Disruption Of Microtubule Dynamics Research Articles

Anticancer role of flubendazole: Effects and molecular mechanisms (Review).

Flubendazole, an anthelmintic agent with a well-established safety profile, has emerged as a promising anticancer drug that has demonstrated efficacy against a spectrum of cancer types over the past decade. Its anticancer properties encompass a multifaceted mechanism of action, including the inhibition of cancer cell proliferation, disruption of microtubule dynamics, regulation of cell cycle, autophagy, apoptosis, suppression of cancer stem cell characteristics, promotion of ferroptosis and inhibition of angiogenesis. The present review aimed to provide a comprehensive overview of the molecular underpinnings of the anticancer activity of flubendazole, highlighting key molecules and regulatory pathways. Given the breadth of the potential of flubendazole, further research is imperative to identify additional cancer types sensitive to flubendazole, refine experimental methodologies for enhancing its reliability, uncover synergistic drug combinations, improve its bioavailability and explore innovative administration methods. The present review provided a foundation for future studies on the role of flubendazole in oncology and described its molecular mechanisms of action.

Sulfamoylated Estradiol Analogs Targeting the Actin and Microtubule Cytoskeletons Demonstrate Anti-Cancer Properties In Vitro and In Ovo.

The microtubule-disrupting agent 2-methoxyestradiol (2-ME) displays anti-tumor and anti-angiogenic properties, but its clinical development is halted due to poor pharmacokinetics. We therefore designed two 2-ME analogs in silico-an ESE-15-one and an ESE-16 one-with improved pharmacological properties. We investigated the effects of these compounds on the cytoskeleton in vitro, and their anti-angiogenic and anti-metastatic properties in ovo. Time-lapse fluorescent microscopy revealed that sub-lethal doses of the compounds disrupted microtubule dynamics. Phalloidin fluorescent staining of treated cervical (HeLa), metastatic breast (MDA-MB-231) cancer, and human umbilical vein endothelial cells (HUVECs) displayed thickened, stabilized actin stress fibers after 2 h, which rearranged into a peripheral radial pattern by 24 h. Cofilin phosphorylation and phosphorylated ezrin/radixin/moesin complexes appeared to regulate this actin response. These signaling pathways overlap with anti-angiogenic, extra-cellular communication and adhesion pathways. Sub-lethal concentrations of the compounds retarded both cellular migration and invasion. Anti-angiogenic and extra-cellular matrix signaling was evident with TIMP2 and P-VEGF receptor-2 upregulation. ESE-15-one and ESE-16 exhibited anti-tumor and anti-metastatic properties in vivo, using the chick chorioallantoic membrane assay. In conclusion, the sulfamoylated 2-ME analogs displayed promising anti-tumor, anti-metastatic, and anti-angiogenic properties. Future studies will assess the compounds for myeloproliferative effects, as seen in clinical applications of other drugs in this class.

Discovery of Novel Diaryl-Substituted Fused Heterocycles Targeting Katanin and Tubulin with Potent Antitumor and Antimultidrug Resistance Efficacy.

Disrupting microtubule dynamics has emerged as a promising strategy for cancer treatment. However, drug resistance remains a challenge hindering the development of microtubule-targeting agents. In this work, a novel class of diaryl substituted fused heterocycles were designed, synthesized, and evaluated, which were demonstrated as effective dual katanin and tubulin regulators with antitumor activity. Following three rounds of stepwise optimization, compound 21b, featuring a 3H-imidazo[4,5-b]pyridine core, displayed excellent targeting capabilities on katanin and tubulin, along with notable antiproliferative and antimetastatic effects. Mechanistic studies revealed that 21b disrupts the microtubule network in tumor cells, leading to G2/M cell cycle arrest and apoptosis induction. Importantly, 21b exhibited significant inhibition of tumor growth in MDA-MB-231 and A549/T xenograft tumor models without evident toxicity and side effects. In conclusion, compound 21b presents a novel mechanism for disrupting microtubule dynamics, warranting further investigation as a dual-targeted antitumor agent with potential antimultidrug resistance properties.

