Elucidation of the anticancer potential and tubulin isotype-specific interactions of β-sitosterol

Beta-sitosterol (β-SITO), a phytosterol present in many edible vegetables, has been reported to possess antineoplastic properties and cancer treatment potential. We have shown previously that it binds at a unique site (the ‘SITO-site’) compared to the colchicine binding site at the interface of α- and β-tubulin. In this study, we investigated the anticancer efficacy of β-SITO against invasive breast carcinoma using MCF-7 cells. Since ‘isotypes’ of β-tubulin show tissue-specific expression and many are associated with cancer drug resistance, using computer-assisted docking and atomistic molecular dynamic simulations, we also examined its binding interactions to all known isotypes of β-tubulin in αβ-tubulin dimer. β-SITO inhibited MCF-7 cell viability by up to 50%, compared to vehicle-treated control cells. Indicating its antimetastatic potential, the phytosterol strongly inhibited cell migration. Immunofluorescence imaging of β-SITO-treated MCF-7 cells exhibited disruption of the microtubules and chromosome organization. Far-UV circular dichroism spectra indicated loss of helical stability in tubulin when bound to β-SITO. Docking and MD simulation studies, combined with MM-PBSA and MM-GBSA calculations revealed that β-SITO preferentially binds with specific β-tubulin isotypes (β_II and β_III) in the αβ-tubulin dimer. Both these β-tubulin isotypes have been implicated in drug resistance against tubulin-targeted chemotherapeutics. Our data show the tubulin-targeted anticancer potential of β-SITO, and its potential clinical utility against β_II and β_III isotype-overexpressing neoplasms.     

Phenethyl isothiocyanate induces cell cycle arrest and reduction of alpha- and beta-tubulin isotypes in human prostate cancer cells

This study was to investigate the effect of phenethyl isothiocyanate (PEITC), a constituent of many edible cruciferous vegetables, on the expression of α- and β-tubulins, which are the main components of microtubules in prostate cancer cells. Flow cytometry, light microscopy and western blot were used to study the cell cycle distribution, morphology changes and the expression of α- and β-tubulins in prostate cancer cells treated with PEITC. The results showed that PEITC-induced G2-M cell phase arrest and inhibited the expression of α- and β-tubulin proteins in a number of human prostatic carcinoma cell lines. Further, it is showed that this inhibitory effect could be reversed by antioxidant N-acetyl cysteine and proteasome inhibitor MG132. Finally, it is concluded that PEITC inhibited the expression of α- and β-tubulins in prostate cancer cells, which is at least related to the oxygen reaction species and protein degradation.     

BMP2 promotes migration and invasion of breast cancer cells via cytoskeletal reorganization and adhesion decrease: an AFM investigation

Bone morphogenetic protein 2 (BMP2) has been shown to modulate the proliferation and differentiation of breast cancer cells. However, the biochemical effects and mechanisms remain unknown. In this paper, the effects of recombinant human BMP2 on the migration of MCF-7 cells—one breast cancer cell line, using transwell and wound healing experiments, as well as on the cellular morphology, cytoskeleton, cell surface adhesion, and stiffness detected at subcellular level by an atomic force microscope, were investigated. After BMP2 treatment, the untreated round-shaped MCF-7 cells transformed to a spindle-like shape with lots of specialized structures, such as lamellipodia, filopodia, membrane protrusions, and others, which are essential for cellular migration or spreading. Moreover, flow cytometry quantitatively detected the BMP2-induced changes in the expression of adhesion molecules, a significant rise of CD44, and a remarkable drop of E-cadherin. The data indicated that BMP2 promoted the migration and invasion of MCF-7 cells by regulating the reorganization of cytoskeleton and the expression of adhesion molecules in/on the cells. Thus, it is very imperative to evaluate the oncogenicity of BMP2 when used in tissue engineering.     

