Danish A. Nagda

A novel chitosan-hydrogel-based nanoparticle delivery system for local inner ear application.

Hypothesis A chitosan-hydrogel-based nanoparticle (nanohydrogel) delivery system can be used to deliver therapeutic biomaterials across the round window membrane (RWM) into the inner ear in a mouse model. Background Delivering therapies to the inner ear has always been a challenge for the otolaryngologist. Advances in biomedical nanotechnology, increased understanding of the RWM diffusion properties, and discovery of novel therapeutic targets and agents, have all sparked interest in the controlled local delivery of drugs and biomaterials to the inner ear using nanoparticles (NPs). Methods Fluorescently-labeled liposomal NPs were constructed and loaded into a chitosan-based hydrogel to form a nanohydrogel, and in vitro studies were performed to evaluate its properties and release kinetics. Furthermore, the nanohydrogel was applied to the RWM of mice, and perilymph and morphologic analysis were performed to assess the NP delivery and distribution within the inner ear. Results NPs with an average diameter of 160 nm were obtained. In vitro experiments showed that liposomal NPs can persist under physiologic conditions for at least two weeks without significant degradation and that the nanohydrogel can carry and release these NPs in a controlled and sustained manner. In vivo findings demonstrated that the nanohydrogel can deliver intact nanoparticles into the perilymphatic system and reach cellular structures in the scala media of the inner ear of our mouse model. Conclusion Our study suggests that the nanohydrogel system has great potential to deliver therapeutics in a controlled and sustained manner from the middle ear to the inner ear without altering inner ear structures.

Dual disruption of DNA repair and telomere maintenance for the treatment of head and neck cancer.

Purpose: Poly(ADP-ribose) polymerases (PARP) and the Mre11, Rad50, and Nbs1 (MRN) complex are key regulators of DNA repair, and have been recently shown to independently regulate telomere length. Sensitivity of cancers to PARPi is largely dependent on the BRCAness of the cells. Unfortunately, the vast majority of cancers are BRCA-proficient. In this study, therefore, we investigated whether a targeted molecular “hit” on the MRN complex, which is upstream of BRCA, can effectively sensitize BRCA-proficient head and neck squamous cell carcinoma (HNSCC) to PARP inhibitor (PARPi). Experimental Design: Human HNSCC cell lines and a mouse model with HNSCC xenografts were used in this study. In vitro and in vivo studies were conducted to evaluate the effects and underlying mechanisms of dual molecular disruption of PARP and the MRN complex, using a pharmacologic inhibitor and a dominant-negative Nbs1 expression vector, respectively. Results: Our findings demonstrate that downregulation of the MRN complex disrupts homologous recombination, and, when combined with PARPi, leads to accumulation of lethal DNA double-strand breaks. Moreover, we show that PARPi and MRN complex disruption induces significantly shortening telomere length. Together, our results demonstrate that dual disruption of these pathways causes significant cell death in BRCA-proficient tumor cells both in vitro and in vivo. Conclusion: Our study, for the first time, elucidates a novel mechanism for MRN complex and PARP inhibition beyond DNA repair, demonstrating the feasibility of a dual disruption approach that extends the utility of PARPi to the treatment of BRCA-proficient cancers. Clin Cancer Res; 20(24); 6465–78. ©2014 AACR.

Leiomyosarcoma of the head and neck: A 17-year single institution experience and review of the National Cancer Data Base

Leiomyosarcoma is a rare neoplasm of the head and neck. The purpose of this study was to present our single‐institution case series of head and neck leiomyosarcoma and a review of cases in the National Cancer Data Base (NCDB).

The Development of Nanoparticles for Targeted Head and Neck Cancer Detection with Molecular Imaging

Objectives: 1) Develop an accurate detection system for head and neck squamous cell carcinoma (HNSCC) using tumor specific nanoparticle-based probes. 2) Apply these probes for real-time non-invasive HNSCC detection using molecular imaging (MI) system. Methods. Fluorescence-labeled LDS nanoparticles targeting Hsp47 (a highly specific biomarker for HNSCC) were constructed. The HNSCC targeted properties of these constructs were evaluated in vitro using human HNSCC tumor cell lines and in a mouse model of HNSCC with a MI system. Results: Fluorescence-labeled LDS nanoparticles demonstrated HNSCC tumor cell targeting properties in vitro. Furthermore, this targeting strategy demonstrated accurate and real time tumor detection using MI system. Conclusions: Our study suggests that these novel nanoparticle-based HNSCC tumor-targeting constructs have the potential to be used clinically for non-invasive, accurate, and real time detection of tumors for patients evaluated for HNSCC.

