Microtubule-stabilizing Agent Research Articles

Epothilones in epithelial ovarian, fallopian tube, or primary peritoneal cancer: a systematic review.

Ovarian cancer is the most lethal gynecologic malignancy; consequently, there is a need for effective therapies. Epothilones are microtubule-stabilizing agents that inhibit cell growth. Currently, patupilone and its four synthetic derivatives ixabepilone, BMS-310705, sagopilone, 20-desmethyl-20-methylsulfanyl epothilone B and epothilone D, as well as its derivative KOS-1584, are under clinical evaluation. This is the first systematic review conducted in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines that synthesizes all available data emerging from trials and evaluates the efficacy and safety of epothilones in epithelial ovarian, primary fallopian tube, and primary peritoneal cancer. Despite the fact that epothilones have proven active in taxane-resistant settings in preclinical models, it is not yet clear from Phase II/III studies reviewed here that their clinical activity is superior to that of taxanes. Nevertheless, responses to epothilones have been observed in platinum-refractory/resistant ovarian cancer patients. Moreover, despite the shared mechanism of action of epothilones, their clinical profile seems clearly different, with diarrhea being the most common dose-limiting toxicity encountered with patupilone, whereas neutropenia and sensory neuropathy are the most common toxic effects observed with the other epothilones. In any case, randomized trials comparing epothilones with standard treatments seem warranted to define further the role of these agents, whereas biomarker analysis might further optimize patient selection.

Peloruside A Inhibits Growth of Human Lung and Breast Tumor Xenografts in an Athymic nu/nu Mouse Model

Peloruside A is a microtubule-stabilizing agent isolated from a New Zealand marine sponge. Peloruside prevents growth of a panel of cancer cell lines at low nanomolar concentrations, including cell lines that are resistant to paclitaxel. Three xenograft studies in athymic nu/nu mice were performed to assess the efficacy of peloruside compared with standard anticancer agents such as paclitaxel, docetaxel, and doxorubicin. The first study examined the effect of 5 and 10 mg/kg peloruside (QD×5) on the growth of H460 non-small cell lung cancer xenografts. Peloruside caused tumor growth inhibition (%TGI) of 84% and 95%, respectively, whereas standard treatments with paclitaxel (8 mg/kg, QD×5) and docetaxel (6.3 mg/kg, Q2D×3) were much less effective (%TGI of 50% and 18%, respectively). In a second xenograft study using A549 lung cancer cells and varied schedules of dosing, activity of peloruside was again superior compared with the taxanes with inhibitions ranging from 51% to 74%, compared with 44% and 50% for the two taxanes. A third xenograft study in a P-glycoprotein-overexpressing NCI/ADR-RES breast tumor model showed that peloruside was better tolerated than either doxorubicin or paclitaxel. We conclude that peloruside is highly effective in preventing the growth of lung and P-glycoprotein-overexpressing breast tumors in vivo and that further therapeutic development is warranted. Mol Cancer Ther; 14(8); 1816-23. ©2015 AACR.

Peloruside A is a microtubule-stabilizing agent with exceptional anti-migratory properties in human endothelial cells.

Peloruside A is a novel antimitotic drug originally isolated from the marine sponge Mycale hentschieli. Previous studies showed that peloruside A stabilizes microtubules by binding to a site on tubulin distinct from paclitaxel, another microtubule stabilizing drug. Peloruside A blocks mitosis, but little is known about the effects on other cellular activities. Here we report that peloruside A is the most potent microtubule inhibitor yet tested for its ability to block endothelial cell migration. Quantitative analysis indicated that it inhibits microtubule dynamics and endothelial cell migration at 1/200(th) of the concentration needed to inhibit cell division (the cytotoxic concentration), indicating that it could potentially have a large margin of safety when used to specifically target angiogenesis. By comparison, paclitaxel, a well-known cancer therapeutic drug, suppresses cell migration at 1/13(th) of its cytotoxic concentration; and vinblastine suppresses cell migration at just slightly below its cytotoxic antimitotic concentration. Thus, different microtubule targeted drugs have varying relative potencies for inhibition of cell migration versus cell division. The results suggest that peloruside A may be an especially useful agent for anti-angiogenesis therapy and point to the likelihood that other antimitotic drugs might be found with an even larger potential margin of safety.