Pathogenic variants in autism gene KATNAL2 cause hydrocephalus and disrupt neuronal connectivity by impairing ciliary microtubule dynamics

Enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles (cerebral ventriculomegaly), the cardinal feature of congenital hydrocephalus (CH), is increasingly recognized among patients with autism spectrum disorders (ASD). KATNAL2, a member of Katanin family microtubule-severing ATPases, is a known ASD risk gene, but its roles in human brain development remain unclear. Here, we show that nonsense truncation of Katnal2 (Katnal2Δ17) in mice results in classic ciliopathy phenotypes, including impaired spermatogenesis and cerebral ventriculomegaly. In both humans and mice, KATNAL2 is highly expressed in ciliated radial glia of the fetal ventricular-subventricular zone as well as in their postnatal ependymal and neuronal progeny. The ventriculomegaly observed in Katnal2Δ17 mice is associated with disrupted primary cilia and ependymal planar cell polarity that results in impaired cilia-generated CSF flow. Further, prefrontal pyramidal neurons in ventriculomegalic Katnal2Δ17 mice exhibit decreased excitatory drive and reduced high-frequency firing. Consistent with these findings in mice, we identified rare, damaging heterozygous germline variants in KATNAL2 in five unrelated patients with neurosurgically treated CH and comorbid ASD or other neurodevelopmental disorders. Mice engineered with the orthologous ASD-associated KATNAL2 F244L missense variant recapitulated the ventriculomegaly found in human patients. Together, these data suggest KATNAL2 pathogenic variants alter intraventricular CSF homeostasis and parenchymal neuronal connectivity by disrupting microtubule dynamics in fetal radial glia and their postnatal ependymal and neuronal descendants. The results identify a molecular mechanism underlying the development of ventriculomegaly in a genetic subset of patients with ASD and may explain persistence of neurodevelopmental phenotypes in some patients with CH despite neurosurgical CSF shunting.

Paclitaxel Overload Supramolecular Oxidative Stress Nanoamplifier with a CDK12 Inhibitor for Enhanced Cancer Therapy.

Combination therapy has emerged as a promising approach for treating tumors, although there is room for improvement. This study introduced a novel strategy that combined the enhancement of apoptosis, ferroptosis, and DNA damage to improve therapeutic outcomes for prostate cancer. Specifically, we have developed a supramolecular oxidative stress nanoamplifier, which was comprised of β-cyclodextrin, paclitaxel, and ferrocene-poly(ethylene glycol). Paclitaxel within the system disrupted microtubule dynamics, inducing G2/M phase arrest and apoptosis. Concurrently, ferrocene utilized hydrogen peroxide to generate toxic hydroxyl radicals in cells through the Fenton reaction, triggering a cascade of reactive oxygen species expansion, reduction of glutathione levels, lipid peroxidation, and ferroptosis. The increased number of hydroxyl radicals and the inhibitory effect of THZ531 on DNA repair mechanisms exacerbated DNA damage within tumor cells. As expected, the supramolecular nanoparticles demonstrated excellent drug delivery ability to tumor cells or tissues, exhibited favorable biological safety in vivo, and enhanced the killing effect on prostate cancer.

