3-Deoxyschweinfurthin B Lowers Cholesterol Levels by Decreasing Synthesis and Increasing Export in Cultured Cancer Cell Lines.

The schweinfurthins have potent antiproliferative activity in multiple glioblastoma multiforme (GBM) cell lines; however, the mechanism by which growth is impeded is not fully understood. Previously, we demonstrated that the schweinfurthins reduce the level of key isoprenoid intermediates in the cholesterol biosynthetic pathway. Herein, we describe the effects of the schweinfurthins on cholesterol homeostasis. Intracellular cholesterol levels are greatly reduced in cells incubated with 3-deoxyschweinfurthin B (3dSB), an analog of the natural product schweinfurthin B. Decreased cholesterol levels are due to decreased cholesterol synthesis and increased cholesterol efflux; both of these cellular actions can be influenced by liver X-receptor (LXR) activation. The effects of 3dSB on ATP-binding cassette transporter 1 levels and other LXR targets are similar to that of 25-hydroxycholesterol, an LXR agonist. Unlike 25-hydroxycholesterol, 3dSB does not act as a direct agonist for LXR α or ß. These data suggest that cholesterol homeostasis plays a significant role in the growth inhibitory activity of the schweinfurthins and may elucidate a mechanism that can be targeted in human cancers such as GBM.

Calcium and P-glycoprotein independent synergism between schweinfurthins and verapamil.

Schweinfurthins are intriguing natural products with anti-cancer activities and as yet incompletely understood mechanisms of action. We investigated whether inhibitors of P-glycoprotein (Pgp), in a manner analogous to other natural products, might enhance schweinfurthins' growth inhibitory actions by increasing intracellular schweinfurthin levels. Both the schweinfurthin-sensitive glioblastoma multiforme cell line SF-295 and relatively insensitive lung carcinoma cell line A549 were treated with 2 schweinfurthin analogs: 3-deoxyschweinfurthin B-p-nitro bis-stilbene (3dSB-PNBS) and 5'-methylschweinfurthin G (methyl-G). There was a synergistic enhancement of growth inhibition with the combination of the Pgp inhibitor verapamil and both analogs in SF-295 cells. Methyl-G, verapamil, and the combination did not result in alterations to intracellular calcium concentration. Verapamil increased the intracellular concentration of 3dSB-PNBS in both SF-295 and A549 cells in a Pgp-independent manner. Methyl-G, verapamil, and the combination do not result in increased ER stress. Methyl-G increased the intracellular concentration of a known Pgp substrate, Rhodamine 123 in SF-295 cells. Reduction of cellular cholesterol leads to the accumulation of Pgp substrates, as Pgp requires cholesterol for proper function. Since 3dSB enhances lovastatin-induced upregulation of the cholesterol efflux pump ABCA1, it is intriguing that co-treatment with cholesterol rescued the methyl-G-induced increase in Rhodamine 123 intracellular concentration. These studies support the hypothesis that verapamil potentiates the schweinfurthin growth inhibitory effect by increasing its intracellular concentration.

In vitro studies in a myelogenous leukemia cell line suggest an organized binding of geranylgeranyl diphosphate synthase inhibitors.

A small set of isoprenoid bisphosphonates ethers has been tested in the K562 chronic myelogenous leukemia cell line to determine their impact on isoprenoid biosynthesis. Five of these compounds inhibit geranylgeranyl diphosphate synthase (GGDPS) with IC50 values below 1 µM in enzyme assays, but in cells their apparent activity is more varied. In particular, the isomeric C-geranyl-O-prenyl and C-prenyl-O-geranyl bisphosphonates are quite different in their activity with the former consistently demonstrating greater impairment of geranylgeranylation in cells but the latter showing greater impact in the enzyme assays with GGDPS. Together, these findings suggest an organized binding of these inhibitors in the two hydrophobic channels of the geranylgeranyl diphosphate synthase enzyme.

Quantitative determination of isopentenyl diphosphate in cultured mammalian cells.

