Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells.

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G0/G1 phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G0/G1 phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G0/G1 phase.

Seasonal biochemical plasticity of a flight muscle in a bat, Murina leucogaster.

Cellular and biochemical responses of the pectoral muscle to variation in seasonal activity were studied in the bat, Murina leucogaster ognevi. We collected bats in mid-hibernation (February), end-hibernation (April), and mid-summer (August) to track major activity periods in their annual cycle. Our findings indicated that myofiber cross-sectional area decreased to 68% between mid- and end-hibernation, but returned to the winter level in mid-summer. Total soluble protein and total RNA concentrations were not altered over these sampling periods. Oxidative potential gauged by citrate synthase activity increased 1.47-fold from mid- to end-hibernation and then remained at the similar level in mid-summer. Glycolytic potential gauged by lactate dehydrogenase activity changed little between mid- and end-hibernation but increased 1.42-fold in summer, compared with the winter level. Thus, the myofibers underwent disuse atrophy during hibernation, while enzymatic catalytic function recovered towards the level of mid-summer.

Effects of KR-30035, a novel multidrug-resistance modulator, on the cardiovascular system of rats in vivo and on the cell cycle of human cancer cells in vitro.

The present study was performed to evaluate the adverse effects of KR-30035, a multidrug-resistance modulator, on the cardiovascular system in vivo, along with its effect on paclitaxel-induced cell cycle arrest in cultured cancer cells. In anesthetized rats, KR-30035 was about 10-fold less potent than verapamil in lowering blood pressure (i.v. ED20: 0.320+/-0.052 and 0.034+/-0.005 mg/kg, respectively) and in producing electrocardiogram changes. In conscious spontaneously hypertensive rats, verapamil caused a significant antihypertensive effects at the doses tested (p.o. ED20, 7.8+/-4.0 mg/kg), whereas KR-30035 did not significantly change either the blood pressure or the heart rate at any doses tested (up to 100 mg/kg). The estimated i.v. LD50 values in mice were 5.9 and 48.9 mg/kg for verapamil and KR-30035, respectively. In the presence of 10 microM KR-30035, paclitaxel (1 microM) when added to cultures of HCT15/CL02 human cancer cells greatly shifted the cell population from the G0/G1 phases towards G2/M phases (from 42.4, 30.3 and 27.3 to 14.6, 21.5 and 63.9% for the G0/G1, S and G2/M phases, respectively), with a similar magnitude to that of 10 microM verapamil (14.0, 15.7 and 70.3%, respectively). These results suggest that KR-30035 has weaker in vivo effects on the cardiovascular system compared with verapamil, while potentiating the G2/M arresting effect of paclitaxel on the cell cycle.

Nuclear factor kappa B-mediated kainate neurotoxicity in the rat and hamster hippocampus.

Administration of the excitotoxin kainate produces seizure activity and selective neuronal death in various brain areas. We examined the degeneration pattern of hippocampal neurons following systemic injections of kainate in the hamster and the rat. As reported, treatment with kainate resulted in severe neuronal loss in the hilus and CA3 in the rat. While the hilar neurons were also highly vulnerable to kainate in the hamster, neurons in the CA1 area, but not CA3, were highly sensitive to kainate. In both animals, immunoreactivity to anti-p50 nuclear factor kappa B antibody was increased in nuclei of the hilar neurons within 4 h following administration of kainate. Kainate treatment also increased the nuclear factor kappa B immunoreactivity in hamster CA1 neurons and rat CA3 neurons 24 h later. Neurons showing intense nuclear factor kappa B signal were stained with acid fuchsin. Kainate also increased DNA binding activity of p50 and p65 nuclear factor kappa B in the nuclear extract of the hippocampal formation as analysed by electrophoretic mobility shift assay in the hamster, suggesting that activation of nuclear factor kappa B may contribute to kainate-induced hippocampal degeneration. Administration of 100 nmol dizocilpine maleate 3 h prior to kainate attenuated kainate-induced activation of nuclear factor kappa B and neuronal death in CA1 in the hamster. The present study provides evidence that the differential vulnerability of neurons in the rat and the hamster hippocampus to kainate is partly mediated by mechanisms involving N-methyl-D-aspartate-dependent activation of nuclear factor kappa B.

