Toshihiro Aiuchi

Involvement of tumor necrosis factor receptor-associated protein 1 (TRAP1) in apoptosis induced by β-hydroxyisovalerylshikonin

β-Hydroxyisovalerylshikonin (β-HIVS), a compound isolated from the traditional oriental medicinal herb Lithospermum radix, is an ATP non-competitive inhibitor of protein-tyrosine kinases, such as v-Src and EGFR, and it induces apoptosis in various lines of human tumor cells. However, the way in which β-HIVS induces apoptosis remains to be clarified. In this study, we performed cDNA array analysis and found that β-HIVS suppressed the expression of the gene for tumor necrosis factor receptor-associated protein 1 (TRAP1), which is a member of the heat-shock family of proteins. When human leukemia HL60 cells and human lung cancer DMS114 cells were treated with β-HIVS, the amount of TRAP1 in mitochondria decreased in a time-dependent manner during apoptosis. A similar reduction in the level of TRAP1 was also observed upon exposure of cells to VP16. Treatment of DMS114 cells with TRAP1-specific siRNA sensitized the cells to β-HIVS-induced apoptosis. Moreover, the reduction in the level of expression of TRAP1 by TRAP1-specific siRNA enhanced the release of cytochrome c from mitochondria when DMS114 cells were treated with either β-HIVS or VP16. The suppression of the level of TRAP1 by either β-HIVS or VP16 was blocked by N-acetyl-cysteine, indicating the involvement of reactive oxygen species (ROS) in the regulation of the expression of TRAP1. These results suggest that suppression of the expression of TRAP1 in mitochondria might play an important role in the induction of apoptosis caused via formation of ROS.

Apoptosis induced by arsenic trioxide in leukemia U937 cells is dependent on activation of p38, inactivation of ERK and the Ca2+-dependent production of superoxide

The mechanism of the induction of apoptosis by arsenic trioxide (As2O3), which was demonstrated recently to be an effective inducer of apoptosis in patients with leukemia, was examined in detail in human leukemia U937 cells. Upon treatment of U937 cells with 50 μM of As2O3, complete inactivation of the kinases ERK1 and ERK2 was detected within 30 min. p38 was activated within 3 hr, and the maximum activity was detected at 6 hr, when DNA fragmentation remained undetectable. Experiments with transfected cells that expressed constitutively activated MEK1 and a specific inhibitor of p38 also suggested that inactivation of ERKs and activation of p38 might be associated with the induction of apoptosis by As2O3. In contrast to the inactivation of ERKs and the activation of p38, activation of JNK by As2O3 appeared to protect cells against the induction of apoptosis. Treatment of U937 cells with As2O3 also caused the Ca2+‐dependent production of superoxide and intracellular acidification and a decrease in the mitochondrial membrane potential at the early stages of induction of apoptosis by As2O3. These changes preceded the release of cytochrome c from mitochondria and the activation of caspase‐3. It should be possible to exploit the unusual characteristics of the mechanism of induction of apoptosis by As2O3 in U937 cells by making use of synergistic effects of this compound with other inducers of apoptosis.

Sophoranone, extracted from a traditional Chinese medicine Shan Dou Gen, induces apoptosis in human leukemia U937 cells via formation of reactive oxygen species and opening of mitochondrial permeability transition pores.

Screening of various natural products in a search for novel inducers of apoptosis in human leukemia cells led us to identify the strong apoptosis‐inducing activity in a fraction extracted with methanol from the roots of Sophora subprostrata Chun et T. Chen. We purified the compound that induced apoptosis in human leukemia cells and identified it as sophoranone. Sophoranone inhibited cell growth and induced apoptosis in various lines of cells from human solid tumors, with 50% inhibition of growth of human stomach cancer MKN7 cells at 1.2 ± 0.3 μM. The growth‐inhibitory and apoptosis‐inducing activities of sophoranone for leukemia U937 cells were very much stronger than those of other flavonoids, such as daidzein, genistein and quercetin. At the early stages of treatment of U937 cells with sophoranone, reactive oxygen species were formed, mitochondrial permeability pores were opened and cytochrome c was released from mitochondria. Cytochrome c was also released upon treatment of isolated mitochondria with sophoranone. Inhibitors of complexes III and IV, but not complexes I and II, of the mitochondrial respiratory chain prevented the release of cytochrome c from isolated mitochondria by sophoranone, as well as the induction of apoptosis in U937 cells in response to sophoranone. Our results indicate that sophoranone might be a unique apoptosis‐inducing anticancer agent that targets mitochondria.

