Renata Jaskula-Sztul

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.

Bone marrow cytotoxicity of benzo[a]pyrene is dependent on CYP1B1 but is diminished by Ah receptor-mediated induction of CYP1A1 in liver

We have previously used CYP1B1-null mice to demonstrate that dimethylbenz(a)anthracene (DMBA) requires CYP1B1 for bone marrow (BM) toxicity. Benzo(a)pyrene (BP), a much more potent Ah receptor ligand, shows very different responses that nevertheless depend on CYP1B1. Wild-type (AhR(b)) mice treated with DMBA for 48 h exhibit a large loss in BM cellularity and disruption of marrow structure that is not seen for BP treatment. In congenic mice with a low affinity AhR (AhR(d)), DMBA and BP are equally toxic to the BM whereas AhR(d) x CYP1B1-null mice are fully protected. In situ hybridization demonstrates that CYP1B1 mRNA is constitutively expressed in marrow cells and is induced by PAHs according to their AhR affinity (BP>DMBA), including lower levels in AhR(d) mice. Importantly, expression of CYP1A1 mRNA was undetectable in BM. In wild-type mice, BP treatment leads to a fivefold greater induction of hepatic CYP1A1 than that of DMBA treatment. Neither induction occurs in AhR(d) mice. Thus, hepatic metabolism may prevent BP from reaching the BM, where it can be bioactivated by CYP1B1. Flow cytometric analyses of BM cells showed that there were decreases in granulocytes and lymphocytes following DMBA treatment, but not after BP treatment. These data suggest that there is an inverse relationship between liver metabolism and BM toxicity resulting from limitations on the delivery of PAH to CYP1B1 present in BM, where only very low constitutive levels are needed.

Chrysin activates Notch1 signaling and suppresses tumor growth of anaplastic thyroid carcinoma in vitro and in vivo

Anaplastic thyroid cancer (ATC) is a very aggressive thyroid gland malignancy with very poor prognosis. It is suspected that the Notch signaling pathway, which is not active in ATC, may have a tumor suppressor function in this neoplasm. However, it remains unknown whether activation of Notch can yield therapeutic efficacies in ATC.

Resveratrol Induces Differentiation Markers Expression in Anaplastic Thyroid Carcinoma via Activation of Notch1 Signaling and Suppresses Cell Growth

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive malignancy with undifferentiated features, for which conventional treatments, including radioactive iodine ablation, are usually not effective. Recent evidence suggests that the Notch1 pathway is important in the regulation of thyroid cancer cell growth and expression of thyrocyte differentiation markers. However, drug development targeting Notch1 signaling in ATC remains largely underexplored. Previously, we have identified resveratrol out of over 7,000 compounds as the most potent Notch pathway activator using a high-throughput screening method. In this study, we showed that resveratrol treatment (10–50 μmol/L) suppressed ATC cell growth in a dose-dependent manner for both HTh7 and 8505C cell lines via S-phase cell-cycle arrest and apoptosis. Resveratrol induced functional Notch1 protein expression and activated the pathway by transcriptional regulation. In addition, the expression of thyroid-specific genes including TTF1, TTF2, Pax8, and sodium iodide symporter (NIS) was upregulated in both ATC cell lines with resveratrol treatment. Notch1 siRNA interference totally abrogated the induction of TTF1 and Pax8 but not of TTF2. Moreover, Notch1 silencing by siRNA decreased resveratrol-induced NIS expression. In summary, our data indicate that resveratrol inhibits cell growth and enhances redifferentiation in ATC cells dependent upon the activation of Notch1 signaling. These findings provide the first documentation for the role of resveratrol in ATC redifferentiation, suggesting that activation of Notch1 signaling could be a potential therapeutic strategy for patients with ATC and thus warrants further clinical investigation. Mol Cancer Ther; 12(7); 1276–87. ©2013 AACR.

Xanthohumol decreases Notch1 expression and cell growth by cell cycle arrest and induction of apoptosis in epithelial ovarian cancer cell lines

OBJECTIVE Notch1 signaling is active in ovarian cancer and is a promising pathway for new therapies in ovarian cancer. We have previously detected high Notch1 expression in ovarian tumors. Xanthohumol has been shown to inhibit cancer cell growth and invasion, including Kaposis sarcoma, which also highly expresses Notch1. We hypothesized that the Notch1 signaling pathway is targeted by xanthohumol leading to decreased ovarian cancer cell growth. METHODS SKOV3 and OVCAR3 cells were utilized. MTT growth assays were conducted following treatment with xanthohumol. Quantitative RT-PCR and Western blot analyses were conducted to assess Notch1 down-regulation. Luciferase reporter assays were performed to assess functional down-regulation of Notch1. Cell cycle analysis was performed by flow cytometry. RESULTS Significant growth inhibition and down-regulation of Notch1 transcription and protein expression were found following xanthohumol treatment. In addition, xanthohumol increased Hes6 transcription and decreased Hes1 transcription, known downstream targets of Notch 1. These observations were associated with cell cycle inhibition as demonstrated by an increase in p21 expression and S and G2/M cell cycle arrest confirmed by an increase in phosphorylated cdc2. Furthermore, an increase in the apoptotic markers, cleaved caspase-3 and cleaved PARP were observed. CONCLUSION Xanthohumol was a potent inhibitor of ovarian cancer cell growth, and our results suggest that xanthohumol may be influencing the Notch1 pathway. These findings suggest that xanthohumol could be useful as a therapeutic agent in ovarian cancer.

