Bipul R. Acharya

The Natural Naphthoquinone Plumbagin Exhibits Antiproliferative Activity and Disrupts the Microtubule Network through Tubulin Binding

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.

Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC50 value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC50 value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10(-7) M(-1) s(-1) and 4.0 × 10(-9) M(-1) s(-1) for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin-apigenin binding was 1:1 and binding the binding constant (Kd) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G2/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α-β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.

Remodeling the zonula adherens in response to tension and the role of afadin in this response

During development, epithelial cells must generate and respond to tension without disrupting epithelial barrier function. The authors use superresolution microscopy in MDCK cells to examine how the zonula adherens (ZA) is remodeled in response to elevated contractility while maintain tissue integrity. They define key roles for zonula occludens family proteins in regulating contractility and for the scaffolding protein afadin in maintaining ZA architecture at tricellular junctions.

Vitamin K3 disrupts the microtubule networks by binding to tubulin: a novel mechanism of its antiproliferative activity.

Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitamin K in the body. Vitamin K is necessary for the production of prothrombin and five other blood-clotting factors in humans. We have examined the effects of menadione on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human cervical epithelial cancer cells (HeLa) and human oral epithelial cancer cells (KB) indicated that the IC(50) values for menadione are 25.6 +/- 0.6 and 64.3 +/- 0.36 microM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody showed a significant irreversible depolymeriztion of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by menadione with an IC(50) value of 47 +/- 0.65 microM. The binding of menadione with tubulin was studied using menadione fluorescence and intrinsic tryptophan fluorescence of tubulin. Binding of menadione to tubulin is slow, taking 35 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 189.12 +/- 17 and 32.44 +/- 21 M(-1) s(-1) at 25 degrees C, respectively. The stoichiometry of menadione binding to tubulin is 1:1 (molar ratio) with a dissociation constant from 2.44 +/- 0.34 to 3.65 +/- 0.25 microM at 25 degrees C. Menadione competes for the colchicine binding site with a K(i) of 2.5 muM as determined from a modified Dixon plot. The obtained data suggested that menadione binds at the colchicine binding site to tubulin. Thus, we can conclude one novel mechanism of inhibition of cancer cell proliferation by menadione is through tubulin binding.

Coronin 1B Reorganizes the Architecture of F-Actin Networks for Contractility at Steady-State and Apoptotic Adherens Junctions

In this study we sought to identify how contractility at adherens junctions influences apoptotic cell extrusion. We first found that the generation of effective contractility at steady-state junctions entails a process of architectural reorganization whereby filaments that are initially generated as poorly organized networks of short bundles are then converted into co-aligned perijunctional bundles. Reorganization requires coronin 1B, which is recruited to junctions by E-cadherin adhesion and is necessary to establish contractile tension at the zonula adherens. When cells undergo apoptosis within an epithelial monolayer, coronin 1B is also recruited to the junctional cortex at the apoptotic/neighbor cell interface in an E-cadherin-dependent fashion to support actin architectural reorganization, contractility, and extrusion. We propose that contractile stress transmitted from the apoptotic cell through E-cadherin adhesions elicits a mechanosensitive response in neighbor cells that is necessary for the morphogenetic event of apoptotic extrusion to occur.

A biosensor of local kinesin activity reveals roles of PKC and EB1 in KIF17 activation

A biosensor of local kinesin activity demonstrates that PKC and EB1 promote the activation of KIF17 on dynamic microtubules, where it contributes to microtubule stabilization in epithelia.

The Nuclear Receptor, RORγ, Regulates Pathways Necessary for Breast Cancer Metastasis

We have previously reported that RORγ expression was decreased in ER − ve breast cancer, and increased expression improves clinical outcomes. However, the underlying RORγ dependent mechanisms that repress breast carcinogenesis have not been elucidated. Here we report that RORγ negatively regulates the oncogenic TGF-β/EMT and mammary stem cell (MaSC) pathways, whereas RORγ positively regulates DNA-repair. We demonstrate that RORγ expression is: (i) decreased in basal-like subtype cancers, and (ii) inversely correlated with histological grade and drivers of carcinogenesis in breast cancer cohorts. Furthermore, integration of RNA-seq and ChIP-chip data reveals that RORγ regulates the expression of many genes involved in TGF-β/EMT-signaling, DNA-repair and MaSC pathways (including the non-coding RNA, LINC00511). In accordance, pharmacological studies demonstrate that an RORγ agonist suppresses breast cancer cell viability, migration, the EMT transition (microsphere outgrowth) and mammosphere-growth. In contrast, RNA-seq demonstrates an RORγ inverse agonist induces TGF-β/EMT-signaling. These findings suggest pharmacological modulation of RORγ activity may have utility in breast cancer.

Direct regulation of microtubule dynamics by KIF17 motor and tail domains.

Background: KIF17 is targeted to microtubule plus-ends by EB1 and promotes microtubule stabilization in epithelial cells. Results: KIF17 motor and tail domains have direct and distinct effects on microtubule polymerization. Conclusion: The KIF17 motor domain is sufficient to regulate microtubules, but catalytic activity is modulated by EB1 and the KIF17 tail. Significance: KIF17 can function as a direct regulator of microtubule dynamics and stability. KIF17 is a kinesin-2 family motor that interacts with EB1 at microtubule (MT) plus-ends and contributes to MT stabilization in epithelial cells. The mechanism by which KIF17 affects MTs and how its activity is regulated are not yet known. Here, we show that EB1 and the KIF17 autoinhibitory tail domain (KIF17-Tail) interacted competitively with the KIF17 catalytic motor domain (K370). Both EB1 and KIF17-Tail decreased the K0.5MT of K370, with opposing effects on MT-stimulated ATPase activity. Importantly, K370 had independent effects on MT dynamic instability, resulting in formation of long MTs without affecting polymerization rate or total polymer mass. K370 also inhibited MT depolymerization induced by dilution in vitro and by nocodazole in cells, suggesting that it acts by protecting MT plus-ends. Interestingly, KIF17-Tail bound MTs and tubulin dimers, delaying initial MT polymerization in vitro and MT regrowth in cells. However, neither EB1 nor KIF17-Tail affected K370-mediated MT polymerization or stabilization significantly in vitro, and EB1 was dispensable for MT stabilization by K370 in cells. Thus, although EB1 and KIF17-Tail may coordinate KIF17 catalytic activity, our data reveal a novel and direct role for KIF17 in regulating MT dynamics.

Bistable front dynamics in a contractile medium: Travelling wave fronts and cortical advection define stable zones of RhoA signaling at epithelial adherens junctions

Mechanical coherence of cell layers is essential for epithelia to function as tissue barriers and to control active tissue dynamics during morphogenesis. RhoA signaling at adherens junctions plays a key role in this process by coupling cadherin-based cell-cell adhesion together with actomyosin contractility. Here we propose and analyze a mathematical model representing core interactions involved in the spatial localization of junctional RhoA signaling. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. This dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behavior of the system when contractility is inhibited are in qualitative agreement with the predictions of the model.

Pli Selon Pli

Cellular contractility, driven by actomyosin networks coupled to cadherin cell-cell adhesion junctions, is a major determinant of cellular rearrangement during morphogenesis. It now emerges that contractility arises as the emergent property of a mechanochemical feedback system that encompasses the signals that regulate contractility and the elements of the actomyosin network itself.