Eriodictyol mediated selective targeting of the TNFR1/FADD/TRADD axis in cancer cells induce apoptosis and inhibit tumor progression and metastasis.

While the anti-inflammatory activities of Eriodictyol, a plant-derived flavonoid is well-known, reports on its anti-cancer efficacy and selective cytotoxicity in cancer cells are still emerging. However, little is known regarding its mechanism of selective anti-cancer activities. Here, we show the mechanism of selective cytotoxicity of Eriodictyol towards cancer cells compared to normal cells. Investigation reveals that Eriodictyol significantly upregulates TNFR1 expression in tumor cells (HeLa and SK-RC-45) while sparing the normal cells (HEK, NKE and WI-38), which display negligible TNFR1 expression, irrespective of the absence or presence of Eriodictyol. Further investigation of the molecular events reveal that Eriodictyol induces apoptosis through expression of the pro-apoptotic DISC components leading to activation of the caspase cascade. In addition, CRISPR-Cas9 mediated knockout of TNFR1 completely blocks apoptosis in HeLa cells in response to Eriodictyol, confirming that Eriodictyol induced cancer cell apoptosis is indeed TNFR1-dependent. Finally, in vivo data demonstrates that Eriodictyol not only impedes tumor growth and progression, but also inhibits metastasis in mice implanted with 4T1 breast cancer cells. Thus, our study has identified Eriodictyol as a compound with high selectivity towards cancer cells through TNFR1 and suggests that it can be further explored for its prospect in cancer therapeutics.

NMK-BH2, a novel microtubule-depolymerising bis (indolyl)-hydrazide-hydrazone, induces apoptotic and autophagic cell death in cervical cancer cells by binding to tubulin at colchicine - site.

BACKGROUND: Microtubules, the key components of the eukaryotic cytoskeleton and mitotic spindle, are one of the most sought-after targets for cancer chemotherapy, especially due to their indispensible role in mitosis. Cervical cancer is a prevalent malignancy among women of developing countries including India. In spite of the remarkable therapeutic advancement, the non-specificity of chemotherapeutic drugs adversely affect the patients' survival and well-being, thus, necessitating the quest for novel indole-based anti-microtubule agent against cervical cancer, with high degree of potency and selectivity. METHODS: For in vitro studies, we used MTT assay, confocal microscopy, fluorescence microscopy, flow cytometry and Western blot analysis. Study in cell free system was accomplished by spectrophotometry, fluorescence spectroscopy and TEM and computational analysis was done by AutodockTools 1.5.6. RESULTS: NMK-BH2 exhibited significant and selective anti-proliferative activity against cervical cancer HeLa cells (IC50 = 1.5 µM) over normal cells. It perturbed the cytoskeletal and spindle microtubules of HeLa cells leading to mitotic block and cell death by apoptosis and autophagy. Furthermore, NMK-BH2 targeted the tubulin-microtubule system through fast and strong binding to the αß-tubulin heterodimers at colchicine-site. CONCLUSION: This study identifies and characterises NMK-BH2 as a novel anti-microtubule agent and provides insights into its key anti-cancer mechanism through two different cell death pathways: apoptosis and autophagy, which are mutually independent. GENERAL SIGNIFICANCE: It navigates the potential of the novel bis (indolyl)-hydrazide-hydrazone, NMK-BH2, to serve as lead for development of new generation microtubule-disrupting chemotherapeutic with improved efficacy and remarkable selectivity towards better cure of cervical cancer.

Skin-to-skin contact at birth for vaginally delivered neonates in a tertiary care hospital: A cross-sectional study.

BACKGROUND: Neonates undergoing skin-to-skin contact (SSC) have shown to have better cardiovascular stability, improved duration of breastfeeding, temperature maintenance and bonding. The primary objective was to estimate the existing prevalence of SSC among mother-infant dyads in the first hour after vaginal delivery, and the secondary objective was to ascertain the factors affecting adherence of SSC after vaginal delivery. METHODS: This cross-sectional study was carried out in a tertiary care teaching hospital between September 2017 and December 2017. All mother-newborn dyads in the labour room were eligible for the study. Mothers with their newborns, who gave multiple births, or with major congenital malformations or those requiring any form of resuscitation or having respiratory distress or requiring observation in neonatal intensive care unit (NICU) were excluded. All included mother-newborn dyads, after exclusion, were observed for SSC, and the duration in each dyad was recorded. All enrolled mothers, the attending doctors and nurses were asked questions based on a prevalidated questionnaire for the possible barriers of implementation. RESULTS: Of a total of 164 mother-infant dyads studied, only 34 (20.7%) carried out SSC for more than 30 min. Sixty-two (37.8%) mother-infant dyads did not participate in any SSC. The commonest reason for non-adherence to SSC was unawareness about the practice in 82.25%, followed by pain and exhaustion after the labour process in 8%. CONCLUSION: The rate of SSC at birth is suboptimal, and the commonest reason is lack of awareness about this important step of essential newborn care.

