Polymerized Tubulin Levels Research Articles

Design, synthesis and anticancer activity of N-aryl indolylsulfoximines: Identification of potent and selective anticancer agents

A facile and efficient approach utilizing copper-mediated cross-coupling reaction of N-boc-3-indolylsulfoximines with aryl iodides was developed to synthesize a diverse range of N-arylated indolylsulfoximines 11a-m in excellent yields (up to 91%). The key precursors, free NH sulfoximines 9 were readily prepared by the treatment of N-boc-3-methylthioindoles 8 with a combination of IBD and ammonium carbamate. Under similar conditions NH-free indolylsulfoximine 9a was successfully prepared in gram-scale quantities. The reaction is highly chemoselective and tolerant of a wide range of functional groups. The process is environmentally friendly and is amenable to scale-up. Among the prepared N-arylated indolylsulfoximines 11a-m, compounds 11i-j (2.68–2.76 μM), 11f-g (1.9–3.7 μM) and 11k (1.28 μM) showed potent and selective cytotoxicity against 22Rv1, C4-2 and MCF7 cells, respectively. Indolylsulfoximine derivative 11l displayed a broad spectrum of activity (1.7–8.2 μM) against the tested cancer cell lines. These compounds were found to be non-cytotoxic to normal HEK293 cells, indicating their potential selectivity for cancer cells. We analysed the impact of 11l on various cellular assays to uncover its mechanism of action. Cellular assay shows that 11l increases the endogenous level of ROS, leading to the increased level of p-53 and c-jun inducing apoptosis. 11l also induced mitochondrial dysfunction, further promoting apoptotic pathways. Besides, 11l also restricts cell invasiveness, indicating that it could serve as an effective anti-metastatic agent. As oxidative stress severe F actin causing tubulin depolymerization, we examined the impact of 11l on tubulin dynamics. Accordingly, 11l treatment decreased the levels of polymerized tubulin in 22Rv1 and C4-2 cells. Although future studies are needed to determine their exact molecular target(s), our data shows that N-aryl indolylsulfoximines could serve as effective anti-cancer agents.

Synthetic cyclopenta[b]indoles exhibit antineoplastic activity by targeting microtubule dynamics in acute myeloid leukemia cells

Acute promyelocytic leukemia (APL) is associated with PML-RARα oncogene, which is treated using all-trans retinoic acid (ATRA)-based chemotherapy. However, chemoresistance is observed in 20–30% of treated patients and represents a clinical challenge, raising the importance of the development of new therapeutic options. In the present study, the effects of three synthetic cyclopenta[b]indoles on the leukemia phenotype were investigated using NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Among the tested synthetic cyclopenta[b]indoles, compound 2, which contains a heterocyclic nucleus, was the most active, presenting time-dependent cytotoxic activity in the μM range in APL cells, without cytotoxicity for normal leukocytes, and was selected for further characterization. Compound 2 significantly decreased clonogenicity, increased apoptosis, and caused cell cycle arrest at S and G2/M phases in a drug concentration-dependent manner. Morphological analyses indicated aberrant mitosis and diffuse tubulin staining upon compound 2 exposure, which corroborates cell cycle findings. In the molecular scenario, compound 2 reduced STMN1 expression and activity, and induced PARP1 cleavage and H2AX and CHK2 phosphorylation, and modulated CDKN1A, PMAIP1, GADD45A, and XRCC3 expressions, indicating reduction of cell proliferation, apoptosis, and DNA damage. Moreover, in the in vivo tubulin polymerization assay, NB4 and NB4-R2 cells showed a reduction in the levels of polymerized tubulin upon compound 2 exposure, which indicates tubulin as a target of the drug. Molecular docking supports this hypothesis. Taken together, these data indicated that compound 2 exhibits antileukemic effects through disrupting the microtubule dynamics, identifying a possible novel potential antineoplastic agent for the treatment of ATRA-resistant APL.

