Microtubule-Targeting agents (MTA) are indispensable for cancer therapeutics. We here report thymoquinone (TQ) as a new MTA that already has been appreciated for its anticancer effects. TQ induced G2/M cell cycle arrest in human non-small lung epithelial cells (A549) and majority of arrested cells were in mitosis. TQ depolymerized the microtubule (MT) network and disrupted mitotic spindle organization of A549 cells. MT depolymerization by TQ was followed by apoptosis and subsequent loss in cell viability (IC 50 value of ∼10 μM). Interestingly, TQ didn't affect the MT network of normal HUVEC cells at and below the IC50 concentration for A549 cells. TQ also inhibited tubulin polymerization in cell-free system with an IC50 of 27 μM and bound to tubulin heterodimers at a single site with a dissociation constant of 1.19 μM at 25 C. Binding of TQ to tubulin quenched the tryptophan fluorescence of protein in a time-dependent manner. The TQ-tubulin binding kinetics was biphasic in nature and equilibrated in 30 min. TQ competed with colchicine for tubulin binding with a Ki of 1.9 μM as determined by modified Dixon plot analysis, this suggests TQ may bind tubulin at or near the colchicine binding site and in silico modeling study supported that. Our results establish a novel antimitotic mechanism of TQ by its direct binding to tubulin-MT network in A549 cells. © 2013 Elsevier Masson SAS. All rights reserved.

GOPAL CHAKRABARTI

Biotechnology and Dr. B. C Guha Centre for Genetic Engineering and Biotechnology

B R ACHARYA

A CHATTERJEE

A GANGULI

S BHATTACHARYA

University of Calcutta (informally known as Calcutta University or CU) is a collegiate public state university located in Kolkata, West Bengal, India.&nbsp;Within India it is recognized as a &quot;Five-Star University&quot; and accredited &quot;A&quot; Grade by National Assessment and Accreditation Council.

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The University of Calcutta has secured the Fifth position among the Universities of India in the prestigious &quot;Indian University Ranking 2019&quot; list, released by the NIRF of the Ministry of Human Resource Development, Government of India.

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It was established on 24 January 1857, and was one of the first institutions in Asia to be established as a multidisciplinary and Western-style university. Its alumni and faculty include several heads of state, heads of government, social reformers, prominent artists, only academy award winner and Dirac medal winner in India, many Fellows of the Royal Society and five Nobel laureates as of 2019 which is highest in South Asia.

University Of Calcutta

Biochimie

Genistein (4′,5,7-trihydroxyisoflavone), an isoflavone, is a major constituent of soyfoods. It has potential antiproliferative activity against several tumor types. We have examined the effect of genistein on cellular microtubules as well as its binding with purified tubulin in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC50 value for genistein is 72 μM. Flow cytometry experiments demonstrated that genistein arrested cell cycle progression at the G2/M phase, but mitotic index data showed that genistein did not arrest cell cycle progression at mitosis. Immunofluorescence studies using an anti-α-tubulin antibody demonstrated a significant depolymerization of the interphase microtubules in a dose-dependent manner, and this was confirmed by the Western blot experiment using genistein-treated A549 cells. In vitro polymerization of purified tubulin into microtubules was inhibited by genistein with an IC50 value of 87 μM. Genistein binding to tubulin quenched protein tryptophan fluorescence in a time- and concentration-dependent manner. Binding of genistein to tubulin was slow, taking ∼45 min for equilibration at 37 °C. The association rate constant was 104.64 ± 20.63 M-1 s-1 at 37 °C. The stoichiometry of genistein binding to tubulin was nearly 1:1 (molar ratio) with a dissociation constant of 15 μM at 37 °C. It was interesting to note that genistein did not recognize either the colchicine site or the vinblastine binding site of tubulin. Surprisingly, genistein inhibited ANS binding and competed for its binding site of tubulin with a Ki of 20μM as determined from a modified Dixon plot. Hence, we conclude that one of the mechanisms of antiproliferative activity of genistein is depolymerization of microtubules through binding of tubulin. © 2010 American Chemical Society.

Biochemistry

Genistein arrests cell cycle progression of A549 cells at the G 2/M phase and depolymerizes interphase microtubules through binding to a unique site of tubulin

Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitaminKin the body. VitaminKis 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 μM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-α-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 μM. 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 °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 ± 21M -1 s -1 at 25 °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 μM at 25 °C. Menadione competes for the colchicine binding site with a K i of 2.5 μM 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. © 2009 American Chemical Society.

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

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 IC50 value for plumbagin is 14.6 μM. 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 IC50 value of 38 ± 0.5 μM. 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°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°C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 ± 0.71 μM at 25°C. Plumbagin competes for the colchicine binding site with a Ki of 7.5 μM 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. © 2008 American Chemical Society.

Journal	Data powered by TypesetBiochimie
Publisher	Data powered by TypesetELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
ISSN	0300-9084
Open Access	No