TH588 and Low-Dose Nocodazole Impair Chromosome Congression by Suppressing Microtubule Turnover within the Mitotic Spindle

Microtubule-targeting agents (MTAs) have been used for decades to treat different hematologic and solid cancers. The mode of action of these drugs mainly relies on their ability to bind tubulin subunits and/or microtubules and interfere with microtubule dynamics. In addition to its MTH1-inhibiting activity, TH588 has been recently identified as an MTA, whose anticancer properties were shown to largely depend on its microtubule-targeting ability. Although TH588 inhibited tubulin polymerization in vitro and reduced microtubule plus-end mobility in interphase cells, its effect on microtubule dynamics within the mitotic spindle of dividing cells remained unknown. Here, we performed an in-depth analysis of the impact of TH588 on spindle-associated microtubules and compared it to the effect of low-dose nocodazole. We show that both treatments reduce microtubule turnover within the mitotic spindle. This microtubule-stabilizing effect leads to premature formation of kinetochore-microtubule end-on attachments on uncongressed chromosomes, which consequently cannot be transported to the cell equator, thereby delaying cell division and leading to cell death or division with uncongressed chromosomes.

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The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules

eLife ◽

10.7554/elife.72202 ◽

2022 ◽

Vol 11 ◽

Author(s):

Thomas S McAlear ◽

Susanne Bechstedt

Keyword(s):

Cell Division ◽

Growth Factor ◽

Mitotic Spindle ◽

Apparent Rate Constant ◽

Microtubule Dynamics ◽

Proliferation Marker ◽

Microtubule Growth ◽

Large Rearrangements ◽

And Function

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.

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Sodium cacodylate as antimitotic agent

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1988.032 ◽

2014 ◽

Vol 57 (3) ◽

pp. 329-340

Author(s):

Jadwiga A. Tarkowska

Keyword(s):

Allium Cepa ◽

Mitotic Spindle ◽

Sodium Cacodylate ◽

Antimitotic Agent ◽

Ultrastructural Studies ◽

Meristematic Cells ◽

Cacodylate Buffer ◽

Root Meristematic Cells ◽

Dividing Cells

The effect of pure sodium cacodylate on dividing cells was studied. The root meristematic cells of Allium cepa L. (the roots were squashed in acetoorcein) and endosperm cells of Haemanthus katherinae Bak. (in vitro observations) were used. Serious disturbances in karyokinesis and cytokinesis were found that led most often to the formation of polyploid or multinucleate (A. cepa) cells. These results point to damage of the mitotic spindle and phragmoplast. Careful use of cacodylate buffer in ultrastructural studies of microtubules is advised.

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Interleukin-2 (hrIL2) Primed T cells display Reduced Alloproliferative Capacity In Vitro Due To Induction Of FOXP3+ Regulatory Cells

Blood ◽

10.1182/blood.v122.21.5433.5433 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5433-5433

Author(s):

Dimitra Kokkinou ◽

Panagiota Stamou ◽

Angeliki Vittoraki ◽

Anne-Lise De Lastic ◽

Spyros Chondropoulos ◽

...

Keyword(s):