Abstract 317: Microtubule polymerizing effects of ellagic acid reduce efficacy of taxane chemotherapy

Abstract Ellagic acid (EA) is a polyphenolic compound found in berries, pomegranates and other foods, which has cancer preventive and therapeutic properties. EA inhibits proliferation, induces apoptosis, blocks cell cycle progression and inhibits ABC drug transporters. Based on these anti-cancer activities of EA, we hypothesized that EA could enhance the efficacy of taxane chemotherapy on prostate cancer cells and inhibit drug resistance. We used castration-resistant 22Rv1, VCaP, and C4-2 prostate cancer cells and androgen-independent PC-3 cells to study effects of EA on efficacy of the taxane drug cabazitaxel (CBZ). Cells were treated with EA (0.1 μM - 30 μM) and after 72 hr proliferation was measured using the SRB assay. EA inhibited proliferation at levels greater than 10 μM. At 30 μM the greatest inhibition was observed in the most proliferative cell lines (22Rv1: 66.7%, PC-3: 82.7%), with a more modest effect on the slower growing cells (VCaP: 25.2%, C4-2: 31.7%). We next tested if growth inhibitory concentrations of EA could enhance the microtubule stabilizing effects of CBZ. In a cell-free assay, CBZ (1 nM - 1 μM) dose dependently increased tubulin polymerization, and EA (10 μM) increased polymerization by 61.0%. However, we observed an antagonistic response with the combination of CBZ and EA, reducing polymerized tubulin by 13.9% compared to CBZ alone. To confirm these results in cell culture, we treated 22Rv1 cells with EA (10 μM) and CBZ (10 nM) and determined levels of polymerized tubulin by western blotting. We observed a 1.6 fold increase in polymerized tubulin by EA, a 5.5 fold increase by CBZ, which was reduced to a 4.6 fold increase by combination of CBZ with EA. Contrary to our hypothesis, these results suggest that EA may interfere with taxanes, leading to a reduction in drug effectiveness. Additionally, the ability of EA alone to induce polymerization suggests the possibility that the growth inhibitory activity of EA is a consequence of its effects on microtubule dynamics, potentially through an interaction with taxane binding site of tubulin. This is consistent with our observation that EA has the greatest growth inhibitory effect on very proliferative cell lines, since microtubule targeting agents are most effective at killing rapidly dividing cells. To our knowledge, this is the first evidence of microtubule targeting as an anticancer mechanism of EA. We also studied the effects of EA on drug efflux to determine if EA could inhibit development of taxane drug resistance. We treated C4-2 cells with EA (0.01 μM - 30 μM) and measured calcein retention as marker of the activity of the P-glycoprotein (P-gp) pump. There was a dose dependent increase in calcein retention by EA, with a 1.9 fold increase at the highest concentration (30 μM), suggesting an inhibition of P-gp pump activity with increasing amounts of EA. Thus, EA may increase intracellular taxane levels, but at the same time reduce their microtubule-polymerization effects. Citation Format: Jillian N. Eskra, Alaina Dodge, Maarten C. Bosland. Microtubule polymerizing effects of ellagic acid reduce efficacy of taxane chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 317.

STK405759 As a Novel Tubulin Active Agent for Multiple Myeloma Therapy

Background: Despite advances in treatment, multiple myeloma (MM) remains incurable due to development of drug resistance in the bone marrow microenvironment. Microtubules (MTs) are dynamic protein biopolymers formed through polymerization of heterodimers of α- and β-tubulins. Disruption of microtubules induces cell cycle arrest in G2-M phase and formation of abnormal mitotic spindles. MTs are also involved in many processes in interphase cells, including intracellular trafficking, cell motility and angiogenesis. The important functions of MT in the cells make them an attractive target for anti-myeloma drug discovery.Furan metotica is a novel class of anti-mitotic spindle drugs that inhibit kinetochore-microtubule binding and trigger a spindle checkpoint mediated arrest in mitosis, which frequently ends in cell death. We evaluated the activity of STK405759, a member of the furan metotica family, as a novel, potential antitubulin drug for MM treatment in preclinical models.Methods: Cytotoxic activity of STK405759 was evaluated by XTT assay. Apoptosis and cell cycle were analyzed by flow cytometry. Tubulin polymerization inhibition was evaluatedusing a biochemical cell free assay and by testingthe levels of soluble and polymerized tubulin in MM-treated cells using Western blot analysis. Efficacy and toxicity of the drug were checked in a murine MM xenograft model. Histochemistry was used to assay tumor apoptosis.Results: STK405759 had a potent cytotoxic activity against a wide variety of MM cell lines and patient-derived MM cells, regardless of their sensitivity to conventional therapy or novel agents. In contrast, the viability of normal peripheral blood mononuclear cells derived from healthy donors and MM patients was not affected. Importantly, STK405759 induced cell death of RPMI MM cells co-cultured with HS-5 bone marrow stromal cells.STK405759 inhibited tubulin polymerizationin a cell free system anddecreased the level of polymerized tubulin in MM treated cells.The STK405759 anti-tubulin activity was supported by demonstration of MM cell cycle arrest followed by activation of an apoptotic default pathway. Activation of pro-caspase-8 and poly (ADP-ribose) polymerase in the cleaved forms, as well as down-regulation of the Mcl-1 anti-apoptotic protein was detected in RPMI treated cells.Combination studies of STK405759 with bortezomib, lenalidomide or dexamethasone showed significant synergistic and additive cytotoxicity in MM cells. In vivo studies revealed decreased MM tumor burden and prolonged survival of STK405759-treated mice compared to controls. STK405759 induced apoptosis of tumors cells from treated mice.Summary/Conclusion: STK405759 is an active, microtubule-targeting agent with potent anti-myeloma activity. These results provide a rationale for further evaluation of STK405759 as monotherapy or part of combination therapy for treating patients with MM. DisclosuresNo relevant conflicts of interest to declare.

