Pallavi Sethi

Aluminium-induced electrophysiological, biochemical and cognitive modifications in the hippocampus of aging rats

Aluminium (Al) is the most abundant metal known for its neurotoxicity in humans. It gains easy access to the central nervous system under normal physiological conditions and accumulates in different brain regions. It has been reported to be involved in the etiology of several neurodegenerative diseases. In this study, we have investigated the effects of long-term intake of aluminium chloride (AlCl(3)) on the electrophysiological, behavioral, biochemical and histochemical functions of hippocampus. Wistar rats were fed with AlCl(3) at a dose of 50mg/(kgday) for 6 months in the drinking water. Effect of long-term intake of Al was studied on the electrical activity of hippocampal CA1 and CA3 regions in brain of young and old rats. Morris water maze and open field tests were performed to investigate the cognitive and anxiety status of aging rats intoxicated with aluminium. Our studies indicate that aluminium intake results in increased multiple unit activity and adversely affect the spatial learning and memory abilities of both young and old rats. Aluminium intake also inflicts oxidative stress-related damage to lipids, membrane associated proteins (Na-K ATPase and PKC) and endogenous antioxidant enzyme activity (SOD, GPx and GST). The compromised antioxidant system might be playing a crucial role in the observed Al-induced alterations. We have observed that the magnitude of AlCl(3)-induced alteration was considerably higher in younger group of rats compared to older group. In conclusion, the results of the present study implicates that aluminium treatment exerts its neurotoxic effects by altering the overall physiology of brain, and the induced changes were strongly correlated with each other.

Curcumin attenuates aluminium-induced functional neurotoxicity in rats

Curcumin is a polyphenol extracted from the rhizome of Curcuma longa and well known as a multi-functional drug with antioxidative, anti-cancerous and anti-inflammatory activities. Curcumins antiaging and neuroprotective potential is widely reported. In the present study, effect of curcumin treatment dose 30 mg kg(-1) day(-1) was investigated against aluminium neurotoxicity in young and old animals. Direct and indirect intakes of aluminium have been reported to be involved in the etiology of several neurodegenerative disorders like Alzheimers and Parkinsons diseases. Long term Al was administered through drinking water at a dose of 50 mg/kg/day for 6 months in both young (4 months) and old (18 months) male Wistar rats. Result obtained demonstrates that curcumin treatment attenuates the Al-induced alterations at biochemical, behavioral and ultrastructural levels which was well reflected in the electrophysiological recordings. Our results indicate that curcumins ability to bind redox active metals and cross the blood-brain barrier could be playing crucial role in preventing against Al-induced neurotoxicity.

Curcumin counteracts the aluminium-induced ageing-related alterations in oxidative stress, Na+, K+ ATPase and protein kinase C in adult and old rat brain regions.

This study investigated the effect of curcumin on aluminium-induced alterations in ageing-related parameters: lipid peroxidation, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-s-transferase (GST), protein kinase C (PKC), Na+, K+-adenosine triphosphatase (Na+, K+-ATPase) and acetylcholinesterase (AChE) in the cerebral cortex and hippocampus of the brain of 10- and 24-month-old rats. Measurements taken from aluminium-fed rats were compared with those from rats in which curcumin and aluminium were co-administered. In aluminium-treated rats the levels of lipid peroxidation, PKC and AChE were enhanced while the activities of SOD, GPx, GST and Na+, K+-ATPase were significantly decreased in both the brain regions of both age-groups. In animals co-administered with curcumin and aluminium, the levels of lipid peroxidation, activities of PKC and AChE were significantly lowered while the activities of SOD, GPx, GST and Na+, K+-ATPase were significantly enhanced in the two brain regions studied indicating curcumin’s protective effects against aluminium toxicity. Though the magnitudes of curcumin-induced alterations varied in young and old animals, the results of the present study also demonstrated that curcumin exerts a protective effect against aluminium-induced elevation of ageing-related changes by modulating the extent of oxidative stress (by upregulating the activities of antioxidant enzymes) and by regulating the activities of Na+, K+ ATPase, PKC and AChE. Therefore, it is suggested that curcumin counters aluminium-induced enhancement in ageing-related processes.

Biomimetic Tissue–Engineered Systems for Advancing Cancer Research: NCI Strategic Workshop Report

Advanced technologies and biomaterials developed for tissue engineering and regenerative medicine present tractable biomimetic systems with potential applications for cancer research. Recently, the National Cancer Institute convened a Strategic Workshop to explore the use of tissue biomanufacturing for development of dynamic, physiologically relevant in vitro and ex vivo biomimetic systems to study cancer biology and drug efficacy. The workshop provided a forum to identify current progress, research gaps, and necessary steps to advance the field. Opportunities discussed included development of tumor biomimetic systems with an emphasis on reproducibility and validation of new biomimetic tumor models, as described in this report.