A Novel Regulated Nanohydrogel Delivery System for Inner Ear Application

Objectives: 1) Develop a novel chitosan-hydrogel-based nanoparticle delivery system (nanohydrogel) for inner ear application and to evaluate its structures and release kinetics in vitro. 2) Evaluate if the nanohydrogel delivery system can be turned off using an enzymatic regulator for inner ear delivery. 3) Evaluate the inner ear distribution of nanoparticles following round window membrane application in a mouse model. Methods: Nanoparticles labeled with fluorescence were constructed and loaded into a chitosan-based hydrogel to form a nanohydrogel delivery system. In vitro studies were performed to evaluate the thermosensitivity, structure, and nanoparticle release kinetics of the nanohydrogel with/without chitosanase enzymatic regulation. Morphologic studies were performed to evaluate the nanoparticle delivery and distribution within the inner ear structures in a mouse model. Results: After obtaining a homogeneous, viscous and thermosensitive nanohydrogel system, in vitro studies showed that the nanohydrogel can carry and release nanoparticles in a controlled and sustained manner, and chitosanase can effectively regulate this release if needed. A matrix-like ultrastructure containing nanosized particles was confirmed. In vivo findings further confirm that the nanohydrogel delivered nanoparticles into the perilymphatic system and reached cellular structures of the inner ear in our mouse model. Conclusions: Our study suggests that the nanohydrogel system has the potential to safely deliver drugs or biomaterials in a controlled and sustained manner for inner ear application. This system could be used for targeted therapy for inner ear diseases that require safe and non-invasive delivery approaches.

Inhibition of Novel Cetuximab Resistance Pathway Leads to Improved Outcomes in Head and Neck Cancer

Objectives: 1) Determine if cetuximab resistance in head and neck squamous cell carcinoma (HNSCC) is regulated by increased nuclear translocation of Epidermal Growth Factor Receptor (EGFR) through an MRN-dependent Akt phosphorylation pathway. 2) Determine if targeted inhibition of the MRN complex, a key mediator of DNA damage response, can improve cetuximab sensitivity in HNSCC. Methods: Two well-characterized human HNSCC tumor cell lines, with differential resistance to cetuximab, were chosen for this study. Mirin, a novel molecular MRN inhibitor was used for this study. MTT and clonogenic assays were used to evaluate in vitro cytotoxicity. Western blot analysis was performed to evaluate protein expression. In vivo tumor growth was evaluated using molecular imaging. Results: As compared with sensitive cells, cetuximab resistant cells demonstrated increased MRN expression, increased Akt phosphorylation, and increased nuclear EGFR. The inhibition of MRN led to a dose-dependent decrease in Akt phosphorylation and nuclear EGFR translocation. Furthermore, MRN inhibition synergistically enhanced the cytotoxic effect of cetuximab in resistant cells (P < 0.01). Conclusions: The findings from this study suggest a novel cetuximab resistance pathway involving MRN-mediated Akt phosphorylation, leading to increased nuclear translocation of EGFR. Furthermore, inhibition of MRN led to decreased Akt-phosphorylation, subsequently decreasing nuclear EGFR translocation, a key molecular mechanism involved in cetuximab resistance. Based on this discovery, inhibition of this pathway may serve as an effective therapeutic approach for HNSCC patients resistant to cetuximab.

Abstract 2263: Biliverdin inhibits head and neck cancer cell growth via activation of retinoblastoma signaling pathway.

Head and neck cancer patients often present with advanced disease and, despite advances in chemoradiation treatment protocols, continue to have a poor prognosis while experiencing severe treatment induced toxicity. There is an increased demand for natural, and non-toxic agents for treatment, which can be either used alone or in combination with our therapies. In this study, we are using biliverdin (BV), an antioxidant bile pigment, which is oxidized from bilirubin. As a potent antioxidant, accumulating data from observations in experimental and human studies indicate that the BV may be protective against certain diseases, including cancer. Based on our own observations that BV suppresses cell proliferation in head and neck squamous cell carcinoma (HNSCC) cells and clinical observations finding a lesser incidence of cancer in healthy individuals with slightly elevated serum bilirubin levels, we hypothesized that BV might suppress tumor cell proliferation. The present study looks to evaluate whether BV treatment can suppress cell proliferation in HNSCC cells in vitro and in vivo, and to elucidate the potential mechanism through which it exerts this effect. HNSCC cell lines, with varying resistance to chemoradiation, and a mouse model with human HNSCC xenografts were used in this study. As potential effectors, we analyzed key proteins that are involved in apoptosis and cell cycle progression. In vitro, we investigated the effect of BV on five HNSCC cell lines and the signaling pathways involved in BV action on tumor cell proliferation using western blots. We also analyzed the antiproliferative effects of the BV by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and cell cycle analysis where BV caused cell cycle arrest by hypophosphorylation (activating) of the retinoblastoma (Rb) tumor suppressor protein in HNSCC cells. Ex vivo, we found significant suppression of xenograft tumor growth in HNSCC cells pretreated with BV before intradermal injection in nude mice as compared to controls. In vivo, tumor growth was assessed in BALB/c nude mice bearing HNSCC xenografts that were treated with BV. The signaling pathway responsible for this action included dephosphorylation of epidermal growth factor receptor (EGFR), Akt, NF-κB, and hypophosphorylation of the Rb tumor suppressor protein as well as caspase activation. Our study provides a rationale for a novel therapeutic approach using exogenous BV, a nontoxic antioxidant product of heme catabolism, as an anti-cancer agent against HNSCC through its effect on the Rb/Akt/NF-κB signal transduction pathway. Our findings indicate that BV9s actions on these key tumorogenic pathways may extend its therapeutic potential to head and neck cancer. Citation Format: Jun Zheng, Danish A. Nagda, Shayanne A. Lajud, Sanjeev Kumar, Anas Mouchli, Orysia Bezpalko, Bert W. O9Malley, Daqing Li. Biliverdin inhibits head and neck cancer cell growth via activation of retinoblastoma signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2263. doi:10.1158/1538-7445.AM2013-2263