Tubulin-binding Agents Research Articles

ECOG 5805: A phase II study of eribulin mesylate (E7389) in patients (pts) with metastatic castration-resistant prostate cancer (CRPC).

4556 Background: Eribulin mesylate (E7389), a synthetic analog of halichondrin B, is a novel tubulin-binding agent that inhibits cancer cell proliferation at low-nanomolar levels. Methods: In a multicenter ECOG trial, pts with progressive metastatic CRPC, ECOG 0-2 were treated with eribulin 1.4 mg/m2 as an IV bolus over 5 minutes on Days 1 and 8 of a 21-day cycle. This noncomparative study stratified pts to either a chemonaive (CN), prior-taxane (Tax) only, or two prior cytotoxic (TCx) chemotherapy arm. The trialwas powered to detect a 50% PSA reduction using Consensus Criteria in at least 40% vs. 20% (90% power, one-sided α=0.10) for the CN stratum and 25% vs. 10% (power 87%, one-sided α=0.10) for the Tax and TCx strata. Results: In total, 119 pts received treatment of which 116 were eligible for the primary response determination in this study. Median age 70 yrs (range 45-88); median number of treatment cycles 4 (range 1-20+); ECOG 0-1 90%. As of this analysis, only one patient is still on treatment. Among the 115 eligible patients who have discontinued treatment, 82 were off treatment due to disease progression. Among eligible pts who received treatment, 65 had dose modifications (both planned and unplanned). PSA response rate in the CN strata was 24% (versus the anticipated 40% rate). Conclusions: Eribulin demonstrated activity in metastatic CRPC with taxane-naive disease. Clinically significant myelosuppression was noted in all strata. Although single agent activity was observed, the overall response rate did not meet predetermined levels of activity sufficient to warrant further study in CRPC. Evaluable pts ChemonaiveN = 41 Prior taxaneN = 51 Two prior cytotoxicN = 24 ≥50% PSA response: % (90% CI) 24 (13.9–37.9) 10 (4.0–19.5) 4 (0.2–18.3) % Stable disease ≥9 weeks 15 20 29 % Measurable disease response* 8 3 8 Median duration PSA response: months 7.1 3.6 – Overall survival months (median) NR** 11.4 13.7 AEs: % Neutropenia G 3/4 52 50 68 Leukopenia G3/4 33 44 52 Neuropathy—sensory G3/4 14 16 4 Fatigue G1/2-G3/4 69–17 66–8 52–12 Anorexia G1/2-G3/4 33–2 32–6 24–4 Nausea G1/2- G3/4 26–2 38–2 36–0 * 26, 38, and 13 pts with measurable disease in the CN, Tax, and TCx arms, respectively. ** Not reached. No significant financial relationships to disclose.

A multicenter, open-label phase Ib/II study to assess the safety and clinical activity of intravenous combretastatin A1 diphosphate (OXi4503) as monotherapy in subjects with primary or secondary hepatic tumor burden.

TPS164 Background: Combretastatin A1 diphosphate (CA1P, OXi4503) is a novel vascular disrupting agent (VDA) that is a reversible tubulin-binding agent with direct cytotoxic activity when bioactivated to its reactive orthoquinone metabolite. Several oxidative enzymes have been shown to catalyze this reaction including myeloperoxidase (MPO), tyrosinase, and hepatic peroxidases. Based on these findings it is expected that OXi4503 should be more active in tissues and tumors containing high levels of these and other oxidative enzymes. Nonclinical blood flow studies in tumor-bearing SCID mice showed that OXi4503 treatment (1 to 50 mg/kg) produced a decrease in tumor-associated blood volume. A dose of 50 mg/kg resulted in approximately 100% reduction in tumor vascular volume, compared with control mice. Tumor growth inhibition has been observed in single- and repeat-dose xenograft studies in multiple tumor models including breast cancer (MDA-MB-231), colorectal, renal (Caki-1), hepatocellular (HEP-G2), melanoma (LOX-IMVI) and AML (HL60). The extent of tumor volume reduction depended on the dose of OXi4503 and the dosing schedule. Repeat-dose studies over an extended period resulted in significant reductions in tumor volumes relative to control animals. Tumor volume reductions were maintained for up to 120 days after completion of treatment. Methods: This is a phase Ib dose escalation study to determine the safety and tolerability of OXi4503 in subjects with relapsed or refractory carcinomas with hepatic tumor burden. MTD will be the recommended phase II dose. The PK profile of OXi4503 and its major metabolites in subjects with hepatic tumor burden will also be determined. An exploratory analysis of the relative response rate of hepatic vs. non-hepatic tumor deposits will also be undertaken. OXi4503 is administered IV on Days 1, 8, and 15 of a 28-day cycle. Subjects can receive up to 6 cycles if they have stable disease or better, and no intolerable AEs. Tumor assessments are performed after every even-numbered cycle. No intra-subject dose escalation is allowed. Current enrollment is in cohort 2 (11 mg/m2). Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration OXiGENE OXiGENE