Abstract PO3-25-10: Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics

Abstract Background: Microtubule-targeting agents (MTAs) have been widely utilized in cancer treatment, but the development of novel MTAs has faced limitations. CMPD1, a highly selective inhibitor of MAP kinase-activated protein kinase 2 (MK2) with dual functions as a microtubule inhibitor, shows promise as a targeted therapy for triple-negative breast cancer (TNBC). This study aimed to investigate the effects of CMPD1, targeting MK2 and acting as a microtubule inhibitor, on cell cycle progression, microtubule dynamics, and TNBC-related processes. Methods: CMPD1 was evaluated in non-transformed retinal pigment epithelial cells (RPE1) and TNBC cell lines, including MDA-MB-231 and MDA-MB-468 cells. Time-lapse microscopy and immunofluorescence staining were employed to observe the effects of CMPD1 on mitotic progression, cellular morphology, activation of the spindle assembly checkpoint (SAC), and microtubule dynamics. Anchorage-independent growth assays and in vivo studies using a TNBC xenograft model were performed to assess the impact of CMPD1 on colony formation, tumor growth, and metastasis. Results: CMPD1 treatment induced prometaphase arrest and mitotic slippage in both non-transformed and TNBC cells. This arrest was associated with SAC activation, evidenced by increased levels of Mad1 at kinetochores. CMPD1 exhibited robust microtubule depolymerization, primarily targeting microtubule plus-ends and inhibiting microtubule polymerization in vitro. Furthermore, CMPD1 disrupted the organization of the microtubule network in interphase cells and perturbed the mitotic spindle during mitosis. Additionally, CMPD1 treatment resulted in the formation of cytoplasmic protrusions, membrane deformations, and dynamic movement of clustered mitotic chromosomes. These morphological alterations were indicative of the impact of CMPD1 on cellular structures and processes during mitosis. Notably, CMPD1 demonstrated significant efficacy in inhibiting anchorage-independent growth in TNBC cells, highlighting its potential as an anti-TNBC agent. In the TNBC xenograft model, CMPD1 exhibited superior anti-tumor activity compared to the standard chemotherapeutic agent Taxol. Tumor growth was markedly suppressed, and the formation of metastatic lesions was significantly reduced upon CMPD1 treatment. Furthermore, CMPD1 treatment effectively inhibited TNBC cell migration and invasion, likely through its modulation of microtubule dynamics. The disruption of microtubule dynamics by CMPD1 impaired the ability of TNBC cells to migrate and invade surrounding tissues, which are crucial processes in cancer metastasis. Importantly, CMPD1 showed high selectivity for TNBC cells, exerting minimal cytotoxic effects on normal cells, and could be efficiently washed out. This selectivity and washout ability enhance its potential as a targeted therapeutic agent for TNBC treatment. Conclusions: CMPD1, a highly selective MK2 inhibitor and microtubule inhibitor, exerts profound effects on cell cycle progression, microtubule dynamics, and TNBC-related processes. It induces prometaphase arrest, mitotic slippage, and microtubule depolymerization, resulting in altered cellular morphology. CMPD1 displays high selectivity for TNBC cells, while sparing normal cells and allowing efficient washout, making it a promising therapeutic agent for TNBC treatment. Its inhibition of anchorage-independent growth, suppression of tumor growth in vivo, and ability to impair TNBC cell migration and invasion further underscore its potential as a targeted anti-TNBC therapy. Citation Format: Mamoru Takada, Yu-Chia Chen, Yu Muhan, Hideyuki Yamada, Junta Sakakibara, Hiroshi Fujimoto, Takeshi Nagashima, Masayuki Otsuka, Motoki Takaku, Aussie Suzuki. Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-10.

Interplay between paclitaxel, gap junctions, and kinases: unraveling mechanisms of action and resistance in cancer therapy.

This comprehensive review elucidates the multifaceted roles of paclitaxel, a key chemotherapeutic agent, in cancer therapy, with a focus on its interactions with gap junctions and related kinases. Paclitaxel, with its complex diterpene structure, mediates its anticancer effects predominantly through specific interactions with β-tubulin, instigating cell cycle arrest and triggering various cell death pathways, including apoptosis, pyroptosis, ferroptosis, and necroptosis. The paper systematically delineates the chemical attributes and action mechanisms of paclitaxel and its analogs, underscoring their capacity to disrupt microtubule dynamics, thereby leading to mitotic arrest and subsequent cell death induction. It also scrutinizes the pivotal role of gap junctions, composed of connexin proteins, in the modulation of cancer cell behavior and chemoresistance, especially in the milieu of paclitaxel administration. The review articulates how gap junctions can either suppress tumors or contribute to cancer progression, thereby influencing chemotherapy outcomes. Furthermore, the paper provides an in-depth analysis of how paclitaxel modulates gap junction-associated kinases via phosphorylation, influencing the drug's therapeutic efficacy and resistance profiles. By integrating insights from numerous key studies, the review offers a comprehensive understanding of the interplay between paclitaxel, gap junctions, and kinases, shedding light on potential approaches to augment paclitaxel's anti-tumor effectiveness and counteract chemoresistance in cancer treatment.

Vinca alkaloids as a potential cancer therapeutics: recent update and future challenges.

Vinca alkaloids including vincristine, vinblastine, vindesine, and vinflunine are chemotherapeutic compounds commonly used to treat various cancers. Vinca alkaloids are one of the first microtubule-targeting agents to be produced and certified for the treatment of hematological and lymphatic neoplasms. Microtubule targeting agents like vincristine and vinblastine work by disrupting microtubule dynamics, causing mitotic arrest and cell death. The key issues facing vinca alkaloids applications include establishing an environment-friendly production technique based on microorganisms, as well as increasing bioavailability without causing harm to patient's health. The low yield of these vinca alkaloids from the plant and the difficulty of meeting their huge colossal demand around the globe prompted researchers to create a variety of approaches. Endophytes could thus be selected to produce beneficial secondary metabolites required for the biosynthesis of vinca alkaloids. This review covers the significant aspects of these vital drugs, from their discovery to the present day, in a concise manner. In addition, we emphasize the major hurdles that must be overcome in the coming years to improve vinca alkaloid's effectiveness.