Transient Increase in Plasma Oxidized LDL During the Progression of Atherosclerosis in Apolipoprotein E Knockout Mice

Background—Plasma level of oxidized low-density lipoprotein (OxLDL) is a risk marker for cardiovascular diseases. The behavior of plasma OxLDL before disease progression has not been studied previously. Methods and Results—In this study, we developed a sensitive ELISA procedure for detecting mouse circulating OxLDL using a monoclonal antibody that recognizes oxidized phosphatidylcholine and a rabbit antimouse apolipoprotein B-48 polyclonal antibody. Apolipoprotein E knockout mice were fed on a chow diet for 40 weeks. Oil red O–positive lesions developed gradually by 20 weeks, and the percentage area covered by the lesions increased dramatically after 28 weeks; it covers 33.4% of the surface area by 40 weeks. The OxLDL level, measured after LDL fraction was isolated from each mouse, at 10 weeks was 0.015 ng/&mgr;g LDL. It increased 3-fold at 20 weeks of age and then decreased to the basal level by 40 weeks of age, suggesting that OxLDL appears before the development of atherosclerotic lesions. The occurrence of lipid peroxidation products, acrolein and oxidized phosphatidylcholines, in aortic tissue were revealed by immunohistochemical staining as early as 10 weeks. Conclusion—These results suggest that OxLDL might be involved in the early stages of progression of atherosclerotic lesions.

Fluorescence changes of rhodamine 6G associated with changes in membrane potential in synaptosomes

The intensity of rhodamine 6G fluorescence was found to be a useful scale for measuring the membrane potential in synaptosomes. The fluorescence of rhodamine 6G in synaptosomal suspensions increases with depolarization in the synaptosomes induced by the replacement of cations in the medium or by the addition of agents known to depolarize the membrane potential. Considering the character of the dye, we have derived an equation which gives the relation between the fluorescence intensity of the dye and the membrane potential. The change in membrane potential (diffusion potential) of synaptosomes was calculated using the equation. The calculated membrane potential was proportional to the logarithm of the K+ concentration above 20 mM, and the slope of membrane potential against log [K+] was about 52 mV per decade of concentration. The permeability ratio (Px/Pk; the ratio of the permeability constants of a given cation, X+, and K+) was estimated from the calculated membrane potential.

Mechanisms Involved in Apoptosis of Human Macrophages Induced by Lipopolysaccharide from Actinobacillus actinomycetemcomitans in the Presence of Cycloheximide

ABSTRACT Actinobacillus actinomycetemcomitans is a major periodontopathic bacterium with multiple virulence factors, including lipopolysaccharide (LPS). Previous reports have demonstrated that LPS induced apoptosis in a murine macrophage-like cell line, J744.1, as well as in peritoneal macrophages from C3H/HeN mice in the presence of cycloheximide (CHX). However, the detailed molecular mechanisms involved in the apoptosis of macrophages induced by LPS and CHX are not well known. To clarify the possible role of LPS in the induction of macrophage apoptosis, we investigated cell death induced by LPS from A. actinomycetemcomitans and CHX in human macrophage-like U937 cells, which were differentiated by 12-O-tetradecanoylphorbol 13-acetate (TPA), and also assessed the molecular mechanisms involved in the process. We found that TPA-differentiated U937 cells usually showed resistance to LPS-induced apoptosis. However, in the presence of CHX, LPS induced release of cytochrome c without modifying steady-state levels of Bcl-2, Bcl-xL, Bax, and Bak. Treatment with LPS in the presence of CHX also led to activation of caspase-3 and apoptosis via, in part, the CD14/toll-like receptor 4 (TLR4). The induction of cytochrome c release may have been due to dephosphorylation of Akt and Bad, which were cooperatively induced by CHX and LPS. However, endogenous tumor necrosis factor alpha- and Fas-induced signals, extracellular signal-regulated kinase kinase/mitogen-activated protein kinases and I-κBα/nuclear factor-κB (NF-κB) were not required for caspase-3-dependent apoptosis. These results emphasize the possible important role of the mitochondrial apoptotic pathway leading to caspase-3 activation in LPS-induced apoptosis of human macrophages in the presence of CHX.

Enhancement by tetraphenylboron of inhibition of mitochondrial respiration induced by 1-methyl-4-phenylpyridinium ion (MPP+)

The effect of tetraphenylboron (TPB(?)), an activator of a membrane transport of lipophilic cations, on the inhibition of mouse liver mitochondrial respiration induced by a neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+)), and by some structurally related compounds was studied. Of the compounds tested, MPP(+) and 4-phenylpyridine (4-PP) significantly inhibited the respiration in an ADP-activated oxidation of substrates (state 3). TPB(?), dose-dependently, shortened the lag time of MPP(+)-induced inhibition and thus lowered the concentrations of MPP(+) for the inhibition. However, TPB(?), even at the high concentration (10 ?M), did not significantly affect 4-PP-induced inhibition. Carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) blocked the respiratory inhibition by MPP(+), independent of K(+) concentration in the medium, and valinomycin blocked the inhibition only in the medium containing high K(+) concentration. Determination of the intramitochondrial MPP(+) concentration revealed about 1000-fold concentrated MPP(+) from that in the medium during the incubation with TPB(?), indicative of potentiation of MPP(+) transport into mitochondria by TPB(?). This might account for the enhancement of respiratory inhibition by MPP(+). In the case of 4-PP, it will penetrate the mitochondrial membrane and intrinsically inhibit the respiration, but cannot accumulate in mitochondria. The present results indicate that, although the inhibitory potency of MPP(+)per se is similar to 4-PP, MPP(+) will be highly concentrated within mitochondria by the membrane potential, as the drive force for its transport.

