Arnab Ganguli

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.

Inhibition of autophagy by chloroquine potentiates synergistically anti-cancer property of artemisinin by promoting ROS dependent apoptosis

Artemisinin (ART) is a well-known anti-malarial drug, and recently it is shown prospective to selectively kill cancer cells. But low potency makes it inappropriate for use as an anticancer drug. In this study, we modulated the ART-induced autophagy to increase Potency of ART as an anticancer agent. ART reduced the cell viability and colony forming ability of non-small lung carcinoma (A549) cells and it was non-toxic against normal lung (WI38) cells. ART induced autophagy at the early stage of treatment. Pre-treatment with chloroquine (CQ) and followed by ART treatment had synergistic combination index (CI) for cell death. Inhibition of autophagy by CQ pre-treatment led to accumulation of acidic vacuoles (AVOs) which acquainted with unprocessed damage mitochondria that subsequently promoted ROS generation, and resulted releases of Cyt C in cytosol that caused caspase-3 dependent apoptosis cell death in ART-treated A549 cells. Scavenging of ROS by antioxidant N-acetyl-cysteine (NAC) inhibited caspase-3 activity and rescued the cells from apoptosis. Similar effects were observed in other cancer cells SCC25 and MDA-MB-231. The appropriate manipulation of autophagy by using CQ provides a powerful strategy to increase the Potency of selective anticancer property of ART.

2,4-Dichlorophenoxyacetic acid induced toxicity in lung cells by disruption of the tubulin-microtubule network

2,4-Dichlorophenoxyacetic acid (2,4-D), the most widely used herbicide in the world, has been previously reported to induce lung damage. Here in this study we have investigated the molecular mechanism of 2,4-D induced lung toxicity in A549 and WI38 cell lines. Cell viability experiments indicate the IC50 values in A549 and WI38 cells for 72 h are 126 ± 2.25 μM and 115 ± 4.39 μM, respectively. Although not arresting a particular phase of the cell cycle of A549 cells, 2,4-D dose dependently increased the subG1 population, indicative of cell death, and the mode of cell death is apoptosis in both cell lines, as observed by annexin V/PI flow cytometric analysis and the expression status of pro and anti apoptotic proteins by western blot analysis. Dose dependent shrinkage of A549 cells indicates that 2,4-D can disrupt the microtubule network, and this is confirmed by immunofluorescence studies in vitro. In a cell free system, we found that 2,4-D depolymerises the microtubule network in vitro (IC50 = 212 ± 1.63 μM). 2,4-D can quench the intrinsic tryptophan fluorescence of tubulin in both a time and a dose dependent manner; the stoichiometry of 2,4-D–tubulin binding is 1:1 and the dissociation constant is 22.82 ± 1.29 μM. In silico studies indicate that 2,4-D binds to tubulin between the α and β subunits, very close to the colchicine binding site, and there is very little conformational change of the tubulin structure, as we also confirmed by circular dichroism studies. So, in brief, these results suggest that disruption of the cellular tubulin-microtubule network is one of the key mechanisms in the induction of lung cytotoxicity by 2,4-D.

NMK-TD-100, a Novel Microtubule Modulating Agent, Blocks Mitosis and Induces Apoptosis in HeLa Cells by Binding to Tubulin

Thiadiazoles are one of the most widely utilized agents in medicinal chemistry, having a wide range of pharmacologic activity. Microtubules (MTs) have always remained a sought-after target in rapidly proliferating cancer cells. We screened for the growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on cancer cells and identified NMK-TD-100, as the most potent agent. Cell viability experiments using human cervical carcinoma cell line (HeLa cells) indicated that the IC50 value was 1.42±0.11 µM for NMK-TD-100 for 48 h treatment. In further study, we examined the mode of interaction of NMK-TD-100 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. NMK-TD-100 induced arrest in mitotic phase of cell cycle, caused decline in mitochondrial membrane potential and induced apoptosis in HeLa cells. Immunofluorescence studies using an anti-α-tubulin antibody showed a significant depolymerization of the interphase microtubule network and spindle microtubule in HeLa cells in a concentration-dependent manner. However, the cytotoxicity of NMK-TD-100 towards human peripheral blood mononuclear cells (PBMC) was lower compared to that in cancer cells. Polymerization of tissue purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC50 value of 17.5±0.35 µM. The binding of NMK-TD-100 with tubulin was studied using NMK-TD-100 fluorescence enhancement and intrinsic tryptophan fluorescence of tubulin. The stoichiometry of NMK-TD-100 binding to tubulin is 1:1 (molar ratio) with a dissociation constant of ~1 µM. Fluorescence spectroscopic and molecular modeling data showed that NMK-TD-100 binds to tubulin at a site which is very near to the colchicine binding site. The binding of NMK-TD-100 to tubulin was estimated to be ~10 times faster than that of colchicine. The results indicated that NMK-TD-100 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided insights into its potential of being a chemotherapeutic agent.

Development of Novel Bis(indolyl)-hydrazide–Hydrazone Derivatives as Potent Microtubule-Targeting Cytotoxic Agents against A549 Lung Cancer Cells

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.

Epigallocatechin-3-gallate shows anti-proliferative activity in HeLa cells targeting tubulin-microtubule equilibrium.

