Microtubule-stabilizing Agent Research Articles

MP14-16 LATE CELL CYCLE SIGNATURES STRATIFY GEMDOCE RESPONSES FOR BCG-UNRESPONSIVE NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP14-16 LATE CELL CYCLE SIGNATURES STRATIFY GEMDOCE RESPONSES FOR BCG-UNRESPONSIVE NON-MUSCLE INVASIVE BLADDER CANCER Andrew Gabrielson, Gabriel Epstein, Mingxiao Feng, Sunil Patel, Max Kates, and Woonyoung Choi Andrew GabrielsonAndrew Gabrielson More articles by this author , Gabriel EpsteinGabriel Epstein More articles by this author , Mingxiao FengMingxiao Feng More articles by this author , Sunil PatelSunil Patel More articles by this author , Max KatesMax Kates More articles by this author , and Woonyoung ChoiWoonyoung Choi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with BCG-unresponsive non-muscle invasive bladder cancer (NMIBC) are commonly treated with intravesical gemcitabine and docetaxel chemotherapy (GEMDOCE). However, studies evaluating biomarkers of GEMDOCE response in BCG-unresponsive NMIBC have been limited. Docetaxel is a microtubule-stabilizing agent that induces cell cycle arrest and apoptosis through mitotic catastrophe. We evaluated whether early and late cell cycle signatures were associated with GEMDOCE response in BCG-unresponsive NMIBC tumors. METHODS: We performed a retrospective study of patients with BCG-unresponsive NMIBC who underwent intravesical GEMDOCE. Monthly maintenance therapy was given for 52% of patients with a 3-month complete response. RNAseq and DNA panel sequencing were performed on FFPE tissues from pre-treatment TURBT samples. Gene set variation analysis (GSVA) was performed to calculate gene set enrichment scores with cell cycle and immune signatures. Cluster analysis was performed to assess whether cell-cycle signatures and other gene signatures were associated with response and recurrence-free survival. RESULTS: A total of 30 patients with BCG-unresponsive NMIBC were included, of whom 63% (19) experienced disease recurrence. Cluster analysis with cell cycle signatures yielded 2 clusters with high expression of either late (cluster 1) or early (cluster 2) cell cycle signatures (Figure 1). Tumors enriched with late cell cycle signatures showed significant recurrence-free survival benefit compared to tumors with high expression of early cell cycle signatures (Figure 2). TP53 alterations were enriched in cluster 1 which demonstrated a clinical benefit from GEMDOCE. In contrast to prior work from our lab on BCG-treated tumors, response in GEMDOCE-treated tumors was not associated with immune signatures. CONCLUSIONS: These early data suggest that there may be molecular signatures that stratify patients with BCG-unresponsive NMIBC treated with GEMDOCE. Further expansion of these cohorts in both BCG-naïve and BCG-unresponsive cohorts is ongoing. Source of Funding: NA © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e188 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Andrew Gabrielson More articles by this author Gabriel Epstein More articles by this author Mingxiao Feng More articles by this author Sunil Patel More articles by this author Max Kates More articles by this author Woonyoung Choi More articles by this author Expand All Advertisement PDF downloadLoading ...

Role of vinca alkaloids and their derivatives in cancer therapy

Vinca alkaloids and its derivatives like Vincristine, Vinblastine etc. isolated from Catharanthus roseus plants are widely used in the treatment of various types of cancers. Mode of action of Vinca alkaloids (vinblastine, vincristine, vinorelbine) is microtubule-stabilizing agents (MTAs) i.e., arrest the cell cycle via disrupting microtubule dynamics. Vincristine major side effect is neurotoxicity. Hoverer, Vincristine induce neuropathy in mice or rat, used as animal model to study effect of drugs or plants by various authors also reported in review literature. Vinca alkaloids and its derivatives were widely used drugs in combination regimens with cyclophosphamide, doxorubicin, procarbazine, Methotrexate and dacarbazine etc. in various types of cancer. In this review, we also discussed major structure modifications position of chemically synthesized vincristine and vinblastine derivatives required for potential anticancer activities. Anticancer mechanism and some major patents on vinca alkaloids derivatives also reported in this review article. The study of Vinca alkaloids and its derivatives a class of an antimitotic compounds, found essential role in anticancer therapies.