T Cells ◽

Low Dose ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Interleukin 2 ◽

Regulatory Cells ◽

Transplant Tolerance ◽

Immune Exhaustion ◽

The Impact

Abstract Introduction Prophylactic donor lymphocyte infusions (pDLI) after allogeneic transplantation contribute to immune restoration and reduce viral infections. Furthermore, we have recently shown that pDLI in patients with high risk leukemia significantly reduces the relapse rate, however, they were associated with a relatively high incidence of Graft versus Host Disease (GvHD)(BBMT 2013;19:75-81). Strategies to minimize GvHD without compromising the effect of pDLI against leukemia are needed. IL2 plays dual role in immune responses, contributing to both the generation of effector T cells and the maintenance of regulatory T cells (Tregs). Recently, low dose interleukin-2 (IL-2) therapy has been advanced as a potential immune modulator able to modulate the immune response to aid transplant tolerance and to suppress GvHD through expansion of Tregs (N Engl J Med. 2011; 2055-66). We investigated the impact of priming DLI with low dose IL2 on the proliferative responses to allo-stimulation in vitro. Methods CD3+ T cells purified from healthy individuals by MACS negative selection were primed (p-T cells) or unprimed (np-T cells), with or without (control) 100 U/ml hrIL2 (Proleukin, Novartis) for 7 days. Composition of T-cell cultures was analyzed by flow cytometry for: a) the percentage of T regulatory cells (CD4+/CD25high/Foxp3+/Helios+, b) their differentiation (CD28/CD27), c) their immune exhaustion (Programmed cell death 1, PD1). In vitro alloproliferative capacity of the p-T cells was analyzed with CFSE cell proliferation assay by using them as responder cells in mixed lymphocyte cultures (MLC), with irradiated allo-PB mononuclear cells as stimulators. Results In vitro priming of T-cells with IL-2 (p-T cells) in contrast to np-T or control cells: 1) increase the numbers of CD4+CD25highFoxp3+/Helios+ cells (n=8, 3.3%±0.7 mean±SEM vs 1.01%±0.22, p=0.004 και 1.4%±0.42, p=0.006). Increased levels of Foxp3 expression was also confirmed by Real Time PCR (n=2,1.25AU±0.15 vs 0.29AU±0.04, p=0.028 και 0.26AU±0.07, p=0.024). 2) did not affect the proportion of CD28+/CD27+ non late-differentiated cells (n=3, 60%±0.15 vs61%±0.04, p=0.91 και 59%±0.08, p=0.024). 3) did not cause immune exhaustion through PD1 expression (n=6, 13.3%±1.9 vs 8.1%±2.1, p=0.76, και 14%±2.2, p=0.68). 4) significantly decreases their response rate to allo-stimulus in MLC (n=8, 45%±0.5 vs65%±0.2, p=0.006 και 64%±0.2, p=0.008). The p-T cells regained their alloproliferative capacity after FACS-sorting removal of CD4+/CD25high Tregs. Conclusions Our results show that ex vivo priming of T cells with low dose of IL-2 reduces their in vitro alloproliferative capacity. This reduction is not due to late differentiation or immune – exhaustion of T cells but to selective induction of Foxp3+ cells with immunomodulatory properties in the culture. It remains to be seen whether IL2-primed DLI is safe and effective in transplant patients. Disclosures: No relevant conflicts of interest to declare.

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O-085 In-depth analysis of embryo development: Differences among monosomic, trisomic and chromosomally chaotic embryos compared to euploid embryos

Human Reproduction ◽

10.1093/humrep/deab125.015 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

F Meseguer Estornell ◽

L Bori ◽

R Maor ◽

I Kottel ◽

D Gilboa ◽

...

Keyword(s):