In this study our main objective was to find out a novel target of the major bioactive green tea polyphenol, Epigallocatechin-3-gallate (EGCG), in cervical carcinoma HeLa cells. We found that EGCG showed antiproliferative activity against HeLa cells through depolymerization of cellular microtubule. EGCG also prevented the reformation of the cellular microtubule network distorted by cold treatment and inhibited polymerization of tubulin in cell-free system with IC50 of 39.6 ± 0.63 μM. Fluorescence spectroscopic analysis showed that EGCG prevented colchicine binding to tubulin and in silico study revealed that EGCG bound to the α-subunit of tubulin at the interphase of the α-and β-heterodimers and very close to colchicine binding site. The binding is entropy driven (ΔS(0) was 18.75 ± 1.48 cal K(-1) mol(-1)) with Kd value of 3.50 ± 0.40 μM. This is a novel mechanism of antipriliferative activity of EGCG.

Smokeless Tobacco Extract (STE)-Induced Toxicity in Mammalian Cells is Mediated by the Disruption of Cellular Microtubule Network: A Key Mechanism of Cytotoxicity

Smokeless tobacco usage is a growing public health problem worldwide. The molecular mechanism(s) underlying smokeless tobacco associated tissue damage remain largely unidentified. In the present study we have tried to explore the effects of aqueous extract of smokeless tobacco (STE) on tubulin-microtubule, the major cytoskeleton protein that maintains cells morphology and participates in cell division. Exposure to STE resulted in dose-dependent cytotoxicity in a variety of mammalian transformed cell lines such as human lung epithelial cells A549, human liver epithelial cells HepG2, and mouse squamous epithelial cells HCC7, as well as non-tumorogenic human peripheral blood mononuclear cells PBMC. Cellular morphology of STE-treated cells was altered and the associated disruption of microtubule network indicates that STE targets tubulin-microtubule system in both cell lines. Furthermore it was also observed that STE-treatment resulted in the selective degradation of cellular tubulin, whereas actin remains unaltered. In vitro, polymerization of purified tubulin was inhibited by STE with the IC50 value∼150 µg/ml and this is associated with the loss of reactive cysteine residues of tubulin. Application of thiol-based antioxidant N-acetyl cysteine (NAC) significantly abrogates STE-mediated microtubule damage and associated cytotoxicity in both A549 and HepG2 cells. These results suggest that microtubule damage is one of the key mechanisms of STE-induced cytotoxity in mammalian cells.

Potential role of autophagy in smokeless tobacco extract-induced cytotoxicity and in morin-induced protection in oral epithelial cells

Toxic components of STE induced serious, adverse human oral health outcomes. In the present study, we observed that STE was involved in oral toxicity by reducing the viability of human squamous epithelial cells, SCC-25, along with the simultaneous induction of both apoptosis and autophagic signaling. STE was also found to induce significant amount ROS generation in SCC-25 cells. The dietary flavonoid morin, found abundantly in a variety of herbs, fruits and wine, has been reported to attenuate ROS-induced pathogenesis including autophagy. In this study we designed three different treatment regimes of morin treatment, such as pre, co, and post - treatment of STE challenged SCC-25 cells. In all cases morin provided cytoprotection to STE challenged SCC-25 cells by augmenting STE induced ROS-dependent cytotoxic autophagy. Hence, morin is a potential option for antioxidant therapy in treatment of STE induced toxicity.

Azadiradione ameliorates polyglutamine expansion disease in Drosophila by potentiating DNA binding activity of heat shock factor 1.

Aggregation of proteins with the expansion of polyglutamine tracts in the brain underlies progressive genetic neurodegenerative diseases (NDs) like Huntingtons disease and spinocerebellar ataxias (SCA). An insensitive cellular proteotoxic stress response to non-native protein oligomers is common in such conditions. Indeed, upregulation of heat shock factor 1 (HSF1) function and its target protein chaperone expression has shown promising results in animal models of NDs. Using an HSF1 sensitive cell based reporter screening, we have isolated azadiradione (AZD) from the methanolic extract of seeds of Azadirachta indica, a plant known for its multifarious medicinal properties. We show that AZD ameliorates toxicity due to protein aggregation in cell and fly models of polyglutamine expansion diseases to a great extent. All these effects are correlated with activation of HSF1 function and expression of its target protein chaperone genes. Notably, HSF1 activation by AZD is independent of cellular HSP90 or proteasome function. Furthermore, we show that AZD directly interacts with purified human HSF1 with high specificity, and facilitates binding of HSF1 to its recognition sequence with higher affinity. These unique findings qualify AZD as an ideal lead molecule for consideration for drug development against NDs that affect millions worldwide.

Novel nano-insulin formulation modulates cytokine secretion and remodeling to accelerate diabetic wound healing

Little is known about insulins wound healing capability in normal as well as diabetic conditions. We here report specific interaction of silver nanoparticles (AgNPs) with insulin by making a ~2 nm thick coat around the AgNPs and its potent wound healing efficacy. Characterization of the interaction of human insulin with silver nanoparticles showed confirmed alteration of amide-I in insulin whereas amide-II and III remained unaltered. Further, nanoparticles protein interaction kinetics showed spontaneous interaction at physiological temperature with ΔG, ΔS, Ea and Ka values -7.48, 0.076, 3.84 kcal mol-1 and 6 × 105 s-1 respectively. Insulin loaded AgNPs (IAgNPs) showed significant improvement in healing activity in vitro (HEKa cells) and in vivo (Wister Rats) in comparison with the control in both normal and diabetic conditions. The underlying mechanism was attributed to a regulation of the balance between pro (IL-6, TNFα) and anti-inflammatory cytokines (IL-10) at the wound site to promote faster wound remodeling.