Jacob Eide

Co-delivery of doxorubicin and siRNA using octreotide-conjugated gold nanorods for targeted neuroendocrine cancer therapy

A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The Au NR was conjugated with (1) DOX, an anticancer drug, via a pH-labile hydrazone linkage to enable pH-controlled drug release, (2) polyarginine, a cationic polymer for complexing siRNA, and (3) octreotide (OCT), a tumor-targeting ligand, to specifically target NE cancer cells with overexpressed somatostatin receptors. The Au NR-based nanocarriers exhibited a uniform size distribution as well as pH-sensitive drug release. The OCT-conjugated Au NR-based nanocarriers (Au-DOX-OCT, targeted) exhibited a much higher cellular uptake in a human carcinoid cell line (BON cells) than non-targeted Au NR-based nanocarriers (Au-DOX) as measured by both flow cytometry and confocal laser scanning microscopy (CLSM). Moreover, Au-DOX-OCT-ASCL1 siRNA (Au-DOX-OCT complexed with ASCL1 siRNA) resulted in significantly higher gene silencing in NE cancer cells than Au-DOX-ASCL1 siRNA (non-targeted Au-DOX complexed with ASCL1 siRNA) as measured by an immunoblot analysis. Additionally, Au-DOX-OCT-ASCL1 siRNA was the most efficient nanocarrier at altering the NE phenotype of NE cancer cells and showed the strongest anti-proliferative effect. Thus, combined chemotherapy and RNA silencing using NE tumor-targeting Au NR-based nanocarriers could potentially enhance the therapeutic outcomes in treating NE cancers.

Tumor-Suppressor Role of Notch3 in Medullary Thyroid Carcinoma Revealed by Genetic and Pharmacological Induction

Notch1-3 are transmembrane receptors that appear to be absent in medullary thyroid cancer (MTC). Previous research has shown that induction of Notch1 has a tumor-suppressor effect in MTC cell lines, but little is known about the biologic consequences of Notch3 activation for the progression of the disease. We elucidate the role of Notch3 in MTC by genetic (doxycycline-inducible Notch3 intracellular domain) and pharmacologic [AB3, novel histone deacetylase (HDAC) inhibitor] approaches. We find that overexpression of Notch3 leads to the dose-dependent reduction of neuroendocrine tumor markers. In addition, Notch3 activity is required to suppress MTC cell proliferation, and the extent of growth repression depends on the amount of Notch3 protein expressed. Moreover, activation of Notch3 induces apoptosis. The translational significance of this finding is highlighted by our observation that MTC tumors lack active Notch3 protein and reinstitution of this isoform could be a therapeutic strategy to treat patients with MTC. We demonstrate, for the first time, that overexpression of Notch3 in MTC cells can alter malignant neuroendocrine phenotype in both in vitro and in vivo models. In addition, our study provides a strong rationale for using Notch3 as a therapeutic target to provide novel pharmacologic treatment options for MTC. Mol Cancer Ther; 14(2); 499–512. ©2014 AACR.

Thiocoraline alters neuroendocrine phenotype and activates the Notch pathway in MTC-TT cell line

Medullary thyroid cancer (MTC) is an aggressive neuroendocrine tumor (NET). Previous research has shown that activation of Notch signaling has a tumor suppressor role in NETs. The potential therapeutic effect of thiocoraline on the activation of the Notch pathway in an MTC cell line (TT) was investigated. Thiocoraline was isolated from a marine bacterium Verrucosispora sp. MTT assay (3‐[4, 5‐dimethylthiazole‐2‐yl]‐2, 5‐diphenyltetrazolium bromide) was used to determine the IC50 value and to measure cell proliferation. Western blot revealed the expression of Notch isoforms, NET, and cell cycle markers. Cell cycle progression was validated by flow cytometry. The mRNA expression of Notch isoforms and downstream targets were measured using real‐time PCR. The IC50 value for thiocoraline treatment in TT cells was determined to be 7.6 nmol/L. Thiocoraline treatment decreased cell proliferation in a dose‐ and time‐dependent manner. The mechanism of growth inhibition was found to be cell cycle arrest in G1 phase. Thiocoraline activated the Notch pathway as demonstrated by the dose‐dependent increase in mRNA and protein expression of Notch isoforms. Furthermore, treatment with thiocoraline resulted in changes in the expression of downstream targets of the Notch pathway (HES1, HES2, HES6, HEY1, and HEY2) and reduced expression of NET markers, CgA, and ASCL1. Thiocoraline is a potent Notch pathway activator and an inhibitor of MTC‐TT cell proliferation at low nanomolar concentrations. These results provide exciting evidence for the use of thiocoraline as a potential treatment for intractable MTC.

Chrysin suppresses achaete-scute complex-like 1 and alters the neuroendocrine phenotype of carcinoids.