βI-tubulin mutations in the laulimalide/peloruside binding site mediate drug sensitivity by altering drug–tubulin interactions and microtubule stability

Peloruside A (PLA) and laulimalide (LAU) are potent microtubule-stabilizing natural products that are effective against a broad spectrum of cancer cells. The interactions of PLA and LAU with tubulin have attracted a great deal of attention, mainly because they bind to β-tubulin at a site that is different from the classical taxoid site. Multiple βI-tubulin amino acid residues have been predicted by computer modelling studies and more recently by protein crystallography to participate in the binding of PLA and LAU to tubulin. The relevance of these residues in determining cellular sensitivity to the compounds, however, remains largely uncertain. To determine the role of four binding site residues, Q291, D295, V333, and N337 on PLA and LAU activity, we introduced single mutations to these sites by site-directed mutagenesis and transfected each mutant tubulin separately into HEK and/or HeLa cells. We found that a Q291M βI-tubulin mutation increased sensitivity of the cells to PLA, but not to LAU, paclitaxel (PTX), or vinblastine (VBL). In contrast, V333W and N337L mutations led to less stable microtubules, with the V333W causing resistance to PLA and PTX, but not LAU, and the N337L causing resistance to PLA, LAU, and PTX. Moreover, cells expressing either W333 or L337 were hypersensitive to the microtubule-destabilizing agent, VBL. The D295I mutation conferred resistance to both PLA and LAU without affecting microtubule stability or sensitivity to PTX or ixabepilone (IXB). This study identifies the first mammalian βI-tubulin mutation that specifically increases sensitivity to PLA, and reports mutations at PLA and LAU binding site residues that can either reduce microtubule stability or impair drug–tubulin binding, conferring resistance to these microtubule-stabilizing agents. This information provides insights on β-tubulin residues important for maintaining microtubule structural integrity and for sensitivity to microtubule-targeting agents, and suggests novel directions for rational structure-based design of new and more potent agents for cancer treatment that target the LAU/PLA site.

A pH-responsive high density lipoprotein-like nanoparticle of epothilone B

Epothilone B (EpoB) is a paclitaxel (PTX)-like microtubule-stabilizing agent that induces mitotic arrest and apoptosis in cells. EpoB is a promising anti-tumor agent, and is thought to have the potential to overcome well-known PTX resistance. However, EpoB is barely soluble in water, and is fatal to normal cells due to extremely potent cytotoxicity. To reduce the unwanted cytotoxicity of EpoB to normal cells, a reconstituted highdensity lipoprotein (rHDL)-like nanoparticle of EpoB (EpoB-rHDL) was assembled using apolipoprotein A-I (apoA-I). The EpoB-rHDL, as well as PTX-rHDL (a HDLlike nanoparticle of paclitaxel), were indeed mild (nontoxic) to certain cell lines such as MCF7, MDA-MB-231, and SK-OV-3, while free EpoB and PTX were very toxic to those same cells. In contrast, the EpoB-rHDL and PTXrHDL were very effective in killing the Caco-2 and ZR-75-1 while free drugs were less toxic to those cells. The susceptibility of cell lines to rHDLs was dependent on the expression of scavenger receptor class B type I (SR-BI), indicating that EpoB-rHDL selectively and efficiently kills only SR-BI-overexpressing cells. Furthermore, the EpoBrHDL released EpoB only at acidic pH, which may facilitate the escape of drugs from acidic endosome. Thus, EpoB-rHDL shown in this study enables safe and targeted delivery of the potent EpoB to cancer cells in SR-BIdependent manner.

Identifying potential therapeutics by molecular profiling of 136 cases of uterine clear cell carcinoma

• Uterine clear cell carcinoma is a rare subtype of endometrial cancer that accounts for only 1-6% of all uterine malignancies.1-2 • Uterine clear cell carcinomas are considered to be Type-II estrogen-independent and exhibit aggressive behavior with worse clinical outcomes compared Type I tumors.3 • Uterine clear cell carcinomas most often occur in post menopausal women with a median age of 66-68 years.4 • Similar to uterine serous cancers, clear cell carcinoma is more prevalent in African American women.5 • While the pathogenesis of many subtypes of uterine cancer have been well studied, the molecular pathways involved in uterine clear cell carcinoma development are poorly understood. • Broader understanding of the molecular and genomic characteristics of uterine clear cell carcinomas could contribute to the growing body of literature this disease and potentially provide treatment options with targeted therapies. Background: Clear cell carcinoma of the endometrium (CCE) is a rare subtype of endometrial cancer (EC) associated with worse prognosis when compared to other high grade EC subtypes. We aim to evaluate molecular, genomic and protein expression patterns in a large cohort of CCEs in order to direct patients to rational therapeutic strategies and clinical trials. Methods: Out of 3133 EC submitted to Caris Life Sciences from March 2011 to July 2014, 136 CCEs were identified based on reported pathology. Testing was ordered per physician request and included a combination of sequencing (Sanger or next generation sequencing), protein expression (immunohistochemistry), and /or gene amplification (FISH/CISH). Results: Of the samples evaluated by sequencing, the most common genetic mutations were P53 (40%) and BRCA2 (33%). Hormone receptor expression was low: ERα (35%), PR (22%) and AR (7%). ERBB2 was mutated in 8%, while its protein product Her2, a biomarker for HER2-directed therapies, was amplified in 12% and expressed in 5% of patients. Aberrations of the PI3K pathway, including 26% PTEN and 25% PIK3CA mutation rate, with 69% loss of PTEN expression, highlight potential utility with inhibitors of this pathway. DNA repair pathway was altered in our CCE cohort as well: low ERCC1 (6%) and MGMT (34%) expression, and high BRCA2 mutation rate, suggesting sensitivity to alkylating agents. Evaluation of the cMET pathway showed 40% IHC expression. TUBB3, a class III β-tubulin, was infrequently expressed (15%), and TLE3 expression was high (29%) compared to other EC subtypes, implicating sensitivity to microtubule-stabilizing agents. Increased TOPO2A expression, associated with anthracycline efficacy, was seen in over 80% of cases. Loss of RRM1, a DNA synthesis protein known to determine efficacy of gemcitabine, was seen in 78% of cases. Conclusions: Our findings highlight the genetic heterogeneity of CCE, and identified altered cellular pathways with potential diagnostic and predictive values for therapeutic intervention. Drugs targeting the pathways for DNA repair, PI3K, and receptor tyrosine kinases, as well as gemcitabine and taxanes, may warrant consideration in selected patients with CCE. • 136 out of 3133 (4.3%) of endometrial cancers submitted to Caris Life Sciences from March 2011 to July 2014 were identified as clear cell carcinoma. • Specific testing was performed per physician request and included a combination of sequencing (Sanger, NGS or pyrosequencing), protein expression (IHC), gene amplification (CISH or FISH), and/or RNA fragment analysis. • IHC analysis was performed on formalin-fixed paraffin-embedded tumor samples using commercially available detection kits, automated staining techniques (Benchmark XT, Ventana, and AutostainerLink 48, Dako), and commercially available antibodies. • Fluorescent in-situ hybridization (FISH) was used for evaluation of the HER-2/neu [HER-2/CEP17 probe], EGFR [EGFR/CEP7 probe], and cMET [cMET/CEP7 probe] (Abbott Molecular/Vysis). HER-2/neu and cMET status were also evaluated by chromogenic in-situ hybridization (INFORM HER-2 Dual ISH DNA Probe Cocktail; commercially available cMET and chromosome 7 DIG probe; Ventana). The same scoring system was applied as for FISH. • Direct sequence analysis was performed on genomic DNA isolated from formalin-fixed paraffin-embedded tumor samples using the Illumina MiSeq platform. Specific regions of 47 genes of the genome were amplified using the Illumina TruSeq Amplicon Cancer Hotspot panel. • Mutation analysis by Sanger sequencing included selected regions of BRAF, KRAS, NRAS, c-KIT, EGFR, and PIK3CA genes and was performed by using M13-linked PCR primers designed to amplify targeted sequences. • Retrospective data analysis; Statistical analysis (unpaired t-tests used to compare biomarker expression across histologic subtypes) performed using PrismTM v6. Biomarker associations were calculated by two-tailed Fisher Exact tests.

Comparing vaginal sarcoma and carcinoma in the Surveillance, Epidemiology and End Results program, 1988 to 2010: An analysis of 4062 patients

highlighted potential utility with inhibitors of this pathway. DNA repair pathway was altered in our CCE cohort as well: low ERCC1 (6%) and MGMT (34%) expression and high BRCA2 mutation rate, suggesting sensitivity to alkylating agents. Evaluation of the cMET pathway showed 40% IHC expression. TUBB3, a class III β-tubulin, was infrequently expressed (15%), and TLE3 expression was high (29%) compared to other EC subtypes, implicating sensitivity to microtubule-stabilizing agents. Increased TOPO2A expression, associated with anthracycline efficacy, was seen in over 80% of cases. Loss of RRM1, a DNA synthesis protein known to determine efficacy of gemcitabine, was seen in 78% of cases. Conclusions: Our findings highlight the genetic heterogeneity of CCE and identified altered cellular pathways with potential diagnostic and predictive values for therapeutic intervention. Drugs targeting the pathways for DNA repair, PI3K, and receptor tyrosine kinases, as well as gemcitabine and taxanes, may warrant consideration in selected patients with CCE.

Inhibition of human vascular endothelial cell migration and capillary-like tube formation by the microtubule-stabilizing agent peloruside A.

