Different analogues of farnesyl pyrophosphate inhibit squalene synthase and protein:farnesyltransferase to different extents.

The inhibitory potency of farnesyl pyrophosphate analogues was investigated on two farnesyl pyrophosphate-consuming enzymes: squalene synthase, a secondary regulation site in the cholesterol synthesis pathway, and protein:farnesyl transferase, which plays a role in the function of Ras-proteins. For the transferase determination a rapid in vitro assay, using Sepharose-bound Ras-peptides, was developed. The distinct farnesyl pyrophosphate analogues showed a different order of potency in the inhibition of these two enzymes. Using the farnesyl transferase assay with pre-p21Ha-ras as substrate the same result was obtained. The difference observed in the in vitro assays was also reflected in the inhibition of cholesterol synthesis, protein prenylation in general and Ha-ras farnesylation in Rat-1.H-ras13 cells, a rat fibroblast cell line that overproduces human p21Ha-ras. This work shows that farnesyl pyrophosphate analogues can be developed for specific inhibition of different processes such as cholesterol synthesis and protein prenylation.

Pravastatin inhibited the cholesterol synthesis in human hepatoma cell line Hep G2 less than simvastatin and lovastatin, which is reflected in the upregulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and squalene synthase.

The possible difference between lovastatin (mevinolin, MK-803), simvastatin (MK-733) and pravastatin (CS-514), all chemically-related competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, were tested in the human hepatoma cell line Hep G2, which is often used as a model for the human hepatocyte. After an 18-hr incubation of the cells with the drugs, pravastatin (IC50 = 1900 nM) was less potent than simvastatin and lovastatin (IC50 = 34 and 24 nM, respectively) in inhibiting the sterol synthesis. As a consequence of this inhibition, the HMG-CoA reductase mRNA levels and squalene synthase activity, both negatively-regulated by sterols, were increased equally by simvastatin and lovastatin, whereas the induction by pravastatin was much less. In contrast, there were fewer differences between the compounds in inhibiting HMG-CoA reductase activity, when assayed directly in Hep G2 cell homogenates (IC50 values = 18, 61 and 95 nM for simvastatin, lovastatin and pravastatin, respectively). Moreover, in experiments with human hepatocytes in primary culture the IC50 values for inhibition of the cholesterol synthesis by simvastatin and pravastatin were of the same order of magnitude (23 and 105 nM, respectively). The results are therefore explained as follows: the three drugs act in the same way within the Hep G2 cell in terms of inhibiting HMG-CoA reductase and their subsequent effect on the feedback regulation of the cholesterol synthesis, i.e. increasing squalene synthase and HMG-CoA reductase mRNA. However, pravastatin seems to be less able to enter the cells compared with simvastatin and lovastatin, possibly because of the higher hydrophobicity of the latter compounds. The observation with human hepatocytes suggests that in Hep G2 cells a specific hepatic transporter is missing. On one hand the human hepatoma cell line Hep G2 has proved to be a good model for the study of the feedback regulation of enzymes of the cholesterol biosynthetic pathway such as HMG-CoA reductase and squalene synthase, but, on the other hand seems to be less suitable as a model for the study of specific uptake of drugs, e.g. the vastatins, in human hepatocytes.

Butyrate stimulates the secretion of apolipoprotein (apo) A-I and apo B100 by the human hepatoma cell line Hep G2. Induction of apo A-I mRNA with no change of apo B100 mRNA.

Addition of sodium butyrate to the culture medium of the human hepatoma cell line Hep G2 resulted in a time- and dose-dependent increase in the secretion of apolipoprotein A-I (apo A-I) and apolipoprotein B100 (apo B100). After a 24 h preincubation period, a 2.4- and 2.2-fold increase in the secretion of apo A-I and apo B100 respectively was obtained during the next 24 h in the presence of 2 mM-sodium butyrate. Secretion of albumin, fibrinogen or [35S]methionine-labelled newly synthesized proteins was unaffected or only marginally affected, indicating that the effect of butyrate on apo A-I and apo B100 is not part of a general effect on protein synthesis and secretion. In structure-function studies, butyrate was found to be the most potent inducer among various straight-chain carboxylic acids. Hydroxylated, aminated and otherwise modified butyrate derivatives were inactive. The enhanced accumulation of apo A-I and apo B100 in the culture medium could not be explained by changes in the uptake and degradation of the synthesized apolipoproteins or by alterations in the secretion of possible intracellular pools. In addition, [35S]methionine incorporation studies indicated that synthesis and/or secretion of newly synthesized apo A-I and apo B100 is enhanced in the presence of butyrate. The apo A-I mRNA level was increased 2.3-fold upon treatment with 2 mM-butyrate for 48 h, suggesting regulation at (post-)transcriptional level. In contrast, no change in the level of apo B100 mRNA in butyrate-treated cells was observed, indicating regulation at translational or co- or post-translational level. We propose that the effect of butyrate on the secretion of apo A-I and apo B100 by Hep G2 results from two different regulatory mechanisms.