Phase I dose-ranging, pharmacokinetic (PK) study of tesetaxel, a novel orally active tubulin-binding agent.

e13075 Background: Currently approved taxanes are given intravenously and associated with marked hypersensitivity and cumulative neurotoxicity. Tesetaxel is a new orally active tubulin-binding agen...

Class III β-Tubulin Shows Unique Expression Patterns in a Variety of Neoplastic and Non-neoplastic Lymphoproliferative Disorders

Class III beta-tubulin (TUBB3) expression in carcinoma is associated with resistance to tubulin-binding chemotherapeutic agents. Recently, follicular dendritic cells (FDCs) were reported to express TUBB3 under physiologic conditions. We investigated TUBB3 expression in a wide range of lymphoproliferative disorders using immunohistochemistry. Dual immunostaining for Bcl-6 and TUBB3 revealed that some germinal center B cells also express TUBB3 in addition to FDCs. In Hodgkin lymphomas (HLs), 47.1% (40/85) expressed TUBB3 in the tumor cells with an all-or-none pattern. TUBB3 expression in HL was more common in mixed cellularity type than nodular sclerosis type (P=0.032). Among non-HLs, 79.3% (23/29) of anaplastic large cell lymphoma (ALCL), 8% (2/25) of extranodal natural killer/T-cell lymphoma, and 75% (21/28) of Burkitt lymphoma showed TUBB3 expression with an all-or-none pattern. Of diffuse large B-cell lymphoma, 15.2% (32/210) expressed TUBB3 in a heterogeneous pattern. In ALCL, TUBB3 expression was more common in systemic ALCL than in primary cutaneous ALCL (P=0.046). Diffuse large B-cell lymphomas with a germinal center B-like subgroup exhibited TUBB3 expression more frequently than non-GCB-like subgroup (P=0.01). Otherwise, none of the 18 angioimmunoblastic T-cell lymphomas; 18 peripheral T-cell lymphomas, not otherwise specified; 12 follicular lymphomas; 62 marginal zone lymphomas; 7 mantle cell lymphomas; 8 small lymphocytic lymphomas; or 2 FDC sarcomas expressed TUBB3. In angioimmunoblastic T-cell lymphoma and Castleman disease, TUBB3 was positive in immunoblasts corresponding to Epstein-Barr virus-infected or Kaposi sarcoma herpes virus-infected cells. A variety of neoplastic and non-neoplastic lymphoproliferative disorders exhibited characteristic TUBB3 expression patterns; these results suggest potential for diagnostic utility, some insight into the pathobiology of TUBB3 expression, and potential therapeutic implications.

Microtubule Dynamics, Mitotic Arrest, and Apoptosis: Drug-Induced Differential Effects of βIII-Tubulin

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

Tumour‐selective antivascular effects of the novel anti‐mitotic compound A‐318315: An in vivo rat regional haemodynamic study