Chemical modulation of microtubule structure through the laulimalide/peloruside site

Taxanes are microtubule-stabilizing agents used in the treatment of many solid tumors, but they often involve side effects affecting the peripheral nervous system. It has been proposed that this could be related to structural modifications on the filament upon drug binding. Alternatively, laulimalide and peloruside bind to a different site also inducing stabilization, but they have not been exploited in clinics. Here, we use a combination of the parental natural compounds and derived analogs to unravel the stabilization mechanism through this site. These drugs settle lateral interactions without engaging the M loop, which is part of the key and lock involved in the inter-protofilament contacts. Importantly, these drugs can modulate the angle between protofilaments, producing microtubules of different diameters. Among the compounds studied, we have found some showing low cytotoxicity and able to induce stabilization without compromising microtubule native structure. This opens the window of new applications for microtubule-stabilizing agents beyond cancer treatment.

Taccalonolides: Structure, semi-synthesis, and biological activity.

Microtubules are the fundamental part of the cell cytoskeleton intimately involving in cell proliferation and are superb targets in clinical cancer therapy today. Microtubule stabilizers have become one of the effectively main agents in the last decades for the treatment of diverse cancers. Taccalonolides, the highly oxygenated pentacyclic steroids isolated from the genus of Tacca, are considered a class of novel microtubule-stabilizing agents. Taccalonolides not only possess a similar microtubule-stabilizing activity as the famous drug paclitaxel but also reverse the multi-drug resistance of paclitaxel and epothilone in cellular and animal models. Taccalonolides have captured numerous attention in the field of medicinal chemistry due to their variety of structures, unique mechanism of action, and low toxicity. This review focuses on the structural diversity, semi-synthesis, modification, and pharmacological activities of taccalonolides, providing bright thoughts for the discovery of microtubule-stabilizing drugs.

In Vivo Evaluation of (-)-Zampanolide Demonstrates Potent and Persistent Antitumor Efficacy When Targeted to the Tumor Site.

Microtubule-stabilizing agents (MSAs) are a class of compounds used in the treatment of triple-negative breast cancer (TNBC), a subtype of breast cancer where chemotherapy remains the standard-of-care for patients. Taxanes like paclitaxel and docetaxel have demonstrated efficacy against TNBC in the clinic, however new classes of MSAs need to be identified due to the rise of taxane resistance in patients. (−)-Zampanolide is a covalent microtubule stabilizer that can circumvent taxane resistance in vitro but has not been evaluated for in vivo antitumor efficacy. Here, we determine that (−)-zampanolide has similar potency and efficacy to paclitaxel in TNBC cell lines, but is significantly more persistent due to its covalent binding. We also provide the first reported in vivo antitumor evaluation of (−)-zampanolide where we determine that it has potent and persistent antitumor efficacy when delivered intratumorally. Future work on zampanolide to further evaluate its pharmacophore and determine ways to improve its systemic therapeutic window would make this compound a potential candidate for clinical development through its ability to circumvent taxane-resistance mechanisms.

P-108 A phase II study of weekly paclitaxel in patients with advanced or recurrent esophageal cancer who had previously received docetaxel-containing chemotherapy

Both docetaxel and paclitaxel are a class of microtubule-stabilizing anticancer agents. Partial cross-resistance between docetaxel and paclitaxel was indicated in breast and ovary cancers. In advanced gastric cancer, retrospective study showed comparable efficacy of subsequent weekly paclitaxel with or without prior docetaxel-based chemotherapy. Therefore, we evaluated the efficacy and safety of weekly paclitaxel in patients with advanced or recurrent esophageal cancer who had previously received docetaxel-containing chemotherapy.

Manganese-enhanced magnetic resonance imaging method detects age-related impairments in axonal transport in mice and attenuation of the impairments by a microtubule-stabilizing compound

