Browsing by Subject "tumors"
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Item HIF: A Key Survival Factor for Serum-Deprived Prostrate Cancer Cells(2008-05-01) Thomas, Rusha; Jamboor Vishwanatha; Harlan Jones; Raghu KrishnamoorthyThomas, Rusha, HIF: A key survival factor for serum-deprived prostate cancer cells. Doctor of Philosophy (Molecular Biology and Immunology), May 2008, 134 pages, 47 illustrations, reference list, 105 titles. The hypoxia-inducible factor (HIF) is central to hypoxic adaptation of tumors, and consists of an oxygen-labile HIF-1α and a constitutively expressed HIF-1β subunit. In specific aim 1, we report that prolonged serum deprivation is a potent inducer of HIF-1α in PC-3 and LNCaP prostate cancer (PCa) cells, despite normal oxygen conditions. In contrast, cells grown in the presence of serum did not upregulate HIF-1α protein levels. Moreover, HIF-1α protein increase during serum deprivation correlated with increased cell survival, while suppression of HIF-1α expression significantly decreased PCa cell viability. Our results further demonstrate that HIF-1α protein increase during serum deprivation is due to increased HIF-1α protein synthesis. First, there was no significant increase in HIF-1α mRNA. Secondly, cycloheximide, a protein synthesis inhibitor, prevented HIF-1α protein increase in serum-deprived PCa cells. Moreover, the expression of HIF-1α-target genes, VEGF and IGF-2, was concomitantly increased in serum-deprived PCa cells, while suppression of HIF-1α expression markedly inhibited their induction. Most interestingly, our study showed a significant decline in PCa cell survival following inhibitor of IGF-2 activity. Taken together, our study demonstrates for the first time that PCa cells counteract the stress of prolonged serum deprivation by upregulating HIF-1α protein which increases IGF-2 expression to promote cell survival. In specific aims 2 and 3, we investigated the molecular mechanism of autocrine regulation of HIF-1α, IGF-2 and cell survival in serum-deprived PC-3 and LNCaP PCa cells. We detected a time-dependent increase in Akt activation during serum deprivation, and inhibition of Akt activation attenuated the serum deprivation-mediated increase in HIF-1α and cell survival. Importantly, IGF-2 secretion significantly increased during serum deprivation, and was accompanied by increased activation of its receptor, insulin-like growth factor-I receptor (IGF-IR). Additionally, inhibition of IGF-2 activity markedly suppressed the serum deprivation-mediated increase in IGF-IR and Akt activation, HIF-1α expression, as well as its own transcription, suggesting autocrine regulation of HIF-1α expression via IGF-2. Reciprocal regulation of the IGF-2/IGF-IR system and P13K-Akt pathway was further demonstrated by findings wherein Akt activation was prevented following suppression of IGF-IR expression, and IGF-IR activation was inhibited following P13K inhibition. Lastly, HIF-1α suppression abolished the serum deprivation-mediated increase in Akt activation, and also resulted in higher IGF-IR protein levels indicating reduced IGF-IR activation. Collectively, our study demonstrates that a HIF-1α-dependent autocrine feedback loop upregulates HIF-1α, and thus promotes survival of normoxic, serum-deprived PCa cells.Item Novel Method to Obtain Contact Angles of Tumor Biopsies(American Chemical Society, 2023-08-07) Rincon, Julio; Kastellorizios, MichailCharacterizing the strength of a solid-liquid interface can be done by depositing a single drop of liquid on a planar solid surface and measuring the angle of the formed semicircle, called the contact angle. The contact angle of pure water is indicative of a surface's hydrophobicity and is a useful metric in biomedical applications such as tissue scaffolding and drug/tissue interactions. However, the roughness and inhomogeneity of most biological surfaces make obtaining accurate contact angles of such materials challenging. Here, we developed an instrument and methodology to obtain contact angles of tissue sections. Breast cancer tumor and nearby healthy tissue sections were used as the model biological surface. The custom instrument was built on existing equipment by improving drop dispensing accuracy in the nanoliter range, an XYZ stage, additional side view cameras, and microscope-based sample visualization. The method takes into account the inherent surface inhomogeneity and topology of tissue and the required method of illumination for contact angle acquisition. As such, the system uses an inverted microscope with a high sensitivity camera, an XYZ stage for accurate droplet placement on tissue, and multiple cameras to obtain contact angles around the entire perimeter of the drop. We tested the system with breast cancer biopsies and adjacent normal tissue from 75 patients and report here a trend of tumor exhibiting higher water contact angles, and thus higher hydrophobicity, compared to their respective normal adjacent tissue. The system described here can be used to characterize any type of biological tissue, which can be sectioned, with any liquid including water or solutions with dissolved or suspended therapeutic molecules and particles.Item Production of Extracellular Matrix-Degrading Proteases by a Rat B Cell Line.CRL-1631(2002-05-01) Badeaux, Kathleen S.; R. GoldfarbBadeaux, K. Production of Extracellular Matrix-Degrading Proteases by a Rat B Cell Line.CRL-1631. Master of Science (Microbiology and Immunology), May 2002. 30 pp., 9 illustrations, 1 table, 16 bibliography titles. Previously B lymphocytes have been reported to accumulate at the site of tumor development and to play a role in immune surveillance against metastatic tumors. Investigating this mechanism, we studied B lymphocyte production of extracellular matrix-degrading proteinases: matrix metalloproteinases (MMPs) and components of the urokinase plasminogen activator (uP A) system. Our studies include RT-PCR of CRL-1631 eDNA revealing mRNA for MMP-2, MMP-9, TIMP-1, TIMP-2 and uPAR. MMP-2 and MMP-9 activity was verified by gelatin zymography. TIMP-1, TIMP-2 and uPAR protein expression was confirmed by Western blot analyses. I also report, for the first time, MT -1 MMP gene and protein expression in B cells by RT-PCR and Western blot, respectively. CRL-1631 invasion through Matrigel model basement membrane was significantly inhibited by BB-94, confirming MMP involvement in this cell line's invasiveness. Therefore, B cells use multiple proteases in the degradation of the extracellular matrix, ECM, and this may be one factor responsible for their accumulation at the site of established tumors.