Isopentenyl diphosphate (IPP), an intermediate of the isoprenoid biosynthetic pathway (IBP), has several important biological functions, yet a method to determine its basal level has not been described. Here, we describe a nonradioactive and sensitive analytical method to isolate and specifically quantify IPP from cultured mammalian cells. This method applies an enzymatic coupling reaction to determine intracellular concentrations of IPP. In this reaction, geranylgeranyl diphosphate synthase catalyzes the formation of geranylgeranyl diphosphate (GGPP) from IPP and farnesyl diphosphate (FPP). Subsequently, geranylgeranyl protein transferase I conjugates GGPP with a fluorescently labeled peptide. The geranylgeranylated peptide can be quantified by high-performance liquid chromatography (HPLC) with a fluorescence detector, thereby allowing for IPP quantification. The detection lower limit of the fluorescence-labeled geranylgeranyl peptide is approximately 5 pg (~0.017 pmol). This method was used to examine the effects of IBP inhibitors such as lovastatin and zoledronate on intracellular levels of IPP. Inhibition of hydroxymethylglutaryl coenzyme A reductase (HMGCR) by lovastatin (50 nM) decreases IPP levels by 78% and 53% in K562 and MCF-7 cells, respectively. Whereas zoledronic acid (10 µM) increased IPP levels 12.6-fold when compared with untreated cells in the K562 cell line, an astonishing 960-fold increase was observed in the MCF-7 cells.

Functional evaluation of a fluorescent schweinfurthin: mechanism of cytotoxicity and intracellular quantification.

Schweinfurthins are potent inhibitors of cancer cell growth, especially against human central nervous system tumor lines such as SF-295 cells. However, the mechanisms through which these compounds impede cell growth are not fully understood. In an effort to understand the basis for the effects of schweinfurthins, we present a fluorescent schweinfurthin, 3-deoxyschweinfurthin B-like p-nitro-bis-stilbene (3dSB-PNBS), which displays biological activity similar to that of 3-deoxyschweinfurthin B (3dSB). These two schweinfurthins retain the unique differential activity of the natural schweinfurthins, as evidenced by the spindle-like morphological changes induced in SF-295 cells and the unaltered appearance of human lung carcinoma A549 cells. We demonstrate that incubation with 3dSB or 3dSB-PNBS results in cleavage of poly-ADP-ribose polymerase (PARP) and caspase-9, both markers of apoptosis. Coincubation of 3dSB or 3dSB-PNBS with the caspase-9 inhibitor (Z)-Leu-Glu(O-methyl)-His-Asp(O-methyl)-fluoromethylketone prevents PARP cleavage. Therapeutic agents that induce apoptosis often activate cellular stress pathways. A marker for multiple stress pathways is the phosphorylation of eukaryotic initiation factor 2α, which is phosphorylated in response to 3dSB and 3dSB-PNBS treatment. Glucose-regulated protein 78 and protein disulfide isomerase, both endoplasmic reticulum chaperones, are up-regulated with schweinfurthin exposure. Using the fluorescent properties of 3dSB-PNBS and dimethoxyphenyl-p-nitro-bis-stilbene (DMP-PNBS), a control compound, we show that the intracellular levels of 3dSB-PNBS are higher than those of Rhodamine 123 or DMP-PNBS in SF-295 and A549 cells.

Pleiotropic effects of a schweinfurthin on isoprenoid homeostasis.

The schweinfurthins, a family of natural products derived from the isoprenoid biosynthetic pathway (IBP), have marked growth inhibitory activity. However, the biochemical basis for the schweinfurthins cellular effects has remained ill-defined. Here, the effects of the synthetic schweinfurthin, 3-deoxyschweinfurthin (3dSB) on multiple aspects of isoprenoid homeostasis are explored. Cytotoxicity assays demonstrate a synergistic interaction between 3dSB and the HMG-CoA reductase inhibitor lovastatin but not with other IBP inhibitors in a variety of human cancer cell lines. The cytotoxic effects of 3dSB were enhanced in cells incubated in lipid-depleted serum. 3dSB was found to enhance the lovastatin-induced decrease in protein prenylation. In addition, 3dSB decreases intracellular farnesyl pyrophosphate and geranylgeranyl pyrophosphate levels in both established cell lines and primary cells. To determine whether 3dSB alters the regulation of expression of genes involved in isoprenoid homeostasis, real-time PCR studies were performed in human cell lines cultured in either lipid-replete or -deplete conditions. These studies demonstrate that 3dSB abrogates lovastatin-induced upregulation of sterol regulatory element-containing genes and lovastatin-induced downregulation of ABCA1. In aggregate, these studies are the first to demonstrate that a schweinfurthin exerts pleiotropic effects on isoprenoid homeostasis.

Biologically active biotin derivatives of schweinfurthin F.

As a prelude to efforts to identify schweinfurthin binding proteins, an ester conjugate and an amide conjugate of schweinfurthin F and biotin have been prepared by chemical synthesis. These compounds maintain activity in SF-295 cells comparable to the parent system, and display the lower potency in A549 cells that is a characteristic of the schweinfurthin pattern of activity.

