BP-M345, a New Diarylpentanoid with Promising Antimitotic Activity

Previously, we reported the in vitro growth inhibitory effect of diarylpentanoid BP-M345 on human cancer cells. Nevertheless, at that time, the cellular mechanism through which BP-M345 exerts its growth inhibitory effect remained to be explored. In the present work, we report its mechanism of action on cancer cells. The compound exhibits a potent tumor growth inhibitory activity with high selectivity index. Mechanistically, it induces perturbation of the spindles through microtubule instability. As a consequence, treated cells exhibit irreversible defects in chromosome congression during mitosis, which induce a prolonged spindle assembly checkpoint-dependent mitotic arrest, followed by massive apoptosis, as revealed by live cell imaging. Collectively, the results indicate that the diarylpentanoid BP-M345 exerts its antiproliferative activity by inhibiting mitosis through microtubule perturbation and causing cancer cell death, thereby highlighting its potential as antitumor agent.     

The Design,Synthesis, and Biological Activities of Pyrrole-Based Carboxamides: The Novel Tubulin Inhibitors Targeting the Colchicine-Binding Site

Microtubule targeting agents (MTAs) that interfere with the dynamic state of the mitotic spindle are well-known and effective chemotherapeutic agents. These agents interrupt the microtubule network via polymerization or depolymerization, halting the cell cycle progression and leading to apoptosis. We report two novel pyrrole-based carboxamides (CAs) (CA-61 and -84) as the compounds exhibiting potent anti-cancer properties against a broad spectrum of epithelial cancer cell lines, including breast, lung, and prostate cancer. The anti-cancer activity of CAs is due to their ability to interfere with the microtubules network and inhibit tubulin polymerization. Molecular docking demonstrated an efficient binding between these ligands and the colchicine-binding site on the tubulin. CA-61 formed two hydrogen bond interactions with THR 179 (B) and THR 353 (B), whereas two hydrogen bonds with LYS 254 (B) and 1 with ASN 101 (A) were identified for CA-84. The binding energy for CA-84 and CA-61 was −9.910 kcal/mol and −9.390 kcal/mol. A tubulin polymerization assay revealed a strong inhibition of tubulin polymerization induced by CA-61 and -84. The immunofluorescence data revealed the disruption of the tubulin assembly in CA-treated cancer cells. As an outcome of the tubulin inhibition, these compounds halted the cell cycle progression in the G2/M phase, leading to the accumulation of the mitotic cells, and further induced apoptosis. Lastly, the in vivo study indicated that CAs significantly inhibited the HCC1806 breast cancer xenograft tumor growth in a nude mouse model. Collectively, we identified the novel CAs as potent MTAs, inhibiting tubulin polymerization via binding to the colchicine-binding site, disrupting the microtubule network, and exhibiting potent pro-apoptotic activities against the epithelial cancer cell lines both in vitro and in vivo.     

Chemistry and chemical biology of taxane anticancer agents

Taxol (paclitaxel) and Taxotere (docetaxel) are currently considered to be among the most important anticancer drugs in cancer chemotherapy. The anticancer activity of these drugs is ascribed to their unique mechanism of action, i.e., causing mitotic arrest in cancer cells, leading to apoptosis through inhibition of the depolymerization of microtubules. Although both paclitaxel and docetaxel possess potent antitumor activity, treatment with these drugs often results in a number of undesirable side effects, as well as multidrug resistance (MDR). Therefore, it has become essential to develop new anticancer agents with superior pharmacological properties, improved activity against various classes of tumors, and fewer side effects. This paper describes an account of our research on the chemistry of paclitaxel and taxoid anticancer agents at the biomedical interface, including: 1. The structure-activity relationship (SAR) study of taxoids leading to the development of the "second-generation" taxoids, which possess exceptional activity against drug-resistant cancer cells expressing the MDR phenotype. 2. Development of fluorinated taxoids to study the bioactive conformation of paclitaxel and photoaffinity labeling taxoids for mapping of the drug-binding domain on both microtubules and P-glycoprotein. 3. The synthesis of novel macrocyclic taxoids for the investigation into the common pharmacophore for microtubule stabilizing anticancer agents.     