Peloruside A is a microtubule-stabilizing agent that is currently under investigation as a potential anticancer agent. Peloruside A binds to a site on β-tubulin that is distinct to that of the taxanes (paclitaxel and docetaxel) and the epothilones. An attractive clinical quality of microtubule-stabilizing agents is their ability to target multiple mechanisms of tumour growth. In addition to inducing tumour cell apoptosis by arresting cells in mitosis, microtubule-stabilizing agents also inhibit angiogenesis, a process needed by tumor cells for growth and metastasis. In this study, the effects of peloruside A on endothelial cell processes important for angiogenesis were assessed in comparison to docetaxel. Both peloruside A and docetaxel potently inhibited the proliferation of human umbilical vein endothelial cells, with IC50 values of 1.4 and 1.7 nM, respectively. Peloruside also potently blocked endothelial cell migration during wound closure and the three-dimensional organization of the endothelial cells into capillary-like tubes. In the wound scratch assay, peloruside A inhibited wound recovery with an IC50 of 6.3 nM after 18 h. Docetaxel was approximately 3-fold more potent than peloruside A. The number of capillary-like tubes that formed after 16 h culture in Matrigel™ was also inhibited in a dose-dependent manner with an IC50 of 4.5 nM. Docetaxel was about 2-fold more potent than peloruside A in preventing tube formation. This inhibition of endothelial cell function occurred at relatively non-cytotoxic concentrations over the 16-18 h incubations for both stabilizing agents, suggesting that anti-angiogenic effects are likely to occur before therapeutically relevant doses begin to inhibit tumor growth or adverse side effects develop.

Microtubule Dynamicity Is More Important than Stability in Memory Formation: an In Vivo Study.

It has been shown that microtubule (MT) activity and dynamics can have huge impacts on synaptic plasticity and memory formation. This is mainly due to various functions of MTs in neurons; MTs are involved in dendritic spine formation, axonal transportation, neuronal polarity, and receptor trafficking. Recent studies from our group and other labs have suggested the possible role of brain MT dynamicity and activity in memory; however, there is a need for more detailed studies regarding this aspect. In this study, we have tried to evaluate the importance of microtubule dynamicity rather than stability in memory formation in vivo. In order to investigate the role of MT stability in memory formation, we treated mice with paclitaxel-a classic microtubule-stabilizing agent. We then studied the behavior of treated animals using Morris water maze (MWM) test. To measure the effect of injected paclitaxel on MT polymerization kinetics, we conducted polymerization assays on brain extracts of the same paclitaxel-treated animals. Our results show that paclitaxel treatment affects animals' memory in a negative way and treated animals behave poorly in MWM compared to control group. In addition, our kinetics studies show that MT stability is significantly increased in brain extracts from paclitaxel-treated mice, but MT dynamics is reduced. Thus, we suggest that dynamicity is a very important feature of MT protein structures, and regarding memory formation, dynamicity is more important than stability and high activity.

Combined treatment strategies for microtubule stabilizing agent-resistant tumors.

Resistance to microtubule-stabilizing agents is a major hurdle for successful cancer therapy. We investigated combined treatment of microtubule-stabilizing agents (MSAs) with inhibitors of angiogenesis to overcome MSA resistance. Treatment regimens of clinically relevant MSAs (patupilone and paclitaxel) and antiangiogenic agents (everolimus and bevacizumab) were investigated in genetically defined MSA-resistant lung (A549EpoB40) and colon adenocarcinoma (SW480) tumor xenografts in nude mice (CD1-Foxn1<nu>, ICRnu; 5-14 per group). Tumor growth delays were calculated by Kaplan-Meier analysis with Holm-Sidak tests. All statistical tests were two-sided. Inhibition of mTOR-kinase by everolimus only minimally reduced the proliferative activity of β tubulin-mutated lung adenocarcinoma cells alone and in combination with the MSA patupilone, but everolimus inhibited expression and secretion of vascular endothelial growth factor (VEGF) from these cells. mTOR-kinase inhibition strongly sensitized tumor xenografts derived from these otherwise MSA-resistant tumor cells to patupilone. Tumors treated with the combined modality of everolimus and patupilone had statistically significantly reduced tumor volume and stronger tumor growth delay (16.2 ± 1.01 days) than control- (7.7 ± 0.3 days, P = .004), patupilone- (10 ± 0.97 days, P = .009), and everolimus-treated (10.6 ± 1.4 days, P = .014) tumors. A combined treatment modality with bevacizumab also resensitized this MSA-refractory tumor model to patupilone. Treatment combination also strongly reduced microvessel density, corroborating the relevance of VEGF targeting for the known antivasculature-directed potency of MSA alone in MSA-sensitive tumor models. Resensitization to MSAs was also probed in P glycoprotein-overexpressing SW480-derived tumor xenografts. Different bevacizumab regimens also sensitized this otherwise-resistant tumor model to clinically relevant MSA paclitaxel. A treatment combination of MSAs with antiangiogenic agents is potent to overcome tumor cell-linked MSA resistance and should be considered as strategy for MSA-refractory tumor entities.

