Intra-tumor ROS amplification by melatonin interferes in the apoptosis-autophagy-inflammation-EMT collusion in the breast tumor microenvironment.

Epidemiological as well as experimental studies have established that the pineal hormone melatonin has inhibitory effects on different types of cancers. Several mechanisms have been proposed for the anticancer activities of melatonin, but the fundamental molecular pathways still require clarity. We developed a mouse model of breast cancer using Ehrlich's ascites carcinoma (injected in the 4th mammary fat pad of female Swiss albino mice) and investigated the possibility of targeting the autophagy-inflammation-EMT colloquy to restrict breast tumor progression using melatonin as intervention. Contrary to its conventional antioxidant role, melatonin was shown to augment intracellular ROS and initiate ROS-dependent apoptosis in our system, by modulating the p53/JNK & NF-κB/pJNK expressions/interactions. Melatonin-induced ROS promoted SIRT1 activity. Interplay between SIRT1 and NF-κB/p65 is known to play a pivotal role in regulating the crosstalk between autophagy and inflammation. Persistent inflammation in the tumor microenvironment and subsequent activation of the IL-6/STAT3/NF-κB feedback loop promoted EMT and suppression of autophagy through activation of PI3K/Akt/mTOR signaling pathway. Melatonin disrupted NF-κB/SIRT1 interactions blocking IL-6/STAT3/NF-κB pathway. This led to reversal of pro-inflammatory bias in the breast tumor microenvironment and augmented autophagic responses. The interactions between p62/Twist1, NF-κB/Beclin1 and NF-κB/Slug were altered by melatonin to strike a balance between autophagy, inflammation and EMT, leading to tumor regression. This study provides critical insights into how melatonin could be utilized in treating breast cancer via inhibition of the PI3K/Akt/mTOR signaling and differential modulation of SIRT1 and NF-κB proteins, leading to the establishment of apoptotic and autophagic fates in breast cancer cells.

microRNA-205 represses breast cancer metastasis by perturbing the rab coupling protein [RCP]-mediated integrin ß1 recycling on the membrane.

During cancer cell invasion, integrin undergoes constant endo/exocytic trafficking. It has been found that the recycling ability of integrin ß1 through Rab11-controlled long loop pathways is directly associated with cancer invasion. Previous studies showed that gain-of-function mutant p53 regulates the Rab-coupling protein [RCP]-mediated integrin ß1 recycling by inactivating tumor suppressor TAp63. So, we were interested to investigate the involvement of miR-205 in this process. In the current study first, we evaluated that the lower expression of miR-205 in MDA-MB-231 cell line is associated with high motility and invasiveness. Further investigation corroborated that miR-205 directly targets RCP resulting in attenuated RCP-mediated integrin ß1 recycling. Overexpression of TAp63 validates our in vitro findings. To appraise the anti-metastatic role of miR-205, we developed two in vivo experimental models- xenograft-chick embryo and xenograft-immunosuppressed BALB/c mice. Our in vivo results support the negative effect of miR-205 on metastasis. Therefore, these findings advocate the tumor suppressor activity of miR-205 in breast cancer cells and suggest that in the future development of miR-205-targeting RNAi therapeutics could be a smart alternative approach to prevent the metastatic fate of the disease.

A mechanistic insight into the potential anti-cancerous property of Nigella sativa on breast cancer through micro-RNA regulation: An in vitro & in vivo study.

Cancer continues to threat mortal alongside scientific community with burgeoning grasp. Most efforts directed to tame Cancer such as radiotherapy or chemotherapy, all came at a cost of severe side effects. The plant derived bioactive compounds on the other hand carries an inevitable advantage of being safer, bioavailable & less toxic compared to contemporary chemotherapeutics. Our strategic approach employed solvent extraction of Black Seed Oil (BSO) to highlight the orchestrated use of its oil soluble phytochemicals - Thymoquinone, Carvacrol & Trans-Anethole when used in cohort. These anti-cancer agents in unbelievably modest amounts present in BSO shows better potential to delineate migratory properties in breast cancer cells as compared to when treated individually. BSO was also observed to have apoptotic calibre when investigated in MDA-MB-231 and MCF-7 cell lines. We performed chemical characterization of the individual phytochemical as well as the oil in-whole to demonstrate the bioactive oil-soluble entities present in whole extract. BSO was observed to have significant anti-cancerous properties in cumulative proportion that is reportedly higher than the individual three components. Besides, this study also reports micro-RNA regulation on BSO administration, thereby playing a pivotal role in breast cancer alleviation. Thus, synergistic action of the integrants serves better combat force against breast cancer in the form of whole extract, hence aiming at a more lucrative paradigm while significantly regulating microRNAs associated with breast cancer migration and apoptosis.

