Inhibits Microtubule Depolymerization Research Articles

Redox-responsive hyaluronic acid-based nanoparticles for targeted photodynamic therapy/chemotherapy against breast cancer

Specific cellular uptake and sufficient drug release in tumor tissues are important for effective cancer therapy. Hyaluronic acid (HA), a skeleton material, could specifically bind to cluster determinant 44 (CD44) receptors highly expressed on the surface of tumor cells to realize active targeting. Cystamine (cys) is sensitive highly reductive environment inside tumor cells and was used as a connecting arm to connect docosahexaenoic acid (DHA) and chlorin e6 (Ce6) to the HA skeleton to obtain redox-sensitive polymer HA-cys-DHA/Ce6 (CHD). Nanoparticles were fabricated and loaded with chemotherapeutic drug docetaxel (DTX) by physical encapsulation. The prepared nanoparticles had significantly increased uptake by MCF-7 cells that overexpressed CD44 receptors, and DTX was effectively released at high reducing condition. Compared with mono-photodynamic therapy (PDT) or mono-chemotherapy, the prepared nanoparticles exhibited superior anti-tumor effect by inhibiting microtubule depolymerization, blocking cell cycle and generating reactive oxygen species (ROS). In vivo anti-tumor experiments proved that DTX/CHD nanoparticles had the best antitumor response versus DTX and CHD nanoparticles under near-infrared (NIR) irradiation. These studies revealed that redox-responsive DTX-loaded CHD nanoparticles held great potential for the treatment of breast cancer.

Knockout of vasohibin-2 reduces tubulin carboxypeptidase activity and increases paclitaxel sensitivity in ovarian cancer.

Vasohibin‐1 (VASH1) is a VEGF‐inducible endothelium‐derived angiogenesis inhibitor, and vasohibin‐2 (VASH2), its homolog, exhibits proangiogenic activity. VASH2 is expressed by various cancer cells and accelerates tumor angiogenesis and progression. VASH2 was recently shown to exhibit tubulin carboxypeptidase (TCP) activity related to microtubule functions. Paclitaxel (PTX), an effective chemotherapeutic agent that is widely used to treat ovarian cancer, inhibits microtubule depolymerization and may interact with VASH2. We herein established several VASH2 knockout ovarian cancer cell lines using the CRISPR/Cas9 genome editing system to examine the intracellular tubulin detyrosination status and PTX chemosensitivity. The knockout of VASH2 did not affect the proliferation or sphere‐forming activity of ovarian cancer cells in vitro. A Western blot analysis of VASH2 knockout cells revealed the weak expression of detyrosinated tubulin and upregulated expression of cyclin B1. The knockout of VASH2 significantly increased chemosensitivity to PTX, but not to cisplatin in ovarian cancer cell lines. The knockout of VASH2 reduced TCP activity and increased cyclin B1 expression, resulting in increased PTX chemosensitivity in ovarian cancer cells. The inhibition of angiogenesis and regulation of microtubule activity may be achieved in ovarian cancer treatment strategies targeting VASH2.

MicroRNA Let-7c Contributes to Paclitaxel Resistance via Aurora-B in Endometrial Serous Carcinoma.

The incidence of endometrial cancer has rapidly risen over recent years. Paclitaxel, a key drug for endometrial cancer treatment, inhibits microtubule depolymerization and induces apoptosis in cancer cells. Endometrial serous carcinoma (ESC) accounts for < 10% of all endometrial carcinomas, but its aggressive nature makes it responsible for close to 40% of cancer deaths. Thus, novel therapeutic targets are required for ESC. To identify microRNAs that promote paclitaxel resistance, we established two paclitaxel-resistant cell lines from USPC1 human ESC cells by exposing paclitaxel to parental cells for 12 weeks. Paclitaxel concentrations were increased every 2 weeks, and after 12 weeks of paclitaxel exposure, two replicate paclitaxel-resistant cell lines were established (USPC1-PTSR1 and USPC1-PTXR2). The microarray analysis was performed using USPC1 cells and USPC1-PTXR1 cells, and eight candidate microRNAs were thus selected as potential mediators of paclitaxel sensitivity. Among these candidate microRNAs, let-7c precursor treatment of paclitaxel-resistant USPC1-PTXR1 cells caused the greatest increase in paclitaxel-mediated cytotoxicity. Let-7c inhibition conversely decreased paclitaxel-induced apoptosis. It is known that let-7a microRNA, a member of the let-7 family, inhibits growth of endometrial carcinoma cells targeting Aurora-B that controls progression through each phase of mitosis. We thus studied whether let-7c mediates Aurora-B expression in ESC cells. The expression levels of Aurora-B mRNA and protein were higher in USPC-PTXR1 cells compared with USPC1 cells. Let-7c inhibition increased Aurora-B expression in USPC1 cells but decreased Aurora-B expression in USPC1-PTXR1 cells. These results indicate that let-7c mediates paclitaxel resistance via inhibition of Aurora-B expression in ESC cells.