In this study a manganese-enhanced magnetic resonance imaging (MEMRI) method was developed for mice for measuring axonal transport (AXT) rates in real time in olfactory receptor neurons, which project from the olfactory epithelium to the olfactory neuronal layer of the olfactory bulb. Using this MEMRI method, two major experiments were conducted: 1) an evaluation of the effects of age on AXT rates and 2) an evaluation of the brain-penetrant, microtubule-stabilizing agent, Epothilone D for effect on AXT rates in aged mice. In these studies, we improved upon previous MEMRI approaches to develop a method where real-time measurements (32 time points) of AXT rates in mice can be determined over a single (approximately 100 min) scanning session. In the age comparisons, AXT rates were significantly higher in young (mean age ∼4.0 months old) versus aged (mean age ∼24.5 months old) mice. Moreover, in aged mice, eight weeks of treatment with Epothilone D, (0.3 and 1.0 mg/kg) was associated with statistically significant increases in AXT rates compared to vehicle-treated subjects. These experiments conducted in a living mammalian model (i.e., wild type, C57BL/6 mice), using a new modified MEMRI method, thus provide further evidence that the process of aging leads to decreases in AXT rates in the brain and they further support the argument that microtubule-based therapeutic strategies designed to improve AXT rates have potential for age-related neurological disorders.

Photopharmacology of Antimitotic Agents.

Antimitotic agents such as the clinically approved vinca alkaloids, taxanes and epothilone can arrest cell growth during interphase and are therefore among the most important drugs available for treating cancer. These agents suppress microtubule dynamics and thus interfere with intracellular transport, inhibit cell proliferation and promote cell death. Because these drugs target biological processes that are essential to all cells, they face an additional challenge when compared to most other drug classes. General toxicity can limit the applicable dose and therefore reduce therapeutic benefits. Photopharmacology aims to avoid these side-effects by introducing compounds that can be applied globally to cells in their inactive form, then be selectively induced to bioactivity in targeted cells or tissue during a defined time window. This review discusses photoswitchable analogues of antimitotic agents that have been developed by combining different photoswitchable motifs with microtubule-stabilizing or microtubule-destabilizing agents.

Antiproliferative and Microtubule-stabilizing Activities of Two Iboga-vobasine Bisindoles Alkaloids from Tabernaemontana corymbosa in Colorectal Adenocarcinoma HT-29 Cells.

Two iboga-vobasine bisindoles, 16'-decarbomethoxyvoacamine (1: ) and its 19,20-dihydro derivative, 16'-decarbomethoxydihydrovoacamine (2: ) from Tabernaemontana corymbosa exhibited potent cytotoxicity against the human colorectal adenocarcinoma HT-29 cells in our previous studies. Bisindoles 1: and 2: selectively inhibited the growth of HT-29 cells without significant cytotoxicity to normal human colon fibroblasts CCD-18Co. Treatment with bisindoles 1: and 2: suppressed the formation of HT-29 colonies via G0/G1 cell cycle arrest and induction of mitochondrial apoptosis. Owing to its higher antiproliferative activity, bisindole 2: was chosen for the subsequent studies. Bisindole 2: inhibited the formation of HT-29 spheroids (tumor-like cell aggregates) in 3D experiments in a dose-dependent manner, while an in vitro tubulin polymerization assay and molecular docking analysis showed that bisindole 2: is a microtubule-stabilizing agent which is predicted to bind at the β-tubulin subunit at the taxol-binding site. The binding resulted in the generation of ROS, which consequently activated the oxidative stress-related cell cycle arrest and apoptotic pathways, viz., JNK/p38, p21Cip1/Chk1, and p21Cip1/Rb/E2F, as shown by microarray profiling.

Epothilone D Modulates Autism-like Behaviors in the BTBR Mouse Model of Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, characterized by impaired social communication, abnormal repetitive behaviors and restricted interests and/or sensory behaviors. It has been widely accepted that ASD involves a complex interplay of both genetic and environmental risk factors. Existing medications are only symptomatic treatments, there are no effective treatments that can improve these core social behavior deficits. Recent studies indicated that synaptic development and abnormal myelination are linked to the pathogenesis of ASD. The stable tubule only polypeptide (STOP) protein, also known as microtubule-associated protein 6, plays an important role in neuronal development and synaptic plasticity. Our previous studies showed that STOP protein was significantly reduced in the plasma of autistic subjects and in the cortex of BTBR T+ Itpr3tf (BTBR) mouse model of ASD. Furthermore, studies have shown that Epothilone D, a taxol-like microtubule-stabilizing agent, could alleviate behavioral and synaptic deficits in STOP-null mice. Here, we further evaluate whether Epothilone D treatment is sufficient to modulate the autism-like behaviors in the BTBR mice, and explore the underlying mechanism. BTBR mice were treated either with Epothilone D dissolved in 99% dimethyl sulfoxide (DMSO) or with 99% DMSO vehicle. Our studies demonstrated that the restricted and repetitive behaviors of BTBR mice were improved after Epothilone D treatment, which could be achieved by improving microtubule stability and further regulating the expression of excitatory synapse-related and myelin-related proteins. These results indicate that microtubule stability may be a new and promising therapeutic target for treating patients with ASD.

