Saudi Cultural Missions Theses & Dissertations
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Item Restricted ANALYZING THE ROLE OF MDMX COMPARED TO MDM2 IN REGULATING CELL DEATH IN PROSTATE CANCER CELLS(Nova Southeastern University, 2024-07) Alsarrani, Ahmed; Rathinavelu, AppuProstate cancer is the most prominent cancer type and the second leading cause of cancer death among men in the USA. In this study, we evaluated the role of MDMX compared to MDM2, the two proto-oncogenes involved in the negative regulation of p53, in causing cell death and cell cycle arrest through p53 dependent and independent mechanisms. We observed that inhibition of MDMX decreased the cell viability of LNCaP prostate cancer cells in response to treatments with both NSC (NSC-207895) and SJ (SJ-172550), which are MDMX specific inhibitors. MDMX inhibition appears to induce necroptosis in LNCaP and PC3 prostate cancer cells by elevating the expression of receptor-interacting protein kinase 1 and receptor-interacting protein kinase 3 (RIP1 and pRIP3) and phosphor-mixed lineage kinase domain-like protein (pMLKL), which are key elements of the necroptotic pathway. These data were consistent with fluorescence images that were obtained using SYTOX® Green which is specific for detecting cells that undergo programmed or other forms of cell death. Moreover, the fluorescence images using DEVD staining, which is a fluorogenic substrate that can bind to caspases 3/7 during apoptosis, revealed that MDM2 inhibition by RG-7388 induced apoptosis through caspase activation. To further confirm the effects of MDMX on cell migration, NSC effectively blocked the cancer cell migration in LNCaP, not PC3 cells during the scratch assay. However, RG-7388 didn’t have a significant effect on cell migration but mainly caused cell death. Moreover, MDM2 inhibition by RG-7388 increases the expression of p21 and induces PARP cleavage, which highlights its role in cell cycle arrest and apoptosis. Analysis of the miRNA has revealed that inhibition of MDMX and MDM2 can significantly upregulate hsa-let-7d-5p, hsa-let- 7g-5p, hsa-miR-15a-5p, and hsa-miR-106b-5p levels, which have tumor suppressor characteristics. Furthermore, oncogenic miRNAs (OncomiRs) such as hsa-miR-10a-5p and hsa-miR-30b-5p were downregulated, which is in favor of the cell cycle arrest and cell death. The overall outcomes of our experiments provide additional knowledge and strategies for making an accurate assessment of the most suitable therapeutic strategies for achieving effective treatments for MDMX-positive prostate cancer.21 0Item Restricted Evaluation of Targeted Selective Inhibitors to Enhance Temozolomide Treatment Sensitivity in Acute Myeloid Leukemia(University of Alberta, 2024-01-04) Basonbul, Asmaa Abdullah; Brandwein, Joseph; Berthiaume, Luc; Weinfeld, MichaelTemozolomide (TMZ) is an alkylating agent with limited activity in acute myeloid leukemia (AML). The DNA repair enzyme O6-methylguanine methyltransferase (MGMT) enhances tumor cell resistance to TMZ. B-cell lymphoma-2 (BCL-2) and mouse double minute 2 homology (MDM2) are proteins involved in AML cell survival and chemotherapeutic resistance. BCL-2 is an antiapoptotic protein that prevents cell apoptosis, and MDM2 is a ubiquitin E3 ligase that mediates p53 degradation. Inhibiting BCL-2 and restoring p53 functions may affect AML treatment. Venetoclax (VEN) is a small molecule that promotes cell apoptosis through inhibition of the BCL-2 protein. Idasanutlin (IDA) is an MDM2-specific inhibitor that disrupts p53:MDM2 interactions and restores p53 functions. This study established the need to enhance TMZ sensitivity in AML cells with high and low MGMT expression levels by incorporating VEN and IDA into TMZ treatment. AML human cell lines and blast-containing mononuclear cells were used to evaluate MGMT and target proteins for each inhibitor. Cell viability, cell apoptosis, and DNA damage were assessed with TMZ alone and in combination with a pre-selected inhibitory concentration of 50% (IC50) of a VEN or IDA inhibitor. Further, VEN inhibitor was applied in vivo using mouse model. Leukemic engrafted mice treated with selected TMZ and VEN concentrations, alone and in combination, were evaluated for survival and CD45+ and CD33+ expression in the bone marrow. In high and low MGMT-expressing AML cell lines, co-incubation of TMZ with VEN at IC50 resulted in a marked enhancement of TMZ sensitivity. In VEN+TMZ treated cell lines, apoptosis was induced and severe DNA damage was observed compared to TMZ alone. AML patient samples were resistant to TMZ alone but became sensitized to TMZ+VEN in combination, including those with high MGMT expression levels. The combination-treated mice had the longest survival among the group; however, by the time of death, no difference was seen in CD45+ cells compared to the single drug or untreated groups. In high and low MGMT-expressing cells, co-incubation of TMZ with IDA at IC50 did not improve TMZ sensitivity, regardless of p53 status. In both high and low MGMT-expressing cells, there was no evidence of DNA damage in the TMZ+IDA combination groups compared to TMZ alone. AML patient samples were resistant to TMZ alone and highly sensitive to IC50 IDA. IDA did not enhance the TMZ sensitivity, as insignificant cell death was observed in IDA alone and TMZ combination. VEN enhances TMZ cytotoxicity in high and low MGMT cells by decreasing cell viability, increasing cell apoptosis, and inducing DNA damage. The opposite was observed in IDA and TMZ combination-treated cells. Overall, we found VEN and TMZ combination to be promising to pursue further for clinical trails.26 0Item Restricted Effects of MDM2 and HMG-CoA Reductase Inhibition on Epithelial to Mesenchymal Transition in Prostate Cancer(2023-07-27) Alsubhi, Samia Mekheer; Rathinavelu, AppuAccording to the American Cancer Society, prostate cancer is the second leading cause of cancer death among men. Aggressive growth and metastasis are the main reasons for high mortality among prostate cancer patients. The proto-oncogene MDM2 controls multiple signaling pathways that are involved in cell cycle regulation and survival. MDM2 also has a major role in imparting stemness and Epithelial to Mesenchymal Transition (EMT) abilities to prostate cancer cells that are inherently less aggressive. During cancer growth, lipid metabolism also plays a pivotal role in cellular reprogramming, which eventually leads to the induction of EMT and the acquisition of stemness. It has been well documented that the intermediates of mevalonate metabolism can significantly affect gene expressions in cancer and influence the tumor microenvironment to support tumor growth and survival. At present, there is a lack of comprehensive knowledge about the use of MDM2 inhibitor RG7388 in cancers with p53 mutation, since most studies report that it is most effective in cancers with wild-type p53. Therefore, our study was designed to investigate the effects of RG7388 in p53 mutant or p53 null prostate cancer cells. Our study also explored the effect of RG7388 in combination with simvastatin, which blocks the mevalonate pathway by inhibiting the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase enzyme. Simvastatin has been reported to have anticancer effects in several cancers but, so far, there is no data available regarding its use in combination with RG7388. Therefore, our study aimed to analyze the effects of RG7388, individually or in combination with simvastatin, on EMT and cancer stemness. Our experiments were conducted using DU145 and PC3 prostate cancer cells which have p53 mutant and null status respectively. Our experimental results showed a decrease in the viability of PC3 and DU145 cells, in a time and concentration-dependent manner, after treatment with RG7388 or simvastatin. The mesenchymal and stem cell markers were also downregulated after RG7388 and simvastatin combination treatment. In addition, individual and combination treatments with the above-mentioned drugs induced the disintegration of spheroids and eventually a reduction in the total number of spheroids. Immunofluorescence staining of spheroids revealed a downregulation of cancer stem cell markers among all treated groups. Furthermore, the scratch assay showed inhibition of migration of the DU145 and PC3 cells, particularly after the combination treatment. Our results have confirmed that RG7388, simvastatin, and their combination treatments can reverse EMT, inhibit migration, and reduce cancer stemness in p53 mutant or null prostate cancer cells. Our study also suggests that RG7388 and simvastatin combination can be effective for treating prostate cancers that are aggressive due to MDM2 expression.18 0