1. It has been shown that tubulin-binding agents can destabilize cellular microtubules and suppress tumour growth; but it has also become apparent that some compounds can exert anti-vascular effects within the neovasculature of a solid tumour. To date, the difficulty with these targets has been the ability to selectivity induce vascular damage to the tumour while leaving normal vasculature unaffected. The data presented here characterizes the in vivo, tumour selective, anti-vascular effects of the novel tubulin-binding agent A-318315. 2. To that purpose, we have used an anaesthetized in vivo rat model designed to quantify acute changes in regional vascular resistance (VR) in both tumour and non-tumour vascular beds, simultaneously. Tissue-isolated tumours (approximately 1.25 gm) with blood flow supplied by a single epigastric artery were grown in the hindlimb of adult male rats. Blood flow to the tumour, mesenteric, renal and normal (non-tumour epigastric) arteries was measured pre-dose and post-dose under anaesthesia. 3. A-318315 was tested at 3, 10 and 30 mg/kg, i.v. These doses produced modest, transient increases in mean arterial pressure with little to no effect on heart rate. At peak effect, tumour VR increased to 175 +/- 47, 337 +/- 77 and 751 +/- 151% above the baseline, for the 3, 10 and 30 mg/kg doses, respectively, whereas VR was only modestly and transiently increased in normal epigastric (88 +/- 19%), mesenteric (33 +/- 3.3%) and renal arteries (17 +/- 8.6%). 4. These data demonstrate that A-318315 produces marked reductions in tumour blood flow in the rat at doses that exert minor effects on normal vascular function.

β-tubuline de classe III et cancer du poumon non à petites cellules

Several investigators have addressed the relationship between expression of class III beta-tubulin and outcome in patients with nonsmall cell lung cancer (NSCLC) treated with tubulin-binding agents (TBA). High expression of class III beta-tubulin has been found to be correlated with low response rates and reduced survival in advanced NSCLC patients treated with taxane/vinorelbine--containing regimens. Two studies have shown that patients receiving paclitaxel whose tumours expressed high levels of class III beta-tubulin had a lower response rate and shorter survival, whereas this variable was not found to be predictive in patients receiving regimens without TBA. Conversely, analysis of samples from operable NSCLC patients in the JBR-10 trial showed that cisplatin/vinorelbine chemotherapy seemed to overcome the negative prognostic effect of high class III beta-tubulin expression and that the greatest benefit from chemotherapy was observed in patients with high class III beta-tubulin expression. In advanced NSCLC, high betaIII tubulin expression may prompt selection of taxane-free regimens as the preferred initial treatment approach. The epothilones may fulfil such a role in the treatment of NSCLC. betaIII tubulin expression is emerging as a valuable biomarker for taxane resistance in advanced disease, as well as offering prognostic information for outcomes among patients with earlier stage disease.

Erratum: Microtubules and resistance to tubulin-binding agents

Nature Reviews Cancer 10, 194–204 (2010) On page 202 of this article, extracellular matrix transforming growth factor-β1 (TGFβ1), should have read extracellular matrix transforming growth factor-β induced (TGFβI). This has been corrected online.

Microtubules and resistance to tubulin-binding agents

Microtubules are dynamic structures composed of alpha-beta-tubulin heterodimers that are essential in cell division and are important targets for cancer drugs. Mutations in beta-tubulin that affect microtubule polymer mass and/or drug binding are associated with resistance to tubulin-binding agents such as paclitaxel. The aberrant expression of specific beta-tubulin isotypes, in particular betaIII-tubulin, or of microtubule-regulating proteins is important clinically in tumour aggressiveness and resistance to chemotherapy. In addition, changes in actin regulation can also mediate resistance to tubulin-binding agents. Understanding the molecular mechanisms that mediate resistance to tubulin-binding agents will be vital to improve the efficacy of these agents.

An in vitro model that can distinguish between effects on angiogenesis and on established vasculature: Actions of TNP-470, marimastat and the tubulin-binding agent Ang-510

In anti-cancer therapy, current investigations explore the possibility of two different strategies to target tumor vasculature; one aims at interfering with angiogenesis, the process involving the outgrowth of new blood vessels from pre-existing vessels, while the other directs at affecting the already established tumor vasculature. However, the majority of in vitro model systems currently available examine the process of angiogenesis, while the current focus in anti-vascular therapies moves towards exploring the benefit of targeting established vasculature as well. This urges the need for in vitro systems that are able to differentiate between the effects of compounds on angiogenesis as well as on established vasculature. To achieve this, we developed an in vitro model in which effects of compounds on different vascular targets can be studied specifically. Using this model, we examined the actions of the fumagillin derivate TNP-470, the MMP-inhibitor marimastat and the recently developed tubulin-binding agent Ang-510. We show that TNP-470 and marimastat solely inhibited angiogenesis, whereas Ang-510 potently inhibited angiogenesis and caused massive disruption of newly established vasculature. We show that the use of this in vitro model allows for specific and efficient screening of the effects of compounds on different vascular targets, which may facilitate the identification of agents with potential clinical benefit. The indicated differences in the mode of action between marimastat, TNP-470 and Ang-510 to target vasculature are illustrative for this approach.