EDI3 links choline metabolism to integrin expression,cell adhesion and spreading

Endometrial carcinoma differential 3 (EDI3) was the first member of the glycerophosphodiesterase (GDE) protein family shown to be associated with cancer. Our initial work demonstrated that endometrial and ovarian cancer patients with primary tumors overexpressing EDI3 had a higher risk of developing metastasis and decreased survival. Further analysis indicated that EDI3 cleaves glycerophosphocholine to choline and glycerol-3-phosphate, increases the levels of active PKC, and enhances the migratory activity of tumor cells. Despite these initial findings, EDI3 remained mainly uncharacterized. Therefore, to obtain an overview of processes in which EDI3 may be involved, gene array analysis was performed using MCF-7 breast cancer cells after EDI3 knockdown compared with a non-targeting control siRNA. Several biological motifs were altered, including an enrichment of genes involved in integrin-mediated signaling. More specifically, silencing of EDI3 in MCF-7 and OVCAR-3 cells was associated with reduced expression of the key receptor subunit integrin β1, leading to decreased cell attachment and spreading accompanied by delayed formation of cell protrusions. To confirm these results, we stably overexpressed EDI3 in MCF-7 cells which led to elevated integrin β1 expression associated with enhanced cell attachment and spreading - two processes critical for metastasis. In conclusion, our data provide further insight into the role of EDI3 during cancer progression.     

Anti-tumor potential of ethanol extract of Curcuma phaeocaulis Valeton against breast cancer cells

Curcuma phaeocaulis Valeton is a commonly prescribed Chinese medical herb for tumor therapy. In this study, an extract of Curcuma phaeocaulis Valeton referred as Cpv was prepared and its anti-tumor effect was evaluated with MCF-7 and MDA-MB-231 cells. Curcuma phaeocaulis Valeton power was extracted with ethanol and the main components of the extract (Cpv) were analyzed with HPLC. The effect of Cpv on MCF-7 cells proliferation, intracellular reactive oxygen species (ROS) formation, mitochondrial membrane potential (ΔΨm), apoptosis, apoptotic related proteins, MDA-MB-231 cell migration, and integrins expression were determined. Furthermore, the effect of Cpv on some key signal transduction molecules was also investigated. Furanodienone, germacrone and furanodiene were identified as the main components of Cpv. Cpv treatment significantly inhibited cell proliferation, increased LDH release, induced intracellular ROS formation, and decreased ΔΨm in a dose-dependent manner in MCF-7 cells. Cpv induced apoptosis without affecting cell migration. Cpv increased protein expression of Bax, PARP, cleaved PARP, caspase-3, 7, JNK1, p-p42/44MAPK, NF-κB, IKKα, IKKβ, decreased protein expression of Bcl-2, Bcl-xL, Bim, Bik, Bad, integrin β5, p42/44MAPK without affecting integrin α5, β1, and p38MAPK protein expression. We concluded that Cpv inhibited MCF-7 cells proliferation by inducing apoptosis mediated by increasing ROS formation, decreasing ΔΨm, regulating Bcl-2 family proteins expression, and activating caspases. Cpv treatment also modulated several signaling transduction pathways. These results might provide some molecular basis for the anti-tumor activity of Curcuma phaeocaulis Valeton.     

Surfactin对人乳腺癌MCF-7细胞增殖、凋亡及细胞骨架的影响

以体外培养的人乳腺癌细胞株MCF-7为研究对象,探讨surfactin对肿瘤细胞增殖、凋亡及细胞骨架的影响。MTT实验表明,surfactin能抑制MCF-7的增殖,并且呈现出浓度和时间的依赖关系,作用48h时的IC50是27.3μmol/L。AO/EB荧光染色法及流式细胞术检测发现,surfactin可诱导MCF-7发生典型的凋亡形态学改变和G2/M期阻滞。免疫荧光和免疫印迹结果表明,surfactin显著抑制了细胞内vimentin的表达,诱导了α-tubulin的解聚和重排,使细胞的骨架系统发生了剧烈的变化。可见,surfactin具有抑制MCF-7细胞增殖,诱导细胞凋亡的作用,其机制可能与细胞骨架蛋白的表达水平有关。     

RhoB regulates cell migration through altered focal adhesion dynamics

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.     