Microtubule-binding protein FOR20 promotes microtubule depolymerization and cell migration

Microtubules are highly dynamic filaments assembled from αβ-tubulin heterodimers and play important roles in many cellular processes, including cell division and migration. Microtubule dynamics is tightly regulated by microtubule-associated proteins (MAPs) that function by binding to microtubules or free tubulin dimers. Here, we report that FOR20 (FOP-related protein of 20 kDa), a conserved protein critical for ciliogenesis and cell cycle progression, is a previously uncharacterized MAP that facilitates microtubule depolymerization and promotes cell migration. FOR20 not only directly binds to microtubules but also regulates microtubule dynamics in vitro by decreasing the microtubule growth rate and increasing the depolymerization rate and catastrophe frequency. In the in vitro microtubule dynamics assays, FOR20 appears to preferentially interact with free tubulin dimers over microtubules. Depletion of FOR20 inhibits microtubule depolymerization and promotes microtubule regrowth after the nocodazole treatment in HeLa cells. In addition, FOR20 knockdown significantly inhibits both individual and collective migration of mammalian cells. Taken together, these data suggest that FOR20 functions as a MAP to promote microtubule depolymerization and cell migration.

The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans.

Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase.

Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage

T cells rapidly reposition their centrosome to the center of the immunological synapse (IS) to drive polarized secretion in the direction of the bound target cell. Using an optical trap for spatial and temporal control over target presentation, we show that centrosome repositioning in Jurkat T cells exhibited kinetically distinct polarization and docking phases and required calcium flux and signaling through both the T cell receptor and integrin to be robust. In "frustrated" conjugates where the centrosome is stuck behind the nucleus, the center of the IS invaginated dramatically to approach the centrosome. Consistently, imaging of microtubules during normal repositioning revealed a microtubule end-on capture-shrinkage mechanism operating at the center of the IS. In agreement with this mechanism, centrosome repositioning was impaired by inhibiting microtubule depolymerization or dynein. We conclude that dynein drives centrosome repositioning in T cells via microtubule end-on capture-shrinkage operating at the center of the IS and not cortical sliding at the IS periphery, as previously thought.

Abstract 4061: A novel CXCR4-mediated mechanism of docetaxel resistance in prostate cancer cells.

Abstract Docetaxel (DTX), a semi-synthetic analog of paclitaxel, has emerged as the standard of care for chemotherapy of hormone-resistant prostate cancer. DTX confers its anti-neoplastic activity by inhibiting microtubule depolymerization, which leads to G2/M cell cycle arrest and subsequent apoptosis. However, most patients treated with DTX ultimately develop resistance to the drug and succumb to the disease. Therefore, understanding the mechanisms underlying DTX resistance is a priority area in prostate cancer research. Increasingly, it is being suggested that cancer cells may use common pathways for disease aggressiveness/metastatic spread and chemotherapeutic resistance. Therefore, we investigated the role of CXCL12/CXCR4 signaling, which is known to promote invasion and metastasis, in DTX-resistance of prostate cancer cells. Our data demonstrated that CXCL12 treatment rescued the prostate cancer cells from DTX-induced cytotoxicity and G2/M mitotic arrest. Furthermore, the cytoprotective effect of CXCL12 was abolished upon pretreatment of prostate cancer cells with AMD3100 (a small molecule antagonist of CXCR4) or siRNA-mediated silencing of CXCR4, thus confirming the role of CXCR4 in DTX-resistance. Immunofluorescence analyses revealed enhanced polymerization of microtubules in DTX-treated prostate cancer cells. In accordance with this, immunoblot analyses demonstrated increased levels of detyrosinated (glu-) and acetylated (ace-) tubulins, specific markers of the polymerized tubulin, in prostate cancer cells treated with DTX. Co-treatment of CXCL12 abrogated DTX-induced stabilization of microtubules in the prostate cancer cells, an effect that was diminished when the cells were pre-treated with AMD3100. Altogether, these initial findings demonstrate the role of CXCL12/CXCR4 signaling axis in DTX-resistance of prostate cancer cells and thus could be an attractive target for therapeutic enhancement of DTX. Citation Format: Arun Bhardwaj, Sanjeev K. Srivastava, Sumit Arora, Stephen J. Hyde, Joel F. Andrews, Steven McClellan, Seema Singh, Ajay P. Singh. A novel CXCR4-mediated mechanism of docetaxel resistance in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4061. doi:10.1158/1538-7445.AM2013-4061