Ethyl acetate extract from marine sponge Hyattella cribriformis exhibit potent anticancer activity by promoting tubulin polymerization as evidenced mitotic arrest and induction of apoptosis.

Background:Marine sponges are important sources of bioactive compounds.Objective:This study investigated the anticancer properties of Hyattella cribriformis ethyl acetate (EA) fraction in various cancer and normal cell lines.Materials and Methods:anticancer assay was carried out in 15 cell lines to evaluate the anticancer potential of the EA fraction. Impact on cell cycle distribution was determined using flow cytometry. The fraction was investigated for interfering microtubules assembly in both in vitro and cellular assay. Further studies were conducted to determine the fraction induced cell death (apoptosis) using calcein/propidium iodide dual staining, activated caspase-3 and phosphorylation of Bcl-2 protein at Ser70. DNA fragmentation assay was performed to confirm the apoptosis.Results:EA fraction exhibited potent inhibition of cancer cell growth and resulted in 50% growth inhibition (GI50) of 0.27 μg/mL in A673 cell line. Sarcoma (MG-63, Saos-2) and ovarian (SK-OV-3 and OVCAR-3) cancer cell lines also showed superior anticancer activity GI50 of 1.0 μg/mL. Colon and breast cancer cell lines exhibited moderate GI compare other cancer cell lines and normal human lung fibroblast showed GI50 of 15.6 μg/mL. EA fraction showed potent G2/M phase arrest in A673 cell line and induced apoptosis at 48 h exposure. EA fraction promoted microtubule polymerization in tubulin polymerization assay and increased level of polymerized tubulin in the HeLa cells. Fraction induced the activation of caspase-3 and phosphorylation of Bcl-2 anti-apoptotic protein. Fraction induced DNA fragmentation in HeLa cells as evidence of apoptosis.Conclusion:Marine sponge H. cribriformis EA fraction exhibited potent anticancer activity through tubulin polymerization and induction of apoptosis.

Alpha-synuclein promotes early neurite outgrowth in cultured primary neurons

We previously showed that alpha-synuclein (α-Syn), a protein implicated in the pathogenesis of several neurodegenerative diseases, is a microtubule-associated protein (MAP), facilitating the polymerization of tubulin into microtubules. Therefore, we hypothesized that α-Syn might promote neurite outgrowth, a process that requires microtubule assembly. To test this hypothesis, recombinant human wild type (WT) and mutant (A30P and A53T) α-Syn proteins were added to cultured primary rat cortical neurons, and their effects on early neurite outgrowth were observed. The WT and mutant α-Syn proteins entered the neurons after 1-4 h of incubation. However, a significant increase in neurite outgrowth was observed only in neurons treated with WT α-Syn. MES23.5 dopaminergic neuronal cells overexpressing WT α-Syn also exhibited enhanced neurite outgrowth, indicating that the ability of α-Syn to promote neurite outgrowth was not due to a direct action on the cell membrane or by the membrane translocation process. Co-immunoprecipitation demonstrated that the recombinant human α-Syn was bound to tubulin. In addition, the α-Syn-treated neurons displayed increased levels of polymerized tubulin. Because α-Syn's MAP functionality is mediated by specific domains, we generated N-terminal (a.a. 1-65), non-amyloid-β (non-Aβ) component (NAC) (a.a. 61-95) and C-terminal (a.a. 96-140) fragments and added them to the primary neurons. After 1-4 h of incubation, the various α-Syn fragments had entered the neurons. However, only the NAC and C-terminal fragments, which have been previously shown to mediate MAP functionality, promoted neurite outgrowth. These results suggest that α-Syn promotes neurite outgrowth by facilitating the polymerization of tubulin into microtubules.