Tubulin targets in the pathobiology and therapy of glioblastoma multiforme. I. class III β‐tubulin

Glioblastoma multiforme (GBM) is the most common and deadliest form of primary brain cancer in adults. Despite advances in molecular biology and genetics of gliomas currently there is no effective treatment or promising molecularly targeted experimental therapeutic strategies for these tumors. In previous studies we have shown aberrant overexpression of the class III beta-tubulin isotype (betaIII-tubulin) in GBM and have proposed that this change may reflect perturbations in microtubule dynamics associated with glioma tumorigenesis, tumor progression and malignant transformation into GBM. This minireview focuses on microtubules and tubulin as emerging targets in potential therapy of GBM using a new class of betaIII-tubulin-targeted drugs in the light of recent developments concerning the function and potential role of this isotype in clinically aggressive tumor behavior, cancer stem cells, tumor hypoxia and chemoresistance to tubulin binding agents, principally taxanes.

Non‐toxic melanoma therapy by a novel tubulin‐binding agent

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

Inclusion complexes of noscapine in β-cyclodextrin offer better solubility and improved pharmacokinetics

Noscapine (NOS) is a unique class of tubulin-binding anticancer agents. Their potential usefulness as anticancer drugs is however limited by the poor bioavailability, thus necessitating administration of a higher dose regime in the range of 300-600 mg/kg for tumor growth inhibition. To augment bioavailability, we prepared an inclusion complex of NOS in beta-cyclodextrin (beta-CD) and evaluated its physico-chemical characteristics. Our phase-solubility analysis shows a 1:1-complexation (Kc approximately 0.454 mM(-1)) of NOS with beta-CD that offers better dissolution properties. We confirmed complex formation in solid state by differential scanning calorimetry, powder X-ray diffractometry, Fourier-transform infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy, rotating frame Overhauser enhancement spectroscopy and by molecular modeling methods. Based upon theoretical calculations in gas phase, we propose O-CH2-O- in orientation of NOS in the beta-CD cavity. The thermal behavior data also provides complementary evidences of complex formation. The pharmacokinetic studies showed a 1.87-fold increase in bioavailability of NOS upon complexation in the beta-CD inclusion complex state as compared to free NOS. Furthermore, the complex retains the anticancer attributes of NOS. Our studies propose for the first time a stable NOS-beta-CD inclusion complex as an effective approach to enhance the solubility and bioavailability of NOS for anticancer therapy.

ABT-751, a novel tubulin-binding agent, decreases tumor perfusion and disrupts tumor vasculature

ABT-751 is an orally bioavailable tubulin-binding agent that is currently under clinical development for cancer treatment. In preclinical studies, ABT-751 showed antitumor activity against a broad spectrum of tumor lines including those resistant to conventional chemotherapies. In this study, we investigated the antivascular properties of ABT-751 in a rat subcutaneous tumor model using dynamic contrast-enhanced magnetic resonance imaging. A single dose of ABT-751 (30 mg/kg, intravenously) induced a rapid, transient reduction in tumor perfusion. After 1 h, tumor perfusion decreased by 57% before recovering to near pretreatment levels within 6 h. In contrast, ABT-751 produced little change in muscle perfusion at either time point. To further elucidate mechanisms of drug action at the cellular level, we examined the effects of ABT-751 on endothelial cells using an in-vitro assay. ABT-751, at concentrations corresponding to plasma levels achieved in vivo, caused endothelial cell retraction and significant loss of microtubules within 1 h. The severity of these morphological changes was dose-dependent but reversible within 6 h after the discontinuation of the drug. Taken together, these results show that ABT-751 is a tubulin-binding agent with antivascular properties. Microtubule disruption and morphological changes in vascular endothelial cells may be responsible, at least in part, for the dysfunction of tumor blood vessels after ABT-751 treatment.

