Antitumour and Apoptotic Effects of a Plant Extract Mixture Containing Rhus verniciflua and Other Herbs in Human Leukaemia Cells.

The apoptotic effects of a novel antitumour agent (Rv-PEM01) prepared from 6 kinds of herbs, including Rhus verniciflua were investigated using flow cytometry and western blot analysis. Rv-PEM01 induced apoptosis but not necrosis in MOLT-3, KG-1, and K562 human leukaemia cell lines. Further, Rv-PEM01-treated cells showed significantly upregulated expression of caspase-3 and 9 and cleaved caspase-3 and 9 compared to the control cells. Taken together, the results suggest that Rv-PEM01 induced apoptosis via the mitochondrial-mediated pathway, and is a potential natural anticancer agent and/or a functional food material.

Characterization of the cytotoxic activity of [2]rotaxane (TRO-A0001), a novel supramolecular compound, in cancer cells.

Rotaxanes comprise a class of interlocked molecules containing a wheel threaded onto an axle with blocking groups on the ends to keep the wheel from sliding off. Here, we show that [2][bis(2-(3,5-dimethylphenylcarbonyloxy)ethyl) ammoniumtrifluoromethanesulfonate]-[dibenzo-24-crown-8] rotaxane (TRO-A0001), a rotaxane compound, exerted a growth inhibitory effect on several human cancer cell lines. An MTT assay revealed an IC50 of 14-830 nM for TRO-A0001 in these cells. Neither the wheel nor the axle part alone inhibited tumor cell growth, suggesting that the complete rotaxane molecule with its unique "intramolecular mobility" is required to inhibit cell growth. Annexin-V/PI staining provided evidence of the induction of apoptosis, which was further confirmed by the observation of poly (ADP-ribose) polymerase cleavage. Furthermore, a cell cycle analysis using flow cytometry showed that TRO-A0001 treatment resulted in G1 arrest in glioblastoma T98G and melanoma G361 cells. An immunoblot analysis revealed that in both cell lines, TRO-A0001 treatment caused the induction of p21/Cip1, thereby down-regulating Cdks 2, 4 and 6 and reducing Cyclins D1 and E. The results presented in this study demonstrate cytotoxicity of the rotaxane compound and its potential as a lead compound for the development of a chemotherapeutic agent against cancer.

Nicotine facilitates reinnervation of phenol-injured perivascular adrenergic nerves in the rat mesenteric resistance artery.

Nicotine has been shown to have neuroprotective and neurotrophic actions in the central nervous system. To elucidate the peripheral neurotrophic effects of nicotine, we determined whether nicotine affected the reinnervation of mesenteric perivascular nerves following a topical phenol treatment. A topical phenol treatment was applied to the superior mesenteric artery proximal to the abdominal aorta in Wistar rats. We examined the immunohistochemistry of the distal small arteries 7 days after the treatment. The topical phenol treatment markedly reduced the density of tyrosine hydroxylase (TH)-LI and calcitonin gene-related peptide (CGRP)-LI fibers in these arteries. The administration of nicotine at a dose of 3 mg/kg/day (1.5 mg/kg/injection, twice a day), but not once a day or its continuous infusion using a mini-pump significantly increased the density of TH-LI nerves without affecting CGRP-LI nerves. A pretreatment with nicotinic acetylcholine receptor antagonists hexamethonium, mecamylamine, and methyllycaconitine, but not dextrometorphan, canceled the TH-LI nerve reinnervation induced by nicotine. Nicotine significantly increased NGF levels in the superior cervical ganglia (SCG) and mesenteric arteries, but not in the dorsal root ganglia, and also up-regulated the expression of NGF receptors (TrkA) in the SCG, which were canceled by hexamethonium. These results suggested that nicotine exhibited neurotrophic effects that facilitated the reinnervation of adrenergic TH-LI nerves by activating α7 nicotinic acetylcholine receptor and NGF in the SCG.

Protons modulate perivascular axo-axonal neurotransmission in the rat mesenteric artery.