Relationship between the Structures of Taxane Derivatives and Their Microtubule Polymerization Activity

Paclitaxel (Taxol), one of the most potent anticancer drugs, is a microtubule-stabilizing compound that inhibits microtubule depolymerization within the cell. The structure of paclitaxel is composed of two key elements, a taxane ring and an N-benzoylphenylisoserine side chain at C-13. A number of natural and artificial compounds with taxane skeletons have been isolated, but almost none of their bioactivities have been evaluated. In this study, we focused on compounds having a taxane skeleton structure and examined their effects on tubulin dynamics. Although none of these compounds had an N-benzoylphenylisoserine side chain, three were found to promote tubulin assembly. On the other hand, one compound inhibited tubluin assembly in a way similar to nocodazole. These compounds exhibited novel structure-activity relationships of taxane compounds.

Cabazitaxel: a new drug for metastatic prostate cancer

Cabazitaxel: a new drug for metastatic prostate cancer

Identification of the Human UDP-Glucuronosyltransferases Involved in the Glucuronidation of Combretastatin A-4

The stilbenic compound (Z)-combretastatin A-4 (CA-4) has been described as a potent tubulin polymerization inhibitor. In vivo, CA-4 binds to tubulin and inhibits microtubule depolymerization, which results in morphological changes in proliferating endothelial cells. Combretastatin A-4 prodrug phosphate is a leading vascular disrupting agent and is currently being evaluated in multiple clinical trials as a treatment for solid tumors. The aim of this study was to identify and characterize the UDP-glucuronosyltransferase (UGT) isoforms involved in CA-4 glucuronidation by incubation with human liver microsomes and a panel of nine liver-expressed recombinant UGT Supersomes (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15, and 2B17). As we observed, the high rate of formation of CA-4 glucuronide (V(max) = 12.78 +/- 0.29 nmol/min/mg protein) and the low K(m) (6.98 +/- 0.65 microM) denoted that UGT1A9 was primarily responsible for the in vitro glucuronidation of CA-4. UGT1A6 was also a significant contributor to CA-4 glucuronidation (V(max) = 3.95 +/- 0.13 nmol/min/mg protein and S(50) = 44.80 +/- 3.54 microM). Furthermore, we demonstrated that the kinetics of CA-4 glucuronidation with liver microsomes but also with a panel of recombinant UGTs is atypical as it fits two different models: the substrate inhibition and also the sigmoidal kinetic model. Finally, experiments conducted to inhibit the glucuronosyltransferase activity in the human liver microsomes assay showed that phenylbutazone, trifluoperazine, propofol, and 1-naphthol effectively inhibited CA-4 glucuronidation.

A Bayesian population PK–PD model for ispinesib/docetaxel combination-induced myelosuppression

Ispinesib, a kinesin spindle protein inhibitor, blocks assembly of a functional mitotic spindle, leading to G2/M arrest. Docetaxel promotes tubulin assembly into microtubules while inhibiting microtubule de-polymerization leading to mitotic arrest. Prolonged (> or =5 days) Gr 4 neutropenia and/or febrile neutropenia were the observed dose-limiting toxicities with both agents. Both agents are substrates and inhibitors of CYP3A4; thus, the potential for a drug-drug interaction exists. The goal was to fit a Bayesian population PK/PD model to characterize the relationship between the ispinesib/docetaxel combination and absolute neutrophil counts (ANC). Escalating doses of docetaxel (60-75 mg/m(2)) were administered over 1 h followed by a 1-h infusion of escalating doses of ispinesib (8-12 mg/m(2)) on a 21-day schedule. At least 3 pts were treated at each dose level. Limited PK samples were obtained. ANC were measured weekly on days 1, 8, 15, and 22. More ANC samples were taken from some subjects. The PK properties of ispinesib and docetaxel, and the relationship of PK with ANC were investigated using nonlinear mixed-effects models and Bayesian methods. With a limited dataset, informative prior distributions for the model parameters were needed. These prior distributions were formed using information from a previous study for ispinesib, and from the literature for docetaxel. Twenty-four pts were treated in this study. The PK of ispinesib and docetaxel were well characterized by a two-compartment model and a three-compartment model, respectively. There is no obvious PK interaction between ispinesib and docetaxel. The model for ANC consisted of a proliferating compartment, three transit compartments that represented maturation, and a compartment of circulating blood cells. This ANC model has been used previously for ispinesib given as monotherapy, and for other chemotherapeutic drugs in the literature. Using Bayesian methods, the model was successfully fit for the PK of both compounds and the PD simultaneously. The PK/PD model developed for ispinesib/docetaxel, may be used to examine different schedules, doses, and infusion times of both agents. Bayesian methods allow for the use of prior information available for the model parameters.

