Cyclin D1, a proto-oncogene, is required for progression from the G1 phase into the S phase of the cell cycle. Over-expression of cyclin D1 causes an increase in cell cycle progression and cell proliferation, implicating it in a variety of cancers including renal cell carcinoma (RCC). The rodent RCC cell model, QTRRE, and human RCC cell models, ACHN, 786-O and Caki-2, exhibit elevated levels of cyclin D1. Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor, is an FDA-approved hemorheologic agent used to treat intermittent claudication, stemming from peripheral vascular diseases, as well as other diseases involving defective locoregional blood flow. Treatment of QTRRE, ACHN, 786-O and Caki-2 with PTX caused a time- (0-24 hrs) and dose- (0-1.0 mg/mL) dependent decrease of cyclin D1 protein and p-Rb levels in whole cell lysate as well as cytosolic and nuclear fractions, albeit, to different extents within the models. Concomitant with cyclin D1 and p-Rb decrease, enhanced G1 phase cell cycle arrest was observed in the RCC models. Mechanistic studies in these RCC cell models were carried out to determine PTXs mechanism of action with regard to cyclin D1 protein level decrease. RT-PCR analysis showed no significant changes in cyclin D1 mRNA copy number in time- (0-24 hrs) and dose- (0-1.0 mg/mL) dependent PTX treatments. However, such treatments caused decrease in p-4EBP1 (Ser65), p-4EBP1 (Thr70), and p-4EBP1 (Thr37/46). Because PTX's ability to decrease cyclin D1 protein was prevented in the presence of the proteasome inhibitor, MG-132, studies were performed to determine whether cyclin D1 stability was decreased during PTX treatment. Cyclin D1 degradation is initiated by phosphorylation of residue Thr286 by GSK-3β. Inhibition of GSK-3β with LiCl or knockdown via siRNA in the presence of PTX failed to block cyclin D1 decrease. Moreover, PTX treatment in the presence of MG-132 revealed no significant increase in cyclin D1 p-Thr286 compared to control. Finally, using the protein synthesis inhibitor, CHX, PTX caused no significant decrease in cyclin D1 t₁/₂ (wt-HA and T286A-HA) compared to control. Sorafenib, a broad-spectrum (cRAF, bRAF, KIT, FLT-3, VEGFR-2, VEGFR-3, and PDGFR-β) kinase inhibitor, is FDA-approved for the treatment of RCC. Studies with sorafenib and PTX in the ACHN cell model were carried out to determine PTXs possible adjuvant role in inhibiting cell growth via cyclin D1 decrease and G1 phase arrest. MTS data showed PTX potentiates the anti-proliferative effects of sorafenib. PTX pre-treatment for 24 hrs was also lowered the effective dose of sorafenib from 50 μM to 5 μM. Further, ACHN xenograft tumor volumes from mice treated with PTX and sorafenib displayed significantly higher tumor growth inhibition compared to either drug treatment alone or vehicle. Finally, drug treated ACHN xenograft tissue displayed significantly lower cyclin D1, p-RB and p-4EBP1 levels. These results demonstrate a novel anti-cancer property of PTX and suggest its use as a possible adjuvant therapy in RCC treatment should be further explored.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/337293 |
| Date | January 2014 |
| Creators | Mastrandrea, Nicholas Joseph |
| Contributors | Lau, Serrine S., Lau, Serrine S., Monks, Terrence J., Zhang, Donna, Vaillancourt, Richard, Tsaprailis, George |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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