The microtubule-stabilizing drug Epothilone D increases axonal sprouting following transection injury in vitro.

Neuronal cytoskeletal alterations, in particular the loss and misalignment of microtubules, are considered a hallmark feature of the degeneration that occurs after traumatic brain injury (TBI). Therefore, microtubule-stabilizing drugs are attractive potential therapeutics for use following TBI. The best-known drug in this category is Paclitaxel, a widely used anti-cancer drug that has produced promising outcomes when employed in the treatment of various animal models of nervous system trauma. However, Paclitaxel is not ideal for the treatment of patients with TBI due to its limited blood-brain barrier (BBB) permeability. Herein we have characterized the effect of the brain penetrant microtubule-stabilizing agent Epothilone D (Epo D) on post-injury axonal sprouting in an in vitro model of CNS trauma. Epo D was found to modulate axonal sprout number in a dose dependent manner, increasing the number of axonal sprouts generated post-injury. Elevated sprouting was observed when analyzing the total population of injured neurons, as well as in selective analysis of Thy1-YFP-labeled excitatory neurons. However, we found no effect of Epo D on axonal sprout length or outgrowth speed. These findings indicate that Epo D specifically affects injury-induced axonal sprout generation, but not net growth. Our investigation demonstrates that primary cultures of cortical neurons are tolerant of Epo D exposure, and that Epo D significantly increases their regenerative response following structural injury. Therefore Epo D may be a potent therapeutic for enhancing regeneration following CNS injury. This article is part of a Special Issue entitled 'Traumatic Brain Injury'.

Microtubule-Depolymerizing Agents Used in Antibody-Drug-Conjugates Induce Antitumor Activity by Stimulation of Dendritic Cells

ABSTRACT Antibody drug conjugates (ADCs) are emerging as powerful treatment strategies with outstanding target specificity and high therapeutic activity in cancer patients. While >30 ADCs are currently being investigated in clinical trials, brentuximabvedotin and T-DM1 represent clinically approved ADCs in cancer patients. We hypothesized that their sustained clinical responses could be related to the stimulation of an antitumor immune response. Indeed, the two microtubule-destabilizing agents Dolastatin 10 and Ansamitocin P3, from which the cytotoxic components of brentuximabvedotin and T-DM1 are derived, may serve as prototypes for a class of agents that induce tumor cell death and convert tumor resident, tolerogenic dendritic cells (DCs) into efficient antigen presenting cells (APCs). The two drugs induced phenotypic and functional maturation of murine splenic as well as human monocyte-derived DCs. In contrast, microtubule-stabilizing agents such as taxanes did not display this feature. In tumor models, both Dolastatin 10 and Ansamitocin P3 efficiently promoted antigen uptake and migration of tumor-resident DCs to tumor-draining lymph nodes, thereby potentiating tumor-specific T cell responses. Underlining the requirement of an intact host immune system for the full therapeutic benefit of these two compounds, their antitumor effect was far less pronounced in mice lacking adaptive immunity or dendritic cells. Combinations with immune checkpoint inhibition (anti-CTLA-4/-PD-1) did further augment antitumor immunity and tumor rejection, which was reflected by reduced Treg numbers and elevated effector function of tumor resident T cells. Ultimately, we were able to demonstrate peripheral immune cell activation and brisk T cell infiltration into tumors in patients previously treated with BrentuximabVedotin. Experiments are currently ongoing to investigate the immunological mode of action of T-DM1 using orthotopic breast cancer models and patients undergoing treatment. Our data reveal a novel mode of action for microtubule-depolymerizing agents and provide a strong rationale for clinical treatment regimens combining these with immune-based therapies. Disclosure All authors have declared no conflicts of interest.

