Gopal Chakrabarti

miR-17-5p Downregulation Contributes to Paclitaxel Resistance of Lung Cancer Cells through Altering Beclin1 Expression

Non- small- cell lung cancer (NSCLC) is one of the most leading causes of cancer-related deaths worldwide. Paclitaxel based combination therapies have long been used as a standard treatment in aggressive NSCLCs. But paclitaxel resistance has emerged as a major clinical problem in combating non-small-cell lung cancer and autophagy is one of the important mechanisms involved in this phenomenon. In this study, we used microRNA (miRNA) arrays to screen differentially expressed miRNAs between paclitaxel sensitive lung cancer cells A549 and its paclitaxel-resistant cell variant (A549-T24). We identified miR-17-5p was one of most significantly downregulated miRNAs in paclitaxel-resistant lung cancer cells compared to paclitaxel sensitive parental cells. We found that overexpression of miR-17-5p sensitized paclitaxel resistant lung cancer cells to paclitaxel induced apoptotic cell death. Moreover, in this report we demonstrated that miR-17-5p directly binds to the 3′-UTR of beclin 1 gene, one of the most important autophagy modulator. Overexpression of miR-17-5p into paclitaxel resistant lung cancer cells reduced beclin1 expression and a concordant decease in cellular autophagy. We also observed similar results in another paclitaxel resistant lung adenosquamous carcinoma cells (H596-TxR). Our results indicated that paclitaxel resistance of lung cancer is associated with downregulation of miR-17-5p expression which might cause upregulation of BECN1 expression.

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.

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.

MiR-16 targets Bcl-2 in paclitaxel-resistant lung cancer cells and overexpression of miR-16 along with miR-17 causes unprecedented sensitivity by simultaneously modulating autophagy and apoptosis.

Non-small cell lung cancer is one of the most aggressive cancers as per as the mortality and occurrence is concerned. Paclitaxel based chemotherapeutic regimes are now used as an important option for the treatment of lung cancer. However, resistance of lung cancer cells to paclitaxel continues to be a major clinical problem nowadays. Despite impressive initial clinical response, most of the patients eventually develop some degree of paclitaxel resistance in the course of treatment. Previously, utilizing miRNA arrays we reported that downregulation of miR-17 is at least partly involved in the development of paclitaxel resistance in lung cancer cells by modulating Beclin-1 expression [1]. In this study, we showed that miR-16 was also significantly downregulated in paclitaxel resistant lung cancer cells. We demonstrated that anti-apoptotic protein Bcl-2 was directly targeted miR-16 in paclitaxel resistant lung cancer cells. Moreover, in this report we showed that the combined overexpression of miR-16 and miR-17 and subsequent paclitaxel treatment greatly sensitized paclitaxel resistant lung cancer cells to paclitaxel by inducing apoptosis via caspase-3 mediated pathway. Combined overexpression of miR-16 and miR-17 greatly reduced Beclin-1 and Bcl-2 expressions respectively. Our results indicated that though miR-17 and miR-16 had no common target, both miR-16 and miR-17 jointly played roles in the development of paclitaxel resistance in lung cancer. miR-17 overexpression reduced cytoprotective autophagy by targeting Beclin-1, whereas overexpression of miR-16 potentiated paclitaxel induced apoptotic cell death by inhibiting anti-apoptotic protein Bcl-2.

Cigarette smoke extract induces disruption of structure and function of tubulin-microtubule in lung epithelium cells and in vitro.

In the present study, we have investigated the effect of the aqueous extract of cigarette smoke (AECS) on tubulin-microtubule, a major cytoskeleton protein that maintains cellular morphology and participates in cell division. We found that treatment of AECS results in the loss of both structural and functional properties of tubulin-microtubule. Disruption of the microtubule network was observed in AECS-treated human lung epithelial (A549) cells and noncarcinoma human lung alveolar epithelium (L132) cells, in a dose and time-dependent manner. Tubulin-microtubule mediated important cellular properties, such as proliferation, migration, and maintenance of the cellular morphology, were affected by AECS in A549 cells. The aqueous extract of cigarette smoke (AECS) was also found to interfere the microtubule dynamics inside the cell and induce tubulin degradation. The structure of microtubules was also disrupted by AECS in the presence of protease inhibitors accompanied by a change of morphology of cells and loss of cell viability. In vitro, the functional properties of tubulin, such as the ability of polymerization, was inhibited by AECS in a dose and time-dependent manner, and it was accompanied by the loss of reactive cysteine residues, destabilization of the secondary structure, and quenching of intrinsic tryptophan fluorescence. Carbonyl content of tubulin was increased after treatment with AECS, indicating that one of the pathways of tubulin damage is protein oxidation. The damage of tubulin by AECS thus may be correlated with the pathogenesis of cigarette smoke induced disorders, which result in cellular apoptosis and tissue damage.

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.