Paclitaxel Induces Epidermal Molecular Changes and Produces Subclinical Alterations in the Skin of Gynecological Cancer Patients.

Simple SummarySkin toxicity is one of paclitaxel’s adverse effects. However, its real impact on the skin could be underestimated as these alterations can also appear asymptomatic. We have observed that paclitaxel modifies gene and protein expression of skin markers in a 3D epidermis model, and impairs physical, physiological, and biomechanical properties of the skin in gynecologic cancer patients. These subclinical alterations might be avoided by using prophylactic measures during treatment to prevent possible future adverse reactions.Background: Paclitaxel is a microtubule-stabilizing chemotherapeutic agent. Despite its widespread use, it damages healthy tissues such as skin. The goal of this study was to prove that the real impact of paclitaxel-induced skin toxicity could be underestimated because the adverse events might appear asymptomatic. Methods: Gynecological cancer patients were recruited. Skin parameters measurements were taken after three and six paclitaxel cycles. Measurements were conducted using specific probes which measure hydration, transepidermal water loss (TEWL), sebum, elasticity and firmness, erythema, roughness, smoothness, skin thickness, and desquamation levels. Further, a 3D epidermis model was incubated with paclitaxel to analyze gene and protein expression of aquaporin 3, collagen type 1, elastin, and fibronectin. Results: Paclitaxel induced alterations in the skin parameters with no visible clinical manifestations. Gynecological cancer patients under paclitaxel treatment had a decrease in hydration, TEWL, sebum, elasticity, and thickness of the skin, while erythema, roughness, and desquamation were increased. The molecular markers, related to hydration and the support of the skin layers, and analyzed in the 3D epidermis model, were decreased. Conclusions: Results suggest that paclitaxel modifies gene and protein expression of skin-related molecular markers, and impairs different physical, physiological, and biomechanical properties of the skin of cancer patients at a subclinical level.

Abstract P4-01-08: Morphological and functional plasticity of mitochondria in chemoresistant triple negative breast cancer

Abstract BACKGROUND: Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in only 45% of patients. TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. Therefore, understanding the mechanisms of resistance to standard chemotherapy is essential. Our previous studies found mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual tumor cells that survived after NACT. Mitochondria are morphologically plastic and dynamic organelles that continuously cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. Mitochondrial fusion is thought to support OXPHOS through maintenance of the mitochondrial genome (mtDNA) which encodes 13 subunits of electron transport chain complexes. On the other hand, mitochondrial fission can be the precursor to mitophagy, the selective degradation of damaged mitochondria. We are investigating how mitochondrial structure dynamics impact metabolic states driving chemoresistance in TNBC. Methods We used a previously characterized orthotopic patient-derived xenograft (PDX) mouse model derived from the primary tumor of a treatment-naïve metastatic TNBC patient. After treatment with a standard NACT regimen, combined Adriamycin (aka doxorubicin) and cyclophosphamide (AC), we observed initial partial response followed by tumor regrowth. We collected PDX tumors at three time points: pre-treatment, post-AC residual (when tumors reached the volume nadir), and post-AC tumors that later regrew to the starting tumor volume. We used transmission electron microscopy (TEM) and immunohistochemical (IHC) analysis of human mitochondria to assess mitochondrial morphology and mass, respectively. For in vitro assays, we assessed mitochondria morphology, mtDNA content, and OXPHOS activity in TNBC cells treated with two different types of chemotherapeutic drugs, DNA-damaging agents (doxorubicin and carboplatin) and a microtubule-stabilizing agent (paclitaxel). To examine metabolic adaptations upon chemotherapy, we conducted 13C-glucose and glutamine flux metabolomics. To pharmaceutically perturb mitochondrial morphology, we used Mdivi-1 and Silibinin, known to induce mitochondrial fusion and fission, respectively. We assessed cell growth by Incucyte real-time imaging. Results: Analyses of residual PDX tumors after in vivo AC treatment revealed increased mitochondrial content, and increased average mitochondrial length in residual tumor cells. Metabolomic analysis of TNBC cells revealed all three chemotherapies induced glucose, but not glutamine, flux through the TCA cycle. Flux through glycolysis was not affected by chemotherapies. Treatment with doxorubicin and carboplatin, increased mitochondrial elongation, mtDNA content, and OXPHOS activity. Conversely, paclitaxel treatment reduced mitochondrial length and OXPHOS activity. We observed Mdivi-1 induced fusion concomitant with increased OXPHOS activity, while treatment with Silibinin reduced fusion and decreased OXPHOS. We next tested if induction of mitochondrial fission enhanced chemosensitivity. Sequential treatment of TNBC cells with DNA-damaging chemotherapeutics followed by Silibinin perturbed OXPHOS and resulted in enhanced cell killing. Conclusion: These findings establish a functional role for mitochondrial structure in chemotherapeutic response and metabolic reprogramming, which may confer a survival advantage to TNBC cells. Given the increased chemosensitivity we observed after pharmacologic inhibition of mitochondrial fusion, our study suggests perturbing mitochondrial structure adaptations may provide an opportunity to overcome TNBC chemoresistance in the neoadjuvant setting. Citation Format: Lily Mokryun Baek, Junegoo Lee, James P. Barrish, Bora Lim, Jeffrey T. Chang, Phillip L. Lorenzi, Weston Porter, Gloria V. Echeverria. Morphological and functional plasticity of mitochondria in chemoresistant triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-08.

