Bile acids have been implicated in the development of colorectal cancers. We investigated the expression of the transcription factor regulated by bile acids, farnesoid X receptor (FXR), as well as other components of this pathway in human colorectal tumors and cell lines. The most significant changes were a decrease in FXR mRNA levels in adenomas (5-fold average) and carcinomas (10 fold average) and an increase in peroxisome proliferator activated receptor-gamma (2-fold average). FXR was not expressed in undifferentiated colon adenocarcinoma SW480 cells and metastasis derived SW620 cells. In Caco-2 and HT-29 cells, the level of FXR expression increased with the degree of differentiation. Intestinal bile acid binding protein was activated by chenodeoxycholic acid and the synthetic FXR agonist GW4064 in Caco-2 and HT-29 but not in SW cells unless FXR was transfected. The down-regulation of the nuclear receptor FXR in colon cancer might be of clinical and pharmacological importance.

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The orphan nuclear receptors FXR and LXRalpha have become challenging targets for the discovery of new therapeutic agents. Bile acids and hydroxysterol intermediates are the respective natural ligands of these two structurally and functionally closely related receptors. Both FXR and LXRalpha; are thought to play a major role in the control of cholesterol catabolism by regulating the expression of cholesterol 7alpha-hydroxylase, the rate limiting enzyme of bile acid synthesis. Reverse cholesterol transport might also be affected by FXR and LXR since they control the expression of PLTP and CETP, two proteins involved in the transfer of phospholipid, cholesterol and cholesteryl esters among plasma lipoproteins. A new class of potent synthetic activators of FXR, the 1,1-bisphosphonate esters, has been discovered which up regulate the Intestinal Bile Acid Binding Protein gene (I-BABP) as demonstrated for chenodeoxycholic acid, however there are no known synthetic activators yet identified for LXRalpha. The evaluation of FXR as a potential target for the development of drugs affecting plasma cholesterol can take advantage of the fact that the activators of FXR (farnesol, bile acids and the 1,1-bisphosphonate esters) have been studied in various in vitro and in vivo models. Administration of chenodeoxycholic acid to animals and man did not result in the increase in plasma cholesterol expected from a decrease in cholesterol 7alpha-hydroxylase expression. Like farnesol, the 1,1-bisphosphonate esters increase the rate of degradation of HMGCoA reductase and have the unexpected property of inducing hypocholesterolemia in normal animals. The natural and synthetic FXR agonists trigger differentiation, inhibit cell proliferation and are potent inducers of apoptosis. The 1,1-bisphosphonate ester SR-45023A (Apomine) is presently being developed as an antineoplastic drug.

Apomine, a novel 1,1-bisphosphonate ester, has been shown to lower plasma cholesterol concentration in several species. Here we show that Apomine reduced the levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the rate-limiting enzyme in the mevalonate pathway, both in rat liver and in cultured cells. Apomine resembles sterols such as 25-hydroxycholesterol in its ability to potently accelerate the rate of HMGR degradation by the ubiquitin-proteasome pathway, a process that depends on the transmembrane domain of the enzyme. The similarity between Apomine and sterols in promoting rapid HMGR degradation extends to its acute requirements for ongoing protein synthesis and mevalonate-derived non-sterol product(s) as a co-regulator. Yet, at suboptimal concentrations, sterols potentiated the effect of Apomine in stimulating HMGR degradation, indicating that these agents act via distinct modes. Furthermore, unlike sterols, Apomine inhibited the activity of acyl-CoA:cholesterol acyltransferase in intact cells but not in cell-free extracts. Apomine stimulated the cleavage of the precursor of sterolregulatory element-binding protein-2 and increased the activity of low density lipoprotein receptor pathway. This Apomine-enhanced activation of sterol-regulatory element-binding protein-2 was prevented by sterols or mevalonate. Taken together, our results provide a molecular mechanism for the hypocholesterolemic activity of Apomine.In mammalian cells, cholesterol homeostasis is maintained by balancing cholesterol uptake and production. Cholesterol uptake is regulated through modulating the levels of the cell surface low density lipoprotein (LDL) 1 receptor (LDLR), and cholesterol synthesis is regulated primarily by changes in levels and activity of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) (1, 2). HMGR is an endoplasmic reticulum (ER) enzyme that catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA into mevalonate (MVA), the first committed step in the MVA pathway that leads to the synthesis of cholesterol as well as of essential non-sterol isoprenoid compounds (1, 2). When cells are starved for cholesterol and/or MVA, levels of HMGR and LDLR are elevated, thus increasing the rate of endogenous sterol synthesis and of LDL uptake (3, 4). Conversely, in cholesterol-replete cells, the levels of HMGR and LDLR decline, thereby lowering sterol production and LDL internalization.HMGR and LDLR are regulated at the transcriptional level by sterol-regulatory elements (SREs) in the promoter of their genes (1, 5). Specific transcription factors, designated SREbinding proteins (SREBPs), bind to these elements and activate transcription (5, 6). The SREBP family comprises three members that control different sets of genes. SREBP-2 regulates the transcription of genes mainly involved in sterol synthesis, whereas SREBP-1a and -1c regulate principally fatty acid biosynthetic genes (7). The nuclear, transcriptionally active SREBPs are derived from the NH 2 -terminal domain of large ER membrane-bound prec...

