HIF-1alpha Activation in 3D Tumor Spheroids and Evaluation of 2-Methoxyestradiol Therapy using Targeted Nanoparticle Formulations

Despite significant advances in new drug discoveries and treatment combinations, the mortality rate due to cancer has not changed significantly over the last fifty years in the United States and in many parts of the world. The inability to detect cancer in the early stage, especially for ovarian cancer, accounts for majority of lethality, which is then associated with poor prognosis as a result of dissemination to other organs. Additionally, the insufficient accumulation of chemotherapeutic agents into the tumor site upon systemic administration and micro-environmental selection pressures lead to development of multidrug resistance.;Irregular blood supply in the growing tumor mass in vivo leads to formation of regions that are deprived of oxygen and nutrients. The cells in this region over a period of time adapt to the aggressive micro-environment, and thereby, contribute to the development of resistance to anti-cancer therapy. HIF-1 alpha is considered to be major regulator of hypoxia-mediated drug resistance in various types of cancers. Thus, it has become a promising target for preventing the progression of this disease. Various HIF-1 alpha inhibitors have been tried and tested for treating cancer and 2-methoxyestradiol (2ME) is one of the potent small molecule inhibitors.;The main objective of this study was to evaluate hypoxia-induced tumor drug resistance using 3D-spheroid model of SKOV-3 human ovarian adenocarcinoma cells and development of targeted polymeric nanoparticle 2ME therapy for inhibition of hypoxia inducing factor-1-alpha. Preliminary studies were carried out to develop poly(ethylene glycol)-modified poly(D,L-lactide-co-glycolide) (PLGA)/poly(epsilon-caprolactone) (PCL) blend nanoparticle formulations for encapsulation and delivery of 2ME in SKOV3 tumor spheroid model. The SKOV3 tumor spheroids were developed using 384-well plate hanging drop method and the level of HIF-1 alpha expression in 3 and 5 day old spheroids was compared to 2D cell culture grown under normoxic and hypoxic conditions. Reverse Transcriptase-PCR and ELISA were used to evaluate baseline HIF-1 alpha mRNA and protein levels respectively in these systems. The permeability of nanoparticle-delivered therapeutics in tumor spheroids was evaluated by scanning confocal microscopy using rhodamine-123 fluorescence dye-encapsualted PEG-PLGA/PCL blend nanoparticles. The nanoparticle based delivery of 2ME was compared to 2ME administered in solution for their ability to inhibit HIF-1 alpha.