Ajitha Dammalapati

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.

Neuroendocrine Tumor-Targeted Upconversion Nanoparticle-Based Micelles for Simultaneous NIR-Controlled Combination Chemotherapy and Photodynamic Therapy, and Fluorescence Imaging.

Although neuroendocrine tumors (NETs) are slow growing, they are frequently metastatic at the time of discovery and no longer amenable to curative surgery, emphasizing the need for the development of other treatments. In this study, multifunctional upconversion nanoparticle (UCNP)-based theranostic micelles are developed for NET-targeted and near-infrared (NIR)-controlled combination chemotherapy and photodynamic therapy (PDT), and bioimaging. The theranostic micelle is formed by individual UCNP functionalized with light-sensitive amphiphilic block copolymers poly(4,5-dimethoxy-2-nitrobenzyl methacrylate)-polyethylene glycol (PNBMA-PEG) and Rose Bengal (RB) photosensitizers. A hydrophobic anticancer drug, AB3, is loaded into the micelles. The NIR-activated UCNPs emit multiple luminescence bands, including UV, 540 nm, and 650 nm. The UV peaks overlap with the absorption peak of photocleavable hydrophobic PNBMA segments, triggering a rapid drug release due to the NIR-induced hydrophobic-to-hydrophilic transition of the micelle core and thus enabling NIR-controlled chemotherapy. RB molecules are activated via luminescence resonance energy transfer to generate 1O2 for NIR-induced PDT. Meanwhile, the 650 nm emission allows for efficient fluorescence imaging. KE108, a true pansomatostatin nonapeptide, as an NET-targeting ligand, drastically increases the tumoral uptake of the micelles. Intravenously injected AB3-loaded UCNP-based micelles conjugated with RB and KE108-enabling NET-targeted combination chemotherapy and PDT-induce the best antitumor efficacy.

Thailandepsin A-loaded and octreotide-functionalized unimolecular micelles for targeted neuroendocrine cancer therapy

Due to the overexpression of somatostatin receptors in neuroendocrine (NE) cancers, drug nanocarriers conjugated with somatostatin analogs, such as octreotide (OCT), for targeted NE cancer therapy may offer increased therapeutic efficacies and decreased adverse effects. In this study, OCT-functionalized unimolecular micelles were prepared using individual hyperbranched polymer molecules consisting of a hyperbranched polymer core (Boltorn(®) H40) and approximately 25 amphiphilic polylactide-poly(ethlyene glycol) (PLA-PEG) block copolymer arms (H40-PLA-PEG-OCH3/OCT). The resulting micelles, exhibiting a uniform core-shell shape and an average hydrodynamic diameter size of 66 nm, were loaded with thailandepsin-A (TDP-A), a relatively new naturally produced histone deacetylase (HDAC) inhibitor. In vitro studies using flow cytometry and confocal laser scanning microscopy (CLSM) demonstrated that OCT conjugation enhanced the cellular uptake of the unimolecular micelles. Consequently, TDP-A-loaded and OCT-conjugated micelles exhibited the highest cytotoxicity and caused the highest reduction of NE tumor markers. Finally, the in vivo studies on NE cancer bearing nude mice demonstrated that TDP-A-loaded and OCT-conjugated micelles possessed superior anticancer activity in comparison with other TDP-A formulations or drug alone, while showing no detectable systemic toxicity. Thus, these TDP-A-loaded and OCT-conjugated micelles offer a promising approach for targeted NE cancer therapy.

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 activates the Notch pathway in carcinoids and reduces tumor progression in vivo.

