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THE ROLE OF THE BAXΔ2 C-TERMINAL STRUCTURE IN CASPASE-8- MEDIATED CELL DEATH OF HUMAN COLORECTAL CARCINOMA CELLS
Title
THE ROLE OF THE BAXΔ2 C-TERMINAL STRUCTURE IN CASPASE-8- MEDIATED CELL DEATH OF HUMAN COLORECTAL CARCINOMA CELLS
Creator
Nelson, Adam
Date
2018, 2018-05
Description
BaxΔ2 is an isoform of the proapoptotic Bcl-2 family member Bax that promotes cell death via caspase-8 activation. The C-terminus of BaxΔ2 has...
Show moreBaxΔ2 is an isoform of the proapoptotic Bcl-2 family member Bax that promotes cell death via caspase-8 activation. The C-terminus of BaxΔ2 has been shown to be crucial for caspase-8 dependent cell death in colon cancer cells. However, it is unknown whether the C-terminal primary sequence or secondary structure is necessary for interaction with caspase-8. In this project, several BaxΔ2 C-terminal mutants were generated based on secondary structure predictions. Models showed that mutating Leu164 and Thr165 to Ala (LT-AA) would increase the probability of alpha helix formation, while mutating Leu164 to Pro (L-P) would decrease the probability of alpha helix formation. Expression of these mutant proteins in colon cancer HCT116 cells, showed that L164P, and not L164A/T165A, significantly impaired BaxΔ2 function. The mutant L164P proteins formed atypical aggregates, and their ability to induce cell death was also significantly decreased when compared to the wild type. These results indicate that the BaxΔ2 C-terminal tridimensional structure, and not the specific primary sequence, is critical for triggering aggregation-mediated cell death.
M.S. in Biology, May 2018
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BAXΔ2: FROM FUNCTIONAL DOMAINS TO CANCER CORRELATION
Title
BAXΔ2: FROM FUNCTIONAL DOMAINS TO CANCER CORRELATION
Creator
MaÑas NÚÑez, Adriana
Date
2018, 2018-05
Description
BaxΔ2 is a functional pro-apoptotic Bax isoform, originally identified in cancer patients with microsatellite instability. Here we performed...
Show moreBaxΔ2 is a functional pro-apoptotic Bax isoform, originally identified in cancer patients with microsatellite instability. Here we performed an extensive study on BaxΔ2, covering the structure-function relationship, the clinical potential, and the human tissue expression profile. Unlike Baxa, BaxΔ2 forms aggregates and triggers non-mitochondrial cell death through caspase 8. However, the functional domain(s) responsible for BaxΔ2 unique behavior were elusive. Here we show that disruption of helix α1 makes Baxα mimic BaxΔ2, but other alterations in the N-terminus have no significant impact. We found that the core region is key for aggregation, but is not sufficient to trigger cell death. The Cterminal helical conformation, not its primary sequence, appears to be critical for caspase 8 activation. As BaxΔ2 shares core and C-terminal with most Bax isoforms, our results indicate an intrinsic potential for aggregate-mediated caspase 8-dependent cell death in other Bax family members. BaxΔ2 has been shown to sensitize cancer cells to chemotherapy, but it is a very unstable protein. Therefore, we screened a panel of proteasome inhibitors in colorectal cancer cells with different Bax statuses. We found that proteasome inhibitors can block BaxΔ2 degradation without affecting the levels of Baxa or Bcl-2. Among the inhibitors tested, bortezomib and carfilzomib were able to induce significantly higher cell death in BaxΔ2-positive cells than in cells with Baxa or no Bax. Furthermore, bortezomib-induced cell death in BaxΔ2-positive cells was predominantly dependent on the caspase 8/3 pathway. These results suggest that BaxΔ2 can selectively sensitize cancer cells to proteasome inhibitors. As many cancers have microsatellite mutations, we screened BaxΔ2 protein expression in 1090 samples of tumor and healthy tissues from several organs. We found that BaxΔ2 is expressed in 1% to 5% of cells in most organs, predominantly in healthy tissues. Production of BaxΔ2 requires a guanine deletion in the microsatellite region, but the great majority of BaxΔ2-positive tissues contained no mutation at genomic or transcript levels. Therefore, we tested Programed Ribosomal Frameshift (PRF) as a possible BaxΔ2 expression mechanism, using a double tagged construct with no mutation. We found that, though rare, PRF can lead to expression of BaxΔ2. In conclusion, BaxΔ2 can be expressed without a genetic mutation. In conclusion, BaxΔ2 is a unique isoform whose expression is the product of the extreme plasticity of the Bax gene and the biochemical circumstances in the cell. As an example of non-traditional expression, it opens the door to a whole new proteome expressed by alternative mechanisms both under physiological and pathological conditions.