3D tumor tissue analogs and their orthotopic implants for understanding tumor-targeting of microenvironment-responsive nanosized chemotherapy and radiation

UNLABELLED An appropriate representation of the tumor microenvironment in tumor models can have a pronounced impact on directing combinatorial treatment strategies and cancer nanotherapeutics. The present study develops a novel 3D co-culture spheroid model (3D TNBC) incorporating tumor cells, endothelial cells and fibroblasts as color-coded murine tumor tissue analogs (TTA) to better represent the tumor milieu of triple negative breast cancer in vitro. Implantation of TTA orthotopically in nude mice, resulted in enhanced growth and aggressive metastasis to ectopic sites. Subsequently, the utility of the model is demonstrated for preferential targeting of irradiated tumor endothelial cells via radiation-induced stromal enrichment of galectin-1 using anginex conjugated nanoparticles (nanobins) carrying arsenic trioxide and cisplatin. Demonstration of a multimodal nanotherapeutic system and inclusion of the biological response to radiation using an in vitro/in vivo tumor model incorporating characteristics of tumor microenvironment presents an advance in preclinical evaluation of existing and novel cancer nanotherapies. FROM THE CLINICAL EDITOR Existing in-vivo tumor models are established by implanting tumor cells into nude mice. Here, the authors described their approach 3D spheres containing tumor cells, enodothelial cells and fibroblasts. This would mimic tumor micro-environment more realistically. This interesting 3D model should reflect more accurately tumor response to various drugs and would enable the design of new treatment modalities.

Investigating the Radioresistant Properties of Lung Cancer Stem Cells in the Context of the Tumor Microenvironment

Lung cancer is the most common cause of cancer-related deaths worldwide and non-small cell lung cancer (NSCLC) accounts for ~85% of all lung cancer. While recent research has shown that cancer stem cells (CSC) exhibit radioresistant and chemoresistant properties, current cancer therapy targets the bulk of the tumor burden without accounting for the CSC and the contribution of the tumor microenvironment. CSC interaction with the stroma enhances NSCLC survival, thus limiting the efficacy of treatment. The aim of this study was to elucidate the role of CSC and the microenvironment in conferring radio- or chemoresistance in an in vitro tumor model for NSCLC. The novel in vitro three-dimensional (3D) NSCLC model of color-coded tumor tissue analogs (TTA) that we have developed is comprised of human lung adenocarcinoma cells, fibroblasts, endothelial cells and NSCLC cancer stem cells maintained in low oxygen conditions (5% O2) to recapitulate the physiologic conditions in tumors. Using this model, we demonstrate that a single 5 Gy radiation dose does not inhibit growth of TTA containing CSC and results in elevated expression of cytokines (TGF-α, RANTES, ENA-78) and factors (vimentin, MMP and TIMP), indicative of an invasive and aggressive phenotype. However, combined treatment of single dose or fractionated doses with cisplatin was found to either attenuate or decrease the proliferative effect that radiation exposure alone had on TTA containing CSC maintained in hypoxic conditions. In summary, we utilized a 3D NSCLC model, which had characteristics of the tumor microenvironment and tumor cell heterogeneity, to elucidate the multifactorial nature of radioresistance in tumors.

Aging accelerates the progression and manifestation of seizures in post-traumatic model of epilepsy.

Traumatic brain injury is a major risk of post-traumatic epilepsy in a large number of individuals of different age groups. Lots of research has been done to elucidate the mechanism of post-traumatic epileptogenesis but age-related vulnerability to develop traumatic seizures is still unknown. Therefore, in the present study investigations were carried out to characterize the electrobehavioral seizure manifestation and associated alterations in young and old epileptic groups. FeCl(3) injection model was used to induce post-traumatic seizures as this model closely resembles human post-traumatic epilepsy. Synchronized video-EEG monitoring was performed to diagnose manifestation of seizures in young (4 months) and old (18 months) rats. Biochemical and ultrastructural studies were performed to determine the mechanism behind the altered age-related vulnerability for post-traumatic seizures. Our result shows that old rats were more vulnerable to post-traumatic epilepsy due to faster seizure spread and lower latency for generalization of electro-clinical seizure activity. The observed biochemical and microscopic alterations associated with old age positively correlate with the altered susceptibility to develop seizures in old epileptic groups.

An in vitro assessment of liposomal topotecan simulating metronomic chemotherapy in combination with radiation in tumor-endothelial spheroids.

Low dose metronomic chemotherapy (LDMC) refers to prolonged administration of low dose chemotherapy designed to minimize toxicity and target the tumor endothelium, causing tumor growth inhibition. Topotecan (TPT) when administered at its maximum tolerated dose (MTD) is often associated with systemic hematological toxicities. Liposomal encapsulation of TPT enhances efficacy by shielding it from systemic clearance, allowing greater uptake and extended tissue exposure in tumors. Extended release of TPT from liposomal formulations also has the potential to mimic metronomic therapies with fewer treatments. Here we investigate potential toxicities of equivalent doses of free and actively loaded liposomal TPT (LTPT) and compare them to a fractionated low dose regimen of free TPT in tumor-endothelial spheroids (TES) with/without radiation exposure for a prolonged period of 10 days. Using confocal microscopy, TPT fluorescence was monitored to determine the accumulation of drug within TES. These studies showed TES, being more reflective of the in vivo tumor microenvironment, were more sensitive to LTPT in comparison to free TPT with radiation. More importantly, the response of TES to low-dose metronomic TPT with radiation was comparable to similar treatment with LTPT. This TES study suggests nanoparticle formulations designed for extended release of drug can simulate LDMC in vivo.

