RESUMO
Glioblastoma (GBM) has poor median survival due to its resistance to chemoradiotherapy, which results in tumor recurrence. Recurrent GBMs currently lack effective treatments. DUSP6 is known to be pro-tumorigenic and is upregulated in GBM. We show that DUSP6 expression is significantly higher in recurrent GBM patient biopsies compared to expression levels in primary GBM biopsies. Importantly, although it has been reported to be a cytoplasmic protein, we found nuclear localization of DUSP6 in primary and recurrent patient samples and in parent and relapse populations of GBM cell lines generated from an in vitro radiation survival model. DUSP6 inhibition using BCI resulted in decreased proliferation and clonogenic survival of parent and relapse cells. Pharmacological or genetic inhibition of DUSP6 catalytic activity radiosensitized primary and, importantly, relapse GBM cells by inhibiting the recruitment of phosphorylated DNAPKcs (also known as PRKDC), subsequently downregulating the recruitment of phosphorylated histone H2AX (γH2AX) and 53BP1 (also known as TP53BP1). This resulted in decreased cell survival and prolonged growth arrest upon irradiation in vitro and significantly increased the progression-free survival in orthotopic mouse models of GBM. Our study highlights a non-canonical function of DUSP6, emphasizing the potential application of DUSP6 inhibitors in the treatment of recurrent GBM.
Assuntos
Neoplasias Encefálicas , Proteína Quinase Ativada por DNA , Glioblastoma , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , DNA , Quebras de DNA de Cadeia Dupla , Fosfatase 6 de Especificidade Dupla , Glioblastoma/genética , Glioblastoma/radioterapia , Humanos , Camundongos , Tolerância a Radiação/genéticaRESUMO
Gold nanoparticles have been investigated extensively for their molecular mechanisms of action and anticancer potential. We report a novel, tubulin-targeted antiproliferative mechanism of action of tryptone-stabilized gold nanoparticles (TsAuNPs). TsAuNPs, synthesized using HAuCl4·3H2O and tryptone and characterized by a variety of spectroscopic methods and transmission electron microscopy, were found to be inhibitory to viability of human pancreatic (PANC-1), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines in a concentration-dependent manner, with highest efficacy against PANC-1 cells. The particles strongly inhibited the clonogenic propagation of PANC-1 cells. TsAuNPs-mediated inhibition of cell viability involved an unusual mode of cell cycle arrest (arrest at both G0/G1 phase and S-phase) followed by apoptosis. In vitro, TsAuNPs bound purified tubulin, competitively inhibited anilinonaphthalene sulfonate binding to tubulin, and suppressed tubulin assembly. In cells, tubulin-TsAuNPs interactions were manifested as a disrupted microtubule network, defective reassembly of cold-disassembled microtubules, and induction of tubulin acetylation. Our data indicate that TsAuNPs inhibit cell viability by inducing differential cell cycle arrest possibly through disrupted dynamicity of cellular microtubules.
Assuntos
Ciclo Celular/efeitos dos fármacos , Ouro/química , Nanopartículas Metálicas/química , Peptonas/química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Estabilidade de Medicamentos , Ouro/farmacologia , Células HeLa , Humanos , Camundongos , Terapia de Alvo Molecular/métodos , Células NIH 3T3 , Peptonas/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
We have synthesized new, biologically active mono- and di-substituted 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives bearing electron withdrawing groups and electron donating groups. These derivative structures were characterized by their spectral and analytical data. The newly synthesized hexahydropyrazole analogues were evaluated for their in vitro anticancer activity against breast and lung cancer cell lines using a cytotoxicity bioassay. To understand their mechanism of action, tubulin binding assays were performed which pointed to their binding to microtubules in a mode similar to but not identical to colchicine, as evidenced by their KD value evaluation. Computational docking studies also suggested binding near the colchicine binding site on tubulin. These results were further confirmed by colchicine-binding assays on the most active compounds, which indicated that they bound to tubulin near but not at the colchicine site. The moderate cytotoxic effects of these compounds may be due to the presence of electron donating groups on the para-position of the phenyl ring, along with the hexahydropyrazole core nucleus. The observed anti-cancer activity based on inhibition of microtubule formation may be helpful in designing more potent compounds with a hexahydropyrazole moiety.
