Head and neck cancer is the sixth leading cancer worldwide. Head and neck squamous cell carcinoma (HNSCC) accounts for more than 90% of incident cases. Despite intense, multimodality treatment regimens for HNSCC including surgery, chemotherapy, and radiation, little progress has been made over the past 30 years in improving overall survival rates. Tumor cell death induced by both conventional and targeted chemotherapy is often mediated by the BCL-2 family-dependent mitochondrial apoptotic pathway. However, initiators of this apoptotic pathway, such as p53, are more than 50% of the time mutated or deleted in HNSCC rendering the disease refractory to treatment. To counter such resistance, direct therapeutic targeting of the BCL-2 family is conceptually appealing. For this purpose, we use three clinically-available drugs: cisplatin, fenretinide, and ABT-263 (navitoclax). Both cisplatin and fenretinide are known to induce a BH3-only pro-apoptotic protein, Noxa, which binds to and inactivates multi-domain anti-apoptotic protein MCL-1 and release from its interaction with multi-domain pro-apoptotic protein BAK, followed by the phosphorylation via CDK2 for the proteasome-mediated degradation. Activated BAK can now go through conformational change for the oligomerization at the outer membrane of the mitochondria to release cytochrome c into the cytosol and induce caspase-dependent apoptotic cell death. ABT-263 directly binds to multi-domain anti-apoptotic proteins, such as BCL-2 and BCL-XL, to inhibit their activity and leads to the activation of multi-domain pro-apoptotic protein BAX to induce apoptosis. We hypothesize that combining the Noxa-inducing drugs (cisplatin or fenretinide) along with ABT-263 can efficiently induce BAX and BAK activation and significantly increase cell death in HNSCC cells by simultaneously inhibiting the activity of MCL-1, BCL-2, and BCL-XL. Combination-induced treatments in four cell lines (HN8, HN30, HN31, and UMSCC1) tested led to significant increase in apoptotic cell death. Cisplatin and ABT-263 combined treatment is inducing the expression of Noxa and leading to increase in apoptosis in HN30, HN31, UMSCC1, but not HN8. Similarly, fenretinide and ABT-263 combined treatment is inducing the expression of Noxa in all four cell lines tested and is largely relying on expression of Noxa.

Head and neck cancer accounts for approximately 3 percent of all cancers in the United States, and over 90% of them are head and neck squamous cell carcinoma (HNSCC). Chemotherapeutic drugs that treat HNSCC can activate BCL-2 family dependent apoptosis. Pro-apoptotic NOXA induced by adenovirus (Ad-NOXA) or fenretinide inactivates anti-apoptotic MCL-1, while ABT-263 can inactivate other anti-apoptotic BCL-2 family members such as BCL-2 and BCL-XL. We used p53 inactive HN8 and HN12, p53 wild-type UMSCC1, and HPV-positive UMSCC47 human HNSCC cell lines and five mouse HNSCC cell lines. Cells were treated with Ad-NOXA, ABT-263, and fenretinide alone or in combinations. Combinational treatment of ABT-263 with Ad-NOXA or fenretinide enhanced cell death among all cell lines we tested regardless of p53 status. These findings support the hypothesis that combinational treatment of Ad-NOXA or fenretinide with ABT-263 increases cell death by simultaneously inhibiting all anti-apoptotic BCL-2 family proteins in HNSCC cells.

The effects of the fenretinide and genistein as single or combined drugs on ovarian cancer proliferation and viability were investigated. Hypothesis: Co-treatment with genistein will enable a lower dose of fenretinide to be effective in inhibiting the proliferation and survival of ovarian cancer cells. Methods: Low and high doses of genistein and fenretinide were tested on A2780s and A2780cp cells using trypan blue viable cell count, MTS assay. Results and conclusions: Unlike low doses of fenretinide, genistein had anti-proliferative effects on both cell lines. There were no additive or synergistic effects of the two compounds. Higher dose treatments induced anti-proliferative effects and apoptotic cell death in both A2780s and A2780cp cells, with a greater sensitivity of A2780s cells to both test compounds. Overall Conclusion: Genistein and higher doses of fenretinide similarly impair cell cycle progression and induce apoptosis. The anti-proliferative effects of genistein can be affected by co-treatment with fenretinide

The effects of the fenretinide and genistein as single or combined drugs on ovarian cancer proliferation and viability were investigated. Hypothesis: Co-treatment with genistein will enable a lower dose of fenretinide to be effective in inhibiting the proliferation and survival of ovarian cancer cells. Methods: Low and high doses of genistein and fenretinide were tested on A2780s and A2780cp cells using trypan blue viable cell count, MTS assay. Results and conclusions: Unlike low doses of fenretinide, genistein had anti-proliferative effects on both cell lines. There were no additive or synergistic effects of the two compounds. Higher dose treatments induced anti-proliferative effects and apoptotic cell death in both A2780s and A2780cp cells, with a greater sensitivity of A2780s cells to both test compounds. Overall Conclusion: Genistein and higher doses of fenretinide similarly impair cell cycle progression and induce apoptosis. The anti-proliferative effects of genistein can be affected by co-treatment with fenretinide