Targeted tumor killing by pomegranate polyphenols: Pro-oxidant role of a classical antioxidant.

One of the key biochemical features that distinguish a cancer cell from normal cells is its persistent pro-oxidative state that leads to intrinsic oxidative stress. Malignant cells have evolved sophisticated adaptation systems that involve high dependency on antioxidant functions and upregulation of pro-survival molecules to counteract the deleterious effects of reactive species and to maintain dynamic redox balance. This situation renders them vulnerable to further oxidative challenges by exogenous agents. In the present study, we advocated that pomegranate polyphenols act as pro-oxidants and trigger ROS-mediated apoptosis in cancer cells. With the help of both in vitro and in vivo models, we have established that pomegranate fruit extract (PFE) can cause a significant reduction in tumor proliferation while leaving normal tissues and cells unharmed. Administration of PFE (0.2% v/v) in Erhlich's ascites carcinoma-bearing mice for 3 weeks, inhibited the nuclear factor (erythroid-derived 2)-like 2-antioxidant response element signaling cascade, increased intracellular reactive oxygen species content, altered glutathione cycle thereby activating reactive oxygen species-induced apoptotic pathway in Erhlich's ascites carcinoma cells. Moreover, PFE mitigated epithelial to mesenchymal transition and migration in triple negative breast cancer cells (MDA-MB 231 cells) by down-regulating nuclear factor kappa light-chain-enhancer of activated B cells. Pre-treatment of tumor cells with N-acetyl cysteine protected these cells from undergoing PFE-induced apoptosis while siRNA-mediated silencing of Nuclear factor (erythroid-derived 2)-like 2 and nuclear factor kappa light-chain-enhancer of activated B cells in tumor cells increased the cytotoxic potential and pro-oxidative activity of PFE, indicating a clear role of these transcription factors in orchestrating the anticancer/pro-oxidative properties of PFE. The seminal findings provided may be exploited to develop potential therapeutic targets for selective killing of malignant cells.

Sustained systemic inflammation increases autophagy and induces EMT/fibrotic changes in mouse liver cells: Protection by melatonin.

The unending lifestyle stressors along with genetic predisposition, environmental factors and infections have pushed the immune system into a state of constant activity, leading to unresolved inflammation and increased vulnerability to chronic diseases. Liver fibrosis, an early-stage liver condition that increases the risk of developing liver diseases like cirrhosis and hepatocellular carcinoma, is among the various diseases linked to inflammation that dominate worldwide morbidity and mortality. We developed a mouse model with low-grade lipopolysaccharide (LPS) exposure that shows hepatic damage and a pro-inflammatory condition in the liver. We show that inflammation and oxidative changes increase autophagy in liver cells, a degradation process critical in maintaining cellular homeostasis. Our findings from in vivo and in vitro studies also show that induction of both inflammation and autophagy trigger epithelial-mesenchymal transition (EMT) and pro-fibrotic changes in hepatocytes. Inhibiting the inflammatory pathways with a naturally occurring NF-κB inhibitor and antioxidant, melatonin, could assuage the changes in autophagy and activation of EMT/fibrotic pathways in hepatocytes. Taken together, this study shows a pathway linking inflammation and autophagy which could be targeted for future drug development to delay the progression of liver fibrosis.

Delivery of gefitinib in synergism with thymoquinone via transferrin-conjugated nanoparticle sensitizes gefitinib-resistant non-small cell lung carcinoma to control metastasis and stemness.