Embryo Development ◽

Chromosomal Abnormalities ◽

Chromosome Abnormality ◽

Automated System ◽

Human Embryos ◽

Developmental Milestones ◽

Preimplantation Genetic Testing ◽

Depth Analysis ◽

The Impact

Abstract Study question Is there any visible variation in the development of aneuploid embryos depending on the type of chromosome abnormality? Summary answer There were significant visible differences in the development of euploid, monosomic, trisomic and, especially, chaotic embryos. What is known already Aneuploidy rates are remarkably high in in vitro fertilized human embryos, with up to 50% of embryos diagnosed as aneuploid based on preimplantation genetic testing for aneuploidies (PGT-A). However, very little is known about the impact of specific aneuploidies during the early human embryo development. A recent publication showed that embryos with single chromosomal gain or loss reached the blastocyst stage later or earlier depending on the chromosome affected (Shahbazi et al., 2020). In our study, we wanted to detect observable differences in embryo behavior between embryos with different chromosomal abnormalities during the entire in vitro development. Study design, size, duration This was a retrospective study including 2,500 blastocysts with PGT-A results. Embryos were cultured in EmbryoScope systems until the fifth/sixth day of development (up to the time of trophectoderm biopsy). Automatic-annotations for division times and quality gradings were supervised routinely by senior embryologists using Guided Annotations Tool. Out of the total, 1,000 were euploid embryos used for reference and 1,500 were aneuploid embryos with one or more defects, including monosomic, trisomic and chromosomally chaotic embryos. Participants/materials, setting, methods Chromosome analysis was performed using next-generation sequence technology. Then, an in-depth analysis of time-lapse videos and supervised-automatic annotations was performed. We calculated the proportion of embryos, in each aneuploid category, that reached one specific event later than the expected value for euploid embryos plus one standard deviation. Later, we calculated the “relative risk” of an embryo of reaching the milestone late. We did the same for the time between milestones and for pairs of milestones. Main results and the role of chance Every aneuploid category was more likely to reach each specific embryo developmental event later than euploid embryos and the time gaps between developmental milestones were also statistically longer in aneuploid embryos (p < 0.0001). The following results were the most interesting relative risks (RR) when we compared the proportion of embryos (in each aneuploid category) to the proportion of euploid embryos (RR for euploid = 1). For reaching the division time to two cells (t2): 1.31 in monosomic embryos, 1.50 in trisomic embryos and 2.43 in chaotic embryos. For the division time to four cells (t4): 1.42 in monosomic embryos, 1.54 in trisomic embryos and 3.07 in chaotic embryos. For the division time to eight cells (t8) and the time of starting blastulation: 1.45 in monosomic embryos, 1.22 in trisomic embryos and 2.74 in chaotic embryos. Combined milestones were stronger indicators than each milestone by itself, the RR were: 1.63 in monosomic embryos, 1.81 in trisomic embryos and 3.35 in chaotic embryos for t2 and t4; 1.50 in monosomic embryos, 1.80 in trisomic embryos and 2.84 in chaotic embryos for t2 and t8; 1.46 in monosomic embryos, 1.90 in trisomic embryos and 3.43 in chaotic embryos for t4 and t8. Limitations, reasons for caution At this stage, we did not go down to specific chromosome abnormality as there were very few cases in each fully detailed category. Also, not all the embryos reached every developmental milestone. Wider implications of the findings Aneuploid embryos were significantly different from euploid embryos in the first five days of development. A large proportion of aneuploid embryos could be rejected because their developmental milestones falling outside the normal range. This could form part of an automated system for determining euploidy/aneuploidy from observation of embryos in vitro. Trial registration number 1902-VLC-018-MM

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Mitotic Spindle Dysfunction Promotes Genomic Instability In Marrow Failure

Blood ◽

10.1182/blood.v116.21.880.880 ◽

2010 ◽

Vol 116 (21) ◽

pp. 880-880 ◽

Cited By ~ 1

Author(s):

Katie Lombardo ◽

Jason Stumpff ◽

Susan Parkhurst ◽

Linda Wordeman ◽

Akiko Shimamura

Keyword(s):

Genomic Instability ◽

Mitotic Spindle ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Microtubule Dynamics ◽

