Heavy water (D2O) induces autophagy-dependent apoptotic cell death in non-small cell lung cancer A549 cells by generating reactive oxygen species (ROS) upon microtubule disruption.

OBJECTIVE: Deuterium oxide (D2O) or heavy water is known to have diverse biological activities and have a few therapeutic applications due to its limited toxicity to human subjects. In the present study, we investigated the mechanism of D2O-induced cytotoxicity in non-small cell lung cancer A549 cells. RESULTS: We found that D2O-treatment resulted in cytotoxicity, cell cycle arrest, and apoptosis in A549 cells in a dose-dependent fashion. In contrast, limited cytotoxicity was observed in lung fibroblasts WI38 cells. Moreover, D2O-treatment resulted in the disruption of the cellular microtubule network, accompanied by the generation of ROS. On further investigation, we observed that the intracellular ROS triggered autophagic responses in D2O-treated cells, leading to apoptosis by inhibiting the oncogenic PI3K/ Akt/ mTOR signaling. D2O-treatment was also found to enhance the efficacy of paclitaxel in A549 cells. SIGNIFICANCE: D2O induces autophagy-dependent apoptosis in A549 cells via ROS generation upon microtubule depolymerization and inhibition of PI3K/ Akt/ mTOR signaling. It augments the efficacy of other microtubule-targeting anticancer drug taxol, which indicates the potential therapeutic importance of D2O as an anticancer agent either alone or in combination with other chemotherapeutic drugs.

Targeting cellular microtubule by phytochemical apocynin exhibits autophagy-mediated apoptosis to inhibit lung carcinoma progression and tumorigenesis.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. Several targets have been identified for lung cancer therapy, amongst which 'Microtubule' and its dynamics are the most widely studied and used in therapy. Tubulin-microtubule polymer dynamics are highly sought after targets in the field of anti-cancer drug designing. Natural compounds are important sources for developing anticancer therapeutics owing to their efficacy and lower cytotoxicity. Evidence suggested that therapeutic targeting of microtubule by natural compounds is amongst the most widely used interventions in numerous cancer therapies including lung cancer. PURPOSE: To determine the efficacy of apocynin (a natural compound) in suppressing the progression of lung carcinoma both in vitro and in vivo, along with the identification of targets and the underlying mechanism for developing a novel therapeutic approach. METHODS: We have demonstrated themicrotubule depolymerizing role of apocynin by established protocols in cellular and cell-free system. The efficacy of apocynin to inhibit lung carcinoma progression was studied on A549 cells.The tumoricidal ability of apocynin was studied in BALB/c mice model as well.Mice were classified into 4 groups namely-group II mice as tumor control; group III-IV mice asalso tumor-induced but treated with differential apocynin doses whereas group I mice were kept as normal. RESULTS: Apocynin, showed selective cytotoxicity towards lung cancer cells rather than normal lung fibroblast cells. Apocynin inhibited oncogenic properties including growth, proliferation (p < 0.05), colony formation (p < 0.05), invasion (p < 0.05) and spheroid formation (p < 0.05) in lung cancer cells. Apart from other established properties, apocynin was found to be a novel and potent component to bind with tubulin and depolymerize cellular microtubule network. Apocynin mediated cellular microtubule depolymerization was the driving mechanism to trigger autophagy-mediated apoptotic cell death (p < 0.05) which in turn retarded lung cancer progression. Furthermore, apocynin showed tumoricidal characteristics to inhibit lung tumorigenesis in mice as well. CONCLUSION: Targeting tubulin-microtubule equilibrium with apocynin could be the key regulator to catastrophe cellular catabolic processes to mitigate lung carcinoma. Thus, apocynin could be a potential therapeutic agent for lung cancer treatment.

Enhanced antifungal activity of fluconazole conjugated with Cu-Ag-ZnO nanocomposite.

Cu-Ag-ZnO nanocomposite (NC) has been successfully synthesized by mechanical alloying the Cu, Zn and Ag powder mixture under Ar atmosphere within 4 h of milling. The nanocomposite is then conjugated with the antifungal drug fluconazole by adding 5 wt% powdered drug to the NC and mechanical alloying the total powder mixture for one more hour. The Rietveld refinement of XRD data and FTIR spectrum analyses reveal the detailed structural and microstructural characterizations of the nanocomposite-drug conjugate (NC-DC). Presence of Cu, Ag, ZnO and drug in the 5 h milled powder are confirmed by analyzing TEM images and FESEM-EDS spectrum. Results obtained from FESEM and TEM images reveal the measure of particle size of the nanocomposite-drug conjugate and it agrees well with the crystallite size obtained from the Rietveld refinement. A significant antifungal activity of NC-DC against Candida sp. fungi has been revealed using disk agar diffusion method. Minimum inhibitory concentration (MIC) test confirms that NC-DC with only 5 wt% fluconazole produces similar antifungal activity of the pure (100 wt%) and conventional fluconazole. Thus, the conjugation of conventional drug to a nanocomposite results in enhancement of drug efficiency by a factor 20 folds. This is very important, particularly, for those antibiotics which are very effective in controlling several epidemic diseases but show intense side effects when used at higher dose and/or for a longer duration.