Identification of TACC3 (Transforming Acidic Coiled-Coil 3) as a novel target of paclitaxel-mediated tumor therapy in cervical cancer cells

Objective : Paclitaxel is currently used in the treatment of ovarian, breast, gastric, colorectal, lung and recurrent cervical cancer. Initial studies on the mechanism of action of paclitaxel have demonstrated that this drug alters microtubule assembly, by inhibiting microtubule depolymerization and changing microtubule dynamics. Although treatment of various tumor cells with paclitaxel induces apoptosis, but early paclitaxel-targeted proteins is not yet known. We tried to search paclitaxel-targeted proteins and to investigate its functions. Methods : The effects of paclitaxel on HeLa cervical cancer cell growth were evaluated by cell proliferation assay, DAPI stain, and FACS analysis. We performed proteome analysis including 2-DE and MALDI-TOF-MS in nontreated- and paclitaxel-treated HeLa cells, as a result, we identified TACC3 protein that is down-regulated with paclitaxel treatment. We tried to characterize TACC3 functions through in vitro treatment of paclitaxel or RNAi technique. Results : Paclitaxel- and TACC3 siRNA-treated cells are unable to proceed normally through the cell cycle and are arrested in G2/M phase and reveal apoptotic morphology. TACC3 levels after paclitaxel treatment decreased as a time- and dosedependent manner both mRNA and protein levels. We confirmed that the role of TACC3 down-regulation for microtubule stabilization was similar to that of paclitaxel. Also, TACC3 is expressed at high levels in various cancer cells and tumor tissues. Conclusion : This study is proposed that the TACC3 protein may be participated in microtubule formation as an oncoprotein during mitosis and be regulated by paclitaxel as a novel target.

Curative Resection of Inoperable, Locally Advanced Gastric Cancer after Neoadjuvant Chemotherapy with Taxotere and Cisplatin

At diagnosis, the majority of patients with gastric cancer are found to have local invasion or distant organ metastasis, even though the sole measure for a complete cure is a curative resection. A curative resection is hardly applicable for those with invasion and metastasis; thus, trials with neoadjuvant chemotherapy for downstaging the cancer should be considered. Docetaxel is a semisynthetic taxane that promotes tubulin polymerization and inhibits microtubule depolymerization. In recent studies, many metastatic gastric cancers were treated using neoadjuvant chemotherapy with docetaxel, and the response rates were reported. We report here two cases of locally advanced, non-resectable gastric cancer that were candidates for a curative resection after induction chemotherapy with docetaxel and cisplatin.

‘Rings’ of F-actin form around the nucleus in cultured human MCF7 adenocarcinoma cells upon exposure to both taxol and taxotere

The anti-cancer taxoids, Taxol® (paclitaxel) and Taxotere® (docetaxel), are the most promising anti-mitotic agents developed for cancer treatment in the past decade. The effectiveness of this new class of compounds lies in their unique mechanism of action on the cytoskeleton. Both taxol and taxotere bind to microtubules and shift the normal equilibrium between monomeric and polymerized tubulin to favor the polymerized form by strongly promoting tubulin assembly and inhibiting microtubule depolymerization. Although very similar in structure, these two compounds have recently demonstrated different in vitro, in vivo, and clinical activities; however, no study to date has effectively compared specific cytoskeletal alterations induced by taxol and taxotere in cultured cells. Using specific staining techniques for both F-actin and α-tubulin, this study provides the first detailed immunohistochemical comparison of the effects of equimolar concentrations of taxol and taxotere on both the microfilament and microtubule networks in a cultured cell line. Using human MCF7 breast adenocarcinoma cells, new observations of taxotere/taxol alterations of the cytoskeleton include: an increased abundance of parallel microtubule ‘bundles’ in taxotere treated cells and a definitive reorganization of the microfilament network which results in novel ring-like formations of F-actin condensed exclusively in the perinuclear zone. Reorganization of the actin cytoskeleton induced by a taxoid disruption of the microtubule equilibrium is indicative of the interdependence between microtubules and microfilaments in this transformed cell line and suggests that the indirect role of the taxoids on the microfilament network may have been overlooked in their mechanism of action as chemotherapeutic agents.