Clinical, Pharmacodynamic, and Pharmacokinetic Evaluation of BNC105P: A Phase I Trial of a Novel Vascular Disrupting Agent and Inhibitor of Cancer Cell Proliferation

To determine the recommended phase II dose and evaluate the safety and toxicity profile and pharmacokinetic (PK) and pharmacodynamic (PD) effects of BNC105P, an inhibitor of tubulin polymerization that has vascular disrupting and antiproliferative effects. BNC105P was administered as a 10-minute infusion on days 1 and 8 of a 21-day cycle in a first-in-human phase I study. A dynamic accelerated dose titration method was used for dose escalation. Plasma concentrations of BNC105P (phosphate prodrug) and BNC105 (active agent) were determined. PD assessments were carried out using dynamic contrast enhanced (DCE)-MRI and analysis of a blood-borne biomarker. Twenty-one subjects with advanced solid tumors were enrolled on 6 dose levels (range: 2.1-18.9 mg/m(2)). The recommended dose level was 16 mg/m(2) and was well tolerated. BNC105P (prodrug) rapidly converted to BNC105 with a half-life of 0.13 hours. Plasma concentrations of BNC105 generally increased in proportion to dose with a half-life of 0.57 hours. Pharmacodymanically active plasma levels were obtained with a dose dependant reduction in the levels of polymerized tubulin (on-target action) being observed in PBMCs. DCE-MRI also indicated blood flow changes in the tumor lesions of a number of subjects. BNC105P has a favorable toxicity profile at the recommended dose of 16 mg/m(2) and is associated with PD changes consistent with its known mechanism of action. Phase II studies in renal cancer and mesothelioma have commenced.

Highlights of This Issue

Bortezomib is the first proteasome inhibitor approved for the treatment of cancer. The determinants of resistance to bortezomib, however, remain poorly defined. Here, Weniger and colleagues used gene expression profiling to investigate the cellular response to proteasome inhibition in mantle celllymphoma. They found that induction of a strong NRF2-regulated antioxidant response in tumor cells during bortezomib treatment identified patients with superior clinical response. In contrast, high basal antioxidant capacity was associated with a reduced stress response and inferior clinical outcome. These results indicate that high cellular antioxidant capacity contributes to bortezomib resistance and may represent a therapeutic target.Malignant pleural mesothelioma (MPM) is an aggressive tumor that has high resistance to chemotherapy and radiotherapy. Genetic alteration of TP53 is rare in MPM, even though MPM exhibits antiapoptosis and cell cycle alterations suggestive of functional p53 deficiency. The microRNA-34s (miR-34s) are direct transcriptional targets of p53. Here,Kubo and colleagues found that miR-34b/c expression was frequently silenced by DNA methylation in MPM, resulting in loss of tumorsuppressive p53 function. This study provides new insights into the molecular pathogenesis of MPM and suggests that miR-34b/c could be a potential therapeutic target.Preclinical data suggest human Vα24Vβ11 natural killer T cells (NKT) cells, activated by α-galactosylceramide presented by dendritic cells (DC), have antitumor potential. In a phase I clinical trial,Nicol and colleagues evaluated the effects of administration route (intravenous vs. intradermal) and α-galactosylceramide-pulsed DC dose on NKT cell stimulation. They found that both administration route and DC dose had a major impact on NKT cell responses and on secondary immune effects consequent on NKT cell activation. Inflammatory flares involving tumor deposits were frequently observed. These data provide important insights into the design of immunotherapy studies using DC-based NKT cell stimulation.BNC105P is an inhibitor of tubulin polymerization that has vascular disrupting and anti-proliferative effects. In this issue, Rischin and colleagues report on a first-in-human phase I trial of this agent. The experimental design incorporated a number of pharmacokinetic and pharmacodynamic assessments.Dynamic contrast enhanced MRI was used to assess tumor blood flow following exposure to drug, and a novel immunoblot densitometry assay was developed that assessed the level of polymerized tubulin in PBMCs isolated from trial subjects.BNC105P was associated with pharmacodynamic changes consistent with its mechanism of action and had a favorable toxicity profile at the recommended dose level.

Leaf Extracts of Calocedrus formosana (Florin) Induce G2/M Cell Cycle Arrest and Apoptosis in Human Bladder Cancer Cells

Calocedrus formosana (Florin) bark acetone/ethylacetate extracts are known to exert an antitumor effect on some human cancer cell lines, but the mechanism is yet to be defined. The aim of this study was to determine the effects of Florin leaf methanol extracts on the growth and apoptosis of human bladder cancer cell lines. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that the growth of these bladder cancer cells was potently inhibited by the Florin leaf extracts. The cell cycle of these extract-treated cells (TCCSUP cells) was arrested at the G2/M phase as determined by flow cytometry. Western blot analysis revealed the increases of cyclin B1 and Cdc2 kinase levels, alone with the decrease of phosphorylated Cdc2 kinase, after treating these cells with the extracts. An immunofluorescence assessment of β-tubulin showed decreased levels of polymerized tubulin in treated cells. However, the proteolytic cleavage of poly ADP-ribose polymerase and the activation of caspase-3/-8/-9 were all increased upon treatments of extracts. The concurrent increase of Bax and decrease of Bcl-2 levels indicated that the extracts could induce apoptosis in these treated cells. Taken together, these results suggest that the Florin leaf extracts may be an effective antibladder cancer agent.

