Identification of a Potent Cytotoxic Pyrazole with Anti-Breast Cancer Activity That Alters Multiple Pathways

In this study, we identified a novel pyrazole-based derivative (P3C) that displayed potent cytotoxicity against 27 human cancer cell lines derived from different tissue origins with 50% cytotoxic concentrations (CC50) in the low micromolar and nanomolar range, particularly in two triple-negative breast cancer (TNBC) cell lines (from 0.25 to 0.49 µM). In vitro assays revealed that P3C induces reactive oxygen species (ROS) accumulation leading to mitochondrial depolarization and caspase-3/7 and -8 activation, suggesting the participation of both the intrinsic and extrinsic apoptotic pathways. P3C caused microtubule disruption, phosphatidylserine externalization, PARP cleavage, DNA fragmentation, and cell cycle arrest on TNBC cells. In addition, P3C triggered dephosphorylation of CREB, p38, ERK, STAT3, and Fyn, and hyperphosphorylation of JNK and NF-kB in TNBC cells, indicating the inactivation of both p38MAPK/STAT3 and ERK1/2/CREB signaling pathways. In support of our in vitro assays, transcriptome analyses of two distinct TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells) treated with P3C revealed 28 genes similarly affected by the treatment implicated in apoptosis, oxidative stress, protein kinase modulation, and microtubule stability.

Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent

Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC50 values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.

Receptor tyrosine kinase ROR1 ameliorates Aβ1–42 induced cytoskeletal instability and is regulated by the miR146a-NEAT1 nexus in Alzheimer’s disease

Alzheimer’s disease (AD) involves severe cytoskeletal degradation and microtubule disruption. Here, we studied the altered dynamics of ROR1, a Receptor Tyrosine Kinase (RTK), and how it could counter these abnormalities. We found that in an Aβ1–42 treated cell model of AD, ROR1 was significantly decreased. Over expressed ROR1 led to the abrogation of cytoskeletal protein degradation, even in the presence of Aβ1–42, preserved the actin network, altered actin dynamics and promoted neuritogenesis. Bioinformatically predicted miRNAs hsa-miR-146a and 34a were strongly up regulated in the cell model and their over expression repressed ROR1. LncRNA NEAT1, an interactor of these miRNAs, was elevated in mice AD brain and cell model concordantly. RNA Immunoprecipitation confirmed a physical interaction between the miRNAs and NEAT1. Intuitively, a transient knock down of NEAT1 increased their levels. To our knowledge, this is the first instance which implicates ROR1 in AD and proposes its role in preserving the cytoskeleton. The signalling modalities are uniquely analyzed from the regulatory perspectives with miR-146a and miR-34a repressing ROR1 and in turn getting regulated by NEAT1.

Microtubule Disruption does not Impair Learning in the Unicellular Organism Paramecium: Implications for Information Processing in Microtubules

Information processing in microtubules is an open question that has not been properly addressed yet. It was suggested that microtubules could store and process information in the nervous system or even support consciousness. The unicellular organism, Paramecium caudatum, that has a microtubular structure but does not have a neuron or neural network, shows intelligent behaviors such as associative learning. This may suggest that the microtubules are involved in intelligent behavior, information storage or information processing in paramecium. To test this hypothesis, we have utilized a paramecium learning task in which the organism associates brightness in its swimming medium with attractive cathodal shocks to study the role of microtubules in paramecium learning. We disrupted the microtubular dynamics in paramecium using an antimicrotubular agent (parbendazole) to see if microtubules are an integral part of information storage and processing in paramecium. We observed that while a partial allosteric modulator of GABA (midazolam) could disrupt the learning process in paramecium, the antimicrotubular agent could not interfere with the learning in paramecium. Therefore, our results suggest that microtubules are probably not vital for the learning behavior in P. caudatum. Consequently, our results call for a further revisitation of the microtubular information processing hypothesis.