P-glycoprotein (Pgp) does not affect the cytotoxicity of flavonoids from Sophora flavescens, which also have no effects on Pgp action.

Sophoraflavanone, kurarinone (GS08), norkurarinol (GS11), kurarinol (GS12) and kushenol K are cytotoxic flavonoids isolated from Sophora flavescens. In this study, we tested the cytotoxicity of those flavonoids to human cancer cells including P-glycoprotein (Pgp)-expressing HCT15 cells and its multidrug resistant subline, HCT15/CL02 cells. HCT15/CL02 cells revealed resistance to GS08, GS11 and GS12 about 2 fold in comparison with HCT15 cells. Nonetheless, verapamil, a Pgp inhibitor, could not increase the cytotoxicity of all the flavonoids tested. We also investigated that the flavonoids could modulate the Pgp action. At nontoxic concentrations, the flavonoids could not effect on the cytotoxicity of paclitaxel, a well-known Pgp-substrate. The flavonoids also had no effects on the accumulation of rhodamine 123 in all the cells tested at 10 microM. From the results, we concluded that Pgp had no effect on the cytotoxicity of the flavonoids, and the flavonoids also had no effect on the action of Pgp. Our results also suggested that HCT15/CL02 cells had additional mechanisms for drug resistance distinct from Pgp overexpression.

Korean red ginseng saponins with low ratios of protopanaxadiol and protopanaxatriol saponin improve scopolamine-induced learning disability and spatial working memory in mice.

The effects of two ginseng saponins having a different ratio of protopanaxadiol (PD) and protopanaxatriol saponins (PT) on the learning impairment induced by scopolamine, and learning and memory in mice were investigated in a passive avoidance task and a Morris water maze task. The ratio of PD and PT was 1.24 and 1.46, respectively. Before training, the ginseng saponins were administered intraperitoneally at doses of 50 and 100 mg/kg. The two saponins improved the scopolamine-induced learning impairment at different dosages in mice, 50 and 100 mg/kg, respectively. However, the two saponins did not show a favorable effect on learning and memory in normal mice. Korean red ginseng saponin with a low PD/PT ratio had an improving effect on spatial working memory, but the saponin with a high PD/PT ratio did not. This finding suggests that the PD/PT ratio of the ginseng saponins may be an important factor in the pharmacological role of red ginseng as a medicinal herb.

Substance P augments nitric oxide production and gene expression in murine macrophages.

We investigated the effects of substance P (SP) on nitric oxide (NO) synthase activity in macrophages by measuring the production of nitrite and the expression of inducible NO synthase (iNOS) mRNA and protein. In LPS-activated macrophages, SP stimulated NO production in time and concentration dependent manners. These SP effects were blocked by a specific NK-1 receptor antagonist. Furthermore, SP stimulation increased the levels of both iNOS mRNA and iNOS protein. These results demonstrate that SP can increase LPS induced NO production in macrophages by augmenting the induction of iNOS expression. We also examined the role of SP on acute-cold stress induced altered production of NO by mouse peritoneal macrophages. SP enhanced the LPS-induced macrophages NO production from stressed mice relative to the non-stressed mice. These results suggest that SP may have an important modulatory role in production of NO by macrophages.

Effects of flavonoids on the growth and cell cycle of cancer cells.

In this study, we investigated the cytotoxicities of flavone (F01), 3-hydroxyflavone (F02), 6- hydroxyflavone (F03), 7-hydroxyflavone (F04), 3,6-dihydroxyflavone (F05), 5,7-dihydroxyflavone (F06) and 5,6,7-trihydroxyflavone (F07) to human cancer cells including P- glycoprotein (Pgp)-expressing HCT15 cells and its multidrug resistant subline, HCT15/CL02 cells. We also examined the effects of those flavonoids on the cell cycle of these cancer cells. HCT15/CL02 cells did not reveal resistance to all the flavonoids tested in comparison with HCT15 cells. In cell cycle analysis, all the flavonoids tested, except F01 and F04, reduced the G0/G1 population of SF295 cells at growth inhibitory concentrations, and increased G2/M (F02, F03 and F06) or S (F05 and F07) populations. In addition, F02 and F03 decreased the G2/M and G0/G1 population, and increased the S and G2/M population in HCT15 cells, respectively. Meanwhile, in HCT15/CL02 cells, F02 and F03 decreased the G0/G1 populations and increased the S population. In conclusion, we deemed that the flavonoids tested had diverse cytotoxic mechanisms, and exerted their cell growth inhibitory or killing activity by distinctive ways in different cells.