Carcinoids are neuroendocrine neoplasms that cause significant morbidity and mortality and for which few effective therapies are available. Given the recent identification of the anticancer flavonoid chrysin, we sought to investigate its therapeutic potential in carcinoids. Here we report chrysin’s ability to modulate the achaete-scute complex-like 1 (ASCL1), a neuroendocrine-specific transcription factor highly implicated in the malignant phenotype of carcinoids and other neuroendocrine cancers. Moreover, we elucidate the role of ASCL1 in carcinoid growth and bioactivity. Treatment of two carcinoid cell lines (BON and H727) with varying chrysin concentrations suppressed cell proliferation, while reducing expression of ASCL1 and the neuroendocrine biomarker chromogranin A (CgA), demonstrated by western blotting. Propidium iodide and phycoerythrin AnnexinV/7-aminoactinomycin D staining and sorting following chrysin treatment revealed S/G2 phase arrest and apoptosis, respectively. This was corroborated by chrysin-induced cleavage of caspase-3 and poly ADP-ribose polymerase and activation of p21Waf1/Cip1. Furthermore, direct ASCL1 knockdown with an ASCL1-specific small interfering RNA inhibited CgA and synaptophysin expression as well as carcinoid proliferation, while also reducing cyclin B1 and D1 and increasing p21Waf1/Cip1 and p27Kip1 expression, suggesting an arrest of the cell cycle. Collectively, these findings warrant the deliberation of targeted ASCL1 suppression by chrysin or other agents as a therapeutic approach for carcinoid management.

PPARγ targeted oral cancer treatment and additional utility of genomics analytic techniques

Peroxisome proliferator‐activated receptor γ (PPARγ) agonists have been shown to have anti‐proliferative, anti‐angiogenic, and proapoptotic effects, leading to interest in their use as cancer therapeutics. Pioglitazone, a U.S. Food and Drug Administration‐approved type II diabetes medication and PPARγ agonist, may have a role in adjuvant head‐and‐neck squamous cell carcinoma treatment or prevention. Therefore, the purpose of this study was: 1) to treat oral cavity cancer cells with the PPARγ activator, pioglitazone, to analyze gene expression changes; and 2) to compare those changes with our preexisting genomic data for development of hypothesis‐driven additional basic and clinical studies.

Abstract 1953: Co-delivery of doxorubicin and siRNA using octreotide-conjugated gold nanorods for targeted neuroendocrine cancer therapy

Introduction: Gastrointestinal (GI) carcinoids are neuroendocrine (NE) tumors that secrete hormones causing carcinoid syndrome. Metastatic carcinoids are not amenable to curative surgery. Therefore, siRNA-mediated gene silencing, in combination with targeted chemotherapy, may be a potential therapeutic strategy for patients with carcinoid neoplasms. We have developed and assessed antitumor effects of a gold nanorod (Au NR)-based nanocarrier conjugated with an anticancer drug (doxorubicin (DOX)) and small interfering RNA (siRNA against carcinoid tumor marker ASCL1, a protein required for NE cancer cell proliferation) specifically targeting the NE cancer cells with overexpressed somatostatin receptors (SSTRs) using octreotide (Oct) as an active - tumor targeting ligand. Methods: Au NR-based nanoparticles (NPs) were synthesized via a multiple step reaction. DOX was conjugated onto the Au NR via hydrazone bonds. siRNA was complexed onto the cationic poly(L-arginine) polymer. Oct targeting ligand was selectively conjugated onto the distal end of PEG arms. A human GI carcinoid cell line (BON) was treated with either non-targeted (Au-DOX) or targeted (Au-DOX-Oct) NPs with or without siRNA, at a concentration of 1 μg/ml for 30′ and 120′ or 24, 48, 72 hours. Based on the doxorubicin fluorescent intensity, the cellular uptake of NPs was analyzed by flow cytometry and confocal laser scanning microscope (CLSM). MTT assay and Western blot were used to determine cell proliferation and assess ASCL1 expression, respectively. Results: DOX release profiles from NPs were dependent on the environmental pH values, which can minimize systemic spread of toxic drugs during circulation. BON cells incubated with Au-DOX-Oct exhibited higher cellular uptake than Au-DOX (14% and 44% increase after 30 min and 120 min, respectively) measured by flow cytometry. The CLSM observations confirmed that the intracellular transport of Au-DOX-Oct is faster than Au-DOX. MTT assay showed that Au-DOX-Oct-ASCL1siRNA has the strongest inhibitory effect on carcinoid cell proliferation (more than 50% of growth reduction). Immunoblot analysis demonstrated that Au-DOX-Oct-ASCL1siRNA reduced the expression of ASCL1 much more than other NPs. Conclusions: We have developed a multifunctional, pH-responsive and tumor-targeting Au NR-based nanoplatform for anticancer drug therapy. We showed that the presence of the surface ligand Oct increased the cellular uptake of the NPs (Au-DOX-Oct) substantially in a BON cell line compared to the non-targeted NPs (Au-DOX). Targeted NPs with ASCL1siRNA were the most efficient in altering the neuroendocrine phenotype of BON cells demonstrating the strongest anti-proliferative effect. Thus, targeted delivery of anticancer drug (DOX) and genetic material (ASCL1siRNA) specifically to the tumor tissue could achieve therapeutic outcomes in treating carcinoid disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1953. doi:1538-7445.AM2012-1953