Radiation-enhanced therapeutic targeting of galectin-1 enriched malignant stroma in triple negative breast cancer

Currently there are no FDA approved targeted therapies for Triple Negative Breast Cancer (TNBC). Ongoing clinical trials for TNBC have focused primarily on targeting the epithelial cancer cells. However, targeted delivery of cytotoxic payloads to the non-transformed tumor associated-endothelium can prove to be an alternate approach that is currently unexplored. The present study is supported by recent findings on elevated expression of stromal galectin-1 in clinical samples of TNBC and our ongoing findings on stromal targeting of radiation induced galectin-1 by the anginex-conjugated arsenic-cisplatin loaded liposomes using a novel murine tumor model. We demonstrate inhibition of tumor growth and metastasis in response to the multimodal nanotherapeutic strategy using a TNBC model with orthotopic tumors originating from 3D tumor tissue analogs (TTA) comprised of tumor cells, endothelial cells and fibroblasts. The ‘rigorous’ combined treatment regimen of radiation and targeted liposomes is also shown to be well tolerated. More importantly, the results presented provide a means to exploit clinically relevant radiation dose for concurrent receptor mediated enhanced delivery of chemotherapy while limiting overall toxicity. The proposed study is significant as it falls in line with developing combinatorial therapeutic approaches for stroma-directed tumor targeting using tumor models that have an appropriate representation of the TNBC microenvironment.

Abstract 5377: Assessing the chemotherapeutic response of nanoliposomal topotecan in conjunction with radiotherapy on tumor and its microenvironment

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Topotecan (TPT), a highly active, water soluble analogue of camptothecin, is currently being used in the treatment of various localized cancers from different tissue origin including the breast. Topotecan is classically known for its ability to inhibit topoisomerase I activity as well as accumulation of HIF-1α to block upregulation of pro-angiogenic factors. Despite its known anticancer and antiangiogenic properties the clinical use of TPT is limited owing to its rapid clearance and hematological toxicity. Liposomal encapsulation of TPT can potentially increase its efficacy by shielding it from systemic clearance, allowing greater uptake and extended tissue exposure in solid tumors. We hypothesize that tumor cell biology and treatment response would be more informative when done in the presence of stromal components, like endothelial cells. To that end we have developed a 3D in-vitro/ in-vivo murine model of Triple Negative Breast Cancer where 4T1-GFP tumor cells are co-cultured with their stromal component (2H11 murine endothelial cells) in ‘hanging drops’ of medium. In the present study we have determined the effective dose of radiation and TPT for 4T1 tumor and 2H11 endothelial cells by cell viability and clonogenic assays. Based on the observed toxicity treatment response of actively loaded liposomal topotecan (ALLTs) and free drug with or without radiation exposure (3Gy) in tumor-endothelial spheroids was determined by monitoring the spheroid size and quantitation of apoptotic changes observed. 100nm DSPC and m-PEG DSPE (95:5 mol: mol) nanoliposomes were prepared in ammonium besylate and then TPT was actively loaded by pH gradient method. Tumor-endothelial spheroids grown in hanging drops, were subsequently transferred to cell repellent plates for high throughput in-vitro analysis. Our studies indicate that endothelial cells are more sensitive to TPT treatment (IC50 of 0.13µM) than tumor cells (IC50=63µM). Combinatorial treatment with topotecan loaded nanoliposome (ALLT) and radiation of tumor endothelial spheroid reveals a decrease in spheroid size and severe disruption of the peripheral layers with time. ALLTs based chemotherapy in conjunction with radiation was thus observed to be more efficacious in disrupting the integrity, more specifically the extracellular matrix of tumor-endothelial spheroids. Unlike the free drug which gets hydrolyzed into its relatively inactive carboxylate form, we expect that the ability of ALLTs for extended release of active TPT (more than 24 hours) in a metronomic fashion, may contribute to the therapeutic efficacy of its combined treatment with radiation. Efforts are underway to implant these spheroids in mammary fat pad and dorsal skin fold window chamber and study the effect of ALLTs in combination with radiation on tumor growth and neovascularization and metastasis. Supported by NCI grants R25CA153954 and CA173609 Citation Format: Amar Jyoti, Pallavi Sethi, Kyle Fugit, Ulrich Langner, William St. Clair, Ronald C. McGarry, Bradley D. Anderson, Meenakshi Upreti. Assessing the chemotherapeutic response of nanoliposomal topotecan in conjunction with radiotherapy on tumor and its microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5377. doi:10.1158/1538-7445.AM2014-5377