Epidermal growth factor receptor (EGFR) normally over-expresses in non-small cell lung cancer (NSCLC) cells. Its mutations act as oncogenic drivers in the cellular signal transduction pathway, and induce the downstream activation of numerous key cellular events involved in cellular proliferation and survival. EGFR tyrosine kinase inhibitors (EGFR-TK inhibitors), such as gefitinib and erlotinib, have been used for a long time in the treatment of NSCLC. However, they fail to overcome the EGFR-TK mutation due to the acquisition of drug resistance. It is strongly believed that the epithelial-to-mesenchymal transition (EMT) is a key player for acquired resistance and consequent limitation of the clinical efficiency of EGFR-TKIs. Therefore, a new strategy needs to be developed to overcome the resistance in NSCLC. In this current study, we have disclosed for the first time the efficiency of transferrin-modified PLGA-thymoquinone-nanoparticles in combination with gefitinib (NP-dual-1, NP-dual-2 and NP-dual-3) towards gefitinib-resistant A549 cells. The gefitinib-resistant A549 cells (A549/GR) showed 12.3-fold more resistance to gefitinib in comparison to non-resistant A549 cells. The phenotypic alteration resembling spindle-cell shape and increased pseudopodia integuments featured the EMT phenomena in A549/GR cells. EMT in A549/GR was later coupled with the loss of Ecad and expansion of Ncad, along with upregulated vimentin expression, as compared to the control A549 cells. Moreover, the invasive nature and migration potential are more amplified in A549/GR cells. Pre-incubation of A549 cells with TGFß1 also initiated EMT, leading to drug resistance. Conversely, treatment of A549 or A549/GR cells with NP-dual-3 effectively retrieved the sensitivity to gefitinib, restricted the EMT phenomenon, and impaired the TGFß1-induced EMT. On unveiling the underlying mechanism of therapeutic action, we found that STAT3 and miR-21 were individually overexpressed in the A549/GR cells by transfection, and followed by treatment with NP-dual-3. Simultaneously, NP-dual-3 fragmented HIF1-α induced EMT in A549/GR cells and reduced the CSCs markers, viz., Oct-4, Sox-2, Nanog, and Aldh1. These data are self-sufficient to suggest that NP-dual-3 re-sensitizes the drug-resistant A549/GR cells to gefitinib, possibly by retrieving MET phenomena via modulation of STAT3/mir-21/Akt/PTEN/HIF1-α axis. Thus, TQ nanoparticles combined with TKI gefitinib may provide an effective platform to treat NSCLC.

Self Nano-Emulsifying Curcumin (SNEC30) attenuates arsenic-induced cell death in mice.

Several precedents have confirmed numerous infirmities caused by arsenic poisoning, including immune suppression and cancer. Exposure to arsenic leads to alterations of the cellular machinery and eventually cell death, depending on the dose and duration of exposure. Oxidative stress induced by arsenic is the major mechanism by which it inflicts cellular toxicity, challenging the survival-support - autophagy and culminating in apoptosis in the thymus and spleen of mice. Curcumin, a potent dietary anti-oxidant with known anti-apoptotic and anti-inflammatory properties, was assessed for therapeutic benefits. However, the major caveat of this polyphenol is its low water solubility and limited bioavailability. Therefore, Self Nano-Emulsifying Curcumin (SNEC30) was used to treat mice exposed to arsenic. When administered, SNEC30 effectively ameliorated the adverse effects of arsenic in mice, by restoring structural alterations and reducing ROS-mediated cell death, thereby endorsing the importance of nutraceuticals in counteracting heavy metal-induced cellular toxicity.

Pomegranate Polyphenols Attenuate Inflammation and Hepatic Damage in Tumor-Bearing Mice: Crucial Role of NF-κB and the Nrf2/GSH Axis.

It has been widely reported that cancer, along with its treatment regimens, cause severe toxicity in the host. A suitable agent having chemopreventive properties as well as capabilities of ameliorating tumor- and drug-induced toxicities is of imminent need. Pomegranate has been projected as an excellent anti-tumor, anti-inflammatory and anti-oxidant agent. In this study, for the first time, we delineated the exact signaling cascade by which dietary supplementation of pomegranate fruit extract (PFE) protects tumor-bearing mice from tumor-induced hepatotoxicity. Increased activities of serum Alanine transaminase, Aspartate transaminase, Lactate dehydrogenase and Alkaline phosphatase, as well as histological studies confirmed the establishment of a state of hepatic dysfunction in tumor-bearers. Further investigations revealed that increased hepatic reactive oxygen species content and glutathione depletion-initiated apoptosis in these hepatocytes as we observed an alteration in the apoptotic proteins. PFE supplementation in tumor-bearing mice, on the other hand, differentially modulated redox-sensitive transcription factors Nrf2 and NF-κB, ultimately decreasing tumor-induced hepatic oxidative damage and cell death. siRNA-mediated inhibition of Nrf2 and NF-κB completely abolished the hepato-protective activities of PFE while pre-treatment of tumor-conditioned hepatocytes with N-acetyl cysteine augmented the cyto-protective properties of PFE. The present study clearly identified Nrf2/NF-κB/glutathione axis as the key factor behind the hepatoprotective potential of PFE. These findings would add to the existing knowledge about cancer chemoprevention by dietary polyphenols and might lead to the application of pomegranate polyphenols as supplement to escalate the effectiveness of cancer therapy by protecting normal cells from cancer related toxicities.