Electrochemical immunoassay on expression of integrin β1 on tumor cells and drug-resistant tumor cells

An electrochemical indirect competitive immunoassay protocol as a promising cytosensing strategy was developed to detect integrin β1 expression on human breast cancer MCF-7 cells and adriamycin-resistant human breast cancer MCF-7 (MCF-7/ADR) cells and quantify the cell number. Integrin α5β1 was adsorbed on the gold-nanoparticle modified glassy carbon electrode to bind integrin β1 monoclonal antibody (anti-CD29 mAb). A sandwich structure was then formed using nanocomposites which consisted of horseradish peroxidase (HRP) labeled anti-antibody and gold nanoparticles. HRP bound on the electrode surface could cause an amperometric response of the hydroquinone-H(2)O(2) system. The assembly of the sandwich structure was inhibited by tumor cells to give decreased enzyme-catalytic signals due to the capture of anti-CD29 mAb by integrin β1 on cell membranes. Under optimal conditions the relative current change (S) was proportional to the cell concentration from 1.6×10(3) to 2.0×10(6)cellsmL(-1) with a detection limit of 700cellsmL(-1). Integrin β1 expression in MCF-7/ADR cells was found to be significantly higher than that in MCF-7 cells, indicating the increased adhesion ability of MCF-7/ADR cells.     

Erythropoietin promotes MCF-7 breast cancer cell migration by an ERK/mitogen-activated protein kinase-dependent pathway and is primarily responsible for the increase in migration observed in hypoxia

Recent studies indicate that cancer cells express erythropoietin receptor (EpoR). In this study, we have shown that erythropoietin (Epo) activates the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and promotes migration in MCF-7 breast cancer cells. Epo-stimulated MCF-7 cell migration was blocked by the MEK inhibitor PD098059 and by dominant negative MEK-1, indicating an essential role for ERK. When MCF-7 cells were exposed to hypoxia (1.0% O(2)) for 3 h, the Epo mRNA level increased 2.4 +/- 0.5-fold, the basal level of ERK activation increased, and cell migration increased 2.0 +/- 0.1-fold. Soluble EpoR and Epo-neutralizing antibody significantly inhibited hypoxia-induced MCF-7 cell migration, suggesting a major role for autocrine EpoR cell signaling. MCF-7 cell migration under hypoxic conditions was also inhibited by PD098059. These experiments identify a novel pathway by which exogenously administered Epo, and Epo that is produced locally by cancer cells under hypoxic conditions, may stimulate cancer cell migration.     

Cytosolic carboxypeptidase 1 is involved in processing α- and β-tubulin

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.     

Hsa_circ_0092276 promotes doxorubicin resistance in breast cancer cells by regulating autophagy via miR-348/ATG7 axis

Previous study has confirmed that hsa_circ_0092276 is highly expressed in doxorubicin (DOX)-resistant breast cancer cells, indicating that hsa_circ_0092276 may be involved in regulating the chemotherapy resistance of breast cancer. Here we attempted to investigate the biological role of hsa_circ_0092276 in breast cancer. We first constructed DOX-resistant breast cancer cells (MCF-7/DOX and MDA-MB-468/DOX). The 50% inhibiting concentration of MCF-7/DOX and MDA-MB-468/DOX cells was significantly higher than that of their parental breast cancer cells, MCF-7 and MDA-MB-46. MCF-7/DOX and MDA-MB-468/DOX cells also exhibited an up-regulation of drug resistance-related protein MDR1. Compared with MCF-7 and MDA-MB-46 cells, hsa_circ_0092276 was highly expressed in MCF-7/DOX and MDA-MB-468/DOX cells. Hsa_circ_0092276 overexpression enhanced proliferation and the expression of LC3-II/LC3-I and Beclin-1, and repressed apoptosis of breast cancer cells. The effect of hsa_circ_0092276 up-regulation on breast cancer cells was abolished by 3-methyladenine (autophagy inhibitor). Hsa_circ_0092276 modulated autophagy-related gene 7 (ATG7) expression via sponging miR-384. Hsa_circ_0092276 up-regulation promoted autophagy and proliferation, and repressed apoptosis of breast cancer cells, which was abolished by miR-384 overexpression or ATG7 knockdown. In addition, LV-circ_0092276 transfected MCF-7 cell transplantation promoted autophagy and tumor growth of breast cancer in mice. In conclusion, our data demonstrate that hsa_circ_0092276 promotes autophagy and DOX resistance in breast cancer by regulating miR-348/ATG7 axis. Thus, this article highlights a novel competing endogenous RNA circuitry involved in DOX resistance in breast cancer.     