Microtubules Promote the Non-cell Autonomy of MicroRNAs by Inhibiting their Cytoplasmic Loading into ARGONAUTE1 in Arabidopsis

Mobile microRNAs (miRNAs) serve as local and long-distance signals in 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 and intermediary cells to inhibit the loading of miR165/6 into ARGONUATE1 (AGO1), which is cell-autonomous, to enable the miRNA's cell exit. Microtubule disruption enhances the association of miR165/6 with AGO1 in the cytosol. These findings suggest that, while cell-autonomous miRNAs load into AGO1 in the nucleus, cytoplasmic AGO1 loading of mobile miRNAs is a key step regulated by microtubules to promote the range of miRNA's cell-to-cell movement.

Microtubule Disruption Reduces Metastasis More Effectively Than Primary Tumor Growth

Background: Clinical breast cancer imaging inevitably focuses on tumor growth rather than the metastatic dissemination that is a greater challenge for patient survival. Emerging preclinical evidence indicates that chemotherapy can elevate levels of circulating tumor cells (CTCs) and increase metastatic recurrence. Targeting metastatic phenotypes of CTCs could reveal therapeutic strategies to reduce metastasis that would be overlooked by measurements of tumor growth.Methods: We investigated how the FDA-approved microtubule-depolymerizing Vinca alkaloid, Vinorelbine, affects three metastatic phenotypes of reattachment, clustering and microtentacles (McTNs). Microfluidic cell tethering technology (TetherChip) was used to measure Vinorelbine effects on McTNs and clustering while xCelligence impedance was used for reattachment assays. Bioluminescence imaging monitored tumor growth and metastasis in mice. ANGLE Parsortix and Vortex Biosciences VTX-1 were used to capture live tumor cells from blood samples for confocal microscopy to rapidly measure tumor cell responses to Vinorelbine.Results: We demonstrate that a focused (1h) Vinorelbine treatment is sufficient to inhibit reattachment, clustering and McTNs in non-adherent breast tumor cells. Quantitative analysis of treated non-adherent cells reveals that McTNs are significantly lower in number (p= 0.012) and shorter in length (p= 0.000034). Treating mice with Vinorelbine (5mg/kg) for only 24h did not significantly affect primary tumor survival. However, median metastatic tumor survival after injection of circulating tumor cells extended from 8 weeks to 30 weeks after a focused 24h treatment with Vinorelbine. Microtentacle inhibition by Vinorelbine was also detectable within 1h, using live tumor cells isolated from blood samples and analyzed with confocal microscopy for McTNs on TetherChip microfluidic surfaces. As few as 11 tumor cells were sufficient to yield 90% power to detect this 1h Vinorelbine drug response, demonstrating feasibility with the small number of tumor cells available from patient biopsies.Conclusions: This study establishes a proof-of-concept that targeted microtubule disruption can selectively inhibit metastasis and reveals that existing FDA-approved therapies could have anti-metastatic actions that are currently overlooked when focusing exclusively on tumor growth. Moreover, the anti-metastatic actions of Vinorelbine could be detected in less than one hour using microfluidic TetherChip technology, establishing a new approach for precision medicine aimed at reducing metastasis.

Preservation of the microtubule network to beat atrial fibrillation: role of SR-mitochondrial contacts