Delivery of novel coumarin-dihydropyrimidinone conjugates through mixed polymeric nanoparticles to potentiate therapeutic efficacy against triple-negative breast cancer.

To date, most of the accessible therapeutic options are virtually non-responsive towards triple-negative breast cancer (TNBC) due to its highly aggressive and metastatic nature. Interestingly, chemotherapy reacts soundly in many TNBC cases compared to other types of breast cancer. However, the side effects of many chemotherapeutic agents are still under cross-examination, and thus prohibit their extensive uses. In this present study, we have developed a series of coumarin-dihydropyrimidinone conjugates (CDHPs) and subsequently their poly(lactic-co-glycolic acid) (PLGA)-PEG4000 mixed copolymer nanoparticles as excellent chemotherapeutic nanomedicine to control TNBC. Among all the synthesized CDHPs, CDHP-4 (prepared by the combination of EDCO with 3,4-difluorobenzaldehyde) showed excellent therapeutic effect on a wide variety of cancer cell lines, including TNBC. Besides, it can control the metastasis and stemness property of TNBC. Furthermore, the nano-encapsulation of CDHP-4 in a mixed polymer nanoparticle system (CDHP-4@PP-NPs) and simultaneous delivery showed much improved therapeutic efficacy at a much lower dose, and almost negligible side effects in normal healthy cells or organs. The effectiveness of the present therapeutic agent was observed both in intravenous and oral mode of administration in in vivo experiments. Moreover, on elucidating the molecular mechanism, we found that CDHP-4@PP-NPs could exhibit apoptotic, anti-migratory, as well as anti-stemness activity against TNBC cell lines through the downregulation of miR-138. We validated our findings in MDA-MB-231 xenograft chick embryos, as well as in 4T1-induced mammary tumor-bearing BALB/c mice models, and studied the bio-distribution of CDHP-4@PP-NPs on the basis of the photoluminescence property of nanoparticles. Our recent study, hence for the first time, unravels the synthesis of CDHP-4@PP-NPs and the molecular mechanism behind the anti-migration, anti-stemness and anti-tumor efficacy of the nanoparticles against the TNBC cells through the miR-138/p65/TUSC2 axis.

Hyaluronic acid engrafted metformin loaded graphene oxide nanoparticle as CD44 targeted anti-cancer therapy for triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with limited treatment modalities. It is associated with high propensity of cancer recurrence. METHODS: UV Spectroscopy, FTIR, DLS, Zeta potential, TEM and SEM were employed to characterize nanoparticles. MTT assay, Wound healing assay, SEM, Immunocytochemistry analysis, Western blot, RT-PCR, mammosphere formation assay were employed to study apoptosis, cell migration and stemness. Tumor regression was studied in chick embryo xenograft and BALB/c mice model. RESULTS: Hylaluronic acid engrafted metformin loaded graphene oxide (HA-GO-Met) nanoparticles exhibited an anti-cancer efficacy at much lower dosage as compared to metformin alone. HA-GO-Met nanoparticles induced apoptosis and inhibited cell migration of TNBC cells by targeting miR-10b/PTEN axis via NFkB-p65. Upregulation of PTEN affected pAKT(473) expression that induced apoptosis. Cell migration was inhibited by reduction of pFAK/integrinß1 expressions. Treatment inhibited epithelial mesenchymal transition (EMT) and reduced stemness as evident from the increase in E-cadherin expression, inhibition of mammosphere formation and low expression levels of stemness markers including nanog, oct4 and sox2 as compared to control. Moreover, tumor regression was studied in chick embryo xenograft and BALB/c mice model. HA-GO-Met nanoparticle treatment reduced tumor load and nullified toxicity in peripheral organs imparted by tumor. CONCLUSIONS: HA-GO-Met nanoparticles exhibited an enormous anti-cancer efficacy in TNBC in vitro and in vivo. GENERAL SIGNIFICANCE: HA-GO-Met nanoparticles induced apoptosis and attenuated cell migration in TNBC. It nullified overall toxicity imparted by tumor load. It inhibited EMT and reduced stemness and thereby addressed the issue of cancer recurrence.