15-Deoxi-Δ 12,14-prostaglandin J 2 is a tubulin-binding agent that destabilizes microtubules and induces mitotic arrest

15-Deoxi-Δ 12,14-prostaglandin J 2 (15d-PGJ 2) is known to play an important role in the pathophysiology of carcinogenesis, however, the molecular mechanisms underlying these effects are not yet fully understood. Recently, we have shown that 15d-PGJ 2 is a potent inducer of breast cancer cell death and that this effect is associated with a disruption of the microtubule cytoskeletal network. Here, we show that treatment of the MCF-7 breast cancer cell line with 15d-PGJ 2 induces an accumulation of cells in the G 2/M compartment of the cell cycle and a marked disruption of the microtubule network. 15d-PGJ 2 treatment causes mitotic abnormalities that consist of failure to form a stable metaphase plate, incapacity to progress through anaphase, and failure to complete cytokinesis. 15d-PGJ 2 binds to tubulin through the formation of a covalent adduct with at least four cysteine residues in α- and β-tubulin, as detected by hybrid triple-quadrupole mass spectrometry analysis. Overall, these results support the hypothesis that microtubule disruption and mitotic arrest, as a consequence of the binding of 15d-PGJ 2 to tubulin, can represent one important pathway leading to breast cancer cell death.

BI 6727, A Polo-like Kinase Inhibitor with Improved Pharmacokinetic Profile and Broad Antitumor Activity

Antimitotic chemotherapy remains a cornerstone of multimodality treatment for locally advanced and metastatic cancers. To identify novel mitosis-specific agents with higher selectivity than approved tubulin-binding agents (taxanes, Vinca alkaloids), we have generated inhibitors of Polo-like kinase 1, a target that functions predominantly in mitosis. The first compound in this series, suitable for i.v. administration, has entered clinical development. To fully explore the potential of Polo-like kinase 1 inhibition in oncology, we have profiled additional compounds and now describe a novel clinical candidate. BI 6727 is a highly potent (enzyme IC(50) = 0.87 nmol/L, EC(50) = 11-37 nmol/L on a panel of cancer cell lines) and selective dihydropteridinone with distinct properties. First, BI 6727 has a pharmacokinetic profile favoring sustained exposure of tumor tissues with a high volume of distribution and a long terminal half-life in mice (V(ss) = 7.6 L/kg, t(1/2) = 46 h) and rats (V(ss) = 22 L/kg, t(1/2) = 54 h). Second, BI 6727 has physicochemical and pharmacokinetic properties that allow in vivo testing of i.v. as well as oral formulations, adding flexibility to dosing schedules. Finally, BI 6727 shows marked antitumor activity in multiple cancer models, including a model of taxane-resistant colorectal cancer. With oral and i.v. routes of administration, the total weekly dose of BI 6727 is most relevant for efficacy, supporting the use of a variety of well-tolerated dosing schedules. These findings warrant further investigation of BI 6727 as a tailored antimitotic agent; clinical studies have been initiated.

A Direct and Efficient Total Synthesis of the Tubulin-Binding Agents Ceratamine A and B; Use of IBX for a Remarkable Heterocycle Dehydrogenation

The total synthesis of the tubulin-binding agents ceratamine A and B is reported, along with des-methyl analogs, via a synthetic route that is high-yielding and operationally efficient. The synthetic route involved a Beckmann rearrangement to form an azepine ring precursor, a Knoevenagel condensation to install the benzylic side chain, and an effective imidazole annulation onto an alpha-aminoketone precursor with a protected S-methylisothiourea. Final dehydrogenation proved remarkably facile using IBX.