A first-in-human, phase I pharmacodynamic (PD) and pharmacokinetic (PK) study of BNC105P, a novel vascular disrupting agent (VDA) and inhibitor of cancer cell proliferation.

3054 Background: BNC105P is a novel agent which inhibits tubulin polymerization and acts as a VDA. BNC105P is a phosphorylated parent compound which rapidly converts to the active agent BNC105. BNC105 exhibits 100-fold specificity for activated endothelial cells (cf. quiescent). Methods: BNC105P was administered IV over 10 min on day 1 and 8 every 21 days to patients (pts) with advanced solid tumors. PD effects were evaluated using DCE-MRI (detection of blood flow and vascular permeability) with two baseline and two post dose assessments (3-6, 24h). In addition, a novel assay for detection of tubulin polymerization in PBMCs was developed as a biomarker of BNC105 on-target action. Furthermore, the effects of BNC105 on the levels of VEGF, PAI-1, and cytokeratin 18 were measured. Response by RECIST was determined at the end of cycle 2. Results: 21 pts (13M; 8F; median 60 yr) were enrolled on six dose levels (2.1, 4.2, 8.4, 12.6, 16, 18.9 mg/m2). Stable disease (SD) was observed in 4 pts, including one pt with mesothelioma (progression at entry) with SD up to week 22 (8.4 mg/m2), one pt at 12.6 mg/m2 and 2 at 16 mg/m2. One pt in the 16 mg/m2 cohort demonstrated significant Ktrans decline at 24h (-42%). At the highest (18.9 mg/m2) dose cohort, DCE-MRI demonstrated significant declines in tumor Ktrans (mean change -39%, range -14 to -60%) at 24h post dosing. Levels of polymerized tubulin detected in PBMCs decreased by >80% from 1h to 7h post dosing (cycle 1), reflecting the on-target activity of BNC105. Changes in blood borne biomarkers were seen at doses >8.4 mg/m2. PK data of BNC105 indicated a generally linear increase in plasma AUC levels and plasma half life of <0.5h. DLTs at 18.9 mg/m2 included one episode of Gr 3 elevated LFT (ALT) and one episode of Gr 4 N-STEMI (observed in an 83 yr old pt exhibiting an AUC >4 times higher than other pts in this cohort). BNC105P was well tolerated without any DLTs at doses below 18.9 mg/m2. Conclusions: Stable disease was observed in 4 pts dosed at 8.4 mg/m2 or higher. Both DCE-MRI and PBMC-polymerized tubulin content confirm that pharmacodynamically active doses have been achieved and correlate well with plasma exposure. The MTD was identified as 16 mg/m2. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bionomics Ltd. Bionomics Ltd. Bionomics Ltd. Bionomics Ltd.

Stathmin, a microtubule-destabilizing protein, is dysregulated in spinal muscular atrophy†

Spinal muscular atrophy (SMA), a motor neuron degeneration disorder, is caused by either mutations or deletions of survival motor neuron 1 (SMN1) gene which result in insufficient SMN protein. Here, we describe a potential link between stathmin and microtubule defects in SMA. Stathmin was identified by screening Smn-knockdown NSC34 cells through proteomics analysis. We found that stathmin was aberrantly upregulated in vitro and in vivo, leading to a decreased level of polymerized tubulin, which was correlated with disease severity. Reduced microtubule densities and beta(III)-tubulin levels in distal axons of affected SMA-like mice and an impaired microtubule network in Smn-deficient cells were observed, suggesting an involvement of stathmin in those microtubule defects. Furthermore, knockdown of stathmin restored the microtubule network defects of Smn-deficient cells, promoted axon outgrowth and reduced the defect in mitochondria transport in SMA-like motor neurons. We conclude that aberrant stathmin levels may play a detrimental role in SMA; this finding suggests a novel approach to treating SMA by enhancing microtubule stability.

