Inhibition Of Microtubule Assembly Research Articles

Study of a Potent Small‐Molecule Benzosuberene Anti‐Cancer Agent

The discovery of a benzosuberene‐based, small‐molecule anti‐cancer agent (KGP18) with remarkable cytotoxicity against selected human cancer cell lines (BMC 2008 16, 8161–8171) provided the inspiration for a collaborative research program to investigate the mechanism of action of this compound and related benzosuberene analogues. The structure of KGP18 and its ability to inhibit tubulin polymerization into microtubules in vitro suggests that it may act as a vascular disrupting agent (VDA). An adequate blood supply is required for tumor growth and metastasis. VDAs represent a promising new class of anti‐cancer agents that function by selectively disrupting the tumor vasculature, thus starving the tumor of nutrients and oxygen via an adequate blood supply. Tubulin‐binding VDAs inhibit microtubule assembly in the activated endothelial cells, lining the tumor vasculature, leading to cytoskeletal reorganization, activation of RhoA and RhoA kinase, increased vascular permeability, cell rounding and detachment, and vessel occlusion. The lead benzosuberene analogue (KGP18) binds to tubulin at the colchicine binding site as determined by a competitive radiometric assay. Flow cytometry indicates that MDA‐MB‐231 cells treated with KGP18 are arrested at the G2/M phase of the cell cycle. (Supported by Oxigene Inc., the NIH, and CPRIT).

Herbicide cross resistance in weeds

With no major new site-of-action herbicide introduced into the marketplace in the last 20 years, the stagnation or decline in available herbicides in the past decade in a number of jurisdictions, and ever-increasing incidence of herbicide-resistant (HR) weeds, more efficient use of our existing herbicide tools will be required to proactively or reactively manage HR weed populations. Herbicide-resistant weed management can be aided by crop cultivars with alternative single or stacked herbicide-resistance traits, such as synthetic auxins, which will become increasingly available to growers in the future. An examination of cross-resistance patterns in HR weed populations may inform proactive or reactive HR weed management through better insights into the potential for HR trait-stacked crops to manage HR weed biotypes as well as identify possible effective alternative herbicide options for growers. Clethodim is the lowest resistance risk acetyl-CoA carboxylase (ACC) inhibiting herbicide, with only two of eleven target-site mutations (amino acid substitutions) in weed populations that confer resistance. However, there are no reduced-risk acetolactate synthase/acetohydroxyacid synthase (ALS/AHAS) herbicides or herbicide classes. Growers will be increasingly reliant on reduced-risk herbicide sites of action (groups), such as microtubule assembly inhibitors (e.g., trifluralin, pendimethalin), synthetic auxins (e.g., 2,4-D, dicamba), some photosystem-II inhibitors (nitriles such as bromoxynil), protoporphyrinogen oxidase (PPO) or hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, glyphosate, or glutamine synthetase inhibitor (glufosinate), used in sequences, mixtures, or rotations, to manage HR weed populations.

The role of microtubules in the maintenance of regular localization and arrangement of Golgi apparatus in root cells of Triticum aestivum L.

In plant cells Golgi apparatus organization, maintenance and distribution differ from that in mammalian cells and the mechanisms for this are not clearly understood. Here we investigate the role of microtubules in the positioning and arrangement of Golgi apparatus in the root cells of Triticum aestivum L. by using dual immunofluorescent labeling and laser confocal microscopy to localize both throughout the cell cycle. We observed that Golgi stacks (i) in interphase cells predominantly occupied the perinuclear region, (ii) during mitosis they redistributed to the spindle periphery and/or areas above spindle poles, and (iii) in telophase accumulated around the phragmoplast and the chromosomes/nuclei of daughter cells. Inhibition of microtubule assembly by colchicine resulted in aggregation of Golgi in the cortical cytoplasm of interphase cells and accumulation around the chromosomes in C-mitotic cells, in stark contrast with the distribution in untreated cells. Electron microscopy revealed that in colchicine treated cells many Golgi units became disorganized, yet others were abnormally enlarged. Overall, our results indicate that in plant cells microtubules play a key role in restricting the position and maintaining the arrangement and structural integrity of the Golgi apparatus.