Bone marrow CFU-GM and human tumor xenograft efficacy of three tubulin binding agents

The dynamic instability of microtubules in cells is one of the key targets of anticancer therapeutics. Microtubule-disrupting agents such as vinca alkaloids and microtubule-stabilizing agents such as taxanes are important antitumor agents. The bone marrow toxicity and human tumor xenograft activity of three tubulin-binding compounds, vincristine, paclitaxel, and tasidotin were compared. Mouse and human bone marrow were subjected to colony-forming (CFU-GM) assays over a 5-log concentration range in culture. In vivo, a range of tasidotin doses was compared with vincristine, paclitaxel, and docetaxel for efficacy in several human tumor xenografts. The IC(90) concentrations for vincristine and paclitaxel for mouse CFU-GM were 30 and 27 nM, and for human CFU-GM were 3 and 9 nM, giving mouse to human differentials of ten- and threefold. Tasidotin produced IC(90)s of >300 nM in mouse and 65 nM in human CFU-GM, thus a >4.6-fold differential between species. In vivo, tasidotin resulted in a dose-dependent increase in tumor growth delay in the RL lymphoma, the RPMI 8226 multiple myeloma, and MX-1 breast carcinoma models. Vincristine and tasidotin were also very effective against these tumors. The PC-3 prostate carcinoma was very responsive to full-dose paclitaxel and docetaxel while tasidotin generated a dose dependent effect. Bringing together bone marrow toxicity data, pharmacokinetic parameters, and human tumor xenograft efficacy provides valuable information for the translation of preclinical findings to the clinic.

Preclinical evaluation of vascular-disrupting agents in Ewing’s sarcoma family of tumours

The effects of the tubulin-binding vascular-disrupting agents (VDAs), combretastatin A4 phosphate (CA4P), OXi4503/CA1P and OXi8007, in subcutaneous mouse models of the Ewing’s sarcoma family of tumours (ESFTs) have been investigated alone and in combination with doxorubicin. Delay in subcutaneous tumour growth was observed following treatment of mice with multiple doses of OXi4503/CA1P but not with CA4P or OXi8007. A single dose of OXi4503/CA1P caused complete shutdown of vasculature by 24 h and extensive haemorrhagic necrosis by 48 h. However, a viable rim of proliferating cells remained, which repopulated the tumour within 10 days following the withdrawal of treatment. Combined treatment with doxorubicin 1 h prior to administration of OXi4503/CA1P enhanced the effects of OXi4503/CA1P causing a synergistic delay in tumour growth ( p < 0.001). This study demonstrates that OXi4503/CA1P is a potent VDA in ESFT and in combination with conventional cytotoxic agents represents a promising treatment strategy for this tumour group.

Tubulin-Targeted Drug Action: Functional Significance of Class II and Class IVb β-Tubulin in Vinca Alkaloid Sensitivity

Aberrant expression of beta-tubulin isotypes is frequently described in tumor tissues and tubulin-binding agent (TBA)-resistant cell lines. There is limited understanding of the role of specific beta-tubulin isotypes in cellular sensitivity to TBAs, and to gain insights into the functional role of betaII- and betaIVb-tubulin, we examined these isotypes in lung cancer cell lines NCI-H460 (H460) and Calu-6. Drug-treated clonogenic assays revealed that small interfering RNA-mediated knockdown of either betaII- or betaIVb-tubulin hypersensitized the lung cancer cell lines to Vinca alkaloids, with the effects more pronounced following betaIVb-tubulin knockdown. In contrast, there was no change in paclitaxel sensitivity following knockdown of either isotype. Cell cycle analysis revealed a greater propensity for the betaII- and betaIVb-tubulin knockdown cells to undergo G2-M cell cycle block following 5 nmol/L vincristine treatment, with the betaIVb knockdown cells being more sensitive than the betaII-tubulin knockdown cells compared with control. In contrast to betaII-tubulin knockdown, betaIVb-tubulin knockdown cells showed a significant increase in the sub-G1 population (cell death) following treatment with both 5 and 40 nmol/L of vincristine compared with controls. Importantly, betaIVb-tubulin knockdown in H460 cells caused a significant dose-dependent increase in Annexin V staining in response to vincristine but not paclitaxel. Therefore, increased sensitivity to induction of apoptosis is one mechanism underlying the Vinca alkaloid hypersensitivity. This study provides direct evidence that betaII- or betaIVb-tubulins have functionally distinct roles and expression of these isotypes may serve as strong predictors of Vinca alkaloid response and resistance.