Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
Thromboxane (TX) A2 is a prostaglandin produced by metabolism of arachidonic acid through cyclooxygenases and thromboxane synthase. TXA2 is biologically active, mainly through activating its cognate, seven transmembrane, G protein coupled receptor. It was previously reported that thromboxane A2 receptor (TP) was expressed in prostate cancer, and further activation of this receptor elicited cell contraction and modulated tumor cell motility through regulating small GTPase RhoA. This study aims to identify G alpha protein(s) involved in thromboxane A2 signaling in tumor cell motility. Methods: The expression of G alpha proteins in the PC3MM cells was studied by real time PCR. Cell contraction assay was performed by staining cells with TRITC phalliodin. Tumor cell motility and invasion were evaluated using wound healing assay and transwell Boyden Chamber assay. To determine the roles of G alpha protein in thromboxane A2 signaling, we expressed different alleles of G alpha proteins (wild type, constitutive active) in PC3MM cells and then the subsequent effects on cell contractions was determined. We also depleted G alpha protein expression by short hairpin RNA and examined subsequent changes in cell contraction and migration after activation of TP. G (alpha) 12 has been reported to play critical role in cell proliferation in vitro and tumor growth in vivo. These findings were validated by performing BrdU Incorporation assay, Cell proliferation assay and cell cycle analysis. Quantitative analysis of the epithelial to mesenchymal transition associated genes was performed by real time PCR. In vivo experiments were performed in order to validate in vitro findings. Results: PC3 MM cell line had the endogenous level of all the G alpha protein (G alpha 12, G alpha i1, G alpha 11, G alpha 13), but not G alpha q. Overexpression of G alpha 12/13 increased cell contraction after activation of thromboxane A2 receptor with U46619. Expression of constitutively active G alpha 12 by itself was sufficient to cause cell contraction, regardless whether TP is activated or not. We have identified two potent shRNAs that can silence the G Alpha 12. Depletion of G alpha 12 via shRNAs reduced cell contraction after TP activation. Tumor cells with depleted G alpha 12 had shown decreased tumor cell motility, invasiveness and cell proliferation. Cell cycle analysis showed that G alpha 12 depleted cells exhibit G1/G0 arrest. Quantitative expression of EMT associated genes was reduced in G alpha 12 depleted cells exhibiting increase in the E cadherin / Vimentin ratio. G alpha 12 depleted cells in comparison to scramble control show reduced rate of cell proliferation reminiscent of in vitro findings. Conclusion: This study presents compelling evidence that G alpha 12 plays a major role in thromboxane A2 regulation of prostate cancer invasion and metastasis. Silencing of G alpha 12 with shRNA may therefore provide a promising therapeutic strategy for prostate cancer patients.
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