Radiosensitization of Breast Cancer Cells with a 2-Methoxyestradiol Analogue Affects DNA Damage and Repair Signaling In Vitro.

Radiation resistance and radiation-related side effects warrant research into alternative strategies in the application of this modality to cancer treatment. Designed in silico to improve the pharmacokinetics and anti-cancer properties of 2-methoxyestradiol, 2-ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16) disrupts microtubule dynamics and induces apoptosis. Here, we investigated whether pre-exposure of breast cancer cells to low-dose ESE-16 would affect radiation-induced deoxyribonucleic acid (DNA) damage and the consequent repair pathways. MCF-7, MDA-MB-231, and BT-20 cells were exposed to sub-lethal doses of ESE-16 for 24 h before 8 Gy radiation. Flow cytometric quantification of Annexin V, clonogenic studies, micronuclei quantification, assessment of histone H2AX phosphorylation and Ku70 expression were performed to assess cell viability, DNA damage, and repair pathways, in both directly irradiated cells and cells treated with conditioned medium. A small increase in apoptosis was observed as an early consequence, with significant repercussions on long-term cell survival. Overall, a greater degree of DNA damage was detected. Moreover, initiation of the DNA-damage repair response was delayed, with a subsequent sustained elevation. Radiation-induced bystander effects induced similar pathways and were initiated via intercellular signaling. These results justify further investigation of ESE-16 as a radiation-sensitizing agent since pre-exposure appears to augment the response of tumor cells to radiation.

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.

Microtubules promote the non-cell autonomous action of microRNAs by inhibiting their cytoplasmic loading onto ARGONAUTE1 in Arabidopsis.

Mobile microRNAs (miRNAs) serve as local and long-distance signals in the developmental patterning and stress responses in plants. However, mechanisms governing the non-cell autonomous activities of miRNAs remain elusive. Here, we show that mutations that disrupt microtubule dynamics are specifically defective for the non-cell autonomous actions of mobile miRNAs, including miR165/6 that is produced in the endodermis and moves to the vasculature to pattern xylem cell fates in Arabidopsis roots. We show that KTN1, a subunit of a microtubule-severing enzyme, is required in source cells to inhibit the loading of miR165/6 into ARGONUATE1 (AGO1), which is cell autonomous, to enable the miRNA to exit the cell. Microtubule disruption enhances the association of miR165/6 with AGO1 in the cytoplasm. These findings suggest that although cell-autonomous miRNAs load onto AGO1 in the nucleus, the cytoplasmic AGO1 loading of mobile miRNAs is a key step regulated by microtubules to promote the range of miRNA cell-to-cell movement.

The effects of microtubule stabilizing and recovery agents on vitrified bovine oocytes

Oocyte vitrification, while beneficial for research and species conservation applications, has limited success due to cryoinjury to the meiotic spindle. This study aimed to improve meiotic spindle recovery in vitrified bovine oocytes by investigating the effects of treatment with either a microtubule stabilizing agent, or a microtubule recovery agent. In the first two experiments, either paclitaxel or epothilone B were used to treat bovine oocytes before vitrification. Both compounds have microtubule stabilizing properties and are known antimitotic compounds used to disrupt microtubule dynamics in rapidly proliferating cancer cells. Paclitaxel treatment at 2.0 μM significantly increased the proportion of oocytes with normal microtubule distribution and chromosome arrangement after warming. Treatment with 1.0 μM had no effect and 0.5 μM had a negative effect on meiotic spindle recovery. Epothilone B treatment at all concentrations significantly increased the proportion of oocytes with meiotic spindle disruption and abnormally dispersed chromosomes. In the second set of experiments, Rho-associated coiled-coil kinase inhibition and glutathione accumulation were investigated as recovery treatments after vitrification. Oocytes were incubated with either Y-27632 or combinations of cysteine and cysteamine for 4 h after warming. Treatment with 5 μM and 10 μM of Y-27632 to inhibit rho-associated coiled-coil kinase activity significantly increased the proportion of vitrified oocytes with normal microtubule distribution and chromosome arrangement. When oocytes were incubated with 20 μM of Y-27632 there was no effect on spindle recovery. Incubation with 100 μM of cysteamine also had no effect on spindle recovery while 0.6 mM of cysteine and both 0.6 mM of cysteine and 100 μM of cysteamine significantly increased oocytes with normal microtubule distribution and chromosome arrangement.