Synthesis and biological evaluation of phenstatin metabolites

Previous investigations on the incubation of phenstatin with rat and human microsomal fractions revealed the formation of nine main metabolites. The structures of eight of these metabolites have been now confirmed by synthesis and their biological properties have been reported. Eaton’s reagent was utilized as a convenient condensing agent, allowing, among others, a simple multigram scale preparation of phenstatin. Synthesized metabolites and related compounds were evaluated for their antiproliferative activity in the NCI-60 cancer cell line panel, and for their effect on microtubule assembly. Metabolite 23 (2′-methoxyphenstatin) exhibited the most potent in vitro cytotoxic activity: inhibition of the growth of K-562, NCI-H322M, NCI-H522, KM12, M14, MDA-MB-435, NCI/ADR-RES, and HS 578T cell lines with GI 50 values <10 nM. It also showed more significant tubulin polymerization inhibitory activity than parent phenstatin ( 3) (IC 50 = 3.2 μM vs 15.0 μM) and induced G2/M arrest in murine leukemia DA1-3b cells. The identification of this active metabolite led to the design and synthesis of analogs with potent in vitro cytotoxicity and inhibition of microtubule assembly.

Wip1 contributes to cell homeostasis maintained by the steady-state level of Wtp53

Wip1, a human protein Ser/Thr phosphatase also called PPM1D, stands for wild type p53 induced phosphatase 1. Emerging evidences indicate that Wip1 can act as an oncogene largely by turning off DNA damage checkpoint responses. Here we report an unrecognized role of Wipl in normally growing cells. Wip1 can be induced by wild type p53 under not only stressed but also non-stressed conditions. It can trigger G2/M arrest in wild type p53 containing cells, which was attributed to the decreased Cdc2 kinase activity resulting at least partly from a high level of inhibitory tyrosine phosphorylation on Cdc2 protein at Tyr-15. Furthermore, we also found that Wip1 not only causes G2/M arrest but also decreases cell death triggered by microtubule assembly inhibitor in mouse fibroblasts when wild type p53 function was restored. These results indicate that Wip1 can provide ample time for wild type p53-containing cells to prepare entry into mitosis and avoid encountering mitotic catastrophe. Therefore, Wipl may play important roles in cell/tissue homeostasis maintained by wild type p53 under normal conditions, enhancing our understanding of how p53 makes cell-fate decisions.

Efficient Synthesis of the Spiroacetal Core of Paecilospirone via Oxidative Radical Cyclisation

The spiroacetal core of the microtubule assembly inhibitor paecilospironehas been prepared using two separate oxidative cyclisation methods.

Microtubule assembly affects bone mass by regulating both osteoblast and osteoclast functions: Stathmin deficiency produces an osteopenic phenotype in mice

Cytoskeleton microtubules regulate various cell signaling pathways that are involved in bone cell function. We recently reported that inhibition of microtubule assembly by microtubule-targeting drugs stimulates osteoblast differentiation and bone formation. To further elucidate the role of microtubules in bone homeostasis, we characterized the skeletal phenotype of mice null for stathmin, an endogenous protein that inhibits microtubule assembly. In vivo micro-computed tomography (µCT) and histology revealed that stathmin deficiency results in a significant reduction of bone mass in adult mice concurrent with decreased osteoblast and increased osteoclast numbers in bone tissues. Phenotypic analyses of primary calvarial cells and bone marrow cells showed that stathmin deficiency inhibited osteoblast differentiation and induced osteoclast formation. In vitro overexpression studies showed that increased stathmin levels enhanced osteogenic differentiation of preosteoblast MC3T3-E1 cells and mouse bone marrow-derived cells and attenuated osteoclast formation from osteoclast precursor Raw264.7 cells and bone marrow cells. Results of immunofluorescent studies indicated that overexpression of stathmin disrupted radial microtubule filaments, whereas deficiency of stathmin stabilized the microtubule network structure in these bone cells. In addition, microtubule-targeting drugs that inhibit microtubule assembly and induce osteoblast differentiation lost these effects in the absence of stathmin. Collectively, these results suggest that stathmin, which alters microtubule dynamics, plays an essential role in maintenance of postnatal bone mass by regulating both osteoblast and osteoclast functions in bone. \