Ph.D. in Biology, May 2018
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Non-invasive quantification of cancer drug targets: Mathematical models for paired-agent molecular imaging
Title
Non-invasive quantification of cancer drug targets: Mathematical models for paired-agent molecular imaging
Creator
Sadeghipour, Negar
Date
2017
Description
Cancer is among the leading causes of death worldwide. Incidence of cancer is rising at a rate that is almost completely nullifying...
Show moreCancer is among the leading causes of death worldwide. Incidence of cancer is rising at a rate that is almost completely nullifying improvements in cancer treatment and the heterogeneity of advanced disease poses significant complications for the development of effective therapies. With more aggressive cancers tending to display abnormally high expression of signaling receptors associated cell proliferation - receptors that tend to be expressed at very low levels by healthy cells in adulthood - many new cancer-specific “molecular therapies” have been developed to target and block these pathways. However, not all cancers overexpress the same proliferation pathways, so many have proposed molecular imaging as a non-invasive means of identifying on a patient-by-patient basis, which specific targets may be overexpressed to tailor therapies to the individual (“precision medicine”). The primary goal of this thesis was to develop and validate some of the first non-invasive means of measuring drug-target concentrations prior to therapy and the first measures of drug-target occupancy during therapy to ultimately predict and monitor the efficacy of cancer molecular therapy. All work was founded on paired-agent molecular imaging protocols that employ co-administration of two imaging agents: one agent that is targeted to the biomolecule of interest (e.g. a cell surface signaling receptor that may be overexpressed by a cancer), and a second, “control” (“untargeted”) agent that is as chemically similar to the targeted agent as possible, but that does not bind to the biomolecule of interest. In all paired-agent imaging strategies, the signal from the control agent is used to account for delivery and nonspecific retention effects that can confound the relationship between the targeted imaging agent concentration in a region-of-interest (ROI) and the targeted biomolecule concentration in that ROI.
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Apalutamide Modulates the Expression of Regulatory Genes for Prostate Cancer Cell Invasion and Migration In Vivo and In Vitro
Title
Apalutamide Modulates the Expression of Regulatory Genes for Prostate Cancer Cell Invasion and Migration In Vivo and In Vitro
Creator
Qualter, Gina E.
Date
2022
Description
The next generation antiandrogen, Apalutamide (Apa), improves both overall survival and metastasis-free survival in men with castration...
Show moreThe next generation antiandrogen, Apalutamide (Apa), improves both overall survival and metastasis-free survival in men with castration-resistant prostate cancer (CRPC). In vitro and in vivo studies were performed to characterize the mechanistic effects of Apalutamide on prostate cancer cell proliferation, invasion, and migration, and the expression of genes that regulate these processes. Apalutamide inhibited the proliferation of LNCaP human prostate cancer cells in both the presence and absence of dihydrotestosterone (DHT), and also inhibited LNCaP cell migration/invasion. At the mRNA level (RT-PCR), Apalutamide down-regulated the expression of androgen receptor (AR), c-Myc, MMP-2, MMP-9, DANCR, and lncRNA, and up-regulated TIMP-2 expression. Similar data were obtained for protein expression (western blot). In the in vivo study, male Hi-Myc mice received daily oral administration of Apalutamide beginning at age 8 weeks for 2 months, 3.5 months, or 5 months. Daily oral administration of Apalutamide reduced accessory sex gland weights by over 50% at all three time points, inhibited the progression of prostatic intraepithelial neoplasms (PIN) to cancer, and significantly affected the expression of genes that regulate invasion and migration. However, in vitro findings indicated that resistance to Apalutamide through the emergence of the AR splice variant 7 (AR-V7) following extended treatment is possible and may be reversed following knockdown of AR-V7 gene expression.In summary, these results suggest that while Apalutamide is an effective inhibitor of prostate cancer invasion/migration, further investigation into the mechanism of AR-V7 mediated Apalutamide-resistance and strategies to overcome resistance may be indicated to improve prostate cancer patient outcomes following extended periods of treatment.
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