Assuntos
Antineoplásicos/síntese química , Pirazóis/química , Antineoplásicos/química , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Ciclopentanos/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Pirazóis/síntese química , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/toxicidadeRESUMO
Glioblastoma (GBM) is the most common primary brain tumor and is highly aggressive with a median survival of 15 months. We have previously shown that residual cells of GBM form multinucleated giant cells (MNGCs) showing a senescent phenotype, but eventually escape from therapy induced senescence (TIS), resulting in GBM recurrence. Here we demonstrate the role of PARP-1 in TIS and its recovery. We show that genetic and pharmacological inhibition of PARP-1 has an anti-proliferative effect on GBM cell lines and primary cultures derived from patient samples. Furthermore, the PARP-1 inhibitor olaparib, in combination with radiation increased MNGCs formation and senescence as assessed by ß-galactosidase activity, and macroH2A1 levels in residual cells. Additionally, we found that reduced PARP-1 activity and not protein levels in residual cells was crucial for MNGCs formation and their maintenance in the senescent state. PARP-1 activity was restored to higher levels in recurrent cells that escaped from TIS. Importantly, olaparib + radiation treatment significantly delayed recurrence in vitro as well in vivo in orthotopic GBM mouse models with a significant increase in overall survival of mice. Overall, this study demonstrates that sustained inhibition of PARP-1 activity during radiation treatment significantly delays GBM recurrence.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Recidiva Local de Neoplasia/enzimologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Neoplasias Encefálicas/enzimologia , Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Glioblastoma/enzimologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Recidiva Local de Neoplasia/patologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/efeitos da radiação , Tolerância a Radiação/efeitos dos fármacos , Radioterapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Noscapine is a non-narcotic, antitussive alkaloid isolated from plants of Papaveraceae family. This benzylisoquinoline alkaloid and its synthetic derivatives, called noscapinoids, are being evaluated for their anticancer potential. METHODS: The structure of a novel analogue, N-(3-bromobenzyl) noscapine (N-BBN) was elucidated by X-ray crystallography. Effect of N-BBN on cancer cell proliferation and cellular microtubules were studied by sulphorhodamine B assay and immunofluorescence, respectively. Binding interactions of the alkaloid with tubulin was studied using spectrofluorimetry. RESULTS: N-BBN, synthesized by introducing modification at site B ('N' in isoquinoline unit) and a bromo group at the 9th position of the parent compound noscapine, was found to be superior to many of the past-generation noscapinoids in inhibiting cancer cell viability and it showed a strong inhibition of the clonogenic potential of an aggressively metastatic breast tumour cell line, MDA-MB-231. The compound perturbed the tertiary structure of purified tubulin as indicated by an anilinonaphthalene sulfonic acid-binding assay. However, substantiating the common feature of noscapinoids, it did not alter microtubule polymer mass considerably. In cells, the drug-treatment showed a peculiar type of disruption of normal microtubule architecture. CONCLUSION: N-BBN may be considered for further investigations as a potent antiproliferative agent.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Microtúbulos/efeitos dos fármacos , Noscapina/farmacologia , Moduladores de Tubulina/farmacologia , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Feminino , Humanos , Microtúbulos/metabolismo , Microtúbulos/patologia , Modelos Moleculares , Estrutura Molecular , Noscapina/análogos & derivados , Noscapina/síntese química , Noscapina/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/metabolismoRESUMO
Triphala (Trl) is an ayurvedic formulation used for treating disorders of the digestive, respiratory, and nervous systems. Its anticancer properties have also been documented. We studied effects of Trl on tubulin, a target protein for several anticancer drugs, and systematically elucidated a possible antiproliferative mechanism of action of Trl. Trl inhibited proliferation of HeLa (cervical adenocarcinoma), PANC-1 (pancreatic adenocarcinoma), and MDA-MB-231 (triple-negative breast carcinoma) cells in microgram quantities and strongly suppressed the clonogenicity of HeLa cells. The formulation disrupted secondary conformation of tubulin and inhibited anilino naphthalene sulfonate binding to tubulin. In cells, Trl-tubulin interactions were manifested as a perturbed microtubule network. Acetylation pattern of Trl-treated cellular microtubules indicated persistent stabilization of microtubule dynamics. In addition, Trl interfered with reassembly of the microtubules. Cells treated with Trl eventually underwent programmed cell death as evidenced by annexin-V staining. Our study shows that the effect of aqueous extract of Trl is potent enough to interfere with the assembly dynamics of microtubules, and that Trl can be investigated further for its antitumor potential.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Anexina A5/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Microtubule-targeted drugs (MTDs) have been on the forefront of breast cancer chemotherapy. Classic MTDs, such as paclitaxel and their semisynthetic derivatives, have achieved considerable success in the clinical management of breast neoplasms. In order to improve the specificity and to reduce undesirable, dose-limiting toxicities of these drugs, a plethora of novel compounds are being synthesized and investigated in laboratories worldwide. Due to their crucial roles during cell division, and to the fact that the suppression of their innate 'dynamic instability' can arrest cell cycle progression, microtubules formed an attractive target for cancer chemotherapy. Kadcyla (ado-trastuzumab emtansine), Halaven (eribulin mesylate), and Ixempra (Ixabepilone) are three relatively-novel, microtubule-targeting antibreast cancer drugs. Kadcyla was developed by conjugating a very potent derivative of the natural product maytansine to trastuzumab, a HER2-targeted monoclonal antibody. Kadcyla is a double-edged weapon, that is, it prevents receptor dimerization to inhibit cell proliferation, and then it enters inside the target tumour cell by receptor-mediated endocytosis and ensures death of the cell. Halaven (eribulin mesylate), created by simplifying the structure of the marine sponge-derived molecule Halichondrin B, works primarily by suppressing the growth rates of microtubules and thereby inducing cell cycle arrest and cell death. Ixabepilone, the semisynthetic analogue of epothilone B, suppresses the shortening rates of dynamic microtubules resulting in cell cycle inhibition and cell death. In order to improve the efficacy and reduce drug-induced side effects, novel therapeutic strategies, including liposome-mediated drug delivery, are being investigated.
Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Drogas Desenhadas/farmacologia , Microtúbulos/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Drogas Desenhadas/síntese química , Drogas Desenhadas/química , Feminino , HumanosRESUMO
Noscapine is an alkaloid present in the latex of Papaver somniferum. It has been known for its anticancer efficacy and lack of severe toxicities to normal tissues. Structural alterations in noscapine core architecture have produced a number of potent analogues of noscapine. Here, we report an unusual activity of a novel noscapine analogue, 9-(4-vinylphenyl)noscapine (VinPhe-Nos) on cancer cells. As we reported earlier, VinPhe-Nos inhibited MDA-MB-231 cell proliferation with an IC50 of 6µM. The present study elucidated a possible antiproliferative mechanism of action of VinPhe-Nos. The noscapinoid significantly inhibited clonogenic propagation of MDA-MB-231 cells. However, unlike the majority of tubulin-binding agents, it did not induce mitotic arrest; instead, it prolonged S-phase. Although prolonged presence of the drug show some disruption of cellular microtubule architecture, it did not affect microtubule recovery after cold-induced depolymerization. VinPhe-Nos, nevertheless, induced acetylation and bundling of microtubules. Our data suggest that rational modification of parent compound can alter its mechanism of action on cell cycle and that VinPhe-Nos can be investigated further as a less-toxic, S-phase-preferred, cytostatic anticancer agent.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Microtúbulos/fisiologia , Noscapina/análogos & derivados , Noscapina/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Acetilação/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Microtúbulos/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacosRESUMO
We have recently reported the synthesis and antiproliferative potential of a series of biaryl type α-noscapine congeners. Among them, 9-(3-pyridyl) noscapine 3f (9-PyNos, henceforth), which was synthesized by adding pyridine unit to the tetrahydroisoquinoline part of natural α-noscapine core, was found to be the most effective one to inhibit proliferation of a variety of cancer cell lines. However, details of its interactions with its cellular target, tubulin, remain poorly understood. In this report, we examined the nature of interactions of 9-PyNos with tubulin based on the methodologies of spectrofluorimetry, circular dichroism, and turbidimetry techniques. Far-UV circular dichroism spectra indicated perturbation of tubulin secondary structure in the presence of 9-PyNos, not amounting, however, to the perturbation induced by noscapine. The noscapinoid nevertheless altered the surface configuration of the protein considerably, as indicated by an anilinonaphthalene sulphonate binding assay, and promoted colchicine binding to tubulin, the latter indicating its adjacent binding site with colchicine. 9-PyNos however, did not alter microtubule assembly considerably. Investigating the possible reason behind this apparent lack of strong inhibition of microtubule assembly, we found that the binding interactions of tubulin with 9-PyNos do not involve modification of cysteine residues of tubulin. Taken together, our data suggest that the antiproliferative mechanism of action of 9-PyNos involves disruption of structural integrity of tubulin without strong inhibition of tubulin assembly.
Assuntos
Noscapina/análogos & derivados , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Humanos , Estrutura Molecular , Noscapina/síntese química , Noscapina/química , Noscapina/farmacologia , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/químicaRESUMO
Βeta-sitosterol (ß-SITO), a phytosterol present in pomegranate, peanut, corn oil, almond, and avocado, has been recognized to offer health benefits and potential clinical uses. ß-SITO is orally bioavailable and, as a constituent of edible natural products, is considered to have no undesired side effects. It has also been considered as a potent anticancer agent. However, the molecular mechanism of action of ß-SITO as a tubulin-binding anticancer agent and its binding site on tubulin are poorly understood. Using a combination of biochemical analyses and molecular dynamic simulation, we investigated the molecular details of the binding interactions of ß-SITO with tubulin. A polymer mass assay comparing the effects of ß-SITO and of taxol and vinblastine on tubulin assembly showed that this phytosterol stabilized microtubule assembly in a manner similar to taxol. An 8-anilino-1-naphthalenesulfonic acid assay confirmed the direct interaction of ß-SITO with tubulin. Although ß-SITO did not show direct binding to the colchicine site on tubulin, it stabilized the colchicine binding. Interestingly, no sulfhydryl groups of tubulin were involved in the binding interaction of ß-SITO with tubulin. Based on the results from the biochemical assays, we computationally modeled the binding of ß-SITO with tubulin. Using molecular docking followed by molecular dynamic simulations, we found that ß-SITO binds tubulin at a novel site (which we call the 'SITO site') adjacent to the colchicine and noscapine sites. Our data suggest that ß-SITO is a potent anticancer compound that interferes with microtubule assembly dynamics by binding to a novel site on tubulin.