MAP/Microtubule Affinity Regulating Kinase 4 Inhibitory Potential of Irisin: A New Therapeutic Strategy to Combat Cancer and Alzheimer's Disease.

Irisin is a clinically significant protein playing a valuable role in regulating various diseases. Irisin attenuates synaptic and memory dysfunction, highlighting its importance in Alzheimer’s disease. On the other hand, Microtubule Affinity Regulating Kinase 4 (MARK4) is associated with various cancer types, uncontrolled neuronal migrations, and disrupted microtubule dynamics. In addition, MARK4 has been explored as a potential drug target for cancer and Alzheimer’s disease therapy. Here, we studied the binding and subsequent inhibition of MARK4 by irisin. Irisin binds to MARK4 with an admirable affinity (K = 0.8 × 107 M−1), subsequently inhibiting its activity (IC50 = 2.71 µm). In vitro studies were further validated by docking and simulations. Molecular docking revealed several hydrogen bonds between irisin and MARK4, including critical residues, Lys38, Val40, and Ser134. Furthermore, the molecular dynamic simulation showed that the binding of irisin resulted in enhanced stability of MARK4. This study provides a rationale to use irisin as a therapeutic agent to treat MARK4-associated diseases.

A new class of cytotoxic agents targets tubulin and disrupts microtubule dynamics

Despite the advances in treatment strategies, cancer is still the second leading cause of death in the USA. A majority of the currently used cancer drugs have limitations in their clinical use due to poor selectivity, toxic side effects and multiple drug resistance, warranting the development of new anticancer drugs of different mechanisms of action. Here we describe the design, synthesis and initial biological evaluation of a new class of antimitotic agents that modulate tubulin polymerization. Structurally, these compounds are chalcone mimics containing a 1-(1H-imidazol-2-yl)ethan-1-one moiety, which was initially introduced to act as a metal-binding group and inhibit histone deacetylase enzymes. Although several analogues selectively inhibited purified HDAC8 with IC50 values in low micromolar range, tissue culture studies suggest that HDAC inhibition is not a major mechanism responsible for cytotoxicity. The compounds demonstrated cell growth inhibition with GI50 values of upper nanomolar to low micromolar potency with significant selectively for cancer over normal cells. Interestingly, several compounds arrested HeLaM cells in mitosis and seem to target tubulin to cause mitotic arrest. For example, when combined with inhibitors of Aurora B kinase, they led to dramatic disassembly of the mitotic spindle. In-vitro tubulin polymerization studies showed that the compounds reduced the rate of polymerization of microtubules during the elongation phase and lowered the amount of polymerized tubulin during the plateau phase. Finally, in silico docking studies identified binding of IPE-7 to the colchicine site with similar affinity as the test compound D64131. These compounds represent a new antimitotic pharmacophore with limited HDAC inhibitory activity.

Design, synthesis and biological evaluation of novel acridine and quinoline derivatives as tubulin polymerization inhibitors with anticancer activities

A series of acridine and quinoline derivatives were designed and synthesized based on our previous work as novel tubulin inhibitors targeting the colchicine binding site. Among them, compound 3b exhibited the highest antiproliferative activity with an IC50 of 261 nM against HepG-2 cells (the most sensitive cell line). In addition, compound 3b was able to suppress the formation of HepG-2 colonies. Mechanism studies revealed that compound 3b effectively inhibited tubulin polymerization in vitro and disrupted microtubule dynamics in HepG-2 cells. Furthermore, compound 3b inhibited the migration of cancer cells in a dose dependent manner. Moreover, compound 3b induced cell cycle arrest in G2/M phase and led to cell apoptosis. Finally, docking studies demonstrated that compound 3b fitted nicely in the colchicine binding site of tubulin and overlapped well with CA-4. Collectively, these results suggested that compound 3b represents a novel tubulin inhibitor deserving further investigation.

