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.

Glucogallin Attenuates the LPS-Induced Signaling in Macrophages and Protects Mice against Sepsis.

The anti-oxidant and anti-inflammatory effect of beta-glucogallin (BGG), a plant-derived natural product, was evaluated in both in vitro and in vivo studies. For the in vitro study, the ability of BGG pre-treatment to quench LPS-induced effects compared to LPS alone in macrophages was investigated. It was found that BGG pre-treatment showed a significant decrease in ROS, NO, superoxide, and pro-inflammatory cytokines (TNF-alpha, IL-4, IL-17, IL-1ß, and IL-6) and increased reduced glutathione coupled with the restoration of mitochondrial membrane potential. Gene profiling and further validation by qPCR showed that BGG pre-treatment downregulated the LPS-induced expression of c-Fos, Fas, MMP-9, iNOS, COX-2, MyD88, TRIF, TRAF6, TRAM, c-JUN, and NF-κB. We observed that BGG pre-treatment reduced nuclear translocation of LPS-activated NF-κB and thus reduced the subsequent expressions of NLRP3 and IL-1ß, indicating the ability of BGG to inhibit inflammasome formation. Molecular docking studies showed that BGG could bind at the active site of TLR4. Finally, in the LPS-driven sepsis mouse model, we showed that pre-treatment with BGG sustained toxic shock, as evident from their 100% survival. Our study clearly showed the therapeutic potential of BGG in toxic shock syndrome.

Glucogallin Attenuates RAW 264.7 Cells from Arsenic Trioxide Induced Toxicity via the NF-Ò¡B/NLRP3 Pathway.

Chronic arsenic (As) poisoning is mostly due to subsoil water contaminated with As and its salts. Exposure to As has been found to cause an elevation in reactive oxygen species (ROS), leading to the damage of DNA and proteins, and it also causes immunotoxicity. Treatment regimens are primarily based on chelation therapy and amino acid and vitamin supplementations. Recent studies have established that natural products display effective and progressive relief from arsenicosis without any side effects. ß-glucogallin (BGG), a gallo-tannin natural product, is reported to possess anti-oxidant and anti-inflammatory properties. In the present study, we aim to observe the protective role of BGG against As-induced cytotoxicity, apoptosis, mitochondrial dysfunction, and the underlying mechanisms in RAW 264.7 macrophage cells. We found that BGG alleviates As-induced ROS, apoptosis, and mitochondrial dysfunction in RAW 264.7 macrophage cells. Thus, BGG can be used therapeutically to prevent As-induced toxicity.

Tetracycline-loaded magnesium oxide nanoparticles with a potential bactericidal action against multidrug-resistant bacteria: In vitro and in vivo evidence.

Worldwide, the emergence of diarrhoea-causing multi-drug resistant (MDR) bacteria has become a crucial problem in everyday life. Tetracycline (TC) is a bacteriostatic agent that has a wide spectrum of antibacterial activity. One potential strategy to enhance the penetration and antibacterial activity of antibiotics is the use of nanotechnology. In this context, this study dealt with the synthesis of TC loading in biocompatible magnesium oxide nanoparticles (MgONPs), its characterization, and the potency of killing against diarrhoea-causing MDR bacteria E. coli and S. flexneri. TC loaded- MgONPs (MgONPs-TC) were characterized by DLS, SEM-EDS, UV-vis spectroscopy, and FTIR techniques with adequate physical properties. Antibacterial and antibiofilm studies indicate that this nanoparticle successfully eradicated both planktonic and sessile forms of those bacteria. It also significantly reduced the production of bacterial EPS, different levels of antioxidant enzymes, and induced reactive oxygen species (ROS) in the bacterial cell as a mode of antibacterial action. In particular, MgONPs-TC were efficient in reducing the colonization of MDR E. coli and S. flexneri in the C. elegans model. Therefore, all these data suggest that MgONPs-TC are a highly promising approach to combating diseases associated with diarrhoea-causing MDR bacteria in the medical field with limited health care budgets.

Exploring the antibacterial, antibiofilm, and antivirulence activities of tea tree oil-containing nanoemulsion against carbapenem-resistant Serratia marcescens associated infections.