Increased Spinal Cord Na+-K+-2Cl− Cotransporter-1 (NKCC1) Activity Contributes to Impairment of Synaptic Inhibition in Paclitaxel-induced Neuropathic Pain

Microtubule-stabilizing agents, such as paclitaxel (Taxol), are effective chemotherapy drugs for treating many cancers, and painful neuropathy is a major dose-limiting adverse effect. Cation-chloride cotransporters, such as Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) and K(+)-Cl(-) cotransporter-2 (KCC2), critically influence spinal synaptic inhibition by regulating intracellular chloride concentrations. Here we show that paclitaxel treatment in rats significantly reduced GABA-induced membrane hyperpolarization and caused a depolarizing shift in GABA reversal potential of dorsal horn neurons. However, paclitaxel had no significant effect on AMPA or NMDA receptor-mediated glutamatergic input from primary afferents to dorsal horn neurons. Paclitaxel treatment significantly increased protein levels, but not mRNA levels, of NKCC1 in spinal cords. Inhibition of NKCC1 with bumetanide reversed the paclitaxel effect on GABA-mediated hyperpolarization and GABA reversal potentials. Also, intrathecal bumetanide significantly attenuated hyperalgesia and allodynia induced by paclitaxel. Co-immunoprecipitation revealed that NKCC1 interacted with β-tubulin and β-actin in spinal cords. Remarkably, paclitaxel increased NKCC1 protein levels at the plasma membrane and reduced NKCC1 levels in the cytosol of spinal cords. In contrast, treatment with an actin-stabilizing agent had no significant effect on NKCC1 protein levels in the plasma membrane or cytosolic fractions of spinal cords. In addition, inhibition of the motor protein dynein blocked paclitaxel-induced subcellular redistribution of NKCC1, whereas inhibition of kinesin-5 mimicked the paclitaxel effect. Our findings suggest that increased NKCC1 activity contributes to diminished spinal synaptic inhibition and neuropathic pain caused by paclitaxel. Paclitaxel disrupts intracellular NKCC1 trafficking by interfering with microtubule dynamics and associated motor proteins.

Abstract 833: Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol

Abstract There are seven β-tubulin isotypes present in distinct quantities in cells of different origin. Altered expression of β-tubulin isotypes were found in cells resistant to microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, ixabepilone and discodermolide, all of which bind to β-tubulin. For example, the level of βIII-tubulin was very low in the human ovarian cancer cell line, Hey, but increased 8- to 15-fold in two drug resistant daughter cell lines, EpoB8 and Ixab80, respectively. The βV-tubulin level was increased by 2-fold in EpoB8 cells. In contrast, levels of βI- and βIV-tubulin were unchanged and decreased, respectively, in EpoB8. Therefore, it is important to measure the binding affinity of MSAs to different β-tubulin isotypes and study their influence on the drug resistant phenotype. To study the relative binding affinities of MSAs, tubulins from HeLa cells, bovine brain, porcine brain and chicken erythrocytes were specifically photolabeled with the tritium labeled 2-(m-azidobenzoyl)taxol, in the presence and absence of Taxol, Taxotere, epothilone B, ixabepilone and discodermolide. β-tubulin isotype content from these sources was different, particularly for chicken erythrocytes that contain only one β-tubulin isotype, βVI. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulins from different sources. Taxol had a minimal inhibitory effect on HeLa, bovine and porcine brain β-tubulin, but a strong inhibitory effect on chicken erythrocyte β-tubulin. Discodermolide exhibited opposite effects on photolabeling, compared to those of Taxol. However, Taxotere appeared to have a stronger inhibitory effect than Taxol on photolabeling of β-tubulins from all sources tested. Ixabepilone had the strongest inhibitory effect on HeLa β-tubulin. The inhibition pattern for β-tubulin from porcine brain correlated very well with that from bovine brain. To determine drug binding affinities for the different β-tubulin isotypes, tubulins from different sources were photolabeled and the isotypes resolved by high resolution isoelectrofocusing (IEF). Approximately 20 bands were seen with bovine and porcine brain tubulin, but only 3-5 bands were resolved with chicken erythrocyte tubulin. Many bands represent posttranslationally modified tubulins. IEF patterns for bovine and porcine brain tubulin were very similar. All bands were analyzed by mass spectrometry following CNBr digestion, and the identity and relative quantity of each β-tubulin isotype determined. After measuring the radioactivity associated with each band, it was found that the binding affinity of 2-(m-azidobenzoyl)taxol to βIII-tubulin was the lowest, compared to other β-tubulin isotypes. Citation Format: Chia-Ping H. Yang, Hui Xiao, Susan Band Horwitz. Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 833. doi:10.1158/1538-7445.AM2014-833

Abstract 835: Fisetin enhances the efficacy of cabazitaxel chemotherapy in prostate metastatic and multidrug- resistant cancer cells