Synthetic peptides of human papillomavirus type 18 E6 harboring HLA-A2.1 motif can induce peptide-specific cytotoxic T-cells from peripheral blood mononuclear cells of healthy donors.

To identify cytotoxic T-cell (CTL) epitopes against human papillomavirus type 18 (HPV 18) E6 protein that might be useful for developing peptide-based vaccine against HPV 18 infection, 18 peptides which possibly contain CTL epitopes were selected on the basis of previously described human leukocyte antigen (HLA)-A2.1-binding motif and chemically synthesized. In the binding assay of the synthetic peptides, 8 out of 18 synthetic peptides enhanced the expression of HLA-A2.1 molecules on T2 cell surface, which implies that these peptides were able to bind the HLA molecules. Those peptides having good binding affinity to HLA-A2.1 were tested for their ability to activate CTLs which were isolated from peripheral blood mononuclear cells (PBMCs) of healthy blood donors and to kill the target T2 cells pulsed with the same peptide. Five out of eight tested peptides activated CTLs and killed the target cells.

The bisbenzylisoquinoline alkaloids, tetrandine and fangchinoline, enhance the cytotoxicity of multidrug resistance-related drugs via modulation of P-glycoprotein.

The occurrence of resistance to chemotherapeutic drugs is a major problem for successful cancer treatment and reducing drug accumulation by P-glycoprotein (P-gp) is one of the major mechanisms of multidrug resistance (MDR). The present study was performed to evaluate the MDR-reversal abilities of two bisbenzylisoquinoline alkaloids, tetrandine (TET) and fangchinoline (FAN), compared with verapamil (VER), a well-known P-gp modulator. TET (3.0 microM), FAN (3.0 microM) and VER (10.0 microM) reduced the paclitaxel (TAX) concentration required to achieve 50% inhibition of cell growth (EC50) to HCT15 (P-gp-positive) cells about 3100-, 1900- and 410-fold, and these compounds also reduced the EC50 value of actinomycin D (AMD) about 36.0-, 45.9- and 18.2-fold in the cells, respectively. Meanwhile, TET, FAN and VER had no effect on the cytotoxicity of the drugs to SK-OV-3 (P-gp-negative) cells. On the other hand, TET (3.0 microM), FAN (3.0 microM) and VER (10.0 microM) similarly enhanced the accumulation rates of rhodamine 123, a well known P-gp substrate, in HCT15 cells (200-250%). After efflux for 2 h with fresh medium, TET and FAN also enhanced the residual rate of rhodamine 123 about 5.0- and 2.6-fold in comparison with control, respectively. TET, FAN and VER could not affect the accumulation and residual rate of rhodamine 123 in SK-OV-3 cells. From the result, we conclude that TET and FAN enhanced the cytotoxicity of MDR-related drugs via modulation of P-gp.

Behavior and muscle performance in heterothermic bats.

Body temperatures of winter-resident Korean bats typically range from 10 degrees to 40 degrees C between August and September and from 3 degrees to 15 degrees C between January and April. To learn how behavior and the motor systems of heterothermic bats respond to this body-temperature variation, we examined whole-organism performance and the temperature-dependence of contractile properties of flight muscle in Murina leucogaster ognevi. In winter and midspring, the lowest limits of body temperature were 8 degrees C for biting and crawling, 16 degrees C for visually observable shivering, 22 degrees C for wing flapping (without powered flight), and 28 degrees C for aerial flight. In summer, the lowest temperature limits changed little for biting and wing flapping, but the temperature limits increased about 3 degrees C for crawling, shivering, and flight. Maximum isometric tetanic tension of the isolated biceps brachii muscle was almost insensitive to tissue temperatures between 10 degrees and 40 degrees C, with an average temperature coefficient of 1.02 in summer and of 0.96 in winter. Rate of tetanic tension production between 10 degrees and 40 degrees C and shortening velocity and power between 15 degrees and 25 degrees C were temperature sensitive, with average temperature coefficients of 1.3-2.3. Seasonal differences in contractile properties within each temperature were not significant, except for maximum tetanic tension at 30 degrees - 40 degrees C. Thus, the motor system of the bats had functional capacity over the range of body temperature experienced in winter to summer. The temperature-dependence of behavior was consistent with muscle physiology. The defensive behaviors, like biting and crawling, observed at 8 degrees - 12 degrees C body temperature could be exerted by using temperature-independent tetanic tension, whereas activities, such as flight, that require power generation would be restricted to higher body temperatures by temperature-sensitive rate properties. Some rate processes appeared to be more temperature sensitive in summer than in winter.

