Masters of Science in Pharmaceutical Sciences Theses
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Item Crosstalk between adipocytes and breast cancer cells with ESR mutations via adipsin enhances tumor growth.(Albany College of Pharmacy and Health Sciences Theses, 2023-08) Belyakov, Artem '23Estrogen receptor positive breast cancer is a prevalent disease traditionally treated with hormone therapy which relies on the expression of the estrogen receptor to starve the tumor of growth signaling due to estradiol. While hormone therapy generally has great outcomes, the development of endocrine resistance remains a huge hurdle, with poorer outcomes and less effective therapeutic options. Endocrine resistant tumors have been found to uniquely contain point mutations in the ligand binding domain of the Estrogen Receptor 1 (ESR1) coding region, predominantly Y537S and D538G. A growing body of literature has shown the stromal involvement in tumorigenesis, which supports resistance, survival, proliferation and eventual metastasis. Adipocytes have been found to promote breast cancer growth and invasion in coculture but without an understood mechanism. The complement system is generally associated with innate immunity but is increasingly found to promote tumor growth by dysregulation of inflammatory pathways. Since Adipsin (also known as Complement Factor D) is preferentially expressed in adipocytes, an investigation was done on the role of complement, specifically the C3a-Adipsin-C3aR axis on the impact of breast cancer cell characteristics. Using RT-qPCR and ELISA, it was found that C3a and Adipsin are both upregulated preferentially in the mutant cells as compared to wild type when cultured in adipocyte condition media. In addition, experimental inhibition of C3aR using the small molecule SB290157, had tumor attenuating effects that were limited to the mutated breast cancer cell lines. Using CyQuant for cell viability, and flow cytometry to investigate apoptosis, cell proliferation and cell cycle analysis, it was found that SB290157 had a drastic effect by decreasing cell viability and cell proliferation, increasing apoptosis, and causing cell cycle arrest at G2/M. Using knockdown of C3 and Adipsin by siRNA for the more sensitive mutant YS led to a marked decrease in cell viability as well. These findings contribute to the idea that endocrine resistant breast cancers are utilizing complement inflammatory pathways as one of their strategies to survive, proliferate and invade. As this pathway is investigated more, it may be possible to therapeutically block it for better breast cancer outcomes.Item The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocytes(Albany College of Pharmacy and Health Sciences Theses, 2023-08) Akume, Ahone Gina '23Hox genes are regulatory genes that encode nuclear proteins acting as transcription factors during normal development and differentiation. One such gene, HOXB7, plays a role in various developmental processes, including hematopoietic differentiation, lymphoid development, and mammary gland development. However, the role of HOX genes in breast cancer development remains largely unexplored. Our previous studies revealed that HOXB7 expression was significantly elevated in primary cancer and distant metastasis. HOXB7 overexpression promoted cell proliferation in SKBR3 breast cancer cells, leading to robustly vascularized xenografts in immunodeficient mice. Furthermore, HOXB7 enhanced tumor growth through TGF-B signaling and recruitment of macrophages, indicating the role of HOXB7 in the crosstalk of stromal components. We found evidence that HOXB7 overexpression in ER+ breast cancer cells promote tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes. By screening secreted factors in adipocytes induced by TCM of MCF-7-HOXB7 cells, it was also found that CCL5 is highly expressed in HOXB7- overexpressing MCF-7 cells. The CCR5 inhibitor, maraviroc, sensitized MCF-7- HOXB7 cells compared to control cells. These findings suggest that blocking the interaction between CCL5 and CCR5 signaling significantly inhibits ER+ breast cancer with HOXB7 overexpression. We aim to further investigate if HOXB7 overexpression in ER+ breast cancer cells promote tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes and the role of CCL5 in breast cancer cell growth and migration.Item Assessment of Hydrolysis of Prodrugs and Co-drugs Derived from 5-Aminolevulinic Acid and Mycophenolic Acid(Albany College of Pharmacy and Health Sciences Theses, 2023-08) Strumski, Kaitlyn '23Mycophenolic acid (MPA) and 5-aminolevulenic acid (ALA) have been known to reduce the symptoms associated with autoimmune skin disease, psoriasis. It is hypothesized that the combination of these two drugs, when delivered simultaneously to the skin, will provide synergistic therapeutic benefit in suppressing the symptoms of psoriasis. This research project aims to evaluate the in vitro efficacy of prodrugs and investigational co-drugs derived from ALA and MPA using an immortalized human keratinocyte cell line (HaCaT) as models for drug behavior in human skin. Previously, it was shown that MPA and ALA do not negatively interact, but rather improve treatment when administered together. This project is specifically aimed at