farnesyl-pyrophosphate and digeranyl-bisphosphonate

Bisphosphonates are diphosphate analogs that inhibit the intermediate enzymes of the mevalonate pathway. Here, we compared the effects of a farnesyl diphosphate synthase inhibitor, zoledronate, and a geranylgeranyl diphosphate synthase (GGDPS) inhibitor, digeranyl bisphosphonate (DGBP), on lymphocytic leukemia cell proliferation and apoptosis. Both zoledronate and DGBP inhibited proliferation with DGBP doing so more potently. DGBP was markedly less toxic than zoledronate toward the viability of healthy human peripheral blood mononuclear cells. Addition of GGPP, but not farnesyl diphosphate (FPP), prevented the anti-proliferative effects of DGBP. Both GGPP and FPP partially rescued the effects of zoledronate. Co-treatment with DGBP and zoledronate was antagonistic. To further assess the effects of the bisphosphonates, we analyzed annexin V and propidium iodide staining via flow cytometry and found that DGBP induced apoptosis more potently than zoledronate. Western blots show that DGBP treatment altered expression and membrane affinity of some but not all geranylgeranylated small GTPases, activated caspases and increased ERK phosphorylation. Importantly, the anti-proliferative effects of DGBP were blocked by treatment with a caspase inhibitor and by treatment with a MEK inhibitor. Together, our findings indicate that DGBP is a more potent and selective compound than zoledronate in inducing apoptosis mediated through pathways that include caspases and MEK/ERK. These findings support the further development of GGDPS inhibitors as anticancer therapeutics.

Topics: Apoptosis; Caspases; Cell Line; Cell Line, Tumor; Cell Proliferation; Diphosphonates; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Imidazoles; Jurkat Cells; Leukocytes, Mononuclear; MAP Kinase Signaling System; Mevalonic Acid; Phosphorylation; Polyisoprenyl Phosphates; Sesquiterpenes; Terpenes; Zoledronic Acid

Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are branch point intermediates of isoprenoid biosynthesis. Inhibitors of isoprenoid biosynthesis, such as the statins and bisphosphonates, are widely used therapeutic agents. However, little is known about the degree to which they alter levels of upstream and downstream isoprenoids, including FPP and GGPP. Therefore, we developed a method to isolate and quantify FPP and GGPP from mammalian tissues. Tissues from mice were collected, snap frozen in liquid nitrogen, and stored at -80 degrees C. FPP and GGPP were isolated by a combined homogenization and extraction procedure and were purified with a C18 solid phase extraction column. Farnesyl protein transferase (FTase) or geranylgeranyl protein transferase I (GGTase I) were used to conjugate FPP and GGPP with fluorescent dansylated peptides. FPP and GGPP were quantified by high-performance liquid chromatography (HPLC). The respective concentrations of FPP and GGPP are as follows: 0.355+/-0.030 and 0.827+/-0.082 units of nmol/g wet tissues in brain, 0.320+/-0.019 and 0.293+/-0.035 units of nmol/g wet tissues in kidney, 0.326+/-0.064 and 0.213+/-0.029 units of nmol/g wet tissues in liver, and 0.364+/-0.015 and 0.349+/-0.023 units of nmol/g wet tissues in heart (means+/-SEM). This method allows for determination of FPP and GGPP concentrations in any tissue type and is sensitive enough to detect changes following treatment with inhibitors of isoprenoid biosynthesis.

Topics: Animals; Calibration; Diphosphonates; Diterpenes; Humans; K562 Cells; Male; Mice; NIH 3T3 Cells; Organ Specificity; Polyisoprenyl Phosphates; Rats; Reproducibility of Results; Sesquiterpenes; Solid Phase Extraction; Terpenes