Fluorescent schweinfurthin B and F analogs with anti-proliferative activity.

The natural tetracyclic schweinfurthins are potent and selective inhibitors of cell growth in the National Cancer Institute's 60 cell-line screen. At this time, the mechanism or cellular target that underlies this activity has not yet been identified, and efforts to illuminate the schweinfurthins' mode of action would benefit from development of potent fluorescent analogs that could be readily visualized within cells. This report describes the synthesis of fluorescent analogs of schweinfurthins B and F, and demonstrates that these compounds retain the potent and differentially toxic activities against select human cancer cells that are characteristic of the natural schweinfurthins. In addition, the synthesis of control compounds that maintain parallel fluorescent properties, but lack the potent activity of the natural schweinfurthin is described. Use of fluorescence microscopy shows differences between the localization of the active and relatively inactive schweinfurthin analogs. The active compounds localize in peripheral puncta which may identify the site(s) of activity.

Structural analogues of schweinfurthin F: probing the steric, electronic, and hydrophobic properties of the D-ring substructure.

The natural tetracyclic schweinfurthins are potent and selective inhibitors of cell growth in the National Cancer Institute's 60-cell line screen. An interest in determination of their cellular or molecular target has inspired our efforts to prepare both the natural products and analogues. In this paper, chemical synthesis of analogues modified in different olefinic positions, and preliminary results from studies of their biological activity, are reported.

Quantitative determination of geranyl diphosphate levels in cultured human cells.

Geranyl diphosphate (GPP), a 10-carbon isoprenoid, is a key intermediate in the isoprenoid biosynthetic pathway. This pathway, in addition to leading to sterol synthesis, results in the synthesis of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which serve as substrates for protein isoprenylation reactions. Basal levels of GPP in mammalian cells previously have been undetectable. Here we present a novel, sensitive, nonradioactive method which allows for measurement of GPP in mammalian cells. This methodology involves extraction of isoprenoids from cultured cells followed by enzymatic conjugation of GPP to a fluorescent dansylated-peptide via farnesyl transferase and quantification with high-performance liquid chromatography (HPLC). The lower limit of detection of GPP is 5 pg, or 0.015 pmol. Basal levels of GPP were determined in three human multiple myeloma cell lines (RPMI-8226, U266, H929). Treatment of cells with inhibitors of the isoprenoid biosynthetic pathway results in marked changes in GPP levels: the HMG-CoA reductase inhibitor lovastatin decreases GPP levels by over 50%, while the FPP synthase inhibitor zoledronic acid increases GPP levels 16- to 107-fold. This method also allows for the simultaneous measurement of GPP, FPP, and GGPP, thus leading to improved understanding of the pathway in a multitude of biological systems. Furthermore, as drugs targeting this pathway are developed, their biological activity can be more directly linked to effects on isoprenoid levels.

Synthesis and biological activity of a fluorescent schweinfurthin analogue.

Most of the natural schweinfurthins are potent and selective inhibitors of cell growth as measured by the National Cancer Institute's 60-cell line screen. Due to the limited supply of these natural products, we have initiated a program aimed at their synthesis. To date, this effort has led to the preparation of three natural schweinfurthins and more than 40 analogues, and assays on these compounds have afforded some understanding of structure-activity relationships in this family. Further development of schweinfurthins as chemotherapeutic agents would benefit from characterization of their mechanism(s) of action. This perspective led to development of a fluorescent schweinfurthin analogue that retains the differential activity of the natural products, and yet has properties that facilitate its visualization within cells.

Synthesis of fluorescently tagged isoprenoid bisphosphonates that inhibit protein geranylgeranylation.

Geminal bisphosphonates can be used for a variety of purposes in human disease including reduction of bone resorption in osteoporosis, treatment of fractures associated with malignancies of the prostate, breast, and lung, and direct anticancer activity against bone marrow derived malignancies. Previous research led to identification of some novel isoprenoid bisphosphonates that inhibit geranylgeranyl pyrophosphate (GGPP) synthesis and diminish protein geranylgeranylation. Described here is the synthesis of fluorescent anthranilate analogues of the most active isoprenoid bisphosphonates and examine their ability to impact post-translational processing of the small GTPases Ras, Rap1a, and Rab6. Similar to their non-fluorescent counterparts, some of these fluorescent isoprenoid bisphosphonates diminish protein geranylgeranylation. Their biological activity and fluorescent character suggest that they may be useful in studies of bisphosphonate localization both in cultured cells and in whole organisms.