Crosslinking of microtubules and actin filaments by S100A8/S100A9

Microtubules (MT) and actin filaments of phagocytes exhibit highly dynamic properties. The underlying molecular mechanisms are not entirely known. Their exceptional dynamic cytoskeleton is the prerequisite of many phagocyte related functions like exocytosis, phagocytosis, and in particular migration. Cell dynamic/migration is a highly complex process strictly regulated by numerous signaling cascades often initiated by changes in cytosolic calcium concentrations. Therefore, calcium-binding proteins are key elements in signal transduction. The major calcium-binding molecules expressed in granulocytes and monocytes are myeloid-related protein 8 (MRP8 [S100A8]) and MRP14 (S100A9), two members of the S100 protein family. MRP8 and MRP14 form heterodimers in the absence and tetrameric complexes composed of two heterodimers in the presence of calcium. We demonstrate that MRP8/MRP14 tetramers promote MT polymerization as well as MT bundling; moreover, the complex crosslinks MTs to F-Actin in a strictly calcium dependent manner. HEK293 cells transfected with MRP8/MRP14 contain significantly more polymerized tubulin in comparison to mock-transfected cells. These results are in line with reduced level of polymerized tubulin in phagocytes isolated from MRP14 -/- mice. Furthermore, phagocytes of MRP14 knockout mice show altered migration rates compared to wildtype cells. In addition to calcium-induced activation the MRP8/MRP14 complex can be specifically phosphorylated by p38 mitogen-activated protein kinase (MAPK), which abrogates MT/F-Actin crosslinking. Thus MRP8/MRP14 integrates signals of at least two independent signaling pathways. Our results provide evidence, that the MRP8/MRP14 complex fulfils a pivotal role in remodeling cytoskeletal structures necessary for migration of leukocytes.

A Role for Altered Microtubule Polymer Levels in Vincristine Resistance of Childhood Acute Lymphoblastic Leukemia Xenografts

The microtubule-depolymerizing drug, vincristine, is effective in the treatment of acute lymphoblastic leukemia (ALL). Although vincristine resistance mechanisms have been extensively characterized in cell lines, their clinical relevance is poorly understood. The aim of the current study was to define clinically relevant mechanisms of vincristine resistance in a panel of childhood ALL xenografts established in immune-deficient (nonobese diabetic/severe combined immunodeficient) mice. We also studied two independent xenograft sublines that were selected by in vivo vincristine exposure. In vitro vincristine sensitivity determined by a stromal coculture, murine bone marrow stromal cell line (MS-5), assay, but not methyl-thiazolyl-tetrazolium metabolic activity assay, significantly correlated (P = 0.05) with the length of the patients' first remission. Investigations into mechanisms of resistance revealed no association with steady-state vincristine accumulation or increased activity and/or expression of ATP-binding cassette transporters, although increased intracellular levels of polymerized tubulin significantly correlated with resistance (r = 0.85; P = 0.0019). Two xenograft sublines selected by in vivo vincristine exposure exhibited a 2-fold increase in polymerized tubulin levels compared with the parental subline (P < 0.05), reflecting their in vivo vincristine resistance. In this study, a vincristine-resistant xenograft with high levels of polymerized tubulin was relatively sensitive to the microtubule-polymerizing drug paclitaxel. These results indicate that the balance between polymerized and nonpolymerized tubulin may be an important determinant of response to Vinca alkaloid-based chemotherapy regimens in childhood ALL.

Neuronal-associated microtubule proteins class III β-tubulin and MAP2c in neuroblastoma: Role in resistance to microtubule-targeted drugs

Abstract Advanced stage neuroblastoma has a poor clinical outcome and microtubule-destabilizing agents, such as the Vinca alkaloids, are an important component in the treatment of this childhood cancer. Vinca alkaloids bind to β-tubulin on the α/β-tubulin heterodimer and disrupt microtubule dynamics, leading to cell death. To date, studies examining the contribution of microtubules and associated proteins to the efficacy of microtubule-destabilizing agents in neuroblastoma have been limited. In this study, BE(2)-C neuroblastoma cells previously selected for resistance to either vincristine (BE/VCR10) or colchicine (BE/CHCb0.2) were found to display significant decreases in neuronal-specific class III β-tubulin. Interestingly, vincristine-selected cells exhibited increased levels of polymerized tubulin that were not due to α-tubulin and class I, II, or III β-tubulin mutations. Expression levels of the microtubule-depolymerizing protein stathmin were significantly increased in BE/VCR10 cells. In contrast, levels of MAP2a and MAP2b were relatively unaltered. A marked decrease in the neuronal protein, MAP2c, was identified in the vincristine-selected cells and, to a lesser extent, in the colchicine-selected cells. This is the first report describing specific microtubule alterations in neuroblastoma cells resistant to tubulin-targeted agents. The results indicate a need to identify the factors responsible for resistance to tubulin-targeted agents in neuroblastoma so that improved and novel treatment strategies can be developed for this drug refractory disease.

