Document Type

Dissertation

Date of Degree

Fall 2010

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmacology

First Advisor

Raymond J. Hohl

Abstract

The isoprenoid biosynthetic pathway is targeted in the treatment of several diseases, including hypercholesteremia and bone related disorders. Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are isoprenoid biosynthetic pathway intermediates that are utilized during post-translational modification of proteins termed farnesylation and geranylgeranylation, respectively, together known as prenylation. The Ras and Rho GTPase family members are examples of proteins that are prenylated. Prenylation is essential for proper membrane localization and function of these small GTPases. Activating mutations or over-expression of these proteins promote oncogenic events, such as increased proliferation and migration.

Studies have demonstrated that farnesyl transferase inhibitors and geranylgeranyl transferase inhibitors possess anti-cancer effects in humans and animal models of cancer, respectively. An alternative way to impair protein prenylation is through the depletion of FPP and GGPP. Statins and nitrogenous bisphosphonates (NBPs) deplete FPP and GGPP leading to impaired protein prenylation by inhibiting HMG-CoA Reductase (HMGCR) and FPP synthase (FDPS), respectively. These drugs have been shown to induce apoptosis, inhibit cancer cell migration, and induce cell cycle arrest. The anti-cancer effects of statins and NBPs can be prevented by GGPP addition, suggesting that GGPP depletion may be the mechanism by which these agents interfere with cancer cell survival.

We and our collaborators have developed bisphosphonate inhibitors of GGPP synthase (GGDPS), an enzyme that produces GGPP from the substrates FPP and isopentenyl pyrophosphate.

The goal of this research was to identify novel GGDPS inhibitors and to assess the effects of specific inhibition of GGDPS on cancer cell survival and function. Two aromatic bisphosphonates were identified as potent inhibitors of GGDPS in enzyme and cellular assays. Apoptosis hallmarks such as PARP cleavage and DNA fragmentation demonstrated that GGDPS inhibition induces apoptosis in K562 chronic myeloid leukemia cells through GGPP depletion and FPP accumulation. Isobologram analysis and enhanced impairment of protein geranylgeranylation showed that GGDPS inhibition is synergistic with the inhibition of HMGCR. Migration assays, transwell assay and large scale digital cell analysis system microscopy, demonstrated that GGDPS inhibition interferes with MDA-MB-231 breast cancer cell migration. Increased LC3-II expression showed that FDPS and GGDPS inhibition induces autophagy in PC3 prostate and MDA-MB-231 breast cancer cells. Inhibition of autophagy enhances the toxic effects of GGDPS inhibition as measured by MTT assay. Propidium iodine staining of DNA and immunostaining of cell cycle proteins such as p27 did not show significant effects of GGDPS inhibition on cell cycle progression. Importantly, exogenous addition of GGPP prevented most of the effects observed with GGDPS inhibition, suggesting specific inhibition of GGDPS by our bisphosphonate inhibitors. The data obtained herein suggest that GGDPS can be targeted to interfere with the progression of cancer cells.

Keywords

bisphosphonate, digeranyl bisphosphonate, geranylgeranyl diphophate, geranylgeranyl diphosphate synthase, statin

Pages

xiv, 148 pages

Bibliography

Includes bibliographical references (pages 136-148).

Copyright

Copyright 2010 Amel Dudakovic

Included in

Pharmacology Commons

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