Abstract 3288: The anti-tumour efficacy of the novel peptide inhibitor of angiogenesis ALM-201

Abstract Neovascularization is critical to tumour growth and metastasis and this has led to the development of a number of marketed anti-angiogenic agents which target VEGF/VEGFR2 receptors, for clinical use in specific types of cancer. However, incomplete responses and/or resistance to these therapies has highlighted the need for new agents targeting alternative pathways. Here we describe the characterisation of a novel peptide derived from the natural protein FKBP-like binding protein (FKBPL), which has extremely potent anti-angiogenic activity, is very effective in mouse xenograft models at low doses, and exerts its effects through microtubule binding using CD44 as a cell-entry mechanism. ALM-201 has been profiled in a range of human microvascular endothelial cell (HMEC-1) assays and potently inhibited migration, tubule formation and microvessel formation in vitro and in vivo. Although the peptide has a marked effect on migration, ALM-201 does not inhibit proliferation in a range of growth factor stimulated proliferation assays. Importantly, there is a significant disconnect between the pharmacokinetic and pharmacodynamic profiles of ALM-201 which allows for 3 q.d. dosing in mouse xenograft models. The peptide is well tolerated with no signs of toxicity observed in mouse xenograft models up to 80 days of dosing. The mechanism by which ALM-201 inhibits angiogenesis involves the cell surface receptor CD44, as determined by siRNA depletion of the receptor in migration assays. Furthermore, the peptide potently inhibits microtubule assembly and downstream signalling, thus promoting the anti-migratory phenotype. In summary, ALM-201 is a novel, targeted microtubule binding agent which exhibits potent anti-angiogenic activity in vitro and in vivo. Pre-clinical development is in progress with a Phase 1 clinical study planned for 2011. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3288. doi:10.1158/1538-7445.AM2011-3288

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one induces cell cycle arrest and apoptosis in HeLa cells by preventing microtubule polymerization

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) is known as a specific inhibitor of soluble guanylyl cyclase (sGC). Previously, however, ODQ was reported to induce cell death via sGC-dependent and sGC-independent means in a variety of cell types. The aim of this study was to investigate the mechanism by which ODQ induces cell death in HeLa cells. Treatment of HeLa cells with ODQ induced a concentration-dependent decrease in cell viability over the range from 10 to 100 μM. DNA fragmentation and fluorescence-activated cell sorting analysis using annexin V and propidium iodide staining revealed that ODQ triggered apoptosis at concentrations of 50 and 100 μM within 24 to 48 h. The addition of 8-Br-cGMP in the presence of ODQ failed to rescue HeLa cells from death, suggesting that the inhibition of sGC was not responsible for the pro-apoptotic action of ODQ. ODQ arrested the cell cycle at the G2/M phase and caused disassembly of the microtubule network. This process was reversed by dithiothreitol. In addition, ODQ was shown to inhibit the polymerization of purified tubulin, and this was also prevented by dithiothreitol. These results indicate that ODQ inhibits microtubule assembly by direct oxidation of tubulin, induces cell cycle arrest at the G2/M phase, and triggers apoptosis in HeLa cells.

A phase I study of MN-029 (denibulin), a novel vascular-disrupting agent, in patients with advanced solid tumors

MN-029 (denibulin HCl) is a novel vascular-disrupting agent that reversibly inhibits microtubule assembly, resulting in disruption of the cytoskeleton of tumor vascular endothelial cells. This study determined the safety, pharmacokinetics, and acute anti-vascular effects of MN-029. Patients were treated with escalating doses of MN-029 (4.0-225mg/m(2)) administered IV at 3-week intervals. This first-in-human study followed an accelerated titration design, with intra-patient dose escalation. Plasma samples were assayed to determine PK parameters. DCE-MRI scans were acquired at baseline and at 6-8h post-dose. Thirty-four patients received 151 infusions of MN-029. The most common toxicities of MN-029 included nausea and vomiting (which appeared to be dose related), diarrhea, fatigue, headache, and anorexia. No clinically significant myelotoxicity, stomatitis or alopecia was observed. There was no evidence of cumulative toxicity in patients receiving multiple courses of therapy. The cohort at 180mg/m(2) was expanded to six patients due to a reversible episode of acute coronary ischemia, without sequelae and with preservation of myocardial function. Two dose-limiting toxicities occurred at 225mg/m(2), a transient ischemic attack and grade 3 transaminitis, thus ending dose escalation. Pharmacokinetic data indicated dose-related increases in C (max) and AUC values, although substantial inter-subject variability was observed. No objective responses were noted; however, five patients had stable disease ≥6months. A significant linear correlation was found between reduction in K (trans) and exposure to MN-029. MN-029 was generally well tolerated and showed decrease in tumor vascular parameters. The maximum tolerated dose was 180mg/m(2).