A human IgM signals axon outgrowth: coupling lipid raft to microtubules

Mouse and human IgMs support neurite extension from primary cerebellar granule neurons. In this study using primary hippocampal and cortical neurons, we demonstrate that a recombinant human IgM, rHIgM12, promotes axon outgrowth by coupling membrane domains (lipid rafts) to microtubules. rHIgM12 binds to the surface of neuron and induces clustering of cholesterol and ganglioside GM1. After cell binding and membrane fractionation, rHIgM12 gets segregated into two pools, one associated with lipid raft fractions and the other with the detergent-insoluble cytoskeleton-containing pellet. Membrane-bound rHIgM12 co-localized with microtubules and co-immuno precipitated with β3-tubulin. rHIgM12-membrane interaction also enhanced the tyrosination of α-tubulin indicating a stabilization of new neurites. When presented as a substrate, rHIgM12 induced axon outgrowth from primary neurons. We now demonstrate that a recombinant human mAb can induce signals in neurons that regulate membrane lipids and microtubule dynamics required for axon extension. We propose that the pentameric structure of the IgM is critical to cross-link membrane lipids and proteins resulting in signaling cascades.     

A novel adhesion molecule in human breast cancer cells: Voltage-gated Na+ channel β1 subunit

Voltage-gated Na⁺ channels (VGSCs), predominantly the ‘neonatal’ splice form of Na_v1.5 (nNa_v1.5), are upregulated in metastatic breast cancer (BCa) and potentiate metastatic cell behaviours. VGSCs comprise one pore-forming α subunit and one or more β subunits. The latter modulate VGSC expression and gating, and can function as cell adhesion molecules of the immunoglobulin superfamily. The aims of this study were (1) to determine which β subunits were expressed in weakly metastatic MCF-7 and strongly metastatic MDA-MB-231 human BCa cells, and (2) to investigate the possible role of β subunits in adhesion and migration. In both cell lines, the β subunit mRNA expression profile was SCN1B (encoding β1) ? SCN4B (encoding β4) > SCN2B (encoding β2); SCN3B (encoding β3) was not detected. MCF-7 cells had much higher levels of all β subunit mRNAs than MDA-MB-231 cells, and β1 mRNA was the most abundant. Similarly, β1 protein was strongly expressed in MCF-7 and barely detectable in MDA-MB-231 cells. In MCF-7 cells transfected with siRNA targeting β1, adhesion was reduced by 35%, while migration was increased by 121%. The increase in migration was reversed by tetrodotoxin (TTX). In addition, levels of nNa_v1.5 mRNA and protein were increased following β1 down-regulation. Stable expression of β1 in MDA-MB-231 cells increased functional VGSC activity, process length and adhesion, and reduced lateral motility and proliferation. We conclude that β1 is a novel cell adhesion molecule in BCa cells and can control VGSC (nNa_v1.5) expression and, concomitantly, cellular migration.     