Delivery of dual miRNA through CD44-targeted mesoporous silica nanoparticles for enhanced and effective triple-negative breast cancer therapy.

The development of new therapeutic strategies to target triple-negative breast cancer (TNBC) is in much demand to overcome the roadblocks associated with the existing treatment procedures. In this regard, therapies targeting the CD44 receptor have drawn attention for more than a decade. MicroRNAs (miRNAs) modulate post-transcriptional gene regulation and thus, the correction of specific miRNA alterations using miRNA mimics or antagomiRs is an emerging strategy to normalize the genetic regulation in the tumor microenvironment. It has been acknowledged that miR-34a is downregulated and miR-10b is upregulated in TNBC, which promotes tumorigenesis and metastatic dissemination. However, there are a few barriers related to miRNA delivery. Herein, we have introduced tailored mesoporous silica nanoparticles (MSNs) for the co-delivery of miR-34a-mimic and antisense-miR-10b. MSN was functionalized with a cationic basic side chain and then loaded with the dual combination to overexpress miR-34a and downregulate miR-10b simultaneously. Finally, the loaded MSNs were coated with an hyaluronic acid-appended PEG-PLGA polymer for specific targeting. The cellular uptake, release profile, and subsequent effect in TNBC cells were evaluated. In vitro and in vivo studies demonstrated high specificity in TNBC tumor targeting, leading to efficient tumor growth inhibition as well as the retardation of metastasis, which affirmed the clinical application potential of the system.

Delivery of thymoquinone through hyaluronic acid-decorated mixed Pluronic® nanoparticles to attenuate angiogenesis and metastasis of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic subtype of breast cancer showing non-responsiveness to most available therapeutic options. Therefore, smart therapeutic approaches to selectively transport and target TNBCs are required. Herein, we developed thymoquinone (TQ)-loaded, hyaluronic acid (HA)-conjugated Pluronic® P123 and F127 copolymer nanoparticles (HA-TQ-Nps) as a selective drug-carrying vehicle to deliver anticancer phytochemical TQ to TNBC cells. The mean size of nanoparticles was around 19.3 ± 3.2 nm. and they were stable at room temperature up to 4 months. HA-TQ-Nps were immensely cytotoxic towards TNBC cells but did not show the toxic effect on normal cells. Detailed investigations also demonstrated its pro-apoptotic, anti-metastatic and anti-angiogenic activity. In-depth mechanistic studies highlighted that HA-TQ-Nps retarded cell migration of TNBC cells through up-regulation of microRNA-361 which in turn down-regulated Rac1 and RhoA mediated cell migration and also perturbed the cancer cell migration under the influence of the autocrine effect of VEGF-A. Moreover, HA-TQ-Np-treatment also perturbed tumor-induced vascularization by reducing the secretion of VEGF-A. The anti-metastatic and anti-angiogenic activity of HA-TQ-Nps was found to be evident in both MDA-MB-231 xenograft chick embryos and 4T1-mammary solid tumor model in syngeneic mice. Thus, an innovative targeted nano-therapeutic approach is being established to reduce the tumor burden and inhibit metastasis and angiogenesis simultaneously for better management of TNBC.