Mitotic slippage in non-cancer cells induced by a microtubule disruptor,disorazole C<Subscript>1</Subscript>

Background

Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C₁ was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C₁ in both non-cancer and cancer cells and compared our results to vinblastine and taxol.

Results

In non-cancer cells, disorazole C₁ induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C₁. On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C₁.

Conclusion

Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C₁. In contrast, cancer cells induce the apoptotic pathway after disorazole C₁ treatment, indicating a possibly significant therapeutic window for this compound.

     

Conformation-dependent ligand regulation of ATP hydrolysis by human KSP: activation of basal hydrolysis and inhibition of microtubule-stimulated hydrolysis by a single, small molecule modulator

Human kinesin spindle protein (KSP)/hsEg5, a member of the kinesin-5 family, is essential for mitotic spindle assembly in dividing human cells and is required for cell cycle progression through mitosis. Inhibition of the ATPase activity of KSP leads to cell cycle arrest during mitosis and subsequent cell death. Ispinesib (SB-715992), a potent and selective inhibitor of KSP, is currently in phase II clinical trials for the treatment of multiple tumor types. Mutations that attenuate Ispinesib binding to KSP in vitro have been identified, highlighting the need for inhibitors that target different binding sites and inhibit KSP activity by novel mechanisms. We report here a small-molecule modulator, KSPA-1, that activates KSP-catalyzed ATP hydrolysis in the absence of microtubules yet inhibits microtubule-stimulated ATP hydrolysis by KSP. KSPA-1 inhibits cell proliferation and induces monopolar-spindle formation in tumor cells. Results from kinetic analyses, microtubule (MT) binding competition assays, and hydrogen/deuterium-exchange studies show that KSPA-1 does not compete directly for microtubule binding. Rather, this compound acts by driving a conformational change in the KSP motor domain and disrupts productive ATP turnover stimulated by MT. These findings provide a novel mechanism for targeting KSP and perhaps other mitotic kinesins.     

Dimethylarsinic acid targets tubulin in mitotic cells to induce abnormal spindles

Dimethylarsinic acid (DMA) is the most effective inducer of cell‐cycle disruption among the arsenic compounds and their metabolites. The present study was conducted to gain further insight into cell‐cycle disruption induced by DMA. The inhibition of cell proliferation and the mitotic arrest induced by DMA were significant and dose‐dependent in Chinese hamster V79 cells and the two seemed to be closely related. At less than 140 µM the DMA did not inhibit the proliferation of cells, but it significantly induced mitotic arrest. An indirect immunofluorescence assay using anti‐α‐tubulin antibodies revealed that DMA induced the formation of abnormal spindles in the metaphase cells even at 350 µM with 5 h of treatment. At 1.4 mM DMA no metaphase cells could form a definite spindle structure. The spindle figures were similar to those induced by colchicine (125 nM ) or vinblastine (110 nM ), major antimitotic agents. In DMA‐treated interphase cells, the microtubule networks were indistinguishable from those of normal cells. With the tubulin‐assembly assay estimated by turbidity, DMA at less than 200 µM suppressed tubulin assembly in a dose‐dependent manner, whereas at more than 700 µM it enhanced tubulin polymerization remarkably with or without addition of excess guanosine‐5′‐triphosphate. According to the above findings, we discussed the possibility that DMA, a primary metabolite of inorganic arsenic in mammals, is related to arsenic carcinogenicity. Copyright © 2001 John Wiley & Sons, Ltd.     

Spirastrellolide A,an antimitotic macrolide isolated from the Caribbean marine sponge Spirastrella coccinea

Sprirastrellolide A, a novel antimitotic macrolide, has been isolated from the Caribbean marine sponge Spirastrella coccinea. It has a 47-carbon linear polyketide backbone incorporated into a highly functionalized 38-membered lactone containing a tetrahydropyran and two spiro bispyran substructures embedded in the macrocycle and a side chain terminating in a carboxylic acid. Sprirastrellolide A's structure was elucidated by detailed spectroscopic analysis of its methyl ester 2. Spirastrellolide A methyl ester 2 shows potent activity in a cell-based assay that detects mitotic arrest, but it does not affect tubulin polymerization in vitro. It has the unusual biological property of being able to accelerate the entry of cells into mitosis from other cell-cycle stages, before it arrests them in mitosis.     