SR-12813 (tetra-ethyl 2-(3,5-di-tert-butyl-4-hydroxyphenyl)ethenyl-1, 1-bisphosphonate) lowers plasma cholesterol in five species. In this paper we investigate the underlying mechanism using Hep G2 cells. SR-12813 inhibited incorporation of tritiated water into cholesterol with an IC50 of 1.2 microM but had no effect on fatty acid synthesis. Furthermore, SR-12813 reduced cellular 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity with an IC50 of 0.85 microM. The inhibition of HMG-CoA reductase activity was rapid with a T1/2 of 10 min. After a 16-h incubation with SR-12813, mRNA levels of HMG-CoA reductase and low density lipoprotein (LDL) receptor were increased. The increased expression of LDL receptor translated into a higher LDL uptake, which can explain the primary hypocholesterolemic effect of SR-12813 in vivo. Western blot analysis indicated that the amount of HMG-CoA reductase protein rapidly decreased in the presence of SR-12813. Pulse-chase experiments with [35S]methionine showed that the T1/2 of HMG-CoA reductase degradation decreased in the presence of SR-12813 from 90 to 20 min. Pre-incubation with 50 microM of lovastatin did not prevent the effects of SR-12813 on HMG-CoA reductase degradation, indicating that the compound does not need mevalonate-derived regulators for its action. It is concluded that SR-12813 inhibits cholesterol synthesis mainly by an enhanced degradation of HMG-CoA reductase.

The liver X> or = receptor alpha (LXRalpha) is a nuclear receptor with a key role in bile acid biosynthesis and cholesterol metabolism. The present study investigated the expression and function of LXRalpha in the normal and malignant human breast. LXRalpha mRNA transcripts were detected by RT-PCR in nine breast carcinoma cell lines. The nucleotide sequence of the cloned PCR product was identical to the corresponding human LXRalpha cDNA sequence. Expression of LXRalpha protein was confirmed by immunoblot analysis of breast cancer cell lysates. LXRalpha mRNA was expressed in 14/15 (93%) of normal human breast mammoplasty specimens and in 11/15 (73%) of primary breast carcinomas. Oxysterol and nonsteroidal LXRalpha agonists at low micromolar concentrations inhibited proliferation of breast carcinoma cell lines in culture. The importance of LXRalpha signaling in cholesterol homeostasis and the observed expression of LXRalpha in normal breast tissue suggest that this nuclear oxysterol receptor has an important physiological function in the breast. LXRalpha gene expression is regulated by dietary fatty acids implicated in breast carcinogenesis and detection of LXRalpha expression in breast cancer cell lines and breast tumors in the present study indicates that LXRalpha may also be important in breast carcinogenesis. Inhibition of breast cancer cell proliferation suggests that pharmacological LXRalpha agonists may have potential preventive and/or therapeutic antitumor activity in breast cancer.

Apomine, a novel 1,1 bisphosphonate ester, increases the rate of degradation of HMG-CoA reductase, inhibiting the mevalonate pathway and thereby blocking cholesterol biosynthesis. We have investigated whether Apomine can induce myeloma cell apoptosis in vitro and modulate myeloma disease in vivo. Apomine induced a dose-dependent increase in apoptosis in NCI H929, RPMI 8226 and JJN-3 human myeloma cells. Apomine, unlike the bisphosphonate, alendronate, had no measurable effect on osteoclastic bone resorption in vitro. To investigate the effect of Apomine in vivo, 5T2MM murine myeloma cells were injected into C57BL/KaLwRij mice. After 8 weeks all animals had a serum paraprotein and were treated with Apomine (200 mg/kg), or vehicle, for 4 weeks. Animals injected with 5T2MM cells and treated with vehicle developed osteolytic bone lesions, reduced cancellous bone area, decreased bone mineral density (BMD) and increased osteoclast number. Apomine caused a decrease in serum paraprotein and a decrease in tumor burden. Apomine inhibited the development of osteolytic lesions and prevented the tumor-induced decreases in BMD. Apomine had no effect on osteoclast number in contrast to what had been seen previously with the bisphosphonate, zoledronic acid, suggesting that these are direct effects of Apomine on myeloma cells. This demonstrates that Apomine is able to promote myeloma cell apoptosis in vitro and inhibit the development of multiple myeloma and lytic bone disease in vivo. The use of bisphosphonate esters such as Apomine represents a novel therapeutic approach in the treatment of myeloma and, indirectly, the associated bone disease. ' 2007 Wiley-Liss, Inc.

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