Parkinson Disease-Modification Encompassing Rotenone and 6-Hydroxydopamine Neurotoxicity by the Microtubule-Protecting Drug Candidate SKIP.

Encompassing live cell imaging and morphometrics at the microscopical level, we showed here, for the first time, protection of neuronal-like cells by thenovel drug candidate, SKIP, against the Parkinson's disease-related neurotoxin, rotenone. Mechanistically, rotenone disrupted microtubule dynamics,which SKIP partially repaired through microtubule end-binding proteins, coupled with increasing neurite branch length. Given the previous association ofrotenone toxicity with increased dopaminergic cell death hallmarking Parkinson's disease, we chose an established rat model of 6-hydroxydopamine (6-OHDA) toxicity to initially evaluate SKIP in vivo. SKIP pretreatment showed protection against nigral dopaminergic cell degeneration and improved motorbehavior in the forelimb asymmetry test. With Parkinson's disease being a major neurodegenerative disorder, afflicting millions of people globally, and withdisease modification challenges, SKIP may hold promise for future therapeutic development.

Absence of SCAPER causes male infertility in humans and Drosophila by modulating microtubule dynamics during meiosis

BackgroundMutation in S-phase cyclin A-associated protein rin the endoplasmic reticulum (SCAPER) have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits,...

Characterization of Signalling Pathways That Link Apoptosis and Autophagy to Cell Death Induced by Estrone Analogues Which Reversibly Depolymerize Microtubules

The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds’ effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.

Discovery of facile amides-functionalized rhodanine-3-acetic acid derivatives as potential anticancer agents by disrupting microtubule dynamics

Microtubule dynamics are crucial for multiple cell functions, and cancer cells are particularly sensitive to microtubule-modulating agents. Here, we describe the design and synthesis of a series of (Z)-2-(5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-N-phenylacetamide derivatives and evaluation of their microtubule-modulating and anticancer activities in vitro. Proliferation assays identified I20 as the most potent of the antiproliferative compounds, with 50% inhibitory concentrations ranging from 7.0 to 20.3 µM with A549, PC-3, and HepG2 human cancer cell lines. Compound I20 also disrupted cancer A549 cell migration in a concentration-dependent manner. Immunofluorescence microscopy, transmission electron microscopy, and tubulin polymerisation assays suggested that compound I20 promoted protofilament assembly. In support of this possibility, computational docking studies revealed a strong interaction between compound I20 and tubulin Arg β369, which is also the binding site for the anticancer drug Taxol. Our results suggest that (Z)-2-(5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-N-phenylacetamide derivatives could have utility for the development of microtubule-stabilising therapeutic agents.

Miro1-mediated mitochondrial positioning supports subcellular redox status

Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular positioning of mitochondria supports subcellular levels of ATP, Ca2+ and reactive oxygen species (ROS, i.e. hydrogen peroxide, H2O2). Previous work from our group showed that deletion of the mitochondrial adapter protein Miro1 leads to perinuclear clustering of mitochondria, leaving the cell periphery devoid of mitochondria which compromises peripheral energy status. Herein, we report that deletion of Miro1 significantly restricts subcellular H2O2 levels to the perinuclear space which directly affects intracellular responses to elevated mitochondrial ROS. Using the genetically encoded H2O2-responsive fluorescent biosensor HyPer7, we show that the highest levels of subcellular H2O2 map to sites of increased mitochondrial density. Deletion of Miro1 or disruption of microtubule dynamics with Taxol significantly reduces peripheral H2O2 levels. Following inhibition of mitochondrial complex 1 with rotenone we observe elevated spikes of H2O2 in the cell periphery and complementary oxidation of mitochondrial peroxiredoxin 3 (PRX3) and cytosolic peroxiredoxin 2 (PRX2). Conversely, in cells lacking Miro1, rotenone did not increase peripheral H2O2 or PRX2 oxidation but rather lead to increased nuclear H2O2 and an elevated DNA-damage response. Lastly, local levels of HyPer7 oxidation correlate with the size and abundance of focal adhesions (FAs) in MEFs and cells lacking Miro1 have significantly smaller focal adhesions and reduced phosphorylation levels of vinculin and p130Cas compared to Miro1+/+ MEFs. Together, we present evidence that the intracellular distribution of mitochondria influences subcellular H2O2 levels and local cellular responses dependent on mitochondrial ROS.