Published Data ◽

Hematopoietic Progenitor ◽

Microtubule Stability ◽

Cd34 Cells

Abstract Abstract 880 Shwachman-Diamond syndrome (SDS) is an autosomal recessively inherited disorder associated with bone marrow failure and leukemia predisposition. The majority of patients harbor biallelic mutations in the SBDS gene. The SBDS protein has been implicated in several cellular functions including ribosome biogenesis and microtubule stabilization during mitosis. We have previously found that SBDS deficiency results in multipolar spindles, centrosome amplification and aneuploidy, implicating a role for SBDS in cell division. The mechanism by which SBDS functions to ensure proper spindle assembly and DNA segregation during mitosis remains unknown. Here we present evidence that SBDS functions to promote mitotic spindle stability both by directly modifying microtubule dynamics and through a microtubule crosslinking activity. Importantly, the microtubule stabilizing effects of SBDS appear to be essential for the growth and differentiation of hematopoietic progenitor cells. Specifically, we found that SBDS deficiency resulted in shortened mitotic spindle length and decreased spindle acetylation, a marker of microtubule stability. The loss of microtubule stability in the absence of SBDS function may be due to changes in microtubule dynamics or reduction in microtubule crosslinking activity, as we found that addition of recombinant purified wild-type SBDS to polymerized microtubules in vitro increases their polymerization rate and strongly promotes microtubule bundling. Interestingly, recombinant patient-derived missense mutant SBDS proteins showed a marked decrease in their microtubule bundling ability. To assess whether spindle destabilization contributes to marrow failure, we modeled hematopoiesis in the absence of SBDS in vitro. When SBDS expression was knocked down in human CD34+ cells, proliferation, differentiation, and hematopoietic progenitor colony formation were impaired, consistent with published data on primary marrow from SDS patients. The addition of taxol at concentrations that significantly impaired hematopoiesis in control CD34+ cells resulted in stable to improved hematopoiesis in the SBDS-deficient CD34+ cells. Based on these data, we hypothesize that spindle destabilization by SBDS loss promotes genomic instability, which in turn, contributes to marrow failure and leukemia predisposition. Disclosures: No relevant conflicts of interest to declare.

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Biological Impact of Monoallelic and Biallelic BIRC3 Loss in Del(11q) Chronic Lymphocytic Leukemia Progression

Blood ◽

10.1182/blood-2020-142658 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 4-4

Author(s):

Miguel Quijada Álamo ◽

Maria Hernandez-Sanchez ◽

Ana E. Rodriguez ◽

Claudia Pérez Carretero ◽

Marta Martín Izquierdo ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Line ◽

Cell Lines ◽

Nuclear Translocation ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Negative Regulator ◽

Depth Analysis ◽

The Impact

Chronic lymphocytic leukemia (CLL) patients harboring 11q22.3 deletion, del(11q), are characterized by a rapid disease progression. One of the suggested genes to be involved in the pathogenesis of this deletion is BIRC3, a negative regulator of NF-κB, which is monoallelically deleted in ~80% of del(11q) CLL cases. In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which accounts for marked reduced survival in CLL. Nevertheless, the biological mechanisms by which monoallelic or biallelic BIRC3 lesions could contribute to del(11q) CLL pathogenesis, progression and therapy response are partially unexplored. We used the CRISPR/Cas9 system to model monoallelic and biallelic BIRC3 loss in vitro. First, we generated an isogenic HG3 CLL cell line harboring monoallelic del(11q) - HG3-del(11q) - by the introduction of 2 guide RNAs targeting 11q22.1 and 11q23.3 (~17 Mb). Loss-of-function BIRC3 mutations (MUT) were introduced in the remaining allele, generating 3 HG3-del(11q) BIRC3MUT clones. In addition, single BIRC3MUT were introduced in HG3 and MEC1 CLL-derived cells for experimental validation (n = 3 clones/cell line). We first questioned whether monoallelic and biallelic BIRC3 loss had an impact in the DNA-binding activity of NF-κB transcription factors. Interestingly, HG3-del(11q) had higher p52 and RelB (non-canonical NF-κB signaling) activity than HG3WT cells (P = 0.005; P = 0.007), being this activity further increased in HG3-del(11q) BIRC3MUT cells (P < 0.001; P < 0.001). In depth analysis of the non-canonical signaling components by immunoblot revealed that HG3-del(11q) and, to a greater extent, HG3-del(11q) BIRC3MUT cells presented NF-κB-inducing kinase (NIK) cytoplasmic stabilization, high p-IKKα levels and p52-RelB nuclear translocation. Besides, HG3-del(11q) BIRC3MUT cells showed increased levels of the anti-apoptotic proteins BCL2 and BCL-xL. We next assessed this pathway ex vivo in stroma and CpG-stimulated primary CLL cells with or without BIRC3 deletion (n = 22; 11 each group). Remarkably, stimulated BIRC3-deleted primary cells showed higher p52 and RelB activity than BIRC3WT cases (P = 0.01; P = 0.07), and the percentage of BIRC3-deleted cells correlated with p52 activity in del(11q) cases (P = 0.04). We further performed western blot analyses in a homogenous cohort of del(11q) cases including (n = 4) or not including (n = 3) BIRC3 within the deleted region. Interestingly, del(11q)/BIRC3 deleted cases presented high levels of stabilized NIK, which correlated with higher p52 processing (P = 0.003). These patients also showed higher BCL2 levels than those del(11q)/BIRC3 undeleted, and we could further observe a correlation between p52 and BCL2 levels (P = 0.01). Given this p52-dependent BCL2 upregulation, we treated the CRISPR/Cas9 edited clones with venetoclax, demonstrating that HG3-del(11q) BIRC3MUT cells were more sensitive upon BCL2 inhibition than HG3WT clones (mean IC50 3.5 vs. 5.75 μM; P = 0.005). In vitro proliferation assays were performed to interrogate the impact of BIRC3 loss in CLL cell growth, revealing that HG3 BIRC3MUT cell lines had higher growth rates than BIRC3WT cells (P = 0.001). HG3-del(11q) BIRC3MUT cells also showed enhanced proliferation in comparison to HG3-del(11q) clones (P = 0.009). We further determined the clonal dynamics of del(11q) and/or BIRC3MUT cell lines in clonal competition experiments, showing that HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells progressively outgrew HG3WT and HG3-del(11q) cells, respectively, overtime (P = 0.02; P = 0.006). Furthermore, we injected these edited cell lines into NSG mice (n = 20) in vivo, showing that mice xenografted with HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells presented, by flow cytometry, an increase of human CD45+ cells in spleen 14 days after injection, compared to HG3WT and HG3-del(11q) cells (P = 0.02; P = 0.015). In summary, this work demonstrates that biallelic BIRC3 deletion through del(11q) and mutation triggers non-canonical NF-κB signaling, driving BCL2 overexpression and conferring clonal advantage, which could account for the negative predictive impact of BIRC3 biallelic inactivation in CLL. Taken together, our results suggest that del(11q) CLL patients harboring BIRC3 mutations should be considered as a CLL subgroup at a high risk of progression that might benefit from venetoclax-based therapies. Funding: PI18/01500 Disclosures No relevant conflicts of interest to declare.