ChemInform Abstract: Paecilospirone, a Unique Spiro[chroman-2,1′(3′H)-isobenzofuran] Derivative Isolated from Tropical Marine Fungus Paecilomyces sp.

A spiroacetal compound named paecilospirone was isolated from a marine fungus Paecilomyces sp. Collected at Yap Island. Paecilospirone was found by the screening assay for detecting inhibitors of microtubule assembly using conidia of Pyricularia oryzae. The structure was assigned based on the spectroscopic data. This is the first report of the isolation and characterization of spiro[chroman-2,1′(3′H)-isobenzofuran] derivative as a natural product.

Substrate degradation by the anaphase promoting complex occurs during mitotic slippage

Microtubule targeting drugs are successful in chemotherapy because they indefinitely activate the spindle assembly checkpoint. The spindle assembly checkpoint monitors proper attachment of all kinetochores to microtubules and tension between the kinetochores of sister chromatids to prevent premature anaphase entry. To this end, the activated spindle assembly checkpoint suppresses the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC). In the continued presence of conditions that activate the spindle assembly checkpoint, cells eventually escape from mitosis by "slippage". It has not been directly tested whether APC activation accompanies slippage. Using cells blocked in mitosis with the microtubule assembly inhibitor nocodazole, we show that mitotic APC substrates are degraded upon mitotic slippage. To confirm that APC is normally activated upon mitotic slippage we have found that knockdown of Cdc20 and Cdh1, two mitotic activators of APC, prevents the degradation of APC substrates during mitotic slippage. Knockdown of Cdc20 and Cdh1 prevents the degradation of APC substrates during mitotic slippage. We provide the first direct demonstration that despite conditions that activate the spindle checkpoint, APC is indeed activated upon mitotic slippage of cells to interphase cells. Activation of the spindle checkpoint by microtubule targeting drugs used in chemotherapy may not indefinitely prevent APC activation.

Mechanisms of rotenone-induced proteasome inhibition

The etiology of Parkinson's disease is unclear but appears to involve mitochondrial dysfunction, proteasome inhibition, and environmental toxins. It has been shown that pesticides, including the complex I inhibitor rotenone, cause proteasome inhibition but the mechanism of rotenone-induced proteasome dysfunction remains largely unknown. In this study, we examined the role of mitochondrial inhibition, oxidative stress, and microtubule dysfunction as potential mediators of rotenone-induced proteasome inhibition. Proteasome activity (26S) was measured in HEK and SK-N-MC cells expressing an EGFP-U degron fusion protein that is selectively degraded by the proteasome. We found that complexes I and III inhibition led to the production of peroxides and decreased proteasome activity. We also found that rotenone increased nitric oxide production and nitric oxide and peroxynitrites led to proteasome inhibition. The effects of rotenone were attenuated by anti-oxidants and nitric oxide synthase inhibition. Since rotenone can also inhibit microtubule assembly, we tested a specific MT inhibitor and found it led to proteasome dysfunction. Rotenone also led to a decrease in 20S proteasome activity and 20S proteasome subunit immunoreactivity without a change in subunit mRNA. Together, these data suggest that rotenone-induced decreases in proteasome activity are due to increased degradation of proteasome components secondary to oxidative damage and possibly microtubule dysfunction.