Effects of bamboo shoots (Bambusa balcooa) on thyroid hormone synthesizing regulatory elements at cellular and molecular levels in thyrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Bamboo shoots (BS) are consumed in various forms and used largely in naturopathy for curing ailments since ancient times to present days. It is eaten in South East Asian countries in several indigenous preparations. In north east India, it is consumed predominantly and used as natural cure to treat various diseases. Although known for its beneficial effects, adverse effects including goitrogenic/antithyroidal potential are emerging. AIM OF THE STUDY: Endemic goiter exists in Manipur, India even after adequate iodine intake for consumption of BS. It is thus important to study the impact of this goitrogenic food on certain thyroid hormone synthesizing regulatory factors at cellular and molecular level in thyrocytes. MATERIALS AND METHODS: Phytochemical analysis of BS - Bambusa balcooa Roxb (BSBR) extract conducted. IC50 of the extract on thyrocytes in culture was determined. To study the antithyroid effects of this goitrogenic food, activity status of Na+-K+-ATPase, TPO and Deiodinase, mRNA and protein expressions of NIS, TPO and PAX8 were investigated with and without extra iodine in culture media. Simultaneously ROS generation in terms of H2O2 and antioxidant status, NO, LPO were assayed. RESULTS: Activities of the studied enzymes decreased depending on dose and time with increased H2O2, decreased antioxidants followed by increased NO with LPO. DNA damage and LDH also increased while mRNA and protein expression of NIS, TPO and PAX8 were downregulated. Extra iodine ameliorated all such effects partially. CONCLUSIONS: Bioactive constituents of the extract imbalances oxidative status of thyrocytes impairing action of hormone synthesizing elements at cellular and molecular level.

Inflammation-induced behavioral changes is driven by alterations in Nrf2-dependent apoptosis and autophagy in mouse hippocampus: Role of fluoxetine.

Inflammation has been associated with the progression of many neurological diseases. Peripheral inflammation has also been vaguely linked to depression-like symptoms in animal models, but the underlying pathways that orchestrate inflammation-induced behavioral or molecular changes in the brain are still elusive. We have recently shown that intraperitoneal injections of lipopolysaccharide (LPS) to Swiss albino mice triggers systemic inflammation, leading to an activated immune response along with changes in monoamine levels in the brain. Herein we pinpoint the fundamental pathways linking peripheral inflammation and depression-like behavior in a mouse model, thereby identifying suitable targets of intervention to combat the situation. We show that LPS-induced peripheral inflammation provoked a depression-like behavior in mice and a distinct pro-inflammatory bias in the hippocampus, as evident from increased microglial activation and elevated levels of pro-inflammatory cytokines IL-6 and TNF-α, and activation of NFκB-p65 pathway. Significant alterations in Nrf2-dependent cellular redox status, coupled with altered autophagy and increased apoptosis were noticed in the hippocampus of LPS-exposed mice. We and others have previously shown that, fluoxetine (an anti-depressant) has effective anti-inflammatory and antioxidant properties by virtue of its abilities to regulate NFκB and Nrf2 signaling. We observed that treatment with fluoxetine or the Nrf2 activator tBHQ (tert-butyl hydroquinone), could reverse depression-like-symptoms and mitigate alterations in autophagy and cell death pathways in the hippocampus by activating Nrf2-dependent gene expressions. Taken together, the data suggests that systemic inflammation potentiates Nrf2-dependent changes in cell death and autophagy pathway in the hippocampus, eventually leading to major pathologic sequelae associated with depression. Therefore, targeting Nrf2 could be a novel approach in combatting depression and ameliorating its associated pathogenesis.

Transferrin-decorated thymoquinone-loaded PEG-PLGA nanoparticles exhibit anticarcinogenic effect in non-small cell lung carcinoma via the modulation of miR-34a and miR-16.