Carcinoids are slow-growing neuroendocrine tumors (NETs) that are characterized by hormone overproduction; surgery is currently the only option for treatment. Activation of the Notch pathway has previously been shown to have a role in tumor suppression in NETs. The marine-derived thiodepsipeptide thiocoraline was investigated in vitro in two carcinoid cell lines (BON and H727). Carcinoid cells treated with nanomolar concentrations of thiocoraline resulted in a decrease in cell proliferation and an alteration of malignant phenotype evidenced by decrease of NET markers, achaete-scute complex like-1, chromogranin A and neurospecific enolase. Western blotting analysis demonstrated the activation of Notch1 on the protein level in BON cells. Additionally, thiocoraline activated downstream Notch targets HES1, HES5 and HEY2. Thiocoraline effectively suppressed carcinoid cell growth by promoting cell cycle arrest in BON and H727 cells. An in vivo study demonstrated that thiocoraline, formulated with polymeric micelles, slowed carcinoid tumor progression. Thus the therapeutic potential of thiocoraline, which induced activation of the Notch pathway, in carcinoid tumors was demonstrated.

AB3-loaded and tumor-targeted unimolecular micelles for medullary thyroid cancer treatment

Medullary thyroid cancer (MTC) is often resistant to standard therapies, emphasizing the need for the development of other treatments. A new histone deacetylase inhibitor, AB3, can effectively inhibit MTC cell proliferation in vitro. However, its poor aqueous solubility and stability, fast clearance, and lack of tumor targeting ability limit its in vivo application. Therefore, multifunctional unimolecular micelles were developed for targeted delivery of AB3 for MTC therapy. The unimolecular micelles exhibited a spherical core-shell structure, uniform size distribution, and excellent stability. AB3 was encapsulated into the hydrophobic core of the unimolecular micelles, thus significantly enhancing its aqueous solubility and stability. KE108, a somatostatin analog possessing high affinity to all five subtypes of SSTR, was used as an MTC-targeting ligand. In vitro cellular uptake analyses demonstrated that the KE108 exhibited superior targeting ability in MTC cells compared to octreotide, the first clinically used somatostatin analog. Moreover, the AB3-loaded and KE108-conjugated unimolecular micelles exhibited the best efficacy in suppressing MTC cell growth and tumor marker expression in vitro. Furthermore, AB3-loaded, KE108-conjugated micelles demonstrated the best anticancer efficacy in vivo without any apparent systemic toxicity, thereby offering a promising approach for targeted MTC therapy.

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.

Histone deacetylase inhibitor thailandepsin-A activates Notch signaling and suppresses neuroendocrine cancer cell growth in vivo

Novel therapies for neuroendocrine (NE) cancers are desperately needed as they frequently present as metastatic disease and cause debilitating symptoms by secreting excessive hormones. Induction of Notch isoforms has a tumor suppressive effect in NE cancer cell lines, and we have observed that histone deacetylase inhibitors (HDACi) potently activate Notch. In this study, we describe the potential for Burkholderia thailandensis-derived class I HDACi thailandepsin A (TDP-A) as a Notch activator and therapeutic agent against NE cancer. IC50 for TDP-A was determined to be 4-6 nM in NE cancer cell lines (BON, MZ-CRC-1, and TT) without cytotoxicity to lung fibroblasts. The binding characteristics of TDP-A to its target HDAC1 was examined using bioluminescence resonance energy transfer (BRET). Western blot and flow cytometry analysis showed that TDP-A induces cell cycle arrest and apoptosis in a dose-dependent manner. TDP-A dose-dependently activated the Notch pathway as measured by increasing functional CBF1-luciferase reporter signal and mRNA and protein expressions of Notch isoforms, which were attenuated by pretreatment with γ-secretase inhibitor DAPT. Furthermore, TDP-A lead to changes in expression level of downstream targets of Notch pathway and reduced expression of NE cancer markers. An in vivo study demonstrated that TDP-A suppressed NE cancer progression. These results show that TDP-A, as a Notch activator, is a promising agent against NE cancers.

Abstract 1014: Thailandepsin A, a new HDAC inhibitor, reduces cellular proliferation and activates the Notch pathway in human carcinoids cancer cells.

