2 μmol/L, with S-methyl-DM4 showing more extensive aggregate formation than S-methyl-DM1. Both maytansine and S-methyl-DM1 bound to tubulin with similar K(D) values (0.86 ± 0.2 and 0.93 ± 0.2 μmol/L, respectively). Tritiated S-methyl-DM1 bound to 37 high-affinity sites per microtubule (K(D), 0.1 ± 0.05 μmol/L). Thus, S-methyl-DM1 binds to high-affinity sites on microtubules 20-fold more strongly than vinblastine. The high-affinity binding is likely at microtubule ends and is responsible for suppression of microtubule dynamic instability. Also, at higher concentrations, S-methyl-DM1 showed low-affinity binding either to a larger number of sites on microtubules or to sedimentable tubulin aggregates. Overall, the maytansine derivatives that result from cellular metabolism of the antibody conjugates are themselves potent microtubule poisons, interacting with microtubules as effectively as or more effectively than the parent molecule.

• Maytansinoid-antibody conjugates induce mitotic arrest by suppressing microtubule dynamic instability.

  Authors: Emin Oroudjev, Manu Lopus, Leslie Wilson, Charlene Audette, Carmela Provenzano, Hans Erickson, Yelena Kovtun, Ravi Chari, Mary Ann Jordan

  Molecular cancer therapeutics. 10/2010; 9(10):2700-13.

  Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoidsMaytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule dynamic instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule dynamic instability.

• Microtubule dynamics, mitotic arrest, and apoptosis: drug-induced differential effects of betaIII-tubulin.

  Authors: Pei Pei Gan, Joshua A McCarroll, Sela T Po'uha, Kathy Kamath, Mary Ann Jordan, Maria Kavallaris

  Molecular cancer therapeutics. 05/2010; 9(5):1339-48.

  Overexpression of betaIII-tubulin is associated with resistance to tubulin-binding agents (TBA) in a range of tumor types. We previously showed that small interfering RNA silencing of betaIII-tubulinOverexpression of betaIII-tubulin is associated with resistance to tubulin-binding agents (TBA) in a range of tumor types. We previously showed that small interfering RNA silencing of betaIII-tubulin expression hypersensitized non-small cell lung cancer cells to TBAs. To determine whether betaIII-tubulin mediates its effect on drug-induced mitotic arrest and cell death by differentially regulating microtubule behavior, the effects of betaIII-tubulin knockdown on microtubule dynamics were analyzed in H460 non-small cell lung cancer cells stably expressing green fluorescent protein-betaI-tubulin. Interphase cells were examined at three vincristine and paclitaxel concentrations that (a) inhibited cell proliferation, (b) induced 5% to 10% mitotic arrest, and (c) induced 30% to 40% mitotic arrest. In the absence of either drug, betaIII-tubulin knockdown caused no significant change in microtubule dynamic instability. At 2 nmol/L vincristine (IC(50)), overall microtubule dynamicity was significantly suppressed in betaIII-tubulin knockdowns (-31.2%) compared with controls (-6.5%). Similar results were obtained with paclitaxel, suggesting that knockdown of betaIII-tubulin induces hypersensitivity by enhancing stabilization of microtubule dynamics at low drug concentrations. At higher drug concentrations (> or =40 nmol/L vincristine; > or =20 nmol/L paclitaxel), betaIII-tubulin knockdown resulted in significantly reduced suppressive effects on microtubule dynamicity with little or no further increase in mitotic arrest, compared with control cells. Importantly, apoptosis was markedly increased by betaIII-tubulin knockdown independent of further suppression of microtubule dynamics and mitotic arrest. These results show that betaIII-tubulin knockdown enhances the effectiveness of TBAs through two mechanisms: suppression of microtubule dynamics at low drug concentrations and a mitosis-independent mechanism of cell death at higher drug concentrations.

• Eribulin binds at microtubule ends to a single site on tubulin to suppress dynamic instability.

  Authors: Jennifer A Smith, Leslie Wilson, Olga Azarenko, Xiaojie Zhu, Bryan M Lewis, Bruce A Littlefield, Mary Ann Jordan

  Biochemistry. 02/2010; 49(6):1331-7.

  Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressingEribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing dynamic instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086-1095]. Using [(3)H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall K(d) of 46 microM, but also showing a real or apparent very high affinity (K(d) = 0.4 microM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 +/- 1.3 molecules per microtubule (K(d) = 3.5 microM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that the binding of a single eribulin molecule at a microtubule end can potently inhibit its growth. Eribulin does not suppress dynamic instability at microtubule minus ends. Preincubation of microtubules with 2 or 4 microM vinblastine induced additional lower-affinity eribulin binding sites, most likely at splayed microtubule ends. Overall, our results indicate that eribulin binds with high affinity to microtubule plus ends and thereby suppresses dynamic instability.

• Determination of drug binding to microtubules in vitro.

  Authors: Jennifer A Smith, Mary Ann Jordan

  Methods in cell biology. 01/2010; 95:289-99.

  Many naturally occurring compounds and their synthetic analogs bind to soluble tubulin or to tubulin in microtubules. These compounds are often important drugs or drug candidates. They can potentlyMany naturally occurring compounds and their synthetic analogs bind to soluble tubulin or to tubulin in microtubules. These compounds are often important drugs or drug candidates. They can potently alter both the dynamics and the polymer mass of microtubules. The binding affinity of a drug for soluble tubulin heterodimers is a common and relatively readily determined biochemical characteristic of a tubulin-targeted drug. However, it is only one important aspect of the drug-tubulin interaction. In solution and in cells, soluble tubulin is in equilibrium with polymerized microtubules. It is as important to determine drug binding to microtubules as it is to determine binding to soluble tubulin, since drug binding to microtubules frequently alters their function. The affinity of a compound for microtubules often differs vastly from its affinity for soluble tubulin. Here, we present detailed instructions for assessing binding stoichiometry and affinity to assembled unstabilized microtubules using radiolabeled drug. In addition, using examples from binding results with several important drugs including vinca alkaloids, colchicine, eribulin, and taxanes, we discuss aspects of the interactions with microtubules that may alter the experimental design of the drug-binding experiments.

• Determination of microtubule dynamic instability in living cells.

  Authors: Kathy Kamath, Emin Oroudjev, Mary Ann Jordan

  Methods in cell biology. 01/2010; 97:1-14.

  The precise regulation of microtubules and their dynamics is critical for cell cycle progression, cell signaling, intracellular transport, cell polarization, and organismal development. For example,The precise regulation of microtubules and their dynamics is critical for cell cycle progression, cell signaling, intracellular transport, cell polarization, and organismal development. For example, mitosis, cell migration, and axonal outgrowth all involve rapid and dramatic changes in microtubule organization and dynamics. Microtubule-associated proteins (MAPs) such as MAP2 and tau (Bunker et al., 2004; Dhamodharan and Wadsworth, 1995) and microtubule-interacting proteins such as stathmin, the kinesin MCAK, and EB1 (Cassimeris, 1999; Moore and Wordeman, 2004; Ringhoff and Cassimeris, 2009; Rusan et al., 2001) as well as numerous clinically approved or experimental anti-mitotic drugs including the taxanes, vinca alkaloids, and colchicine-like compounds modulate microtubule dynamic in cells (Jordan, 2002; Jordan and Kamath, 2007). In this chapter, we describe methods to analyze the dynamic instability of microtubules in living cells by microscopy of microinjected or expressed fluorescent tubulin, time-lapse microscopy, and analysis of time-dependent microtubule length changes.

• The neuroprotective peptide NAP does not directly affect polymerization or dynamics of reconstituted neural microtubules.

  Authors: Mythili Yenjerla, Nichole E LaPointe, Manu Lopus, Corey Cox, Mary Ann Jordan, Stuart C Feinstein, Leslie Wilson

  Journal of Alzheimer's disease : JAD. 01/2010; 19(4):1377-86.

  NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is a neuroprotective peptide that shows cognitive protection in patients with amnestic mild cognitive impairment, a precursor to Alzheimer's disease. NAPNAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is a neuroprotective peptide that shows cognitive protection in patients with amnestic mild cognitive impairment, a precursor to Alzheimer's disease. NAP exhibits potent neuroprotective properties in several in vivo and cellular models of neural injury. While NAP has been found in many studies to affect microtubule assembly and/or stability in neuronal and glial cells at fM concentrations, it has remained unclear whether NAP acts directly or indirectly on tubulin or microtubules. We analyzed the effects of NAP (1 fM-1 microM) on the assembly of reconstituted bovine brain microtubules in vitro and found that it did not significantly (p< 0.05) alter polymerization of either purified tubulin or of a mixture of tubulin and unfractionated microtubule-associated proteins. NAP also had no significant effect (p < 0.05) on the growing and shortening dynamics of steady-state microtubules at their plus ends, nor did it alter the polymerization or dynamics of microtubules assembled in the presence of 3-repeat or 4-repeat tau. Thus, the neuroprotective activity of NAP does not appear to involve a direct action on the polymerization or dynamics of purified tubulin or microtubules.

• Hesperidin suppressed proliferations of both Human breast cancer and androgen-dependent prostate cancer cells.

  Authors: Choong Jae Lee, Leslie Wilson, Mary Ann Jordan, Vy Nguyen, Jessica Tang, Gregory Smiyun

  Phytotherapy research : PTR. 06/2009;

  Hesperidin, a flavonoid derived from citrus fruits, has been reported to show various biological effects including anticancer activity. This study investigated whether hesperidin affected theHesperidin, a flavonoid derived from citrus fruits, has been reported to show various biological effects including anticancer activity. This study investigated whether hesperidin affected the proliferation of MCF-7 human breast cancer cells transfected with green fluorescent protein (GFP)/alpha-tubulin (MCF-7-GFP-Tubulin cells), androgen-independent PC-3 and DU-145 prostate cancer cells, and androgen-dependent LNCaP prostate cancer cells. The results were as follows. (1) Hesperidin inhibited the proliferation of MCF-7-GFP-Tubulin cells, probably not through an antimitotic mechanism. (2) Hesperidin also inhibited both basal and testosterone-induced proliferation of LNCaP cells. (3) However, hesperidin did not significantly affect the cell proliferation of two hormone-independent prostate cancer cells, PC-3 and DU-145. It is concluded that hesperidin can inhibit the proliferation of breast cancer cells through mechanisms other than antimitosis and it is suggested that hesperidin be further investigated for the possible interaction with androgenic receptors and involvement in signaling pathway after receptor binding in prostate cancer cells through future research. Copyright (c) 2009 John Wiley & Sons, Ltd.

• Carbendazim inhibits cancer cell proliferation by suppressing microtubule dynamics.

  Authors: Mythili Yenjerla, Corey Cox, Leslie Wilson, Mary Ann Jordan

  The Journal of pharmacology and experimental therapeutics. 12/2008;

  Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of humanCarbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells including drug- and multidrug-resistant and p53-deficient cell lines. Due to its promising preclinical antitumor activity, it has undergone Phase I clinical trials and is under further clinical development. While it weakly inhibits polymerization of brain microtubules and induces G2/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC50: 10 microM) and half-maximally arrests mitosis at a similar concentration (8 microM), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 microM with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (Kd, 42.8 +/- 4.0 microM). Unlike some benzimidazoles that bind to the colchicine-site in tubulin, carbendazim does not compete with colchicine nor does it compete with vinblastine for binding to brain tubulin. Thus carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest.

• FTDP-17 mutations in tau alter the regulation of microtubule dynamics - an "Alternative Core" model for normal and pathological tau action.