Non-small cell lung carcinoma (NSCLC) is a highly lethal type of cancer with limited therapeutic avenues available to date. In the present study, we formulated PEGylated PLGA thymoquinone nanoparticles (TQ-Np) for improved TQ delivery to NSCLC cells. Transferrin (TF), a biodegradable, non-immunogenic and non-toxic protein, is well known to bind to TFR (transferrin receptor) over-expressed in non-small cell lung carcinoma A549 cells. Thus, the further decoration of the PEGylated PLGA thymoquinone nanoparticles with transferrin (TF-TQ-Np) enhanced the internalization of the nanoparticles within the A549 cells and the activity of TQ. We established TF-TQ-Np as a potent anti-tumorigenic agent through the involvement of p53 and the ROS feedback loop in regulating the microRNA (miRNA) circuitry to control apoptosis and migration of NSCLC cells. TF-TQ-Np-mediated p53 up-regulation favored the potential simultaneous activation of miR-34a and miR-16 targeting Bcl2 to induce apoptosis in the A549 cells. Additionally, TF-TQ-Np also restricted the migration through actin de-polymerization via activation of the p53/miR-34a axis. Further studies in chick CAM xenograft models confirmed the anti-cancer activity of TF-TQ-Np by controlling the p53/miR-34a/miR-16 axis. Furthermore, in vivo experiments conducted in a xenograft model in immunosuppressed Balb/c mice also proved the efficacy of the nanoparticles as an antitumor agent against NSCLC. Thus, our findings cumulatively suggest that the transferrin-adorned TQ-Np successfully coupled two distinct miRNA pathways to potentiate the apoptotic death cascade in the very lethal NSCLC cells and also restricts the migration of these cells without imparting any significant toxicity, which occurs in the widely used chemotherapeutic combinations. Thereby, our findings rekindle new hopes for the development of improved targeted therapeutic options with specified molecular objectives for combating the deadly NSCLC.

Dietary pomegranate supplement alleviates murine pancreatitis by modulating Nrf2-p21 interaction and controlling apoptosis to survival switch.

Dietary supplementation of polyphenol-rich pomegranate extract (POMx) has been shown to have anti-oxidant and anti-inflammatory activities. Here, we evaluate the efficacy of POMx in mitigating pancreatitis in mice and provide a mechanistic outline of the process. Age-matched male Swiss albino mice were injected with Lipopolysaccharide (LPS) and given POMx supplement alone or in combination with LPS. After 4 weeks of treatment histological scoring for pancreatic edema and vacuolization was performed. Serum insulin levels were estimated and the glucose tolerance test (IPGTT) data revealed that POMx reduced inflammation induced hyperglycemia in mice. Analysis of TLR4, IκB expression, and NF-κB nuclear translocation, and concentrations of IL-6 and TNFα showed that POMx is able to modulate the molecular instigators of inflammatory responses. Annexin V assay indicated that POMx protects against inflammation-mediated apoptosis in the pancreas. Expression profile of SAPK/JNK pathway, p53, Bax, Bcl-2 and Caspase-3 validate an apoptotic to survival shift in POMx treatment group. Co-immunoprecipitation studies show that POMx stabilizes p21 and Nrf2 interaction and increases its nuclear translocation. The study also proves that the nuclear fraction of Nrf2 is able to bind to the Bcl-2 promoter and activate an anti-apoptotic program. The findings of our study underline an anti-inflammatory, anti-oxidative and anti-apoptotic role of POMx and provide a mechanistic idea of how POMx confers protection during pancreatitis.

Fluoxetine triggers selective apoptosis in inflammation-induced proliferating (Ki-67high ) thymocytes.

Inappropriate functioning of the immune system is observed during sustained systemic inflammation, which might lead to immune deficiencies, autoimmune disorders and cancer. Primary lymphoid organs may progress to a deregulated proliferative state in response to inflammatory signals in order to intensify host defense mechanisms and exacerbate an inflammatory niche. Fluoxetine, a selective serotonin reuptake inhibitor, has recently been projected as an anti-inflammatory agent. This study had been designed to evaluate the potential novel role of fluoxetine in reversing inflammation-induced immune dysfunction. Lipopolysaccharide (LPS) administration in Swiss albino mice potentiated a systemic inflammatory response, along with increased proliferation of thymocytes and peripheral blood mononuclear cells, as evident from increased Ki-67 expression. The proliferative changes in the immune system were mainly associated with increased phosphorylation of PI3k, AKT and IκB along with elevated NFκB-p65 nuclear translocation. The Ki-67high thymocytes obtained from LPS administered mice demonstrated significantly low p53 nuclear activity, which was established to be mediated by NFκB through reduced nuclear translocation of p53 during LPS-induced proliferative conditions, thereby blocking p53-dependent apoptosis. Fluoxetine supplementation not only reversed the proinflammatory condition, but also induced selective apoptosis in the proliferation-dictated Ki-67high thymocytes possibly by modulating the hypothalamus-pituitary-adrenal axis and inducing glucocorticoid receptor activation and apoptosis in these proliferation-biased immune cells, authenticating a novel antiproliferative role of an established drug.