Background Atrial fibrillation (AF), the most common tachyarrhythmia, is a progressive disease characterized by electrophathology, which is defined as the structural remodeling of atrial cardiomyocytes that underlies electrophysiological remodeling and contractile dysfunction and consequently AF progression. We recently discovered that disruption of microtubule network, by HDAC6 activation, is a key factor underlying structural remodeling and AF promotion in experimental models for AF and patients with AF. However, the molecular mechanism how microtubule disruption induces contractile dysfunction in AF is unclear. Sarcoplasmic reticulum (SR) and mitochondria are the central organelles for normal cardiomyocyte contraction by controlling the Ca2+ and energy (ATP) homeostasis. The crosstalk between SR and mitochondria via contacts, termed SR-mitochondria contacts (SMCs), is essential for normal mitochondrial and cardiac function. Interestingly, functional SMCs are highly dependent on intact microtubule network. This study aims to whether preservation of microtubule protects against AF via SMC pathway. Methods and results Tachypacing of HL-1 cardiomyocytes significantly impaired calcium transient (CaT) amplitude compared to normal paced cardiomyocytes. Pretreatment with microtubule stalilizer taxol and acetyl-CoA inducer β-hydroxybutyrate (βOHB) significantly protected against tachypacing-induced CaT loss. Moreover, by using immunoflurecent staining, mitochondrial associated membrane isolation and seahorse, we showed that tubacin, Taxol and βOHB also significantly inhibited the SMC reduction, attenuated tachypacing-induced mitochondrial dysfunction. Consistently, these microtubule stabilizer drugs also prevented tachypacing-induced contractile dysfunction in the Drosophila model for AF. Conclusions Preservation of microtubule network prevented the reduction of SMCs and the consequent mitochondrial and contractile dysfunction in HL-1 cardiomyocytes and Drosophila models for AF. Therefore, the microtubule-SMC pathway is a novel central therapeutic target for AF. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Dutch Heart Foundation

Chemometric and Transcriptomic Profiling, Microtubule Disruption and Cell Death Induction by Secalonic Acid in Tumor Cells

Nature is an indispensable source of new drugs, providing unique bioactive lead structures for drug discovery. In the present study, secalonic acid F (SAF), a naturally occurring ergochrome pigment, was studied for its cytotoxicity against various leukemia and multiple myeloma cells by the resazurin assay. SAF exhibited cytotoxic activity on both leukemia and multiple myeloma cells. Generally, multiple myeloma cells were more sensitive to SAF than leukemia cells. NCI-H929 cells were the most affected cells among the tested panel of multiple myeloma cell lines and were taken for further studies to assess the mode of action of SAF on those cells. Cell cycle analysis revealed that SAF induced S and G2/M arrest in NCI-H929 cells. SAF-associated apoptosis and necrosis resulted in cytotoxicity. SAF further inclined the disassembly of the tubulin network, which may also account for its cytotoxicity. COMPARE and hierarchical cluster analyses of transcriptome-wide expression profiles of the NCI tumor cell line panel identified genes involved in numerous cellular processes (e.g., cell differentiation, cell migration, and other numerous signaling pathways) notably correlated with log10IC50 values for secalonic acid. In conclusion, the present study supports the therapeutic potential of SAF to treat multiple myeloma.

Neuropeptide Y Expression Confers Benzo[a]pyrene Induced DNA Damage and Microtubule Disruption in Human Neuroblastoma SH-SY5Y Cells

Benzo[a]pyrene (B[a]P), is a family member of polycyclic aromatic hydrocarbons and a widespread environmental pollutant and neurotoxicant that contribute to the development of cancer. Microtubules are polymers of tubulin that form part of the cytoskeleton and target for anticancer drugs. Furthermore, NPY significantly increased the percentage of cells in S and G2/M phases. However, little is known about the specific role of NPY in proliferation and the underlying protective mechanism remains unclear. Hence, the aim of this work was to investigate the effect of B[a]P on SH-SY5Y neuroblastoma cells and to explore the potential mechanism for alteration of tubulin-microtubule equilibrium causing mitotic arrest and NPY expression. The present findings showed B[a]P treatment significantly increase number of SH-SY5Y cells in S and G2/M phase as compared to G1 phase and provokes cell cycle arrest that correlated with significant decrease in G0/G1 cells. Immunofluorescence study showed significantly distorted tubulin arrangement from metaphasic plate in formation of bipolar mitotic spindle apparatus. Further, higher doses of B[a]P treatment lead to chromosomal abnormalities accompanied by DNA damage due ROS causing oxidative stress showing significant decrease in tubulin protein around spindle. The results of present study demonstrated that NPY exerts a proliferative and protective effect on B[a]P-induced oxidative stress in a dose-dependent manner in vitro and importantly, these effects may be mediated via mitotic arrest and involved in spindle arrangement during cell division. Our findings addresses a novel pathological outcomes of B[a]P-induced NPY expression by oxidative stress through spindle abnormalities leading to microtubule disruption.