Induction of mitotic arrest and apoptosis by evodiamine in human leukemic T-lymphocytes

Leukemias are a heterogenous group of diseases characterized by uncontrolled proliferation of abnormal blood cells of hematopoietic system. Evodiamine, a characteristic alkaloid extracted from Evodia fruits, has been reported to exhibit inhibitory effect on cell proliferation and migration in several types of cancer cells. However, there is no report elucidating the action target and anti-cancer mechanism of this potential natural compound. In this study, we have defined the anti-proliferative and apoptotic mechanisms of evodiamine in human acute leukemia CCRF-CEM cells. According to the MTT assay, the cell viability was inhibited by evodiamine in a concentration-dependent manner with an IC 50 of 0.57 ± 0.05 μM. Flow cytometry analysis showed that the apoptotic cell death proceeded by evodiamine was accompanied with a cell cycle arrest at the G2/M phase. Using Wright-Giemsa staining, we observed that evodiamine caused the cells to arrest in mitosis. It also profoundly caused an increase in polymerized tubulin levels and Bcl-2 phosphorylation on serine 70 in these cells. These data imply that the microtubular cytoskeleton appears to be one of the cellular targets in response to evodiamine. Moreover, treatment of CCRF-CEM cells with evodiamine was associated with increased levels of pro-apoptotic protein Bax, activation of caspase-3, and proteolytic cleavage of poly (ADP-ribose) polymerase, an endogenous caspase-3 substrate. Taken together, we demonstrate that evodiamine causes the mitotic arrest and a consequent apoptosis in CCRF-CEM cells through the enhancement of polymerized tubulin levels. Furthermore, several biological events including the Bcl-2 phosphorylation, Bax up-regulation and increase of caspase-3 activity could explain evodiamine-induced cell apoptosis.

Signals from the AT2 (angiotensin type 2) receptor of angiotensin II inhibit p21ras and activate MAPK (mitogen-activated protein kinase) to induce morphological neuronal differentiation in NG108-15 cells.

In a previous study, we had shown that activation of the AT2 (angiotensin type 2) receptor of angiotensin II (Ang II) induced morphological differentiation of the neuronal cell line NG108-15. In the present study, we investigated the nature of the possible intracellular mediators involved in the AT2 effect. We found that stimulation of AT2 receptors in NG108-15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108-15 cells with Ang II induced a decrease in GTP-bound p21ras but a sustained increase in the activity of p42mapk and p44mapk as well as neurite outgrowth. Similarly, neurite elongation, increased polymerized tubulin levels, and increased mitogen-activated protein kinase (MAPK) activity were also observed in a stably transfected NG108-15 cell line expressing the dominant-negative mutant of p21ras, RasN17. These results support the observation that inhibition of p21ras did not impair the effect of Ang II on its ability to stimulate MAPK activity. While 10 microM of the MEK inhibitor, PD98059, only moderately affected elongation, 50 microM PD98059 completely blocked the Ang II- and the RasN17-mediated induction of neurite outgrowth. These results demonstrate that some of the events associated with the AT2 receptor-induced neuronal morphological differentiation of NG108-15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.

Growth Hormone Promotion of Tubulin Polymerization Stabilizes the Microtubule Network and Protects Against Colchicine-Induced Apoptosis

We have investigated the effect of GH on microtubular physiology in Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR1–638). We show here that after 30 min of human GH (hGH) treatment of CHO-GHR1–638 cells, there was a significant increase in the level of polymerization of all four tubulin isoforms (α-, β-,γ -, and tyrosinated α-tubulin) compared with the serum-deprived state. However, this transient increase in the levels of polymerized tubulin after hGH treatment was particularly pronounced for β- and tyr α-tubulin. For α- and γ-tubulin, the hGH-induced increase in polymerization state lasted to approximately 3 h and then declined by 7 h, whereas for β- and tyr α-tubulin there was a decrease in the polymerization state at 1–2 h after hGH treatment compared with the level at 30 min (but still greater than the serum-deprived state) followed by a second but lesser wave of increased polymerization lasting to 7 h. The changes in the polymerization state of the tubulins were not accompanied by comparative changes in the level of total cellular tubulin. The proline rich box 1 region of the GH receptor was required for hGH to stimulate tubulin polymerization indicative that this event is JAK dependent. Increased tubulin polymerization still occurred in response to hGH in a receptor truncation lacking the carboxyl terminal half of the intracellular domain of the GH receptor indicative that hGH induced changes in intracellular calcium concentration is not required for tubulin polymerization. Prior treatment of CHO-GHR1–638 cells with hGH retarded colchicine induced microtubule depolymerization and also prevented colchicine induced apoptotic cell death. The integrity of the microtubule network was not required for GH-induced STAT5 mediated transcription as treatment of cells with colchicine, vincristine, or vinblastine did not alter the fold stimulation of the STAT5 mediated transcriptional response to GH. Thus one consequence of cellular treatment with GH is alteration in microtubule physiology.