Abstract Natural compounds that target microtubules and disrupt the normal function of the mitotic spindle have proven to be the drug of choice for cancer chemotherapeutic in clinics. The development of primary and acquired drug resistance to chemotherapy, however, remains a significant clinical concern. Microtubule targeting agents are typically susceptible to drug resistance that is mediated by P-gp, a broad-spectrum efflux pump that removes these compounds from cells, thus counteracting their anticancer effects. Cabazitaxel, a semi-synthetic derivative of natural Taxanes, well known microtubule-stabilizing agents, has been approved for the treatment of metastatic prostate cancer (PCa); however, most patients progress and become chemoresistant, which remains a major challenge in the management of advanced PCa. The development and discovery of new drugs, and exploring new treatment strategies that reduce side effects and circumvent drug resistance could provide more effective therapeutic options for PCa patients. Here, we investigated whether fisetin, a dietary flavonoid, could sensitize PCa cells and a P-gp-overexpressing NCI/ADR-RES, a well-known multidrug-resistant cancer cell line to cabazitaxel treatment. The effect of fisetin in enhancing the response of cancer cells to cabazitaxel chemotherapy was evaluated in several cancer cells. Fisetin exhibited an anti-proliferative effect by enhancing tubulin polymerization and mitotic arrest, which ultimately led to apoptotic cell death. Fisetin increased the expression of microtubule-associated proteins MAP-2 and MAP-4 and the PC-3 and DU-145 cells were enriched in acetylated alpha tubulin, an indication of stabilization of microtubules. Also, fisetin inhibited the migration of PC-3 cell in scratch wound assay. Further, we examined the susceptibility of NCI/ADR-RES cells to fisetin treatment. Fisetin (10-80 µM) induced apoptosis in a dose and time dependent manner. Treatment of cells with a combination of fisetin (10-80 µM) and cabazitaxel (10-120 nM) significantly retarded the growth of PC-3, C4-2, 22Rv1 and multidrug-resistant cancer cells when compared to vehicle control or cabazitaxel or fisetin alone treated cells. Moreover, we found that activation and cleavage of Caspase-3 and PARP are significantly enhanced in cells treated with combination of fisetin and cabazitaxel than with\ each agent alone. This response was also associated with increase in the expression of pro-apoptotic protein Bax and down-regulation of anti-apoptotic Mcl-1. These findings highlight the novel effect of fisetin as a microtubule-stabilizing agent with poor susceptibility to P-gp and provide the first evidence supporting that fisetin could be further developed for sensitizing resistant cancer cells and used as an adjuvant with cabazitaxel for therapy of PCa and potentially other cancer types. Citation Format: Eiman Mukhtar, Vaqar Mustafa Adhami, Hasan Mukhtar. Fisetin enhances the efficacy of cabazitaxel chemotherapy in prostate metastatic and multidrug- resistant cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 835. doi:10.1158/1538-7445.AM2014-835

Thiamet-G-mediated inhibition of O-GlcNAcase sensitizes human leukemia cells to microtubule-stabilizing agent paclitaxel

Although the microtubule-stabilizing agent paclitaxel has been widely used for treatment of several cancer types, particularly for the malignancies of epithelia origin, it only shows limited efficacy on hematological malignancies. Emerging roles of O-GlcNAcylation modification of proteins in various cancer types have implicated the key enzymes catalyzing this reversible modification as targets for cancer therapy. Here, we show that the highly selective O-GlcNAcase (OGA) inhibitor thiamet-G significantly sensitized human leukemia cell lines to paclitaxel, with an approximate 10-fold leftward shift of IC50. Knockdown of OGA by siRNAs or inhibition of OGA by thiamet-G did not influence the cell viability. Furthermore, we demonstrated that thiamet-G binds to OGA in competition with 4-methylumbelliferyl N-acetyl-β-d-glucosaminide dehydrate, an analogue of O-GlcNAc UDP, thereby suppressing the activity of OGA. Importantly, inhibition of OGA by thiamet-G decreased the phosphorylation of microtubule-associated protein Tau and caused alterations of microtubule network in cells. It is noteworthy that paclitaxel combined with thiamet-G resulted in more profound perturbations on microtubule stability than did either one alone, which may implicate the underlying mechanism of thiamet-G-mediated sensitization of leukemia cells to paclitaxel. These findings thus suggest that a regimen of paclitaxel combined with OGA inhibitor might be more effective for the treatment of human leukemia.

Ixabepilone-induced mitochondria and sensory axon loss in breast cancer patients.