Correction to: Autophagy inhibition with chloroquine reverts paclitaxel resistance and attenuates metastatic potential in human nonsmall lung adenocarcinoma A549 cells via ROS mediated modulation of ß-catenin pathway.

The original version of this article unfortunately contained an error in acknowledgment text. The authors would like to include a statement: "Moumita Dasgupta is supported by Junior Research Fellowship from University Grant Commission, India." in acknowledgment section.

Autophagy inhibition with chloroquine reverts paclitaxel resistance and attenuates metastatic potential in human nonsmall lung adenocarcinoma A549 cells via ROS mediated modulation of ß-catenin pathway.

Paclitaxel is one of the most commonly used drugs for the treatment of nonsmall cell lung cancer (NSCLC). However acquired resistance to paclitaxel, epithelial to mesenchymal transition and cancer stem cell formation are the major obstacles for successful chemotherapy with this drug. Some of the major reasons behind chemoresistance development include increased ability of the cancer cells to survive under stress conditions by autophagy, increased expression of drug efflux pumps, tubulin mutations etc. In this study we found that inhibition of autophagy with chloroquine prevented development of paclitaxel resistance in A549 cells with time and potentiated the effect of paclitaxel by increased accumulation of superoxide-producing damaged mitochondria, with elevated ROS generation, it also increased the apoptotic rate and sub G0/ G1 phase arrest with time in A549 cells treated with paclitaxel and attenuated the metastatic potential and cancer stem cell population of the paclitaxel-resistant cells by ROS mediated modulation of the Wnt/ß-catenin signaling pathway, thereby increasing paclitaxel sensitivity. ROS here played a crucial role in modulating Akt activity when autophagy process was hindered by chloroquine, excessive ROS accumulation in the cell inhibited Akt activity. In addition, chloroquine pre-treatment followed by taxol (10 nM) treatment did not show significant toxicity towards non-carcinomas WI38 cells (lung fibroblast cells). Thus autophagy inhibition by CQ pre-treatment can be used as a fruitful strategy to combat the phenomenon of paclitaxel resistance development as well as metastasis in lung cancer.

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.

Paclitaxel resistance development is associated with biphasic changes in reactive oxygen species, mitochondrial membrane potential and autophagy with elevated energy production capacity in lung cancer cells: A chronological study.

Paclitaxel (Tx) is one of the first-line chemotherapeutic drugs used against lung cancer, but acquired resistance to this drug is a major challenge against successful chemotherapy. In this work, we have focused on the chronological changes of various cellular parameters and associated effect on Tx (10 nM) resistance development in A549 cell line. It was observed, at initial stage, the cell death percentage due to drug treatment had increased up to 20 days, and thereafter, it started declining and became completely resistant by 40 days. Expressions of ßIII tubulin and drug efflux pumps also increased over the period of resistance development. Changes in cellular autophagy and reactive oxygen species generation showed a biphasic pattern and increased gradually over the course of upto 20 days, thereafter declined gradually; however, their levels remained higher than untreated cells when resistance was acquired. Increase in extracellular acidification rates and oxygen consumption rates was found to be directly correlated with acquisition of resistance. The depolarisation of mitochondrial membrane potential was also biphasic; first, it increased with increase of cell death up to 20 days, thereafter, it gradually decreased to normal level along with resistance development. Increase in activity of catalase, glutathione peroxidase and glutathione content over these periods may attribute in bringing down the reactive oxygen species levels and normalisation of mitochondrial membrane potential in spite of comparatively higher reactive oxygen species production by the Tx-resistant cells.

A Facile and Microwave-assisted Rapid Synthesis of 2-Arylamino-4-(3'-indolyl)- thiazoles as Apoptosis Inducing Cytotoxic Agents.

