The biological significance of microtubules makes them a validated target of cancer therapy. In this study, we have utilized indole, an important pharmacological scaffold, to synthesize novel bis(indolyl)-hydrazide-hydrazone derivatives (NMK-BH compounds) and recognized NMK-BH3 as the most effective one in inhibiting A549 cell proliferation and assembly of tissue-purified tubulin. Cell viability experiments showed that NMK-BH3 inhibited proliferation of human lung adenocarcinoma (A549) cells, normal human lung fibroblasts (WI38) and peripheral blood mononuclear cells (PBMC) with IC50 values of -2, 48.5, and 62 μM, respectively. Thus, the relatively high cytotoxicity of NMK-BH3 toward lung carcinoma (A549) cells over normal lung fibroblasts (WI38) and PBMC confers a therapeutic advantage of reduced host toxicity. Flow cytometry, Western blot, and immunofluorescence studies in the A549 cell line revealed that NMK-BH3 induced G2/M arrest, mitochondrial depolarization, and apoptosis by depolymerizing the cellular interphase and spindle microtubules. Consistent with these observations, study in cell free system revealed that NMK-BH3 inhibited the microtubule assembly with an IC50 value of -7.5 μM. The tubulin-ligand interaction study using fluorescence spectroscopy indicated that NMK-BH3 exhibited strong and specific tubulin binding with a dissociation constant of -1.4 μM at a single site, very close to colchicine site, on β-tubulin. Collectively, these findings explore the cytotoxic potential of NMK-BH3 by targeting the microtubules and inspire its development as a potential candidate for lung cancer chemotherapy. © 2016 American Chemical Society.

GOPAL CHAKRABARTI

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

MUKHERJEE D DAS

N M KUMAR

M P TANTAK

A DAS

A GANGULI

S DATTA

D KUMAR

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

Biochemistry

Paclitaxel is one of the most commonly used drugs for the treatment of nonsmall cell lung cancer (NSCLC). However acquired resistance to paclitaxel, epithelial to mesenchymal transition and cancer stem cell formation are the major obstacles for successful chemotherapy with this drug. Some of the major reasons behind chemoresistance development include increased ability of the cancer cells to survive under stress conditions by autophagy, increased expression of drug efflux pumps, tubulin mutations etc. In this study we found that inhibition of autophagy with chloroquine prevented development of paclitaxel resistance in A549 cells with time and potentiated the effect of paclitaxel by increased accumulation of superoxide-producing damaged mitochondria, with elevated ROS generation, it also increased the apoptotic rate and sub G0/ G1 phase arrest with time in A549 cells treated with paclitaxel and attenuated the metastatic potential and cancer stem cell population of the paclitaxel-resistant cells by ROS mediated modulation of the Wnt/β-catenin signaling pathway, thereby increasing paclitaxel sensitivity. ROS here played a crucial role in modulating Akt activity when autophagy process was hindered by chloroquine, excessive ROS accumulation in the cell inhibited Akt activity. In addition, chloroquine pre-treatment followed by taxol (10 nM) treatment did not show significant toxicity towards non-carcinomas WI38 cells (lung fibroblast cells). Thus autophagy inhibition by CQ pre-treatment can be used as a fruitful strategy to combat the phenomenon of paclitaxel resistance development as well as metastasis in lung cancer. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.

Apoptosis

Autophagy inhibition with chloroquine reverts paclitaxel resistance and attenuates metastatic potential in human nonsmall lung adenocarcinoma A549 cells via ROS mediated modulation of Î²-catenin pathway

In the development of small-molecule drug candidates, naphthalimide-based compounds hold a very important position as potent anticancer agents with considerable safety in drug discoveries. Being synthetically and readily accessible, naphthalimide compounds with planar architecture have been developed mostly as DNA-targeting intercalators. However, in this article, it is demonstrated, for the first time, that an unfused naphthalimide-benzothiazole bichromophoric compound 2-(6-chlorobenzo[d] thiazol-2-yl)-1H-benzo[de] isoquinoline-1,3(2H)-dione (CBIQD), seems to expand the bioactivity of naphthalimide as anti-mitotic agent also. Preliminary studies demonstrate that CBIQD interferes with human lung cancer (A549) cell proliferation and growth and causes cellular morphological changes. However, the underlying mechanism of its antitumor action and primary cellular target in A549 cells remained skeptical. Confocal microscopy in A549 cells revealed disruption of interphase microtubule (MT) network and formation of aberrant multipolar spindle. Consistent with microscopy results, UV-vis, steady-state fluorescence, and time-resolved fluorescence (TRF) studies demonstrate that CBIQD efficiently binds to tubulin (K b = 2.03 × 10 5 M -1 ± 1.88%), inhibits its polymerization, and depolymerizes preformed microtubules (MTs). Low doses of CBIQD have also shown specificity toward tubulin protein in the presence of a nonspecific protein like bovine serum albumin as well as other cytoskeleton component, actin. The in vitro determination of binding site coupled with in silico studies suggests that CBIQD may prefer to occupy the colchicine binding site. Further, CBIQD perturbed tubulin conformation to some extent and protected ∼1.4 cysteine residues toward chemical modification by 5,5′-dithiobis-2-nitrobenzoic acid. We also suggest the possible mechanism underlying CBIQD-induced cancer cell cytotoxicity: CBIQD, when bound to tubulin, may prevent it to maintain a straight conformation; consequently, the α- and β-heterodimers might be no longer available for MT growth. Thus, the consolidated spectroscopic research described herein explores the potential of CBIQD as a new paradigm in the design and development of novel unfused or nonring-fused naphthalimide-based antimitotic cancer therapeutics in medicinal chemistry research. © 2018 American Chemical Society.