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Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition

Cancers ◽

10.3390/cancers13225699 ◽

2021 ◽

Vol 13 (22) ◽

pp. 5699

Author(s):

Amélie Foucault ◽

Noémie Ravalet ◽

Joevin Besombes ◽

Frédéric Picou ◽

Nathalie Gallay ◽

...

Keyword(s):

Stromal Cells ◽

Low Dose ◽

Low Doses ◽

Aldehyde Dehydrogenase 2 ◽

Primitive Hematopoiesis ◽

In Vitro Effects ◽

High Doses ◽

Chlorpyrifos Ethyl ◽

The Impact

(1) Background: The impact of occupational exposure to high doses of pesticides on hematologic disorders is widely studied. Yet, lifelong exposure to low doses of pesticides, and more particularly their cocktail effect, although poorly known, could also participate to the development of such hematological diseases as myelodysplastic syndrome (MDS) in elderly patients. (2) Methods: In this study, a cocktail of seven pesticides frequently present in water and food (maneb, mancozeb, iprodione, imazalil, chlorpyrifos ethyl, diazinon and dimethoate), as determined by the European Food Safety Authority, were selected. Their in vitro effects at low-doses on primary BM-MSCs from healthy volunteers were examined. (3) Results: Exposure of normal BM-MSCs to pesticides for 21 days inhibited cell proliferation and promoted DNA damage and senescence. Concomitantly, these cells presented a decrease in aldehyde dehydrogenase 2 (ALDH2: mRNA, protein and enzymatic activity) and an increase in acetaldehyde levels. Pharmacological inhibition of ALDH2 with disulfiram recapitulated the alterations induced by exposure to low doses of pesticides. Moreover, BM-MSCs capacity to support primitive hematopoiesis was significantly altered. Similar biological abnormalities were found in primary BM-MSCs derived from MDS patients. (4) Conclusions: these results suggest that ALDH2 could participate in the pathophysiology of MDS in elderly people long exposed to low doses of pesticides.