Protein tyrosine phosphatase-1B and T-cell protein tyrosine phosphatase regulate IGF-2-induced MCF-7 cell migration

The protein tyrosine phosphatase-1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) have been implicated in down-regulation of tyrosine kinase receptors, conferring anti-oncogenic functions to these PTPases. However, recent work has shown that PTP1B is positively implicated in oncogenic properties of breast cancer cells by regulating the ERK pathway. Here, we studied the function of PTP1B and TC-PTP in IGF-2-induced growth, survival and migration of MCF-7 breast cancer cells. Using siRNA, we showed that reduction in the expression of these PTPases decreased cell growth and ERK phosphorylation. Reduction in the expression of these PTPases did not impair IGF-2 effects on cell survival to acute treatment with 4-OH Tamoxifen. In contrast, IGF-2-induced MCF-7 cell migration was markedly impaired by reduction of PTP1B or TC-PTP expression, independently of the ERK pathway. This novel finding reinforces the potential role of these PTPases as therapeutic targets for treatment of breast cancer.     

<Emphasis Type="Italic">Ulva pertusa</Emphasis> lectin 1 delivery through adenovirus vector affects multiple signaling pathways in cancer cells

Ulva pertusa lectin 1 (UPL1) is a N-acetyl-D-glucosamine (GlcNAc) binding lectin in marine green alga Ulva pertusa. Exogenous UPL1 colocalized with protein arginine methyltransferase 5 (PRMT5), methylosome protein 50 (MEP50), β-actin and β-tubulin, indicating the interaction of UPL1 with the methylosome and cytoskeleton. UPL1 delivery through adenovirus vector (Ad-UPL1) dramatically induced extracellularly regulated protein kinases 1/2 (ERK1/2) phosphorylation in liver cancer cell lines BEL-7404 and Huh7. Signaling pathways including p38 mitogen-activated protein kinase (MAPK), and Akt were also affected by Ad-UPL1 in a cell type dependent manner. MEK1/2 inhibitor U0126, as well as to a lesser extent p38 MAPK inhibitor SB203580 and phosphoinositide 3-kinase (PI3K) inhibitor LY294002, completely eliminated a higher molecular weight isoform of β-tubulin induced by Ad-UPL1, and significantly enhanced the cytotoxicity of Ad-UPL1 in Huh7 cells, suggesting that the inhibition of MEK1/2, p38 MAPK, and PI3K enhanced antiproliferative effect of Ad-UPL1 possibly through regulating the modification of β-tubulin. Ad-UPL1 completely inhibited the expression of autophagy-related factor Beclin1, but induced LC3-II expression in Huh7 cells. In addition, Ad-UPL1 significantly enhanced starvation induced survival suppression in Huh7 cells. Our data elucidated intracellular signaling pathways affected by exogenous UPL1, and may provide insights into a novel way of UPL1 delivery through adenovirus vectors combined with survival signaling inhibitors for cancer treatment.     

Depletion of JMJD5 sensitizes tumor cells to microtubule-destabilizing agents by altering microtubule stability

Microtubules play essential roles in mitosis, cell migration, and intracellular trafficking. Drugs that target microtubules have demonstrated great clinical success in cancer treatment due to their capacity to impair microtubule dynamics in both mitotic and interphase stages. In a previous report, we demonstrated that JMJD5 associated with mitotic spindle and was required for proper mitosis. However, it remains elusive whether JMJD5 could regulate the stability of cytoskeletal microtubules and whether it affects the efficacy of microtubule-targeting agents. In this study, we find that JMJD5 localizes not only to the nucleus, a fraction of it also localizes to the cytoplasm. JMJD5 depletion decreases the acetylation and detyrosination of α-tubulin, both of which are markers of microtubule stability. In addition, microtubules in JMJD5-depleted cells are more sensitive to nocodazole-induced depolymerization, whereas JMJD5 overexpression increases α-tubulin detyrosination and enhances the resistance of microtubules to nocodazole. Mechanistic studies revealed that JMJD5 regulates MAP1B protein levels and that MAP1B overexpression rescued the microtubule destabilization induced by JMJD5 depletion. Furthermore, JMJD5 depletion significantly promoted apoptosis in cancer cells treated with the microtubule-targeting anti-cancer drugs vinblastine or colchicine. Together, these findings suggest that JMJD5 is required to regulate the stability of cytoskeletal microtubules and that JMJD5 depletion increases the susceptibility of cancer cells to microtubule-destabilizing agents.