Abstract 4391: 4-Hydroxy-2-nonenal causes tetraploidy through a macrophage-induced bystander effect triggered by a commensal bacterium

Abstract Tetraploidy can be an intermediate condition for cells undergoing tranformation from diploidy to aneuploidy. We previously showed that Enterococcus faecalis, a human intestinal commensal, generated tetraploidy and aneuploidy in colonic epithelial cells via a macrophage-induced bystander effect. To explore potential mediators for this effect, supernatants from E. faecalis-infected macrophages were analyzed by HPLC coupled to a coulometric detection system. Significantly increased concentrations of 4-hydroxy-2-nonenal (4-HNE), a breakdown product of polyunsaturated fatty acids, were detected in macrophage supernatants. This system was used to purify macrophage-generated 4-HNE for further experiments. To investigate aneugenic effects of 4-HNE, a near-diploid human colon cancer cell line (HCT116) and non-transformed diploid mouse colonic epithelial cell line (YAMC) were exposed to 4-HNE and ploidy analyzed by FACS. For HCT116 cells the percent tetraploidy significantly increased following exposure to 4-HNE at 2.5 µM compared to untreated controls (9.90% ± 0.09 vs. 0.21% ± 0.08, P &amp;lt; 0.001). In comparison, the percent tetraploidy was increased to 0.68% ± 0.10 for YAMC cells exposed to 1 µM 4-HNE compared to 0.12% ± 0.06 for untreated controls (P &amp;lt; 0.001). This showed that 4-HNE generated tetraploidy in both transformed and non-transformed cells. To address potential mechanisms by which 4-HNE produced tetraploidy, HCT116 cells exposed to 4-HNE were stained with anti-α/β tubulin antibody and microtubules analyzed by confocal microscopy. We found cells exposed to 4-HNE had degraded microtubules and abnormal mitotic patterns, implicating the induction of tetraploidy through inhibition of microtubule assembly. Moreover, for HCT116 and YAMC cells, 4-HNE caused a G2/M cell cycle arrest with increased γH2AX foci suggesting DNA double-strand breaks. Next, we silenced mGSTα4-4, a key enzyme in the detoxification of 4-HNE, by constitutively expressing RNAi in cells. FACS analysis showed that γH2AX positivity remarkably increased in YAMC cells with silenced mGSTα4-4 that were exposed to E. faecalis-infected macrophages compared to non-infected macrophages. No changes were seen in wildtype YAMC cells. Similarly, γH2AX foci increased in mGSTα4-4-silenced YAMC cells exposed to 4-HNE compared to the wildtype control (41.8% ± 1.3 vs. 24.6% ± 6.5, P = 0.01). Finally, percent tetraploidy also significantly increased for mGSTα4-4-silenced YAMC cells exposed to 4-HNE, supporting the hypothesis that 4-HNE can specifically generate tetraploidy. In sum, E. faecalis-infected macrophages produce 4-HNE that, in turn, induces tetraploidy, DNA double-strand breaks and G2/M arrest in colonic epithelial cells. Inactivation of mGSTα4-4, a detoxifying enzyme for 4-HNE, exacerbated these effects. We propose 4-HNE as a mediator for the E. faecalis-infected macrophage bystander effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4391.

Abstract 4425: Oral efficacy and preclinical ADME evaluation of CKD-516, a novel tubulin polymerization inhibitor

Abstract CKD-516 is a potent anticancer agent that inhibits microtubule assembly and disrupts tumor vasculature (VDA). CKD-516 is an amino acid prodrug of S516 releasing S516 in vivo. Previously we reported the discovery and its antitumor efficacy of CKD-516 as well as various in vitro studies (Abstract 5561, 2009 AACR). As a continuation of the investigation, a detailed ADME evaluation of CKD-516 in both rats and dogs and its potential as an oral agent is described in this report. Following intravenous administration to rats and dogs, CKD-516 rapidly became the active metabolite S516 in plasma and the half-lives of S516 were 1.5 hrs in rats, and 8 hrs in dogs. Protein binding was high with an unbound fraction less than or equal to 20% in all species tested. In distribution studies in rats dosed intravenously, S516 was mainly distributed in thymus, lung and stomach and had a short half-life less than 2 hrs in all tissues tested. In rats, the excretion of CKD-516 in urine, bile and feces amounted to 0.22%, 8.22% and 4.92%, respectively. S516 was found to have comparable metabolic stabilities both in dog and human liver microsomes where there was no difference between male and female microsomes. However, the rate of metabolism in rats from male and female liver microsomes were markedly different. In general, S516 is expected to have low drug-drug interaction with IC50 values of &amp;gt;10 μM against most CYP450 enzymes but had a moderate inhibition against CYP450 1A2 and 2C8. The intestinal absorption of CKD-516 was predicted to be moderate to high as the apparent permeabilities (×106 cm/sec) of CKD-516 and its active metabolite S516 in Caco-2 permeability assay were 15.1 and 35.4, respectively. Thus, CKD-516 was found to have an acceptable oral bioavailability in rats, supporting its potential efficacy as an oral agent. In vivo antitumor experiments were performed in the HCT116 human tumor xenograft model. When the established tumor reached the size of 150 mm3, CKD-516 was administered to mice orally. Groups of animals were received CKD-516 with doses of 10 mg/kg or 20 mg/kg (Q4d × 5). The treated group showed a tumor growth suppression (IR = 50∼60%). Drug related toxicity and body weight decrease were not detected at doses tested. In summary, a complete ADME characterization of CKD-516, a novel tubulin polymerization inhibitor and VDA, both in vitro and in vivo was described. Moreover, CKD-516 was found to have a good potential as an oral agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4425.