Paclitaxel-Induced Epidermal Alterations: An In Vitro Preclinical Assessment in Primary Keratinocytes and in a 3D Epidermis Model.

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent approved for the treatment of ovarian, non-small cell lung, head, neck, and breast cancers. Despite its beneficial effects on cancer and widespread use, paclitaxel also damages healthy tissues, including the skin. However, the mechanisms that drive these skin adverse events are not clearly understood. In the present study, we demonstrated, by using both primary epidermal keratinocytes (NHEK) and a 3D epidermis model, that paclitaxel impairs different cellular processes: paclitaxel increased the release of IL-1α, IL-6, and IL-8 inflammatory cytokines, produced reactive oxygen species (ROS) release and apoptosis, and reduced the endothelial tube formation in the dermal microvascular endothelial cells (HDMEC). Some of the mechanisms driving these adverse skin events in vitro are mediated by the activation of toll-like receptor 4 (TLR-4), which phosphorylate transcription of nuclear factor kappa B (NF-κb). This is the first study analyzing paclitaxel effects on healthy human epidermal cells with an epidermis 3D model, and will help in understanding paclitaxel’s effects on the skin.

Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes

Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and the subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within the microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end.

A comprehensive assessment of a new series of 5',6'-difluorobenzotriazole-acrylonitrile derivatives as microtubule targeting agents (MTAs).

Microtubules (MTs) are the principal target for drugs acting against mitosis. These compounds, called microtubule targeting agents (MTAs), cause a mitotic arrest during G2/M phase, subsequently inducing cell apoptosis. MTAs could be classified in two groups: microtubule stabilising agents (MSAs) and microtubule destabilising agents (MDAs). In this paper we present a new series of (E) (Z)-2-(5,6-difluoro-(1H)2H-benzo[d] [1,2,3]triazol-1(2)-yl)-3-(R)acrylonitrile (9a-j, 10e, 11a,b) and (E)-2-(1H-benzo[d] [1,2,3]triazol-1-yl)-3-(R)acrylonitrile derivatives (13d,j), which were recognised to act as MTAs agents. They were rationally designed, synthesised, characterised and subjected to different biological assessments. Computational docking was carried out in order to investigate the potential binding to the colchicine-binding site on tubulin. From this first prediction, the di-fluoro substitution seemed to be beneficial for the binding affinity with tubulin. The new fluorine derivatives, here presented, showed an improved antiproliferative activity when compared to the previously reported compounds. The biological evaluation included a preliminary antiproliferative screening on NCI60 cancer cells panel (1-10μM). Compound 9a was selected as lead compound of the new series of derivatives. The invitro XTT assay, flow cytometry analysis and immunostaining performed on HeLa cells treated with 9a showed a considerable antiproliferative effect, (IC50=3.2μM), an increased number of cells in G2/M-phase, followed by an enhancement in cell division defects. Moreover, β-tubulin staining confirmed 9a as a MDA triggering tubulin disassembly, whereas colchicine-9a competition assay suggested that compound 9a compete with colchicine for the binding site on tubulin. Then, the co-administration of compound 9a and an extrusion pump inhibitor (EPI) was investigated: the association resulted beneficial for the antiproliferative activity and compound 9a showed to be client of extrusion pumps. Finally, structural superimposition of different colchicine binding site inhibitors (CBIs) in clinical trial and our MDA, provided an additional confirmation of the targeting to the predicted binding site. Physicochemical, pharmacokinetic and druglikeness predictions were also conducted and all the newly synthesised derivatives showed to be drug-like molecules.