β‐Hydroxyisovalerylshikonin Is a Novel and Potent Inhibitor of Protein Tyrosine Kinases

β‐Hydroxyisovalerylshikonin (β‐HIVS), a compound isolated from Lithospermium radix, most efficiently induced cell‐death in two lines of lung cancer cells, namely, NCI‐H522 and DMS114, whereas shikonin was effective against a wide variety of tumor cell lines. During our studies of the mechanism of action of β‐HIVS on tumor cells, we found that this compound inhibited protein tyrosine kinase (PTK) activity. The tyrosine kinase activities of a receptor for EGF (EGFR) and v‐Src were strongly inhibited and that of KDR/Flk–1 was weakly inhibited by β‐HIVS. The inhibition by β‐HIVS of the activities of EGFR and v‐Src was much stronger than that by shikonin. The IC50values of β‐HIVS for EGFR and v‐Src were approximately 0.7 μM and 1 μM, respectively. Moreover, the inhibition of v‐Src by β‐HIVS was non‐competitive with respect to ATP. These results strongly suggest that the action of β‐HIVS, as well as that of shikonin, involves the inhibition of PTK, and they also suggest the possibility of producing a novel group of PTK inhibitors based on shikonin as the parent compound.

Activation of protein kinase C prevents induction of apoptosis by geranylgeraniol in human leukemia HL60 cells

In a previous study, we showed that geranylgeraniol (GGO) is a potent inducer of apoptosis in human leukemia cells, including HL60 promyelocytic leukemia cells. The present study describes the effects of activators of protein kinase C (PKC) on GGO‐induced apoptosis in various lines of leukemia cells. Both 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and diacylglycerol (DG) inhibited the GGO‐induced morphological changes that are characteristic of apoptosis and the DNA fragmentation. Similar effects were observed with other lines of human and murine leukemia cells such as MLI, U937, MI and P388. Flow cytometric analysis also revealed that both TPA and DG prevented GGO‐induced DNA degradation in a dose‐dependent manner. These inhibitory effects of TPA and DG were antagonized by inhibitors of PKC such as H‐7 and staurosporin, and by amiloride, an inhibitor of Na+/H+ antiporter. In contrast to the inhibitory effects of TPA and DG on GGO‐induced apoptosis, 4α‐TPA, which is unable to activate PKC, failed to prevent GGO‐induced DNA fragmentation. However, the selective activator of PKC‐β, 12‐deoxyphorbol 13‐phenylacetate 20‐acetate, significantly inhibited GGO‐induced DNA fragmentation. Our results suggest that PKC, and in particular the PKC‐β isoenzyme, might be involved in the process of GGO‐induced apoptosis. Int. J. Cancer 71:691‐697, 1997.

Fluorescence changes of rhodamine 6G associated with chemotactic responses in Tetrahymena pyriformis

1. 1. The chemotactic responses in Tetrahymena to various inorganic salts and octanol were examined quantitatively. Tetrahymena exhibited negative chemotaxis to the salts and octanol above respective threshold concentrations. The order of the threshold of salts was NH4Cl ≅ NaCl > LiCl > KCl > CaCl2 ≅ MgCl2 > LaCl3. 2. 2. Fluorescence intensity of rhodamine 6G added to Tetrahymena suspension increased with increasing concentration of stimuli above respective thresholds. The fluorescence changes were closely correlated with the chemotactic responses for all the chemical stimuli examined. 3. 3. Measurements of the fluorescence intensity of the supernatant of dy-Tetrahymena suspension showed that the dye was taken up by Tetrahymena and the addition of salts led to a release of the dye into an external medium. 4. 4. The adsorption of dye on the liposome also led to the quenching of its fluorescence. Addition of salts to the dye liposome suspension brought about an increase of the fluorescence. The order of the effectiveness was mono-valent < divalent < trivalent. The fluorescence intensity changed linearly with log[K]out in the presence of valinomycin. It was concluded that the changes in fluorescence on rhodamine 6G reflect both changes in the surface potential and those in the intramembrane diffusion potential, which in turn reflect changes in the total membrane potential across the membrane. We suggest that the surface potential as well as the diffusion potential contributes to the changes in the membrane potential in Tetrahymena associated with the chemotactic responses.