BACKGROUND AND OBJECTIVE: The clinical success of the chemotherapeutic drugs is restricted by the nonspecific toxicity-related adverse side effects. The diverse implication of indoles and thiazoles in medicinal chemistry prompted us to develop a new series of novel 2-aryl-amino-4-(3'-indolyl)thiazoles as more effective and less toxic anti-cancer compounds. METHOD AND RESULTS: One-pot microwave-assisted rapid and high yielding synthesis of 2-arylamino-4-(3'- indolyl)thiazoles involved the reaction of easily available α-tosyloxy-ketones with N-arylthioureas in polyethylene glycol-400 (PEG-400). In vitro cytotoxicity study of 2-arylamino-4-(3'-indolyl)thiazoles against a panel of human cancer cell lines by MTT assay revealed IC50 values in the low micromolar range. Of the fifteen synthesized arylaminothiazoles, compounds 17b, 17d, 17g and 17il showed significant anti-proliferative activity against the selected cancer cell lines with IC50 < 10 µM. The compound 17b was identified as the most potent ligand of the series, which exhibited good cytotoxic activity against MCF-7 breast cancer cells with an IC50 value of 1.86 µM but minimal toxicity on normal human cells. Investigation of the underlying mechanism by flow cytometry indicated that 17b induced ROS-mediated apoptosis in MCF-7 cells in a dose-dependent manner as supported by upregulation of Bax and caspase-3 and down-regulation of Bcl-2 (by Western blot). CONCLUSION: Developed an efficient and eco-friendly synthesis for 2-arylamino-4-(3'-indolyl)thiazoles, and their in vitro cytotoxicity studies demonstrate that compound 17b exhibits significant anti-proliferative activity against MCF-7 (breast cancer) cells by activating ROS-mediated apoptosis through the mitochondrial apoptosis pathway.

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 ß-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.

Leishmania donovani infection enhances lateral mobility of macrophage membrane protein which is reversed by liposomal cholesterol.

BACKGROUND: The protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment. METHODOLOGY/PRINCIPAL FINDINGS: We determined the lateral mobility of a membrane protein in normal, LD infected and liposome treated LD infected cells using GFP-tagged PLCδ1 as a probe. The mobility of PLCδ1 was computationally analyzed from the time lapse experiment using boundary distance plot and radial profile movement. Our results showed that the lateral mobility of the membrane protein, which is increased in infection, is restored to normal upon liposomal cholesterol treatment. The results of FRAP experiment lent further credence to the above notion. The membrane proteins are intimately linked with cellular actin and alteration of cellular actin may influence lateral mobility. We found that F-actin is decreased in infection but is restored to normal upon liposomal cholesterol treatment as evident from phalloidin staining and also from biochemical analysis by immunoblotting. CONCLUSIONS/SIGNIFICANCES: To our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.

Modulatory role of quercetin against gamma radiation-mediated biochemical and morphological alterations of red blood cells.

PURPOSE: The present work was intended to evaluate the radioprotective effect of quercetin against gamma radiation-induced oxidative stress on red blood cells (RBC). MATERIALS AND METHODS: Swiss albino male mice were treated with quercetin (100 mg/kg body wt) for three consecutive days prior to 5 Gy (60)Co-gamma irradiation. RBC was isolated to estimate the level of intracellular reactive oxygen species (ROS), membrane lipid peroxidation (LPO), intracellular reduced glutathione (GSH), mean corpuscular hemoglobin concentration (MCHC), osmotic fragility and morphological alterations by atomic force microscope (AFM). RESULTS: Irradiation increased intracellular ROS and membrane LPO whereas it decreased the intracellular GSH. Quercetin pretreatment ameliorated these alterations. The MCHC value decreased after irradiation whereas quercetin pretreatment restored it. The average osmotic fragility (H50) and the maximum rate of hemolysis (dH/dC)max increased after irradiation. Quercetin pretreatment decreased the H50 and (dH/dC)max. The AFM study showed that irradiation transformed RBC from biconcave to echinocytes, increased their surface roughness and decreased the vertical distance whereas pretreatment of quercetin significantly prevented both the alterations. CONCLUSIONS: Gamma radiation produced ROS and LPO which rendered oxidative stress and ultimately damaged RBC whereas quercetin ameliorated these changes and protected RBC from radiation-mediated damage.