UPLC‐PDA coupled HR‐TOF ESI/MS 2 ‐based identification of derivatives produced by whole‐cell biotransformation of epothilone A using Nocardia sp. CS692 and a cytochrome P450 overexpressing strain

Epothilone A, a microtubule-stabilizing agent used as therapeutics for the treatment of cancers, was biotransformed into three metabolites using Nocardia sp. CS692 and recombinant Nocardia overexpressing a cytochrome P450 from Streptomyces venezuelae (PikC). Among three metabolites produced in the biotransformation reaction mixtures, ESI/MS2 analysis predicted two metabolites (M1 and M2) as novel hydroxylated derivatives (M1 is hydroxylated at the C-8 position and M2 is hydroxylated at C-10 position), each with an opened-epoxide ring in their structure. Interestingly, metabolite M3 lacks an epoxide ring and is known as deoxyepothilone A, which is also called epothilone C. Metabolite M1 was produced only in PikC overexpressing strain. The endogenous enzymes of Nocardia sp. catalyzed hydroxylation of epothilone A to produce metabolite M2 and removed epoxide ring to produce metabolite M3. All the metabolites were identified based on UV-vis analysis and rigorous ESI/MS2 fragmentation based on epothilone A standard. The newly produced metabolites are anticipated to display novel cytotoxic effects and could be subjects of further pharmacological studies.

Randomized phase II trial of weekly ixabepilone with or without biweekly bevacizumab for platinum-resistant or refractory ovarian/fallopian tube/primary peritoneal cancer

<h3>Objectives:</h3> Ixabepilone is a microtubule-stabilizing agent that may retain activity in paclitaxel-treated patients. The goal of this multicenter randomized phase II study was to assess the activity and safety of ixabepilone with bevacizumab compared to ixabepilone alone in patients with platinum-resistant/refractory ovarian, fallopian tube, or primary peritoneal cancer. An exploratory objective was to examine the role of prior treatment with bevacizumab and tumor expression of class III ß-tubulin (TUBB3) by immunohistochemistry as a predictive biomarker. <h3>Methods:</h3> Participants were randomly assigned to receive ixabepilone 20 mg/m² days 1, 8, 15 with (IXA+BEV) or without (IXA) bevacizumab 10 mg/kg days 1, 15 every 28 days. Patients were stratified by receipt of prior BEV. The primary endpoint was progression-free survival (PFS). Overall survival (OS), safety, and response rates served as secondary endpoints. <h3>Results:</h3> A total of 78 patients were randomized from March 2017-July 2020. Among 76 evaluable patients who received IXA+BEV (n=39) compared to IXA (n=37), the objective response rate was 33% (n=13) versus 8% (n=3) (<i>P</i>=0.004), with clinical benefit durable at 6 months in 37% (n=14) and 3% (n=1) (<i>P</i><0.001). The addition of BEV significantly improved both PFS (median 5.5 vs 2.2 months, HR=0.33, 95% CI 0.19-0.55, <i>P</i><0.001) [Fig. 1a] and OS (median 10.0 vs 6.0 months, HR=0.52, 95% CI 0.31-0.87, <i>P</i>=0.006) [Fig. 1b]. Both regimens were well-tolerated. TUBB3 expression did not predict response in either arm. Subgroup analyses revealed minimal effect of prior BEV on response to IXA+BEV [Fig 1c/d]. <h3>Conclusions:</h3> IXA+BEV is a well-tolerated, effective combination for treatment of platinum/taxane-resistant/refractory ovarian cancer that extends both PFS/OS relative to IXA monotherapy. Prior receipt of BEV should not preclude use of IXA+BEV. TUBB3 is not a predictive biomarker for response to IXA+BEV.