Radiosensitizing effect of ellagic acid on growth of Hepatocellular carcinoma cells: an in vitro study.

Failure of treatment for cancer in clinic by radio/chemotherapy is generally attributed to tumour resistance. Therefore, it is important to develop strategies to increase the cytotoxicity of tumour cells by radiation in combination with unique tumour selective cytotoxic agents. We evaluated the potential of ellagic acid (EA) as an enhancer of oxidative stress in cancer cells. HepG2 cells were treated with EA (10 µM) for 12 h prior to exposure of single 7.5 Gy dose of irradiation. Treatment of HepG2 cells with EA and gamma radiation showed increased reactive oxygen species generation, up regulation of p53 protein expression, decreased survival markers level like p-Akt, p-NF-kB and p-STAT3 which were significantly higher after radiation treatment alone. We also found that combination treatment increased G2/M phase cell population, decreased IL-6, COX-2 and TNF-α expression and caused a loss in mitochondrial membrane potential with decreased level of angiogenesis marker MMP-9. Over expression of Bax and activation of caspase 3 indicated the apoptosis of the cells. The results provided a strong unique strategy to kill cancer cells HepG2, using less radiation dose along with effective pro-oxidant dose of EA.

Activating the AKT2-nuclear factor-κB-lipocalin-2 axis elicits an inflammatory response in age-related macular degeneration.

Age-related macular degeneration (AMD) is a complex and progressive degenerative eye disease resulting in severe loss of central vision. Recent evidence indicates that immune system dysregulation could contribute to the development of AMD. We hypothesize that defective lysosome-mediated clearance causes accumulation of waste products in the retinal pigmented epithelium (RPE), activating the immune system and leading to retinal tissue injury and AMD. We have generated unique genetically engineered mice in which lysosome-mediated clearance (both by phagocytosis and autophagy) in RPE cells is compromised, causing the development of features of early AMD. Our recent data indicate a link between lipocalin-2 (LCN-2) and the inflammatory responses induced in this mouse model. We show that nuclear factor-κB (NF-κB) and STAT-1 may function as a complex in our animal model system, together controlling the upregulation of LCN-2 expression in the retina and stimulating an inflammatory response. This study revealed increased infiltration of LCN-2-positive neutrophils in the choroid and retina of early AMD patients as compared with age-matched controls. Our results demonstrate that, both in our animal model and in human AMD, the AKT2-NF-κB-LCN-2 signalling axis is involved in activating the inflammatory response, making this pathway a potential target for AMD treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pomegranate protects against arsenic-induced p53-dependent ROS-mediated inflammation and apoptosis in liver cells.

Molecular mechanisms involved in arsenic-induced toxicity are complex and elusive. Liver is one of the most favored organs for arsenic toxicity as methylation of arsenic occurs mostly in the liver. In this study, we have selected a range of environmentally relevant doses of arsenic to examine the basis of arsenic toxicity and the role of pomegranate fruit extract (PFE) in combating it. Male Swiss albino mice exposed to different doses of arsenic presented marked hepatic injury as evident from histological and electron microscopic studies. Increased activities of enzymes alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and alkaline phosphatase corroborated extensive liver damage. It was further noted that arsenic exposure initiated reactive oxygen species (ROS)-dependent apoptosis in the hepatocytes involving loss of mitochondrial membrane potential. Arsenic significantly increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor-κB (NF-κB), coupled with increase in phosphorylated Iκ-B, possibly as adaptive cellular survival strategies. Arsenic-induced oxidative DNA damage to liver cells culminated in p53 activation and increased expression of p53 targets like miR-34a and Bax. Pomegranate polyphenols are known to possess remarkable antioxidant properties and are capable of protecting normal cells from various stimuli-induced oxidative stress and toxicities. We explored the protective role of PFE in ameliorating arsenic-induced hepatic damage. PFE was shown to reduce ROS generation in hepatocytes, thereby reducing arsenic-induced Nrf2 activation. PFE also inhibited arsenic-induced NF-κB-inflammatory pathway. Data revealed that PFE reversed arsenic-induced hepatotoxicity and apoptosis by modulating the ROS/Nrf2/p53-miR-34a axis. For the first time, we have mapped the possible signaling pathways associated with arsenic-induced hepatotoxicity and its rescue by pomegranate polyphenols.