Docetaxel: an Active New Drug for Treatment of Advanced Epithelial Ovarian Cancer

Because of the relative scarcity of natural paclitaxel (Taxol), which has been recently recognized as a highly cytotoxic agent for use in platinum-refractory ovarian cancer, synthetic and semisynthetic substitutes have been actively pursued. Docetaxel (Taxotere), a new semisynthetic taxoid, has been selected for clinical development because it is twice as potent as paclitaxel in promoting assembly of tubulin and in inhibiting microtubule depolymerization. Docetaxel also shows equal or greater cytotoxicity in relevant preclinical models. Because docetaxel has produced consistent antitumor responses in ovarian cancer patients in phase I trials, we planned and conducted a phase II clinical trial to evaluate the drug's effectiveness and its toxic effects. The present trial, which started in May 1992 and ended in December 1992, involved 97 patients with advanced epithelial ovarian cancer. The target study population had disease relapse or disease progression within 12 months of the last administration of a first-line or second-line platinum-based regimen with at least one bidimensionally measurable target lesion. The patients received docetaxel at a dose of 100 mg/m2 given as a 1-hour infusion every 3 weeks without premedication for minimizing potential hypersensitivity. Docetaxel-induced side effects were graded according to the National Cancer Institute's Common Toxicity Criteria. The overall response rate was 23.6% in 76 assessable patients versus 20% if all 90 eligible patients were included in the comparison (95% confidence interval [CI] = 11%-29%). Among 34 eligible patients whose tumor progressed on the most recent platinum treatment, the response rate was 23.5% (95% CI = 8%-39%). The median progression-free survival for all eligible patients was 3.9 months, and the median overall survival was 8.4 months. Docetaxel-associated toxicity in 90 assessable patients consisted of short-lived neutropenia in 81 (90%) patients, which was complicated by fever and hospitalization in seven (8%); hypersensitivity reactions were seen in 29 (31%) patients, with significant reactions seen in seven (8%); and neurotoxicity in 43 (48%) patients, with grade 3 or above toxicity seen in only three (3%). The treatment also produced skin reactions in 58 (64%) patients, of whom only four (4%) showed the intensity of grade 3. Eleven (12%) patients experienced pleural effusions, which were the effects of the drug considered to be of greatest concern. Peripheral edema and weight gain due to fluid retention were reported in 40 (44%) and 17 (19%) patients, respectively. Docetaxel appears to be effective in the treatment of platinum-refractory ovarian cancer patients.

Tubulin-colchicine complex (TC) inhibits microtubule depolymerization by a capping reaction exerted preferentially at the minus end.

The effects of colchicine and tubulin-colchicine complex (TC) on microtubule depolymerization were studied using the axoneme-subunit system described previously [Bergen LG, Borisy GG; J Cell Biol 84:141-150, 1980]. This system allows the independent analysis of the polymerization kinetics at both the plus and minus ends of a microtubule. Depolymerization was induced by isothermal dilution with 10 volumes of an experimental solution containing colchicine, TC, or buffer alone. Colchicine alone (5-100 microM) blocked depolymerization at the minus end, whereas depolymerization at the plus end occurred at almost control rates. A similar effect was produced by TC (0.4:1-1:1 molar ratio to free tubulin). High molar ratios of TC to tubulin (10:1) blocked depolymerization at both plus and minus ends, and intermediate molar ratios of TC:T allowed depolymerization of the plus ends but at attenuated rates. The blockage was not readily reversible; TC-affected ends neither shortened upon dilution nor grew longer upon incubation with additional tubulin. We conclude that TC at suprastoichiometric ratios to tubulin inhibits microtubule depolymerization by a capping reaction and that this effect is exerted preferentially at the minus end.