Growth hormone promotion of tubulin polymerization stabilizes the microtubule network and protects against colchicine-induced apoptosis.

We have investigated the effect of GH on microtubular physiology in Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR(1-638)). We show here that after 30 min of human GH (hGH) treatment of CHO-GHR(1-638) cells, there was a significant increase in the level of polymerization of all four tubulin isoforms (alpha-, beta-, gamma-, and tyrosinated alpha-tubulin) compared with the serum-deprived state. However, this transient increase in the levels of polymerized tubulin after hGH treatment was particularly pronounced for beta- and tyr alpha-tubulin. For alpha- and gamma-tubulin, the hGH-induced increase in polymerization state lasted to approximately 3 h and then declined by 7 h, whereas for beta- and tyr alpha-tubulin there was a decrease in the polymerization state at 1-2 h after hGH treatment compared with the level at 30 min (but still greater than the serum-deprived state) followed by a second but lesser wave of increased polymerization lasting to 7 h. The changes in the polymerization state of the tubulins were not accompanied by comparative changes in the level of total cellular tubulin. The proline rich box 1 region of the GH receptor was required for hGH to stimulate tubulin polymerization indicative that this event is JAK dependent. Increased tubulin polymerization still occurred in response to hGH in a receptor truncation lacking the carboxyl terminal half of the intracellular domain of the GH receptor indicative that hGH induced changes in intracellular calcium concentration is not required for tubulin polymerization. Prior treatment of CHO-GHR(1-638) cells with hGH retarded colchicine induced microtubule depolymerization and also prevented colchicine induced apoptotic cell death. The integrity of the microtubule network was not required for GH-induced STAT5 mediated transcription as treatment of cells with colchicine, vincristine, or vinblastine did not alter the fold stimulation of the STAT5 mediated transcriptional response to GH. Thus one consequence of cellular treatment with GH is alteration in microtubule physiology.

A role for p21ras in the angiotensin II AT2 receptor transduction pathway

We have previously shown that the activation of the AT2 receptor of Ang II induced neurite outgrowth in NG108-15 cells. We also found that stimulation of NG108-15 cells with Ang II induced a rapid decrease in GTP-bound p21ras. In order to investigate the possible role of p21ras in Ang II-induced neuronal differentiation, we have established NG108-15 sublines which inducibly express a dominant inhibitory form of p21ras (p21N17Ras). We observed that IPTG-induced expression of p21N17Ras in these NG108-15 sublines induced the same morphological changes as does Ang II in control untransfected cells. Immunofluorescence labeling of β-tubulin showed that expression of p21N17Ras induced neurite outgrowth and elongation. These observations were supported by Western blot analysis of the level of polymerized tubulin. These results strongly support the hypothesis that AT2 receptor-induced neuronal differentiation in NG108-15 cells is mediated by the inhibition of p21ras.

Changes of depolymerized and polymerized tubulin proteins in the liver after taurocholic acid administration with or without colchicine pretreatment and cholestasis due to cytochalasin B and bile duct ligation in rats

Microtubules, which are components of the cytoskeleton, are the organelles involved in vesicular transport. In the hepatocyte, the microtubular network is presumed to be concerned with bile acid secretion, and there may also be some structural changes in cholestatic conditions. The polymerized tubulin levels were determined as microtubules in the liver by the radiochemical assay in rats after the administration of taurocholic acid, colchicine, taurocholic acid following the treatment of colchicine, cytochalasin B and common bile duct ligation. The polymerized tubulin levels were increased after the high dose administration of taurocholic acid, but were decreased after the treatment with colchicine. In cholestasis models induced by cytochalasin B and common bile duct ligation, the polymerized tubulin levels were markedly increased. These findings suggest the microtubule system plays an important role in the excretion of a large amount of bile acids into the bile, and that growth of the microtubular network occurs in cholestasis in order to promote the vesicular transport and to maintain the cell shape.