The effects of the fenretinide and genistein as single or combined drugs on ovarian cancer proliferation and viability were investigated. Hypothesis: Co-treatment with genistein will enable a lower dose of fenretinide to be effective in inhibiting the proliferation and survival of ovarian cancer cells. Methods: Low and high doses of genistein and fenretinide were tested on A2780s and A2780cp cells using trypan blue viable cell count, MTS assay. Results and conclusions: Unlike low doses of fenretinide, genistein had anti-proliferative effects on both cell lines. There were no additive or synergistic effects of the two compounds. Higher dose treatments induced anti-proliferative effects and apoptotic cell death in both A2780s and A2780cp cells, with a greater sensitivity of A2780s cells to both test compounds. Overall Conclusion: Genistein and higher doses of fenretinide similarly impair cell cycle progression and induce apoptosis. The anti-proliferative effects of genistein can be affected by co-treatment with fenretinide

The effects of the fenretinide and genistein as single or combined drugs on ovarian cancer proliferation and viability were investigated. Hypothesis: Co-treatment with genistein will enable a lower dose of fenretinide to be effective in inhibiting the proliferation and survival of ovarian cancer cells. Methods: Low and high doses of genistein and fenretinide were tested on A2780s and A2780cp cells using trypan blue viable cell count, MTS assay. Results and conclusions: Unlike low doses of fenretinide, genistein had anti-proliferative effects on both cell lines. There were no additive or synergistic effects of the two compounds. Higher dose treatments induced anti-proliferative effects and apoptotic cell death in both A2780s and A2780cp cells, with a greater sensitivity of A2780s cells to both test compounds. Overall Conclusion: Genistein and higher doses of fenretinide similarly impair cell cycle progression and induce apoptosis. The anti-proliferative effects of genistein can be affected by co-treatment with fenretinide

Studies were conducted using cultured immortalized wild type (non-CF) and mutant (CF) DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) tracheal epithelial cells on the anti-inflammatory impact of agents that may alter ceramide and glutathione (GSH) metabolism. The CF cells demonstrated abnormally high levels of GSH and glutathione disulfide (GSSG), which could diminish intracellular production of ceramide, a key modulator of inflammation and apoptosis. Hence, additional cell culture studies were carried out with a known inducer of in situ ceramide synthesis, N-4(4-hydroxyphenyl) retinamide (fenretinide) on interleukin (IL)-8 release, intracellular ceramide content, and cellular proliferation in both the basal state and following the inflammatory stimuli of tumor necrosis factor (TNF) -alpha. Fenretinide treatment was associated with a dose-dependent increase in the cellular content of ceramide in both CF and non CF cells. Also, an inhibition of IL-8 release in the inflammatory condition of TNF-alpha treatment was observed following fenretinide treatment in the CF cells. As hyperbaric treatment of whey proteins was previously associated with improved survivability and higher GSH content in a Pseudomonas aeruginosa murine model of cystic fibrosis (CF), the anti-inflammatory role of low molecular weight peptides (< 1kDa) generated from enzymatic hydrolysis of native and pressurized whey protein isolates (WPI) was examined. Pressure treatment of WPI was associated with an enhanced protein digestibility and an altered peptide profile following in vitro digestion. The whey peptides were tested CF and non-CF lung epithelial cells to identify for their effects on GSH metabolism. The impact of the combined treatment of fenretinide and WPH was also tested in terms of apoptosis and cytokine release in cell culture. As opposed to non-CF cells, CF cells showed a strong downtrend in release of IL-8 following the combined fenretinide and whey peptide treatment. In addition, whey peptides protected wild type epithelial cells from the apoptotic effect of fenretinide. Our results suggest the usefulness of these agents as a pharmacological treatment in CF.

Discovering novel compounds that stimulate or abrogate angiogenesis can lead to development of new therapeutic agents that may effectively treat diseases with pathological angiogenesis. The zebrafish model allows for a whole-organism approach to drug discovery. Advantages over other animal models include small embryo size, fecundity, rapid embryonic development, optical clarity and easy accessibility of the embryos. My goal is to validate the therapeutic efficacy and identify the molecular mechanisms of action of three compounds identified from our previous chemical genetic screens. Fenretinide promoted angiogenesis in zebrafish embryos but inhibited the angiogenesis-dependent process of fin regeneration. The pro-angiogenic effects of fenretinide appear secondary to the stimulation of somitogenesis. I3M potently inhibited angiogenesis and fin regeneration, and may act partially through the notch pathway. Lastly, I validated the anti-angiogenic effect of a novel compound DHM. Comprehensively, my studies support the utility of zebrafish as a versatile tool for anti-angiogenic drug discovery.

Discovering novel compounds that stimulate or abrogate angiogenesis can lead to development of new therapeutic agents that may effectively treat diseases with pathological angiogenesis. The zebrafish model allows for a whole-organism approach to drug discovery. Advantages over other animal models include small embryo size, fecundity, rapid embryonic development, optical clarity and easy accessibility of the embryos. My goal is to validate the therapeutic efficacy and identify the molecular mechanisms of action of three compounds identified from our previous chemical genetic screens. Fenretinide promoted angiogenesis in zebrafish embryos but inhibited the angiogenesis-dependent process of fin regeneration. The pro-angiogenic effects of fenretinide appear secondary to the stimulation of somitogenesis. I3M potently inhibited angiogenesis and fin regeneration, and may act partially through the notch pathway. Lastly, I validated the anti-angiogenic effect of a novel compound DHM. Comprehensively, my studies support the utility of zebrafish as a versatile tool for anti-angiogenic drug discovery.

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