JNK inhibitor SP600125 promotes the formation of polymerized tubulin, leading to G2/M phase arrest, endoreduplication, and delayed apoptosis

The JNK inhibitor SP600125 strongly inhibits cell proliferation in many human cancer cells by blocking cell-cycle progression and inducing apoptosis. Despite extensive study, the mechanism by which SP600125 inhibits mitosis-related effects in human leukemia cells remains unclear. We investigated the effects of SP600125 on the inhibition of cell proliferation and the cell cycle, and on microtubule dynamics in vivo and in vitro. Treatment of synchronized leukemia cells with varying concentrations of SP600125 results in significant G₂/M cell cycle arrest with elevated p21 levels, phosphorylation of histone H3 within 24 h, and endoreduplication with elevated Cdk2 protein levels after 48 h. SP600125 also induces significant abnormal microtubule dynamics in vivo. High concentrations of SP600125 (200 µM) were required to disorganize microtubule polymerization in vitro. Additionally, SP600125-induced delayed apoptosis and cell death was accompanied by significant poly ADP-ribose polymerase (PARP) cleavage and caspase-3 activity in the late phase (at 72 h). Endoreduplication showed a greater increase in ectopic Bcl-2-expressing U937 cells at 72 h than in wild-type U937 cells without delayed apoptosis. These results indicate that Bcl-2 suppresses apoptosis and SP600125-induced G₂/M arrest and endoreduplication. Therefore, we suggest that SP600125 induces mitotic arrest by inducing abnormal spindle microtubule dynamics.     

A new type of biomechanical driver

Segregation of chromosomes and their distribution to opposite ends of the cell is mediated by a mitotic spindle comprised of microtubules (polymeric structures). The forces produced by depolymerizing microtubules, responsible for chromosome motility, were measured, and the mechanochemical mechanism of their development was demonstrated.     

A Novel Docetaxel-Biotin Chemical Conjugate for Prostate Cancer Treatment

A novel conjugate of docetaxel and biotin (designated as IDD-1010) was designed and chemically synthesized via an ester linkage at position 2’ carbon in docetaxel. The synthesized pure IDD-1010 exhibits a potent anti-cancer activity in in vitro and in vivo studies. At 10 nM, IDD-1010 has induced increased apoptosis and mitotic arrest of PC3-Luc prostate cancer cells, causing aneuploidy and cell death at higher concentrations. Toxicology studies indicate that the maximal tolerated dose (MTD) of IDD-1010 is 150 mg/kg in mice; equivalent to about 12.2 mg/kg of body weight, or to about an 850 mg dose for a patient weighing 70 kg. The MTD-treated mice exhibited weight gain similar to that of the control group, with no gross pathological signs at 14 days post-dosing. At a lower dose, IDD-1010 treatment did not lead to any significant weight loss in mice, although decreased the tumor volume stemming from injecting cancer cells into the dorsal loop of mouse prostate, and it was found to be more potent than Paclitaxel (reference drug). Similarly, IDD-1010 treatment significantly reduced tumor weight and thereby increased the percentage of mice survival as compared to reference drug-treated and control groups. To summarize, the described experiments using IDD-1010, as compared to the reference drug, strongly suggest a potential treatment utility with a wider therapeutic window for prostate cancer. Henceforth, clinical research on such a novel drug candidate would be greatly worthwhile.     

