Diptiman Choudhury

Aqueous extract of ginger shows antiproliferative activity through disruption of microtubule network of cancer cells

Ginger has a long history of use as traditional medicine for varied human disease. Our present study has shown that the aqueous extract of ginger (GAE) interacts directly with cellular microtubules and disrupts its structure and induces apoptosis of cancer cells as well. The IC(50) values of GAE, as determined from cell viability experiment on human non-small lung epithelium cancer (A549) cells and human cervical epithelial carcinoma (HeLa), were 239.4+7.4 and 253.4+8.9 μg/ml, respectively. It has been found that the apoptosis of A549 cells by GAE is mediated by up regulation of tumor suppressor gene p53 and alteration of the normal Bax/Bcl-2 ratio followed by down regulation of cellular pro-caspase3. The morphological change of cells upon GAE treatment has also been demonstrated. Both the structural and functional properties of tubulin and microtubule were lost, as confirmed by both ex vivo and invitro experiments. The major component of GAE is poly-phenols (around 2.5%), which consist of ∼ 80% flavones and flavonols. Poly-phenolic compounds are well known to have anti-mitotic properties, and may be further screened for the development of a potential anti-cancer agent.

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.

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

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 IC(50) value (approximately 7.5 microM) 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 (IC(50) approximately 5 microM). 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.

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.

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.

A complex of Co(II) with 2-hydroxyphenyl-azo-2'-naphthol (HPAN) is far less cytotoxic than the parent compound on A549-lung carcinoma and peripheral blood mononuclear cells: Reasons for reduction in cytotoxicity.

Cytotoxic studies using an azo compound HPAN and its Co(II) complex were carried out on non-small lung epithelium carcinoma (A549) cells and peripheral blood mononuclear (PBM) cells. The results obtained suggest that the Co(II) complex is much less toxic toward both cell lines and the decreased toxicity due to the complex was more pronounced with carcinoma A549 cells. An attempt was made to correlate the findings related to cytotoxicity with the interaction of the compounds with DNA using calf thymus DNA as the target. The study was able to conclude that the complex was a relatively weak binder to calf thymus DNA. This information was used to explain the interaction of azo compounds with DNA in peripheral blood mononuclear cells and A549 lung carcinoma cells. It was concluded that the Co(II) complex interacts with DNA to a much lesser extent than HPAN alone. Cyclic voltammetry experiments carried out with HPAN and the Co(II) complex further showed that the presence of the metal ion in the complex prevents reduction of the azo group to such species that are responsible for inducing cytotoxicity. The overall finding was that complex formation with azo compounds might serve as a possible route to curb their toxicities.

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.

Gingival fibroblasts resist apoptosis in response to oxidative stress in a model of periodontal diseases

Periodontal diseases are classified as inflammation affecting the supporting tissue of teeth, which eventually leads to tooth loss. Mild reversible gingivitis and severe irreversible periodontitis are the most common periodontal diseases. Periodontal pathogens initiate the diseases. The bacterial toxin, lipopolysaccharide (LPS), triggers the inflammatory response and leads to oxidative stress. However, the progress of oxidative stress in periodontal diseases is unknown. The purpose of this study is to examine oxidative stress and cell damage in gingivitis and periodontitis. Our results showed that LPS increases reactive oxygen species (ROS) accumulation in gingival fibroblast (GF). However, oxidative stress resulting from excessive ROS did not influence DNA damage and cell apoptosis within 24 h. The mechanism may be related to the increased expression of DNA repair genes, Ogg1, Neil1 and Rad50. Detection of apoptosis-related proteins also showed anti-apoptotic effects and pro-apoptotic effects were balanced. The earliest damage appeared in DNA when increased γH2AX, an early biomarker for DNA damage, was detected in the LPS group after 48 h. Later, when recurrent inflammation persisted, 8-OHdG, a biomarker for oxidative stress was much higher in periodontitis model compared to the control in vivo. Staining of 8-OHdG in human periodontitis specimens confirmed the results. Furthermore, TUNEL staining of apoptotic cells indicated that the periodontitis model induced more cell apoptosis in gingival tissue. This suggested GF could resist early and acute inflammation (gingivitis), which was regarded as reversible, but recurrent and chronic inflammation (periodontitis) led to permanent cell damage and death.

Identification of a novel E-box binding pyrrole-imidazole polyamide inhibiting MYC-driven cell proliferation

The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and overexpression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E‐box (5′‐CACGTGT‐3′) sequence at gene promoters contributes to more than 4000 MYC‐dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence‐specific DNA‐binding pyrrole–imidazole (PI) polyamide, Myc‐5, that recognizes the E‐box consensus sequence. Bioinformatics analysis revealed that the Myc‐5 binding sequence appeared in 5′‐ MYC binding E‐box sequences at the eIF4G1, CCND1, and CDK4 gene promoters. Furthermore, ChIP coupled with detection by quantitative PCR indicated that Myc‐5 has the ability to inhibit MYC binding at the target gene promoters and thus cause downregulation at the mRNA level and protein expression of its target genes in human Burkitts lymphoma model cell line, P493.6, carrying an inducible MYC repression system and the K562 (human chronic myelogenous leukemia) cell line. Single i.v. injection of Myc‐5 at 7.5 mg/kg dose caused significant tumor growth inhibition in a MYC‐dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of E‐box‐mediated MYC downstream gene expression and is a model for showing that phenotype‐associated MYC downstream gene targets consequently inhibit MYC‐dependent tumor growth.

Acenaphthenequinone induces cell cycle arrest and mitochondrial apoptosis via disruption of cellular microtubules

Acenaphthenequinone (AcQ) is a polycyclic aromatic hydrocarbon, present in diesel exhausts. In this report, mechanism(s) of cytotoxicity of AcQ in human lung epithelial cells (A549) and human peripheral blood mononuclear cells (PBMC) have been investigated. Treatment with AcQ resulted in the disruption of the microtubule network in A549 cells in time and concentration-dependent manners and caused cell cycle arrest in the G2/M phase and apoptosis, with an IC50 value of ∼35 and ∼14 μM for 24 h and 48 h respectively. AcQ induced apoptosis in PBMC cells (IC50 ≈ 15 μM, 24 h). We found up-regulation of cyclin B1 and down-regulation of cyclin D1 caused G2/M arrest in treated cells. Up-regulation of pro-apoptotic proteins like p53 and Bax, and down-regulation of anti-apoptotic protein Bcl-2 were observed in treated A549 cells. Loss of mitochondrial membrane potential and activation of caspase-3 as well as release of mitochondrial cytochrome c was also obtained. AcQ also inhibited the tubulin polymerization in cell free system (IC50 ≈ 10 μM). The stoichiometry of AcQ binding to purified tubulin was nearly 1 : 1 with a dissociation constant of 11.0 ± 0.8 μM at 25 °C. Interaction of AcQ with tubulin resulted in conformational changes, monitored by quenching of tryptophan fluorescence, tubulin–colchicine and tubulin–ANS binding and CD experiments.