Journal of Physical Chemistry B

Unveiling the Potential of Unfused Bichromophoric Naphthalimide to Induce Cytotoxicity by Binding to Tubulin: Breaks Monotony of Naphthalimides as Conventional Intercalators

A novel library of C2-substituted tryptamines (based on diverse C2-aroyl/arylimino indoles and indole-diketopiperazine hybrids) possessing antimitotic properties were designed, synthesized and screened for their inhibitory activity against tubulin polymerization, and against proliferation of A549 lung cancer, HeLa cervical cancer, MCF7 breast cancer and HePG2 liver cancer cell lines. The design of molecules were inspired from known antimitotic compounds and natural products. The molecular docking of the designed compounds indicated that they bind to the colchicin binding site of tubulin. They were synthesized by a unique iodine catalysed oxidative ring opening reaction of 1-aryltetrahydro-β-carbolines. Among the compounds synthesized quite a few compounds induced cytotoxicity on the cancer cells by disrupting the tubulin polymerization. They were found to be non-toxic for healthy cells. Immuno Fluorescence study for the most active molecules (between ~6 μM concentration) against A549 and HeLa cells demonstrated complete disruption and shrinkage of the microtubule structures. These compounds also inhibited indoleamine-2, 3-dioxygenase with low micromolar IC50. © 2018 Elsevier B.V.

European Journal of Pharmaceutical Sciences

Design, synthesis and biological evaluation of a novel library of antimitotic C2-aroyl/arylimino tryptamine derivatives that are also potent inhibitors of indoleamine-2, 3-dioxygenase (IDO)

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.

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

Parabenzoquinone (1,4-benzoquinone) (PBQ) is a bioactve quinone present in cigarette smoke and diesel smoke, which causes severe genotoxic effects both in vitro and in vivo. In the previous study, we showed that the microtubules are one of the major targets of cigarette smoke-induced damage of lung epithelium cells. In the present study, we have investigated the effect of PBQ on cellular microtubules using human type II lung epithelial cells (A549) and also on purified tubulin. Cell viability experiments using A549 cells indicated a very low IC50 value (∼7.5 μM) for PBQ. PBQ inhibited cell cycle progression and induced apoptosis of A549 cells. PBQ also induced the contraction and shrinkage of the A549 cells in a time- and concentration- dependent manner, which is proved to be a direct effect of the damage of the microtubule cytoskeleton network, and that was demonstrated by a immunofluorescence study. PBQ also inhibited the assembly of tubulin in lung cells and a in cell free system (IC50 ∼5 μM). Treatment with PBQ resulted in the degradation of tubulin in lung cells without affecting the actin network, and this was confirmed by a Western blot experiment. Upregulation of pro-apoptotic proteins such as p53 and Bax and downregulation of antiapoptotic protein Bcl-2 were observed in PBQ-treated A549 cells. Simultaneously, loss of mitochondrial membrane potential and activation of caspase-3 were also observed in the PBQ treated lung epithelium cells. Fluorescence and circular dichroism studies demonstrated that the denaturation of tubulin in a cell free system was caused by PBQ. However, in the presence of N-acetyl cysteine (NAC), damage of the microtubule network in A549 cells by PBQ was prevented, which led to a significant increase in the viability of A549 cells. These results suggest that microtubule damage is one of the key mechanisms of PBQ induced cytotoxity in lung cells. © 2010 American Chemical Society.

Chemical Research in Toxicology

1,4-benzoquinone (PBQ) induced toxicity in lung epithelial cells is mediated by the disruption of the microtubule network and activation of caspase-3

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 TypesetBiochemistry
Publisher	Data powered by TypesetAmerican Chemical Society
ISSN	0006-2960
Open Access	No