Reversal of multidrug resistance by novel verapamil analogs in cancer cells.

The present study was performed to evaluate the ability of KR-30032 and KR-30035 to overcome multidrug resistance (MDR) by measuring the cytotoxicity and the accumulation rate of rhodamine. Additionally, the adverse cardiac toxicity of KR-30032 and KR-30035 was evaluated by measuring the changes of tension in isolated rat aorta and left ventricular pressure (LVP) in guinea pig heart. KR-30035 potentiated the paclitaxel-induced cytotoxicity to HCT15 [P-glycoprotein (P-gp)-expressed cells] to over 15-fold greater than that of verapamil and KR-30032 was equipotent with verapamil (EC50: 0.07, 5.0 and 3.3 nM at 1.0 microg/ml). KR-30032 and KR-30035 were without effect on cytotoxicity to SK-OV-3 cells (P-gp-non-expressing cells), as well as to tamoxifen-induced cytotoxicity in the above cell types. Maximal rhodamine accumulation rates with KR-30032, KR-30035 and verapamil were 290, 291 and 271% in HCT15 cells; and 451, 970 and 440% in HCT15/CL02 cells, respectively. KR-30032 and KR-30035 were 20- to 25-fold less potent than verapamil in relaxing aorta (EC50: 8.13, 6.40 and 0.32 microM, respectively) and were 12- to 35-fold less potent than verapamil in decreasing LVP in isolated hearts (EC50: 41.8, 14.1 and 1.2 microM, respectively). The results of this study suggest that KR-30032 and KR-30035 are active modulators of MDR with potentially minimal cardiovascular toxicity.

Novel multidrug-resistance modulators, KR-30026 and KR-30031, in cancer cells.

The present study was performed to evaluate the ability of KR-30026 and KR-30031 to overcome multidrug resistance (MDR) by measuring the cytotoxicity of paclitaxel and the rate of rhodamine accumulation, which were then compared with verapamil. KR-30026 potentiated the paclitaxel-induced cytotoxicity of HCT15 to over 60 fold greater than that of verapamil, and KR-30031 was equipotent with verapamil (EC50: 0.00066, 0.04 and 0.05 nM at 4.0 micrograms/ml, respectively). KR-30026 and KR-30031 were without effect on paclitaxel-induced cytotoxicity to SK-OV-3 cells, as well as on tamoxifen-induced cytotoxicity to HCT15, HCT15/CL02 and SK-OV-3 cells. Maximal rhodamine accumulation by KR-30026, KR-30031 and verapamil were similar in HCT15 cells, while KR-30026 was more potent than verapamil in HCT15/CL02 cells (721 and 440%, respectively). To evaluate the cardiac toxicity of KR-30026 and KR-30031, the changes of tension in isolated rat aorta and left ventricular pressure (LVP) in guinea pig heart were determined; KR-30026 and KR-30031 were 15-40 and 25-70 fold less potent than verapamil, respectively. These results suggest that KR-30026 and KR-30031 are active modulators of MDR with potentially minimal cardiovascular toxicity.

Hypothermia-induced changes of afferent sensory transmission to the SI cortex of rats and hamsters.

The effects of acute lowering of body temperature on afferent sensory transmission to the primary somatosensory cortex were determined quantitatively in anaesthetized rats and hamsters. Rats showed no change in afferent sensory transmission until 27 degrees C, but dramatic suppression between 26 degrees C and 22 degrees C, reaching 100% inhibition at 21 degrees C. Hamsters exhibited gradual suppression of sensory transmission from 34 degrees C to 18 degrees C, reaching 95% inhibition at 18 degrees C. Differential effects were also observed during rewarming up to 37 degrees C. Response latencies were also differentially affected during hypothermia in rats and hamsters. These results suggest the presence of inherently different neural mechanisms to process somatosensory information during transient lowering of body temperature between hibernators and non-hibernators.