Introduction: Carcinoids are neuroendocrine (NE) tumors that secrete hormones causing the carcinoid syndrome. Metastatic carcinoids are not amenable to curative surgery. Our previous research has shown that Notch signaling has a tumor suppressor role in NE tumors. In the present study, we sought to examine Thailandepsin A (TDP-A), a newly discovered HDAC inhibitor as a Notch activating drug and carcinoid cancer agent. Methods: TDP-A is a bacterial natural product isolated from the fermentation broth of Burkholderia thailandensis E264. The cytotoxicity of TDP-A on human pancreatic (BON) carcinoid cells was measured by determining the IC50 value.The antiproliferative effect of TDP-A on BON cells growth was assessed by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay.The mechanism of growth inhibition was determined for cell cycle and apoptosis markers by Western blot and flow cytometry analyses. Expression of Notch isoforms 1-3 was assessed at the transcriptional level (real time RT-PCR) and protein level (Western blot) from parental and TDP-A treated BON cells. Functional analysis of Notch Intracellular Domain (NICD) was done by measuring the degree of luciferase activity by CBF1 binding assay. The gene expression of HES and HEY (Notch signaling mediators) was quantified by real time RT-PCR. Effect of TDP-A on NE markers -chromogranin A (CgA) and achaete-scute complex-like1 (ASCL1) - was assessed by Western blot analysis. Results: The IC50 value for TDP-A treatment of BON cells was determined to be 7 nM. TDP-A treatment decreased cell proliferation in a dose and time dependent manner. Western blot analysis and flow cytometry experiments indicated that the growth inhibition was due to cell cycle arrest (at G2/M phase) followed by apoptosis.Treatment of BON cells with TDP-A led to an induction of Notch isoforms in a dose-dependent manner. Functional Notch signaling was demonstrated by an increase in the CBF1 binding activity and upregualtion of transcriptional levels of HES and HEY families. More importantly, Notch activation led to a dose dependent reduction of NE markers ASCL1 and CgA. Conclusions: We demonstrated that TDP-A is a potent Notch pathway activator and an inhibitor of BON carcinoid cell proliferation at low nanomolar concentrations. TDP-A suppressed carcinoid cell growth by promoting cell cycle arrest and apoptosis and decreased NET marker expression (ASCL1 and CgA). These results indicate that TDP-A has therapeutic potential for carcinoid cancer. Citation Format: Renata Jaskula-Sztul, Ajitha Dammalapati, Colin Korlesky, Shaoqin Gong, Yi-Qiang Cheng, Herbert Chen. Thailandepsin A, a new HDAC inhibitor, reduces cellular proliferation and activates the Notch pathway in human carcinoids cancer cells. [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 1014. doi:10.1158/1538-7445.AM2013-1014

Dysadherin specific drug conjugates for the treatment of thyroid cancers with aggressive phenotypes

Background EDC1 is a novel type of antibody-drug conjugate which binds and inhibits the Na,K-ATPase on the surface of cancer cells expressing dysadherin. The purpose of this study was to determine the expression of dysadherin in different types of thyroid carcinoma, and evaluate the therapeutic potential of EDC1 for thyroid carcinomas. Methods Thyroid tissues from 158 patients were examined for dysadherin expression and correlation with clinicopathological features. Thyroid cancer cell lines were examined for the expression of dysadherin and effective dose range of EDC1. RESULTS One in 53 benign thyroid tissues and 62% of thyroid cancers expressed dysadherin. All anaplastic and a majority of papillary thyroid cancers overexpressed dysadherin, while 25% of follicular thyroid cancers was found to be positive for dysadherin. Dysadherin expression significantly correlated with extrathyroidal extension and lymph node metastases in papillary thyroid cancer. Five of six human thyroid cancer cell lines analyzed expressed high levels of dysadherin. Of those cells lines sensitive to EDC1, half maximal effective concentrations (EC50) were observed to be between 0.125 nM and 1 nM. Conclusions EDC1 showed selective inhibition of growth in thyroid cancer cells with moderate to high expression of dysadherin, thus could be a specific and effective treatment.