Regulation of Microtubule Dynamics by Bim1 and Bik1, the Budding Yeast Members of the EB1 and CLIP-170 Families of Plus-End Tracking Proteins

Microtubule dynamics are regulated by plus-end tracking proteins (+TIPs), which bind microtubule ends and influence their polymerization properties. In addition to binding microtubules, most +TIPs physically associate with other +TIPs, creating a complex web of interactions. To fully understand how +TIPs regulate microtubule dynamics, it is essential to know the intrinsic biochemical activities of each +TIP and how +TIP interactions affect these activities. Here, we describe the activities of Bim1 and Bik1, two +TIP proteins from budding yeast and members of the EB1 and CLIP-170 families, respectively. We find that purified Bim1 and Bik1 form homodimers that interact with each other to form a tetramer. Bim1 binds along the microtubule lattice but with highest affinity for the microtubule end; however, Bik1 requires Bim1 for localization to the microtubule lattice and end. In vitro microtubule polymerization assays show that Bim1 promotes microtubule assembly, primarily by decreasing the frequency of catastrophes. In contrast, Bik1 inhibits microtubule assembly by slowing growth and, consequently, promoting catastrophes. Interestingly, the Bim1-Bik1 complex affects microtubule dynamics in much the same way as Bim1 alone. These studies reveal new activities for EB1 and CLIP-170 family members and demonstrate how interactions between two +TIP proteins influence their activities.

Stathmin expression in breast cancer patients treated with anti‐tubulin drugs

ObjectiveStathmin is a protein that inhibits microtubule assembly and is over expressed in highly aggressive and tubulin‐drug resistant breast cancers. This study examines stathmin expression in breast cancers from patients treated with anti‐tubulin chemotherapy.MethodsWe reviewed breast cancers from 44 women enrolled in a randomized trial of neoadjuvant doxorubicin and cyclophosphamide +/− docetaxel, including 39 with invasive ductal carcinoma and 5 with invasive lobular carcinoma. Microarray blocks were constructed from pre‐ and post‐treatment tissues and assayed for stathmin by IHC. Analyses of the stained slides were performed using Aperio Scanscope CS/GL® and TMALab® software. Tissues were evaluated for % staining of strong positive cells, with 4% used as a cut‐off value for high vs. low expression.ResultsThe percentage of strong positive staining of stathmin in invasive ductal carcinomas ranged from 0.5% to 62% with mean 14.4%. Invasive lobular carcinomas had significantly less strong positive stathmin staining, from 0.07% to 3.3% (p = 0.00016, Student's t‐test, two‐tailed). Comparison of the recurrences in high vs. low stathmin was 47% to 21%. There was no correlation with survival or response to chemotherapy.ConclusionInvasive lobular carcinomas had lower intensity stathmin expression than ductal carcinomas. Stathmin expression tended to be higher in tumors that recurred.