Carbapenem-resistant Serratia marcescens (CRE-S. marcescens) has recently emerged as an opportunistic human pathogen that causes various nosocomial and respiratory tract infections. The prognosis for CRE-S. marcescens-related infections is very poor and these infections are difficult to treat. This study investigated the synthesis of tea tree oil nanoemulsion (TTO-NE) and its impact on CRE-S. marcescens both in vitro and in vivo. TTO-NE was characterized by dynamic light scattering (DLS) and effectively eradicated bacterial planktonic and sessile forms, reduced bacterial virulence factors, and generated reactive oxygen species (ROS) in the bacterial cell. Notably, TTO-NE was efficient in reducing the colonization of CRE-S. marcescens in a C. elegans in vivo model. The data suggest that TTO-NE might be an excellent tool to combat infections associated with CRE-S. marcescens.

Natural flavonoid morin showed anti-bacterial activity against Vibrio cholera after binding with cell division protein FtsA near ATP binding site.

BACKGROUND: Increasing antibiotic-resistance in bacterial strains has boosted the need to find new targets for drug delivery. FtsA, a major bacterial divisome protein can be a potent novel drug-target. METHODS AND RESULTS: This study finds, morin (3,5,7,2',4'-pentahydroxyflavone), a bio-available flavonoid, had anti-bacterial activities against Vibrio cholerae, IC50 (50 µM) and MIC (150 µM). Morin (2 mM) kills ~20% of human lung fibroblast (WI38) and human intestinal epithelial (HIEC-6) cells in 24 h in-vitro. Fluorescence studies showed morin binds to VcFtsA (FtsA of V. cholerae) with a Kd of 4.68 ± 0.4 µM, inhibiting the protein's polymerization by 72 ± 7% at 25 µM concentration. Morin also affected VcFtsA's ATPase activity, recording ~80% reduction at 20 µM concentration. The in-silico binding study indicated binding sites of morin and ATP on VcFtsA had overlapping amino acids. Mant-ATP, a fluorescent ATP-derivative, showed increased fluorescence on binding to VcFtsA in absence of morin, but in its presence, Mant-ATP fluorescence decreased. VcFtsA-S40A mutant protein did not bind to morin. CONCLUSIONS: VcFtsA-morin interaction inhibits the polymerization of the protein by affecting its ATPase activity. The destabilized VcFtsA assembly in-turn affected the cell division in V. cholerae, yielding an elongated morphology. GENERAL SIGNIFICANCE: Collectively, these findings explore the anti-bacterial effect of morin on V. cholerae cells targeting VcFtsA, encouraging it to become a potent anti-bacterial agent. Low cytotoxicity of morin against human cells (host) is therapeutically advantageous. This study will also help in synthesizing novel derivatives that can target VcFtsA more efficiently.

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.

FtsA-FtsZ interaction in Vibrio cholerae causes conformational change of FtsA resulting in inhibition of ATP hydrolysis and polymerization.

Proper interaction between the divisome proteins FtsA and FtsZ is important for the bacterial cell division which is not well characterized till date. In this study, the objective was to understand the mechanism of FtsA-FtsZ interaction using full-length recombinant proteins. We cloned, over-expressed, purified and subsequently characterized FtsA of Vibrio cholerae (VcFtsA). We found that VcFtsA polymerization assembly was dependent on Ca2+ ions, which is unique among FtsA proteins reported until now. VcFtsA also showed ATPase activity and its assembly was ATP dependent. Binding parameters of the interaction between the two full-length proteins, VcFtsA, and VcFtsZ determined by fluorescence spectrophotometry yielded a Kd value of around 38 µM. The Kd value of the interaction was 3 µM when VcFtsA was in ATP bound state. We found that VcFtsZ after interacting with VcFtsA causes a change of secondary structure in the later one leading to loss of its ability to hydrolyze ATP, subsequently halting the VcFtsA polymerization. On the other hand, a double-mutant of VcFtsA (VcFtsA-D242E,R300E), that does not bind to VcFtsZ, polymerized in the presence of VcFtsZ. Though FtsA proteins among different organisms show 70-80% homology in their sequences, assembly of VcFtsA showed a difference in its regulatory processes.

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.

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

Little is known about insulin's 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.

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).

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-ß-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.

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.

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.