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Dysfunctional platelet membrane receptors: from humans to mice

Thrombosis and Haemostasis ◽

10.1160/th04-05-0308 ◽

2004 ◽

Vol 92 (09) ◽

pp. 478-485 ◽

Cited By ~ 28

Author(s):

Jerry Ware

Keyword(s):

Biochemical Characterization ◽

Ex Vivo ◽

Human Platelets ◽

Membrane Receptors ◽

Protease Activated Receptors ◽

In Vivo Models ◽

Depth Analysis ◽

The Impact

SummaryInsights into hemostasis and thrombosis have historically benefited from the astute diagnosis of human bleeding and thrombotic disorders followed by decades of careful biochemical characterization. This work has set the stage for the development of a number of mouse models of hemostasis and thrombosis generated by gene targeting strategies in the mouse genome. The utility of these models is the in depth analysis that can be performed on the precise molecular interactions that support hemostasis and thrombosis along with efficacy testing of various therapeutic strategies. Already the mouse has proven to be an excellent model of the processes that support hemostasis and thrombosis in the human vasculature. A brief summary of the salient phenotypes from knockout mice missing key platelet receptors is presented, including the glycoprotein (GP) Ib-IX-V and GP IIb/IIIa (αIIb/β3) receptors; the collagen receptors, GP VI and α2/β1; the protease activated receptors (PARs); and the purinergic receptors, P2Y1 and P2Y12. A few differences exist between mouse and human platelets and where appropriate those will be highlighted in this review. Concluding remarks focus on the importance of understanding the power and limitations of various in vitro, ex vivo and in vivo models currently being used and the impact of the mouse strain on the described platelet phenotype.

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Characterization of proteins immunologically related to brain microtubule-associated protein MAP-1B in non-neural cells

Journal of Cell Science ◽

10.1242/jcs.92.4.607 ◽

1989 ◽

Vol 92 (4) ◽

pp. 607-620

Author(s):

J. Diaz-Nido ◽

J. Avila

Keyword(s):

Mitotic Spindle ◽

Polyclonal Antibodies ◽

Microtubule Assembly ◽

Neural Cells ◽

Microtubule Network ◽

Cytoplasmic Microtubule ◽

Protein Map ◽

Dividing Cells

Brain microtubule-associated protein MAP-1 is composed of at least two polypeptides, MAP-1A and MAP-1B, which are among the main components of the neural cytoskeleton. Specific monoclonal and polyclonal antibodies against MAP-1B stain nuclei, mitotic spindles, centrosomes and the cytoplasmic microtubule network of different non-neural cells studied by immunofluorescence microscopy. It appears that these cells contain two proteins of 325K and 220K (K = 10(3) Mr), which are immunologically related to brain MAP-1B. The 325K protein, which is localized to the cytoplasmic microtubule network, the centrosome and the mitotic spindle, seems to be structurally related to the neural MAP-1B, as judged from their similar peptide maps and phosphorylation patterns. The 220K protein, which is localized to the nuclear matrix in interphase cells and to the mitotic spindle in dividing cells, has a proteolytic profile different from that of neural MAP-1B and is phosphorylated to a much lesser extent than the 325K protein. Both proteins bind tubulin in vitro, which suggests that they may participate in microtubule assembly in vivo; the 325K protein could perform such a role during the entire cell cycle, while the 220K protein could be implicated in the formation of the mitotic spindle.

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Two Antagonistic Microtubule Targeting Drugs Act Synergistically to Kill Cancer Cells

Cancers ◽

10.3390/cancers12082196 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2196 ◽

Cited By ~ 2

Author(s):

Lauralie Peronne ◽

Eric Denarier ◽

Ankit Rai ◽

Renaud Prudent ◽

Audrey Vernet ◽

...

Keyword(s):

Low Dose ◽

Mechanistic Explanation ◽

Chemical Library ◽

Cancer Treatments ◽

Cytotoxic Effects ◽

Colchicine Binding Site ◽

Microtubule Targeting Agents ◽

Microtubule Stabilizing Agent

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

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