UA62784 Is a cytotoxic inhibitor of microtubules, not CENP-E

A recent screen for compounds that selectively targeted pancreatic cancer cells isolated UA62784. We found that UA62784 inhibits microtubule polymerization in?vitro. UA62784 interacts with tubulin dimers ten times more potently than colchicine, vinblastine, or nocodazole. Competition experiments revealed that UA62784 interacts with tubulin at or near the colchicine-binding site. Nanomolar doses of UA62784 promote the accumulation of mammalian cells in mitosis, due to aberrant mitotic spindles, as shown by immunofluorescence and live cell imaging. Treatment of cancerous cell lines with UA62784 is lethal, following activation of apoptosis signaling. By monitoring mitotic spindle perturbations and apoptosis, we found that the effects of UA62784 and of some known microtubule-depolymerizing drugs are additive. Finally, high content screening of H2B-GFP HeLa cells revealed that low doses of UA62784 and vinblastine potentiate each other to inhibit proliferation.     

Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis

BACKGROUND: Understanding the molecular mechanisms of complex cellular processes requires unbiased means to identify and to alter conditionally gene products that function in a pathway of interest. Although random mutagenesis and screening (forward genetics) provide a useful means to this end, the complexity of the genome, long generation time and redundancy of gene function have limited their use with mammalian systems. We sought to develop an analogous process using small molecules to modulate conditionally the function of proteins. We hoped to identify simultaneously small molecules that may serve as leads for the development of therapeutically useful agents. RESULTS: We report the results of a high-throughput, phenotype-based screen for identifying cell-permeable small molecules that affect mitosis of mammalian cells. The predominant class of compounds that emerged directly alters the stability of microtubules in the mitotic spindle. Although many of these compounds show the colchicine-like property of destabilizing microtubules, one member shows the taxol-like property of stabilizing microtubules. Another class of compounds alters chromosome segregation by novel mechanisms that do not involve direct interactions with microtubules. CONCLUSIONS: The identification of structurally diverse small molecules that affect the mammalian mitotic machinery from a large library of synthetic compounds illustrates the use of chemical genetics in dissecting an essential cellular pathway. This screen identified five compounds that affect mitosis without directly targeting microtubules. Understanding the mechanism of action of these compounds, along with future screening efforts, promises to help elucidate the molecular mechanisms involved in chromosome segregation during mitosis.     

Chromosomes are target sites for photodynamic therapy as demonstrated by subcellular laser microirradiation

The present investigation has been undertaken to examine the possibility that the cell nucleus, and specifically the genetic material, is a target site for photodynamic therapy. PTK2 and Hep-2 cells are pretreated with a medium containing 15 microg/ml (0.09 mM) 5-aminolevulinic acid (ALA). Individual fluorescence images are recorded for each selected cell using a cooled charge-coupled device (CCD). A laser microbeam system generating 630 nm is used for subcellular-region irradiation of specific targets: chromosomes, the mitotic spindle, the perispindle region and the peripheral cytoplasm. Nuclei of interphase cells are also irradiated. Data comparing the sensitivities of the different subcellular microirradiation sites in ALA-treated mitotic cells demonstrate that under the irradiation conditions used, the chromosome is the most sensitive subcellular target followed by the perispindle region, the peripheral cytoplasm and spindle, and, lastly, the interphase nucleus.     

Expeditious Total Synthesis of Hemiasterlin through a Convergent Multicomponent Strategy and Its Use in Targeted Cancer Therapeutics

Hemiasterlin is an antimitotic marine natural product with reported sub-nanomolar potency against several cancer cell lines. Herein, we describe an expeditious total synthesis of hemiasterlin featuring a four-component Ugi reaction (Ugi-4CR) as the key step. The convergent synthetic strategy enabled rapid access to taltobulin (HTI-286), a similarly potent synthetic analogue. This short synthetic sequence enabled investigation of both hemiasterlin and taltobulin as cytotoxic payloads in antibody–drug conjugates (ADCs). These novel ADCs displayed sub-nanomolar cytotoxicity against HER2-expressing cancer cells, while showing no activity against antigen-negative cells. This study demonstrates an improved synthetic route to a highly valuable natural product, facilitating further investigation of hemiasterlin and its analogues as potential payloads in targeted therapeutics.     

