A beating heart cell model to predict cardiotoxicity: effects of the dietary supplement ingredients higenamine, phenylethylamine, ephedrine and caffeine.

Some dietary supplements may contain cardiac stimulants and potential cardiotoxins. In vitro studies may identify ingredients of concern. A beating human cardiomyocyte cell line was used to evaluate cellular effects following phenylethylamine (PEA), higenamine, ephedrine or caffeine treatment. PEA and higenamine exposure levels simulated published blood levels in humans or animals after intravenous administration. Ephedrine and caffeine levels approximated published blood levels following human oral intake. At low or midrange levels, each chemical was examined plus or minus 50 µM caffeine, simulating human blood levels reported after consumption of caffeine-enriched dietary supplements. To measure beats per minute (BPM), peak width, etc., rhythmic rise and fall in intracellular calcium levels following 30 min of treatment was examined. Higenamine 31.3 ng/ml or 313 ng/ml significantly increased BPM in an escalating manner. PEA increased BPM at 0.8 and 8 µg/ml, while 80 µg/ml PEA reduced BPM and widened peaks. Ephedrine produced a significant BPM dose response from 0.5 to 5.0 µM. Caffeine increased BPM only at a toxic level of 250 µM. Adding caffeine to PEA or higenamine but not ephedrine further increased BPM. These in vitro results suggest that additional testing may be warranted in vivo to further evaluate these effects.

K-ras 4A and 4B mRNA levels correlate with superoxide in lung adenocarcinoma cells, while at the protein level, only mutant K-ras 4A protein correlates with superoxide.

The K-ras gene is frequently mutated in lung and other cancers. K-ras protein includes two splice variants, K-ras 4A and 4B. While K-ras 4B is more widely expressed, recent evidence implicates K-ras 4A in lung tumorigenesis. We found that K-ras 4A protein has a wide range of expression in a large panel of human lung adenocarcinoma cell lines. In cell lines with mutant K-ras, but not those with wildtype K-ras, the K-ras 4A protein had a strong positive correlation with levels of cellular superoxide. We investigated whether K-ras 4A protein was involved in superoxide production, or alternatively was modulated by elevated superoxide. Experiments with small interfering RNA targeting K-ras 4A did not confirm its role in superoxide generation. However, decreasing cellular superoxide with the scavenger Tiron tended to reduce levels of K-ras 4A protein. K-ras 4A and 4B mRNA were also quantified in a number of NSCLC cell lines. 4A mRNA correlated with 4A protein only in K-ras-mutant cells. K-ras 4A mRNA also correlated with superoxide, but with no difference between cell lines with mutant or wildtype K-ras. K-ras 4B mRNA correlated with 4A mRNA and with superoxide, in both K-ras mutant and wildtype cells. The results are consistent with superoxide directly or indirectly up-regulating expression of all K-ras genes, and also increasing the stability of K-ras 4A mutant protein selectively.

The hypervariable region of K-Ras4B is responsible for its specific interactions with calmodulin.

K-Ras4B belongs to the family of p21 Ras GTPases, which play an important role in cell proliferation, survival, and motility. The p21 Ras proteins, such as K-Ras4B, K-Ras4A, H-Ras, and N-Ras, share 85% sequence homology and activate very similar signaling pathways. Only the C-terminal hypervariable regions differ significantly. A growing body of literature demonstrates that each Ras isoform possesses unique functions in normal physiological processes as well as in pathogenesis. One of the central questions in the field of Ras biology is how these very similar proteins achieve such remarkable specificity in protein-protein interactions that regulate signal transduction pathways. Here we explore specific binding of K-Ras4B to calmodulin. Using NMR techniques and isothermal titration calorimetry, we demonstrate that the hypervariable region of K-Ras4B contributes in a major way to the interaction with calmodulin, while the catalytic domain of K-Ras4B provides a way to control the interaction by nucleotide binding. The hypervariable region of K-Ras4B binds specifically to the C-terminal domain of Ca(2+)-loaded calmodulin with micromolar affinity, while the GTP-gamma-S-loaded catalytic domain of K-Ras4B may interact with the N-terminal domain of calmodulin.

Elevated K-ras activity with cholestyramine and lovastatin, but not konjac mannan or niacin in lung--importance of mouse strain.

Our previous work established that hypocholesterolemic agents altered K-ras intracellular localization in lung. Here, we examined K-ras activity to define further its potential importance in lung carcinogenesis. K-ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeks, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% konjac mannan (KM), or were injected with lovastatin 25mg/kg three or five times weekly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeks, serum cholesterol was assayed with a commercial kit. Activated K-ras protein from lung was affinity precipitated with a Raf-1 ras binding domain-glutathione-S-transferase fusion protein bound to glutathione-agarose beads, followed by Western blotting, K-ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-ras activity increased 1.8-fold versus control (p=0.009). In normal lung with wild-type K-ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-ras activity increased 2.1-fold (p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-ras activity. These data are consistent with the concept that K-ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.