Nuclear import of RNA polymerase II requires the conserved GPN‐loop GTPase XAB1/GPN1

Over the past 3 decades, many efforts have been made to identify and characterize the factors that regulate the activity of RNA polymerase II (RNAPII), the enzyme that synthesizes all the mRNA and many small nuclear RNA in eukaryotes. Quite surprisingly, very little is known about the cell machinery that regulates the fate of RNAPII before or after transcription. In this study, we report that XAB1/GPN1, a member of a unique class of GTPases containing a conserved GPN‐loop, is involved in the biogenesis of RNAPII by regulating its nuclear import. XAB1/GPN1 is physically associated with RNAPII in the soluble cell fraction and is essential for cell growth. Accumulation of RNAPII in the cytoplasm of the cell was induced upon (i) depletion of XAB1/GPN1 by siRNA silencing, (ii) overexpression of a XAB1‐GFP dominant negative fusion protein, (iii) mutation of the GTP‐binding or GPN motifs of the XAB1/GPN1 homologue in yeast, NPA3, and (iv) treatment of cells with leptomycin B, an inhibitor of XPO1/CRM1‐dependent nuclear export. Yeast strains with mutations in the NPA3 gene showed hypersensitivity to benomyl, a specific inhibitor of microtubule assembly, suggesting a role for microtubules in RNAPII biogenesis. Together, these results indicate that XAB1/GPN1 shuttles between the cytoplasm and the nucleus to mediate nuclear import of RNAPII.

Novel combretastatin A-4 derivative XN0502 induces cell cycle arrest and apoptosis in A549 cells

Combretastatin A-4 (CA-4) is a tubulin-binding compound currently in phase II trial as a tumor vascular-targeting agent. The present study evaluates the anti-tumor activities and establishes the mechanism of the action of 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3-amine(XN0502), a novel synthesized CA-4 analogue, in an effort towards finding the favorable therapeutics of CA-4 derivatives. XN0502 is characterized by its more potent anti-proliferative activities against non-small cell lung cancer A549 cells (IC(50): 1.8 +/- 0.6 microM), than that on the normal human liver HL-7702 cells (IC(50): 9.1 +/- 0.4 microM). Of note, using tubulin polymerization assay, western blot and immuofluorescence analyses, XN0502 was showed to inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further studies indicated that XN0502 induced time- and dose-dependent G2/M arrest, accompanying with the reduction of CDC2/p34 expression and the downregulation of CDK7. The protein level alteration and the nuclear translocation of cyclinB1 were observed, denoting the M phase arrest in XN0502-treated cells. Moreover, XN0502 caused caspase-mediated apoptosis, as indicated by the cleavage of PARP, the reduction of procaspase-3 and procaspase-9, and the down-regulation of XIAP. Taken together, the current study demonstrates that the novel CA-4 analogue XN0502 is a promising anti-cancer agent with potent G2/M arrest- and apoptotic-inducing activities via targeting tubulin deserving further research and development, and helps provide data for exploiting new CA-4 analogues.

HMBA depolymerizes microtubules, activates mitotic checkpoints and induces mitotic block in MCF-7 cells by binding at the colchicine site in tubulin

10-[(3-Hydroxy-4-methoxybenzylidene)]-9(10H)-anthracenone (HMBA), a synthetic compound, has been reported to have a potent antitumor activity. In this study, we found that HMBA depolymerized microtubules in MCF-7 cells and produced aberrant spindles in the MCF-7 cells. It also reduced the distance between the centrosomes and activated the mitotic checkpoint proteins BubR1 and Mad2. Further, HMBA inhibited the progression of MCF-7 cells in mitosis and induced apoptotic cell death involving p53 pathway. In vitro, HMBA bound to purified brain tubulin with a dissociation constant of 4.1 ± 0.9 μM. It inhibited microtubule assembly and increased the GTP hydrolysis rate of microtubule assembly. The compound did not alter the binding of 2′(or 3′)- O-(trinitrophenyl) guanosine 5′-triphosphate (TNP–GTP), a fluorescent analogue of GTP, to tubulin suggesting that it did not inhibit the binding of GTP to tubulin. However, we obtained evidence indicating that HMBA perturbed the conformation of the GTP binding site in tubulin. In addition, an analysis of the modified Dixon plot suggested that HMBA competitively inhibited the binding of colchicine to tubulin. A computational analysis of the binding of HMBA to tubulin supported the finding that HMBA shared its binding site with colchicine in tubulin and indicated that the binding of HMBA to tubulin was primarily stabilized through hydrogen bonding.