  Authors: Adria C Leboeuf, Sasha F Levy, Michelle Gaylord, Arnab Bhattacharya, Ambuj K. Singh, Mary Ann Jordan, Leslie Wilson, Stuart C Feinstein

  The Journal of biological chemistry. 11/2008;

  Mutations affecting either the structure or regulation of the microtubule associated protein tau cause neuronal cell death and dementia. However, the molecular mechanisms mediating these deleteriousMutations affecting either the structure or regulation of the microtubule associated protein tau cause neuronal cell death and dementia. However, the molecular mechanisms mediating these deleterious effects remain unclear. Among tau's most characterized activities is the ability to regulate microtubule dynamics, known to be essential for proper cell function and viability. Here, we have tested the hypothesis that tau mutations causing neurodegeneration also alter the ability of tau to regulate the dynamic instability behaviors of microtubules. Using in vitro microtubule dynamics assays to assess average microtubule growth rates, microtubule growth rate distributions and catastrophe frequencies, we found that all tested mutants possessing amino acid substitutions or deletions mapping to either the repeat or inter-repeat regions of tau do indeed compromise its ability to regulate microtubule dynamics. Further mutational analyses suggest a novel mechanism of tau regulatory action based on an "alternative core" of microtubule binding and regulatory activities composed of two repeats and the inter-repeat between them. In this model, the inter-repeat serves as the primary regulator of microtubule dynamics while the flanking repeats serve as tethers to properly position the inter-repeat on the microtubule. Importantly, since there are multiple inter-repeats on each tau molecule, there are also multiple cores on each tau molecule, each with distinct mechanistic capabilities, thereby providing significant regulatory potential. Taken together, the data are consistent with a microtubule misregulation mechanism for tau mediated neuronal cell death and provide a novel mechanistic model for normal and pathological tau action.

• Suppression of microtubule dynamic instability and turnover in MCF7 breast cancer cells by sulforaphane.

  Authors: Olga Azarenko, Tatiana Okouneva, Keith W Singletary, Mary Ann Jordan, Leslie Wilson

  Carcinogenesis. 11/2008;

  Sulforaphane, a prominent isothiocyanate present in cruciferous vegetables, is believed to be responsible along with other isothiocyanates for the cancer preventive activity of such vegetables.Sulforaphane, a prominent isothiocyanate present in cruciferous vegetables, is believed to be responsible along with other isothiocyanates for the cancer preventive activity of such vegetables. Sulforaphane arrests mitosis, possibly by affecting spindle microtubule function. A critical property of microtubules is their rapid and time-sensitive growth and shortening dynamics (dynamic instability), and suppression of dynamics by antimitotic anticancer drugs (e.g., taxanes and the vinca alkaloids) is central to the anticancer mechanisms of such drugs. We found that at concentrations that inhibited proliferation and mitosis of MCF7-GFP-alpha-tubulin breast tumor cells by approximately 50% ( approximately 15 muM), sulforaphane significantly modified microtubule organization in arrested spindles without modulating the spindle microtubule mass, in a manner similar to that of much more powerful antimitotic drugs. By using quantitative fluorescence video microscopy, we determined that at its mitotic IC(50), sulforaphane suppressed the dynamic instability of the interphase microtubules in these cells, strongly reducing the rate and extent of growth and shortening and decreasing microtubule turnover, without affecting the polymer mass. Sulforaphane suppressed the dynamics of purified microtubules in a similar fashion at concentrations well below those required to depolymerize microtubules, indicating that the suppression of dynamic instability by sulforaphane in cells is due to a direct effect on the microtubules. The results indicate that sulforaphane arrests proliferation and mitosis by stabilizing microtubules in a manner weaker than but similar to more powerful clinically used antimitotic anticancer drugs, and strongly support the hypothesis that inhibition of mitosis by microtubule stabilization is important for sulforaphane's chemopreventive activity.

• Inhibition of centromere dynamics by eribulin (E7389) during mitotic metaphase.

  Authors: Tatiana Okouneva, Olga Azarenko, Leslie Wilson, Bruce A Littlefield, Mary Ann Jordan

  Molecular cancer therapeutics. 07/2008; 7(7):2003-11.