Optimization of a nonradioactive method for consistent and sensitive determination of activated K-ras protein.

Accurate measurement of activity of wild-type K-ras protein is important due to its tumor suppressor action in tissues such as lung. A published method by Taylor and co-workers uses plasmid-containing Escherichia coli cells to produce a glutathione-S-transferase/raf-1 ras binding domain (GST-RBD) fusion protein attached to glutathione beads to isolate activated ras protein. We systematically optimized the method before use on lung tissues. Changing the GST-RBD protein induction temperature from the original 37 to 30 degrees C produced a consistently greater yield of fusion protein. To improve stability of the GST-RBD beads so as to perform large-scale experiments, 0.1% NaN(3) was added. NaN(3)-treated beads retained full affinity for at least 24 days. Sensitivity was improved by using a polyvinylidene difluoride membrane rather than nitrocellulose for immunoblotting. We also compared our GST-RBD beads with two commercial assay kits and found that our beads had both superior sensitivity and reduced variability. In summary, our modification of the GST-RBD affinity method to recover activated K-ras greatly increased the yield of fusion protein, prolonged the useful life of GST-RBD beads to at least 24 days, and enhanced detection sensitivity.

Treatment with lovastatin, cholestyramine or niacin alters K-ras membrane association in mouse lung in a strain-dependent manner: results in females.

Hypocholesterolemic drugs may themselves increase (cholestyramine, CS) or decrease (lovastatin, Lov) peripheral tissue de novo cholesterol biosynthesis. This will alter the abundance of prenyl groups and potentially increase (CS) or decrease (Lov) K-ras membrane localization, with possible pro- or anti-carcinogenic effects (K-ras is a proto-oncogene frequently mutated in lung cancer). Female A/J, Swiss, and C57BL/6 mice were fed 2 or 4% CS, 1% niacin, or injected with Lov three (Lov-3x) or five (Lov-5x) times per week. After three weeks, serum cholesterol and triglycerides were determined enzymatically. Total, membrane, and cytoplasmic K-ras proteins were determined in lung homogenates by immunoprecipitation followed by Western blotting with a K-ras specific antibody. CS feeding increased membrane K-ras as hypothesized in A/J and C57BL/6 mice, but had no effect in Swiss mice. Lov failed in all three strains to reduce membrane K-ras, and resulted in an increase in total K-ras in A/J and C57BL/6 mice, while again lacking effect in Swiss mice. Niacin had no effect on K-ras protein in any mouse strain. These results differ from our published results for male mice of the same strains, particularly for A/J mice. Increased amounts of K-ras protein in the membrane fraction of A/J females (but not males) treated with either Lov or CS imply that if K-ras were to become mutated, CS could result in increased lung tumorigenesis and Lov would be less likely to be protective in females. In the light of these data, both sexes should be included in future animal and human chemoprevention trials.

Alterations in membrane-bound and cytoplasmic K-ras protein levels in mouse lung induced by treatment with lovastatin, cholestyramine, or niacin: effects are highly mouse strain dependent.

Agents that either increase (cholestyramine, CS) or decrease (lovastatin, Lov) de novo peripheral cholesterol synthesis may increase (CS) or decrease (Lov) ras protein membrane localization by altering protein prenylation, and potentially have pro- or anti-carcinogenic effects. Male A/J, Swiss, and C57/BL6 mice were treated with 2 or 4% CS, 1% dietary niacin, or 25mg/kg of Lov three times per week (Lov-3X) or five times per week (Lov-5X). After 3 weeks, serum cholesterol and triglycerides were determined enzymatically. Membrane and cytoplasmic K-ras proteins in lung were determined by immunoprecipitation followed by western blotting with a K-ras specific antibody. Results confirmed the hypothesis only in isolated instances. A/J mice had a significant 30% increase in cytoplasmic K-ras and a 40% decrease in membrane K-ras from Lov treatment, as predicted. C57/BL6 mice had a significant 77% increase in membrane K-ras, as expected from CS feeding. At variance with the hypothesis, Swiss mice had increased levels (3-28%) of membrane K-ras with all treatments (including Lov), and C57/BL6 mice treated with Lov had a 58-78% increase in cytoplasmic K-ras without any reduction in the levels of membrane K-ras. Niacin, predicted to have no effect on ras membrane localization, decreased cytoplasmic K-ras in A/J mice, increased both membrane and cytoplasmic K-ras in Swiss mice, and had no effect in C57/BL6 mice. Results may have differed from those predicted because of strain-dependent differences in response to the cholesterol-lowering agents. A difference in response among the mouse strains suggests that individual genetic differences may alter the effect of hypocholesterolemic agents on K-ras membrane localization, and potentially the risk of ras-dependent cancer.