A novel triazole, NMK-T-057, induces autophagic cell death in breast cancer cells by inhibiting γ-secretase-mediated activation of Notch signaling.

Notch signaling is reported to be deregulated in several malignancies, including breast, and the enzyme γ-secretase plays an important role in the activation and nuclear translocation of Notch intracellular domain (NICD). Hence, pharmacological inhibition of γ-secretase might lead to the subsequent inhibition of Notch signaling in cancer cells. In search of novel γ-secretase inhibitors (GSIs), we screened a series of triazole-based compounds for their potential to bind γ-secretase and observed that 3-(3'4',5'-trimethoxyphenyl)-5-(N-methyl-3'-indolyl)-1,2,4-triazole compound (also known as NMK-T-057) can bind to γ-secretase complex. Very interestingly, NMK-T-057 was found to inhibit proliferation, colony-forming ability, and motility in various breast cancer (BC) cells such as MDA-MB-231, MDA-MB-468, 4T1 (triple-negative cells), and MCF-7 (estrogen receptor (ER)/progesterone receptor (PR)-positive cell line) with negligible cytotoxicity against noncancerous cells (MCF-10A and peripheral blood mononuclear cells). Furthermore, significant induction of apoptosis and inhibition of epithelial-to-mesenchymal transition (EMT) and stemness were also observed in NMK-T-057-treated BC cells. The in silico study revealing the affinity of NMK-T-057 toward γ-secretase was further validated by a fluorescence-based γ-secretase activity assay, which confirmed inhibition of γ-secretase activity in NMK-T-057-treated BC cells. Interestingly, it was observed that NMK-T-057 induced significant autophagic responses in BC cells, which led to apoptosis. Moreover, NMK-T-057 was found to inhibit tumor progression in a 4T1-BALB/c mouse model. Hence, it may be concluded that NMK-T-057 could be a potential drug candidate against BC that can trigger autophagy-mediated cell death by inhibiting γ-secretase-mediated activation of Notch signaling.

Theaflavin and epigallocatechin-3-gallate synergistically induce apoptosis through inhibition of PI3K/Akt signaling upon depolymerizing microtubules in HeLa cells.

Theaflavin (TF) and epigallocatechin-3-gallate (EGCG) both have been reported previously as microtubule depolymerizing agents that also have anticancer effects on various cancer cell lines and in animal models. Here, we have applied TF and EGCG in combination on HeLa cells to investigate if they can potentiate each other to improve their anticancer effect in lower doses and the underlying mechanism. We found that TF and EGCG acted synergistically, in lower doses, to inhibit the growth of HeLa cells. We found the combination of 50 µg/mL TF and 20 µg/mL EGCG to be the most effective combination with a combination index of 0.28. The same combination caused larger accumulation of cells in the G 2 /M phase of the cell cycle, potent mitochondrial membrane potential loss, and synergistic augmentation of apoptosis. We have shown that synergistic activity might be due to stronger microtubule depolymerization by simultaneous binding of TF and EGCG at different sites on tubulin: TF binds at vinblastine binding site on tubulin, and EGCG binds near colchicines binding site on tubulin. A detailed mechanistic analysis revealed that stronger microtubule depolymerization caused effective downregulation of PI3K/Akt signaling and potently induced mitochondrial apoptotic signals, which ultimately resulted in the apoptotic death of HeLa cells in a synergistic manner.

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

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)-1 H-benzo[ de] isoquinoline-1,3(2 H)-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 ( Kb = 2.03 × 105 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 ß-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.

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 ß-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 SCC7, [corrected] 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.

Theaflavins depolymerize microtubule network through tubulin binding and cause apoptosis of cervical carcinoma HeLa cells.

Here we studied the antiproliferative activity of theaflavins in cervical carcinoma HeLa cells by investigating their effects on cellular microtubules and purified goat brain tubulin. Theaflavins inhibited proliferation of HeLa cells with IC(50) value of 110 ± 2.1 µg/mL (p = < 0.01), caused cell cycle arrest at G(2)/M phase and induced apoptosis with alteration of expression of pro- and antiapoptotic proteins. Along with these antiproliferative activities, theaflavins act as microtubule depolymerizers. Theaflavins disrupted the microtubule network accompanied by alteration of cellular morphology and also decreased the polymeric tubulin mass of the cells. The polymerization of cold treated depolymerized microtubules in HeLa cells was prevented in the presence of theaflavins. In vitro polymerization of purified tubulin into microtubules was also inhibited by theaflavins with an IC(50) value of 78 ± 2.43 µg/mL (P < 0.01). Thus, disruption of cellular microtubule network of HeLa cells through microtubule depolymerization may be one of the possible mechanisms of antiproliferative activity of theaflavins.

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.