  Eribulin (E7389), a synthetic analogue of halichondrin B in phase III clinical trials for breast cancer, binds to tubulin and microtubules. At low concentrations, it suppresses the growth phase ofEribulin (E7389), a synthetic analogue of halichondrin B in phase III clinical trials for breast cancer, binds to tubulin and microtubules. At low concentrations, it suppresses the growth phase of microtubule dynamic instability in interphase cells, arrests mitosis, and induces apoptosis, suggesting that suppression of spindle microtubule dynamics induces mitotic arrest. To further test this hypothesis, we measured the effects of eribulin on dynamics of centromeres and their attached kinetochore microtubules by time-lapse confocal microscopy in living mitotic U-2 OS human osteosarcoma cells. Green fluorescent protein-labeled centromere-binding protein B marked centromeres and kinetochore-microtubule plus-ends. In control cells, sister chromatid centromere pairs alternated under tension between increasing and decreasing separation (stretching and relaxing). Eribulin suppressed centromere dynamics at concentrations that arrest mitosis. At 60 nmol/L eribulin (2 x mitotic IC(50)), the relaxation rate was suppressed 21%, the time spent paused increased 67%, and dynamicity decreased 35% (but without reduction in mean centromere separation), indicating that eribulin decreased normal microtubule-dependent spindle tension at the kinetochores, preventing the signal for mitotic checkpoint passage. We also examined a more potent, but in tumors less efficacious antiproliferative halichondrin derivative, ER-076349. At 2 x IC(50) (4 nmol/L), mitotic arrest also occurred in concert with suppressed centromere dynamics. Although media IC(50) values differed 15-fold between the two compounds, the intracellular concentrations were similar, indicating more extensive relative uptake of ER-076349 into cells compared with eribulin. The strong correlation between suppression of kinetochore-microtubule dynamics and mitotic arrest indicates that the primary mechanism by which eribulin blocks mitosis is suppression of spindle microtubule dynamics.

• Microtubule Assembly of Isotypically Purified Tubulin and Its Mixtures.

  Authors: Vahid Rezania, Olga Azarenko, Mary Ann Jordan, Hannes Bolterauer, Richard F Ludueña, J Torin Huzil, Jack A Tuszynski

  Biophysical journal. 06/2008;

  Numerous isotypes of the structural protein tubulin have now been characterized in various organisms and their expression offers a plausible explanation for observed differences affecting microtubuleNumerous isotypes of the structural protein tubulin have now been characterized in various organisms and their expression offers a plausible explanation for observed differences affecting microtubule function in vivo. While this is an attractive hypothesis, there are only a handful of studies demonstrating a direct influence of tubulin isotype composition on the dynamic properties of microtubules. Here, we present the results of experimental assays on the assembly of microtubules from bovine brain tubulin using purified isotypes at various controlled relative concentrations. A novel data analysis is developed using recursive maps which are shown to be related to the master equation formalism. We have found striking similarities between the three isotypes of bovine tubulin studied in regard to their dynamic instability properties, except for subtle differences in their catastrophe frequencies. When mixtures of tubulin isotypes are analyzed, their nonlinear concentration dependence is modeled and interpreted in terms of lower affinities of tubulin dimers belonging to the same isotype than those that represent different isotypes indicating hitherto unsuspected influences of tubulin dimers on each other within a microtubule. Finally, we investigate the fluctuations in microtubule assembly and disassembly rates and conclude that the inherent rate variability may signify differences in the GTP composition of the growing and shortening microtubule tips. It is the main objective of this paper to develop a quantitative model of tubulin polymerization for individual isotypes and their mixtures. The possible biological significance of the observed differences is addressed.

• Exploring the mechanisms of action of the novel microtubule inhibitor vinflunine.

  Authors: Mary Ann Jordan, Susan Band Horwitz, Sharon Lobert, John J Correia

  Seminars in oncology. 06/2008; 35(3 Suppl 3):S6-S12.

  Microtubules have been identified as a suitable target for anticancer therapy, primarily based on their biological importance in coordinating chromosomal segregation at mitosis. Two main classes ofMicrotubules have been identified as a suitable target for anticancer therapy, primarily based on their biological importance in coordinating chromosomal segregation at mitosis. Two main classes of microtubule-targeted agents, the taxanes and vinca alkaloids, suppress the dynamic behavior of spindle microtubules, inducing mitotic arrest and subsequent apoptotic cell death. Clinical activity of taxanes and first-generation vinca alkaloids in the treatment of solid tumors and hematologic malignancies, respectively, has prompted further research for novel analogs with improved clinical efficacy and safety. Such efforts have led to the development of vinflunine, a bifluorinated vinca alkaloid endowed with unique antitumor properties. Highlighted in this review are the key features of vinflunine that lead to effective suppression of microtubule dynamics and induction of cell death in cancer cells.