Microtubule Stabilization Protects Hypothermia-Induced Damage to the Cytoskeleton and Barrier Integrity of the Corneal Endothelial Cells.

Purpose: To determine the impact of hypothermia on the barrier function of donor corneal endothelium, thereby enhancing the success of corneal transplantation. Methods: Primary cultures of porcine endothelial cells were subjected to hypothermia (15 h; 4°C). The impact on microtubule assembly, peri-junctional actomyosin ring (PAMR), and ZO-1 was assessed by immunocytochemistry with and without pretreatment with a microtubule-stabilizing agent (Epothilone B; EpoB; 100 nM) and a p38 MAP kinase inhibitor (SB-203580; 20 μM). In addition, EpoB-loaded PLGA nanoparticles (ENPs) prepared by nanoprecipitation technique and coated with poly-L-lysine (PLL-ENPs) were administered one-time for sustained intracellular delivery of EpoB. Results: Exposure to hypothermia led to microtubule disassembly concomitant with the destruction of PAMR and the displacement of ZO-1 at the cellular periphery, suggesting a loss in barrier integrity. These adverse effects were attenuated by pretreatment with EpoB or SB-203580. PLL-ENPs possessed a zeta potential of ∼26 mV and a size of ∼110 nm. Drug loading and entrapment efficiency were 5% (w/w) and ∼87%, respectively, and PLL-ENPs showed a biphasic release in vitro: burst phase (1 day), followed by a sustained phase (∼4 weeks). Pretreatment with PLL-ENPs (0.4 mg/mL) for 24 h stabilized the microtubules and opposed the hypothermia-induced damage to PAMR and the redistribution of ZO-1. Conclusions: Hypothermia induces microtubule disassembly via activation of p38 MAP kinase and subsequently breaks down the barrier function of the endothelium. Sustained intracellular delivery of EpoB using nanoparticles has the potential to overcome endothelial barrier failure during prolonged cold storage of donor cornea.

The Bisindole Alkaloids Angustilongines M and A from Alstonia penangiana Induce Mitochondrial Apoptosis and G0/G1 Cell Cycle Arrest in HT-29 Cells through Promotion of Tubulin Polymerization.

A new linearly fused macroline-sarpagine bisindole, angustilongine M (1), was isolated from the methanolic extract of Alstonia penangiana. The structure of the alkaloid was elucidated based on analysis of the spectroscopic data, and its biological activity was evaluated together with another previously reported macroline-akuammiline bisindole from the same plant, angustilongine A (2). Compounds 1 and 2 showed pronounced in vitro growth inhibitory activity against a wide panel of human cancer cell lines. In particular, the two compounds showed potent and selective antiproliferative activity against HT-29 cells, as well as strong growth inhibitory effects against HT-29 spheroids. Cell death mechanistic studies revealed that the compounds induced mitochondrial apoptosis and G0/G1 cell cycle arrest in HT-29 cells in a time-dependent manner, while in vitro tubulin polymerization assays and molecular docking analysis showed that the compounds are microtubule-stabilizing agents, which are predicted to bind at the β-tubulin subunit at the Taxol-binding site.

Anti-cancer effects of Fenbendazole and Paclitaxel combination on HL-60 cells

Fenbendazole (FBZ) is one of the safest anthelmintic drugs. FBZ has been found to have anti-cancer effects by destabilizing microtubules. In this study, a synergistic effect of paclitaxel (PA), a microtubule-stabilizing anti-cancer agent, and FBZ was investigated on HL-60 cells, a human leukemia cell line. The metabolic activity of cells significantly decreased and the nucleus morphology upon the treatment of FBZ and PA based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and Hoechst 33342 staining, respectively. To investigate the involvement of reactive oxygen species (ROS), the metabolic activity of the cells after treatment of N-acetyl-L-cysteine (NAC) was measured. Indeed, NAC significantly increased the metabolic activity of the cells treated with FBZ and PA, suggesting that both drugs affect at least in part via ROS. Furthermore, FBZ and PA increased cell death in an annexin V- fluorescein isothiocyanate/propidium iodide staining assay. Taken together, FBZ and PA have a synergistic anti-cancer activity on HL-60 cells at a certain concentration. These results may provide researchers and clinicians in cancer-related fields with some valuable information to broaden the use of FBZ.