(+)-Discodermolide binds to microtubules in stoichiometric ratio to tubulin dimers,blocks taxol binding and results in mitotic arrest

Background: The marine natural product (+)-discodermolide has potent immunosuppressive activity. It inhibits proliferation of a wide range of human and murine cells, induces cell cycle arrest in the G2 or M phase and was recently shown to stabilize microtubules. Total synthesis of discodermolide has made it possible to generate variants of the compound to study its intracellular function in detail.Results: We have determined that (+)-discodermolide arrests MG63 cells at M phase, and has a stabilizing effect on microtubules. In vitro studies show that discodermolide induces polymerization of purified tubulin in the absence of microtubule-associated proteins, and that it binds to tubulin dimers in microtubules at 1:1 stoichiometry. Discodermolide binds taxol-polymerized microtubules at near stoichiometric level, whereas taxol binds discodermolide-induced microtubules poorly. Competition data show that the binding of microtubules by discodermolide and taxol are mutually exclusive; discodermolide binds with higher affinity than taxol. The results of binding assays carried out in vivo or in cell lysates also suggest that the microtubule network is discodermolide's cellular target.Condusions: (+)-Discodermolide causes cell cycle arrest at the metaphase-anaphase transition in mitosis, presumably due to its stabilizing effect on microtubules. In vitro, discodermolide polymerizes purified tubulin potently in the absence of MAPs. It binds microtubules at one molecule per tubulin dimer with a higher affinity than taxol, and the binding of microtubules by discodermolide and taxol are mutually exclusive. In total cell lysates discodermolide displays binding activity that is consistent with its effects on microtubules.     

Synthesis of a novel polycyclic ring scaffold with antimitotic properties via a selective domino Heck–Suzuki reaction

The synthesis of a previously undescribed sp³-rich 6-5-5-6 tetracyclic ring scaffold using a palladium catalysed domino Heck–Suzuki reaction is reported. This reaction is high-yielding, selective for the domino process over the direct Suzuki reaction and tolerant towards a variety of boronic acids. The novel scaffold can also be accessed via domino Heck–Stille and radical cyclisations. Compounds based around this scaffold were found to be effective antimitotic agents in a human cancer cell line. Detailed phenotypic profiling showed that the compounds affected the congression of chromosomes to give mitotic arrest and apoptotic cell death. Thus, a novel structural class of antimitotic agents that does not disrupt the tubulin network has been identified.     

Impaired phosphorylation and mis-localization of Bub1 and BubR1 are responsible for the defective mitotic checkpoint function in Brca2-mutant thymic lymphomas

Breast cancer susceptibility gene, BRCA2, is a tumor suppressor and individuals who inherit one defected copy of BRCA2 allele experience early onset breast cancer or ovarian cancer accompanied by the loss of the wild type allele. Mouse model for Brca2 mutation shows growth retardation and paradoxical occurrence of thymic lymphomas. Thymic lymphomas from Brca2-mutant mice harbor mutations in p53, Bub1, and BubR1, which function as mitotic checkpoint proteins. Therefore, interplay between Brca2 and mitotic checkpoint has been suggested in the maintenance of genetic fidelity, although it has not been assessed whether the unique mutations in Bub1 and BubR1 found in Brca2-mutant mice are responsible for the abolishment of mitotic checkpoint function. This report demonstrates that Bub1 and BubR1 mutant proteins from Brca2-/- thymic lymphomas have defects in the phosphorylation and kinetochore localization after spindle damage. Thus, the mutations of Bub1 and BubR1 found in Brca2- mutant mice indeed are responsible for the chromosome instability in Brca2-mutated tumors.     