A Pilot Phase II Study of Valproic Acid for Treatment of Low-Grade Neuroendocrine Carcinoma

INTRODUCTION Notch1 has been shown to be a tumor suppressor in neuroendocrine tumors (NETs). Previous in vitro studies in NET cell lines have also suggested that valproic acid (VPA), a histone deacetylase inhibitor, can induce Notch1 and that Notch1 activation correlates with a decrease in tumor markers for NETs. Thus, this study aimed to evaluate the role of VPA in treating NETs and to determine whether VPA induced the Notch signaling pathway signaling in vivo. PATIENTS AND METHODS Eight patients with low-grade NETs (carcinoid and pancreatic) were treated with 500 mg of oral VPA twice a day with dosing adjusted to maintain a goal VPA level between 50 and 100 μg/mL. All patients were followed for 12 months or until disease progression. RESULTS Notch1 signaling was absent in all tumors prior to treatment and was upregulated with VPA. One patient had an unconfirmed partial response and was noted to have a 40-fold increase in Notch1 mRNA levels. Four patients had stable disease as best response. Tumor markers improved in 5 out of 7 patients. Overall, treatment with VPA was well tolerated. CONCLUSION . VPA activates Notch1 signaling in vivo and may have a role in treating low-grade NETs.

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.

Identification and validation of Notch pathway activating compounds through a novel high-throughput screening method

Carcinoids are neuroendocrine (NE) tumors with limited treatment options. Notch activation has been shown to suppress growth and hormone production in carcinoid cells.

Suberoyl bishydroxamic acid inhibits cellular proliferation by inducing cell cycle arrest in carcinoid cancer cells.

Carcinoid cancers arise from the neuroendocrine cell system of the gastrointestinal tract, lungs, and other organs. Hepatic metastases are common, and patients often suffer from endocrinopathies secondary to tumor secretion of various hormones and peptides. As complete surgical resection is often not possible because of widespread disease, new therapeutic and palliative treatments are needed. In this study, we characterized the effects of suberoyl bishydroxamic acid (SBHA), a histone deacetylase inhibitor, on the growth and neuroendocrine phenotype of carcinoid cancer cells. SBHA treatment of human gastrointestinal and pulmonary carcinoid cancer cells resulted in a dose-dependent inhibition of cell proliferation. Western blot analysis showed a decrease in cyclin D1 and an increase in p21 and p27, indicating that the mechanism of this growth inhibition is cell cycle arrest. Furthermore, SBHA treatment suppressed two neuroendocrine tumor markers, chromogranin A and achaete-scute complex-like 1. These changes in the growth and neuroendocrine phenotype of carcinoid cells were associated with activation of the Notch1 signaling cascade. We conclude that SBHA shows promise as a potential anticancer agent for the treatment of patients with advanced carcinoid tumor disease.

Octreotide-functionalized and resveratrol-loaded unimolecular micelles for targeted neuroendocrine cancer therapy.

Medullary thyroid cancer (MTC) is a neuroendocrine tumor (NET) that is often resistant to standard therapies. Resveratrol suppresses MTC growth in vitro, but it has low bioavailability in vivo due to its poor water solubility and rapid metabolic breakdown, as well as lack of tumor-targeting ability. A novel unimolecular micelle based on a hyperbranched amphiphilic block copolymer was designed, synthesized, and characterized for NET-targeted delivery. The hyperbranched amphiphilic block copolymer consisted of a dendritic Boltorn® H40 core, a hydrophobic poly(l-lactide) (PLA) inner shell, and a hydrophilic poly(ethylene glycol) (PEG) outer shell. Octreotide (OCT), a peptide that shows strong binding affinity to somatostatin receptors, which are overexpressed on NET cells, was used as the targeting ligand. Resveratrol was physically encapsulated by the micelle with a drug loading content of 12.1%. The unimolecular micelles exhibited a uniform size distribution and spherical morphology, which were determined by both transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cellular uptake, cellular proliferation, and Western blot analyses demonstrated that the resveratrol-loaded OCT-targeted micelles suppressed growth more effectively than non-targeted micelles. Moreover, resveratrol-loaded NET-targeted micelles affected MTC cells similarly to free resveratrol in vitro, with equal growth suppression and reduction in NET marker production. These results suggest that the H40-based unimolecular micelle may offer a promising approach for targeted NET therapy.