using HaCaT cells as a viable “skin” model to determine if the prodrugs and the investigational co-drugs derived from ALA and MPA are sufficiently metabolized to release therapeutically effective doses of the parent compounds that elevate protoporphyrin IX (PpIX) and inhibit inosine-5′-monophosphate dehydrogenase (IMPDH). HaCaT cells were grown, and cultures were treated with ALA-BE and ALA separately to evaluate the increase in PpIX which is hypothesized to correlate with hydrolysis of ALA-BE into the active compound ALA. MPA-ME was synthesized, HaCaT cell lysate was prepared, and the lysates were treated with MPA and MPA-ME. The MPA treated lysates were analyzed using HPLC to determine the extraction efficiency of MPA and the MPA-ME treated lysates were analyzed using HPLC to determine the percent hydrolysis of MPA-ME into the active compound MPA. This research establishes that HaCaT cells contain hydrolytic enzymes capable of promoting hydrolysis of ALA-/MPA- prodrugs and co-drugs that results in release of the parent drugs, ALA and MPA. The activity of the parent compounds correlates with the extent to which the prodrugs and co-drugs hydrolyze in epithelial cells.Item Structural and Functional Basis of Drug Binding to Human Cytochrome P450 2C9*14 Genetic Variant(Albany College of Pharmacy and Health Sciences Theses, 2022-12) Edara, Sreeja '22Cytochrome P450 (CYP) enzymes are a group of enzymes encoded by P450 genes and are expressed as membrane-bound proteins mostly found in the endoplasmic reticulum of the liver cells. About 17 of the 57 CYP proteins have a significant role in drug metabolism. CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 are the major enzymes involved in drug metabolism. The highly polymorphic CYP2C9 is responsible for the metabolism of up to 15% of clinical drugs that include the anticoagulant S-Warfarin, the anti-hypertensive losartan, the anti-diabetic tolbutamide, and the analgesic ibuprofen, and others. The CYP2C9*14 allele, which results in the amino acid substitution from arginine to histidine at position 125 (R125H), is found in 3-5% of population, predominantly southeast Asians. Recent studies have illustrated that the CYP2C9*14 variant results in decreased enzyme function leading to the \342\200\234poor metabolizer\342\200\235 phenotype. For example, patients with this allele required a lower S-Warfarin dose compared to those with the normal or wild type allele. However, the implications of the CYP2C9*14 allele on the metabolism of losartan, an anti-hypertensive drug substrate of CYP2C9 is less clear. In this study, we aim to determine the impact of the amino acid change R125H (CYP2C9*14) on the binding of losartan using structural, functional, and biophysical analysis that include X-ray crystallography, enzymatic assays, and isothermal titration calorimetry, respectively. The results of this research will help advance our understanding of the effect of genetic polymorphisms and inter-individual differences in losartan response and may help implementing clinical guideline for patients on losartan that possesses the *14 allelic variation.Item Investigating the HIV-1 Latency Promoting Properties of Sulforaphane(Albany College of Pharmacy and Health Sciences Theses, 2022-12) Paudel, Anisha '22HIV remains incurable due to its ability to exist as an undetectable latent or silent reservoir in different tissues that are insensitive to antiretroviral therapies and invisible to the immune clearance. So far, efforts to completely eradicate the latent reservoir have been a difficult and unsuccessful task. \342\200\234Block and Lock\342\200\235 is a novel approach for HIV cure that aims to permanently silence the latent reservoir using the latency-promoting agents (LPAs) to block the virus transcription and lock the virus promoter to keep it in the latent phase via epigenetic modifications. Recent reports have suggested Sulforaphane (SFN), an inducer of Nrf-2 (nuclear erythroid 2-related factor) mediated antioxidative signaling, to have an additional anti-HIV property by restricting HIV replication at initial stages. For the reactivation of provirus, activation of NF-kB is indispensable. Existing literature suggests that Nrf-2 may negatively regulate NF-kB activation. Considering these independent observations, we propose SFN may function as a latency promoting agent by promoting Nrf-2 signaling in cells latently infected with HIV-1. In our study, we noticed that SFN treatments significantly reduced the TNF\316\261 induced HIV-1 reactivation in the latently infected monocytic (THP89-GFP) as well as T-cell lines (J89-GFP). Viral RNA copy numbers released in the supernatant as well as HIV transcription initiation and elongation-specific transcripts detected by RT-qPCR were remarkably diminished. HIV p24 levels were also decreased by SFN treatment. SFN mediated Nrf-2 prevent the activation of NF-kB induced with TNF-\316\261 treatment that is necessary for the reactivation of the HIV-1 provirus evident with the immunoblot. This shows that SFN can render the latent cells resistant to reactivation. Thus, our study provides compelling evidence that SFN possesses latency promoting properties that can be utilized in block-and lock approach to achieve a functional cure for HIV-1.