• How do microtubule-targeted drugs work? An overview.

  Authors: Mary Ann Jordan, Kathy Kamath

  Current cancer drug targets. 01/2008; 7(8):730-42.

  The importance of microtubules in mitosis makes them a superb target for a group of highly successful, chemically diverse anticancer drugs. Knowledge of the mechanistic differences among the manyThe importance of microtubules in mitosis makes them a superb target for a group of highly successful, chemically diverse anticancer drugs. Knowledge of the mechanistic differences among the many drugs of this class is vital to understanding their tissue and cell specificity, the development of resistance, the design of novel improved drugs, optimal scheduling of treatment, and potential synergistic combinations. This overview covers microtubule assembly dynamics, the exquisite regulation of microtubule dynamics in cells by endogenous regulators, the important role of microtubule dynamics in mitosis, the diversity and number of microtubule-targeted drugs undergoing clinical development, the antimitotic mechanisms of microtubule-targeted drugs with emphasis on suppression of microtubule dynamics by vinblastine and taxol, the role of drug uptake and retention in the efficacy of microtubule-targeted drugs, and the anti-angiogenic and vascular-disrupting mechanisms of microtubule targeted drugs. In view of the success of this class of drugs, it has been argued that microtubules represent the single best cancer target identified to date, and it seems likely that drugs in this class will continue to remain an important chemotherapeutic class of drugs even as more selective chemotherapeutic approaches are developed.

• Effects of tetramethoxystilbene on hormone-resistant breast cancer cells: biological and biochemical mechanisms of action.

  Authors: Hoyong Park, Sarah E Aiyar, Ping Fan, Jiping Wang, Wei Yue, Tatiana Okouneva, Corey Cox, Mary Ann Jordan, Laurence Demers, HyungJun Cho, Sanghee Kim, Robert X-D Song, Richard J Santen

  Cancer research. 07/2007; 67(12):5717-26.

  Secondary resistance to hormonal therapy for breast cancer commonly develops after an initial response to tamoxifen or aromatase inhibitors. Agents to abrogate these adaptive changes wouldSecondary resistance to hormonal therapy for breast cancer commonly develops after an initial response to tamoxifen or aromatase inhibitors. Agents to abrogate these adaptive changes would substantially enhance the long-term benefits of hormonal therapy. Our studies with a stilbene derivative called TMS (2,3',4,5'-tetramethoxystilbene) identified unexpected effects with potential utility for treatment of breast tumors secondarily resistant to hormonal therapy. TMS was originally developed as an inhibitor of cytochrome P450 1B1 to block the conversion of estradiol to 4-OH-estradiol. While studying this agent in three models of hormone resistance, we detected direct antitumor effects not related to its role as an inhibitor of catecholestrogens. During examination of the mechanisms involved, we showed that treatment with 3 micromol/L TMS for 24 h inhibited tubulin polymerization and microtubule formation, caused a cell cycle block at the G2-M phase, and induced apoptosis. TMS also inhibited activated focal adhesion kinase (FAK), Akt, and mammalian target of rapamycin (mTOR) and stimulated c-jun-NH2-kinase and p38 mitogen-activated protein kinase activity. With respect to antitumor effects, TMS at a concentrations of 0.2 to 0.3 micromol/L inhibited the growth of long-term tamoxifen-treated MCF-7 cells by 80% and fulvestrant-treated MCF-7 cells by 70%. In vivo studies, involving 8 weeks of treatment with TMS via a 30-mg s.c. implant, reduced tumor volume of tamoxifen-resistant MCF-7 breast cancer xenografts by 53%. Our data suggest that TMS is a promising therapeutic agent because of its unique ability to block several pathways involved in the development of hormone resistance.