Xanthatin induces cell cycle arrest at G2/M checkpoint and apoptosis via disrupting NF-κB pathway in A549 non-small-cell lung cancer cells

Xanthatin, a natural sesquiterpene lactone, has significant antitumor activity against a variety of cancer cells, yet little is known about its anticancer mechanism. In this study, we demonstrated that xanthatin had obvious dose-/time-dependent cytotoxicity against the human non-small-cell lung cancer (NSCLC) cell line A549. Flow cytometry analysis showed xanthatin induced cell cycle arrest at G2/M phase. Xanthatin also had pro-apoptotic effects on A549 cells as evidenced by Hoechst 33258 staining and annexin V-FITC staining. Mechanistic data revealed that xanthatin downregulated Chk1, Chk2, and phosphorylation of CDC2, which contributed to the cell cycle arrest. Xathatin also increased total p53 protein levels, decreased Bcl-2/Bax ratio and expression of the downstream factors procaspase-9 and procaspase-3, which triggered the intrinsic apoptosis pathway. Furthermore, xanthatin blocked phosphorylation of NF-κB (p65) and IκBa, which might also contribute to its pro-apoptotic effects on A549 cells. Xanthatin also inhibited TNFa induced NF-κB (p65) translocation. We conclude that xanthatin displays significant antitumor effects through cell cycle arrest and apoptosis induction in A549 cells. These effects were associated with intrinsic apoptosis pathway and disrupted NF-κB signaling. These results suggested that xanthatin may have therapeutic potential against NSCLC.     

Synthesis and identification of small molecules that potently induce apoptosis in melanoma cells through G1 cell cycle arrest

Late-stage malignant melanoma is a cancer that is refractory to current chemotherapeutic treatments. The average survival time for patients with such a diagnosis is 6 months. In general, the vast majority of anticancer drugs operate through induction of cell cycle arrest and cell death in either the DNA synthesis (S) or mitosis (M) phase of the cell cycle. Unfortunately, the same mechanisms that melanocytes possess to protect cells from DNA damage often confer resistance to drugs that derive their toxicity from S or M phase arrest. Described herein is the synthesis of a combinatorial library of potential proapoptotic agents and the subsequent identification of a class of small molecules (triphenylmethylamides, TPMAs) that arrest the growth of melanoma cells in the G1 phase of the cell cycle. Several of these TPMAs are quite potent inducers of apoptotic death in melanoma cell lines (IC(50) approximately 0.5 muM), and importantly, some TPMAs are comparatively nontoxic to normal cells isolated from the bone marrow of healthy donors. Furthermore, the TPMAs were found to dramatically reduce the level of active nuclear factor kappa-B (NFkappaB) in the cell; NFkappaB is known to be constitutively active in melanoma, and this activity is critical for the proliferation of melanoma cells and their evasion of apoptosis. Compounds that reduce the level of NFkappaB and arrest cells in the G1 phase of the cell cycle can provide insights into the biology of melanoma and may be effective antimelanoma agents.     

Novel Semi-Synthetic Cu (II)–Cardamonin Complex Exerts Potent Anticancer Activity against Triple-Negative Breast and Pancreatic Cancer Cells via Inhibition of the Akt Signaling Pathway

Cardamonin is a polyphenolic natural product that has been shown to possess cytotoxic activity against a variety of cancer cell lines. We previously reported the semi-synthesis of a novel Cu (II)–cardamonin complex (19) that demonstrated potent antitumour activity. In this study, we further investigated the bioactivity of 19 against MDA-MB-468 and PANC-1 cancer cells in an attempt to discover an effective treatment for triple-negative breast cancer (TNBC) and pancreatic cancer, respectively. Results revealed that 19 abolished the formation of MDA-MB-468 and PANC-1 colonies, exerted growth-inhibitory activity, and inhibited cancer cell migration. Further mechanistic studies showed that 19 induced DNA damage resulting in gap 2 (G2)/mitosis (M) phase arrest and microtubule network disruption. Moreover, 19 generated reactive oxygen species (ROS) that may contribute to induction of apoptosis, corroborated by activation of caspase-3/7, PARP cleavage, and downregulation of Mcl-1. Complex 19 also decreased the expression levels of p-Akt and p-4EBP1, which indicates that the compound exerts its activity, at least in part, via inhibition of Akt signalling. Furthermore, 19 decreased the expression of c-Myc in PANC-1 cells only, which suggests that it may exert its bioactivity via multiple mechanisms of action. These results demonstrate the potential of 19 as a therapeutic agent for TNBC and pancreatic cancer.