BackgroundWe sought to define the clinical and ultrastructure effects of ixabepilone (Ix), a microtubule-stabilizing chemotherapy agent on cutaneous sensory nerves and to investigate a potential mitochondrial toxicity mechanism.MethodsTen breast cancer patients receiving Ix underwent total neuropathy score clinical (TNSc) assessment, distal leg skin biopsies at cycle (Cy) 3 (80–90 mg/m2), Cy5 (160–190 mg/m2), and Cy7 (>200 mg/m2) and were compared to 5 controls. Skin blocks were processed for EM and ultrastructural morphometry of Remak axons done.ResultsAt baseline, Ix-treated subjects had higher TNSc values (4.5 ± 0.8 vs. 0.0 ± 0.0), greater percentage of empty (denervated) Schwann cells (29% vs. 12%), altered axonal diameter (422.9 ± 17 vs. 354.9 ± 14.8 nm, P = 0.01), and axon profiles without mitochondria tended to increase compared to control subjects (71% vs. 70%). With increasing cumulative Ix exposure, an increase in TNSc values (Cy3: 5.4 ± 1.2, Cy7: 10 ± 4, P < 0.001), empty Schwann cells (39% by Cy7), and dilated axons (in nm, Cy3: 506.3 ± 22.1, Cy5: 534.8 ± 33, Cy7: 527.8 ± 24.4; P < 0.001) was observed. In addition, axon profiles without mitochondria (Cy3:74%, Cy7:78%) and mitochondria with abnormal morphology (grade 3 or 4) increased from 24% to 79%. Schwann cells with atypical mitochondria and perineuronal macrophage infiltration in dermis were noted.InterpretationThis study provides functional and structural evidence that Ix exposure induces a dose-dependent toxicity on small sensory fibers with an increase in TNSc scores and progressive axonal loss. Mitochondria appear to bear the cumulative toxic effect and chemotherapy-induced toxicity can be monitored through serial skin biopsy-based analysis.

Axonal transport defects in Alzheimer's disease.

A large body of evidences indicates that axonal transport (AT) defects play an important role in the pathogenesis of Alzheimer' disease (AD). AT, a critical cellular process for the maintenance and function of a neuron, requires components of the cytoskeletons as "tracks", motor proteins and ATP as "driving force", adaptor proteins to ensure the specific connection of the transported cargoes and motor proteins as well as active regulation. In AD pathology, AD-linked pathologic factors respectively perturb the four basic components of AT through different signaling pathways to cause AT defects. Mitochondrial transport, which is different from other transport cargoes, is also impaired via special pathways in AD. In this paper, we review the inhibitory effects of those factors on AT and their possible pathways, indicating these factors act in overlapping, synergistic, and circulating ways. Given the contributions of AT defects to AD, recent therapeutic studies focus on microtubule-stabilizing (MT-stabilizing) agents and alteration in phosphotransferase activities, and we propose more therapeutic strategies targeting AT defects.

Nanotube interactions with microtubules: implications for cancer medicine.

Carbon nanotubes (CNTs) and microtubules are both hollow nanofibers and have similar dimensions; they both self-assemble and form bundles. These common features prompt their association into biosynthetic polymers in vitro and in vivo. Unlike CNTs, microtubules are highly dynamic protein polymers essential for cell proliferation and migration. Interaction between these filaments inside live cells leads to microtubule dysfunction, mitotic arrest and cell death. Thus, CNTs behave as spindle poisons, same as taxanes, vinca alkaloids or epotilones. Recent findings support the idea that CNTs represent a ground-breaking type of synthetic microtubule-stabilizing agents that could play a pivotal role in future cancer treatments in combination to traditional antineoplastic drugs. Here we review the potential use of CNTs in cancer medicine.

Insulin-like growth factor 2 silencing restores taxol sensitivity in drug resistant ovarian cancer.

Drug resistance is an obstacle to the effective treatment of ovarian cancer. We and others have shown that the insulin-like growth factor (IGF) signaling pathway is a novel potential target to overcome drug resistance. The purpose of this study was to validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and to determine the efficacy of targeting IGF2 in vivo. An analysis of The Cancer Genome Atlas (TCGA) data in the serous ovarian cancer cohort showed that high IGF2 mRNA expression is significantly associated with shortened interval to disease progression and death, clinical indicators of drug resistance. In a genetically diverse panel of ovarian cancer cell lines, the IGF2 mRNA levels measured in cell lines resistant to various microtubule-stabilizing agents including Taxol were found to be significantly elevated compared to the drug sensitive cell lines. The effect of IGF2 knockdown on Taxol resistance was investigated in vitro and in vivo. Transient IGF2 knockdown significantly sensitized drug resistant cells to Taxol treatment. A Taxol-resistant ovarian cancer xenograft model, developed from HEY-T30 cells, exhibited extreme drug resistance, wherein the maximal tolerated dose of Taxol did not delay tumor growth in mice. Blocking the IGF1R (a transmembrane receptor that transmits signals from IGF1 and IGF2) using a monoclonal antibody did not alter the response to Taxol. However, stable IGF2 knockdown using short-hairpin RNA in HEY-T30 effectively restored Taxol sensitivity. These findings validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and show that directly targeting IGF2 may be a preferable strategy compared with targeting IGF1R alone.