BACKGROUND AND PURPOSE: Previous studies have demonstrated that nicotine releases protons from adrenergic nerves via stimulation of nicotinic ACh receptors and activates transient receptor potential vanilloid-1 (TRPV1) receptors located on calcitonin gene-related peptide (CGRP)-containing (CGRPergic) vasodilator nerves, resulting in vasodilatation. The present study investigated whether perivascular nerves release protons, which modulate axon-axonal neurotransmission. EXPERIMENT APPROACH: Perfusion pressure and pH levels of perfusate in rat-perfused mesenteric vascular beds without endothelium were measured with a pressure transducer and a pH meter respectively. KEY RESULTS: Periarterial nerve stimulation (PNS) initially induced vasoconstriction, which was followed by long-lasting vasodilatation and decreased pH levels in the perfusate. Cold-storage denervation of the preparation abolished the decreased pH and vascular responses to PNS. The adrenergic neuron blocker guanethidine inhibited PNS-induced vasoconstriction and effects on pH, but not PNS-induced vasodilatation. Capsaicin (CGRP depletor), capsazepine and ruthenium red (TRPV1 inhibitors) attenuated the PNS-induced decrease in pH and vasodilatation. In denuded preparations, ACh caused long-lasting vasodilatation and lowered pH; these effects were inhibited by capsaicin pretreatment and atropine, but not by guanethidine or mecamylamine. Capsaicin injection induced vasodilatation and a reduction in pH, which were abolished by ruthenium red. The use of a fluorescent pH indicator demonstrated that application of nicotine, ACh and capsaicin outside small mesenteric arteries reduced perivascular pH levels and these effects were abolished in a Ca(2+) -free medium. CONCLUSION AND IMPLICATION: These results suggest that protons are released from perivascular adrenergic and CGRPergic nerves upon PNS and these protons modulate transmission in CGRPergic nerves.

Quantum chemical study for radical-induced DNA effects and damage.

Differences in molecular interaction between bases (adenine (A), guanine (G), and cytosine (C)) and the methyl (Me)-radical were investigated by perturbation analysis using the quantum chemical method. Part of the source of damage to the DNA was elucidated at the molecular level. In the reaction of each of the saccharide derivatives (dA, dG, and dC) with Me-radical, the reactivity of dG (≈dA) is more than about 10 times larger than that of dC. Therefore, it is expected that the base G (and A) was more than about 10 times than the base C in radical-reactivity of the base. For the reaction of dA and dG with the radical, the C(8) site of the partial purine ring of dA and dG, and the C(5) site of the pyrimidine ring of dC were the main reaction sites for methylation. In the reaction of DNA composed of hydrogen-bonded base pairs G-C and A-T with the radical, the purine ring in the constituent base G reacted preferentially with the radical to yield 8-methyl-guanines.

Influence of novel supramolecular substance, [2] rotaxane, on the caspase signaling pathway in melanoma and colon cancer cells in vitro.

We studied the influence of novel supramolecular substance, [2] rotaxane (TRO-A0001), on caspase signaling and cell viability in cancer cell lines. TRO-A0001 suppressed concentration-dependently cell proliferation. Expression of the cleaved-form caspase-3 and PARP was significantly increased in cells exposed to TRO-A0001. The expression of Bax was increased by TRO-A0001. Furthermore, the down-regulation of Bax by siRNA resulted in growth activation significantly. The morphological analysis demonstrated that TRO-A0001 increased the levels of apoptotic cells in human cancer cell lines. These results suggest that TRO-A0001 induces apoptosis in cancer cells and holds potential as a new anti-tumor medicine.

[Antitumor effects of a plant extract mixture].

Cancer is the most common cause of death in Japan. Fundamental and clinical studies on cancer were conducted from the viewpoint of Western medicine so far. However, a sustained complete remission has not been achieved yet. In order to alleviate the side effects of anticancer drugs, some traditional herbal medicines (Kampo medicines) have been prescribed to cancer patients. We have been studying on antitumor substances in medicinal herbs and found an antitumor medicinal herb named Rhus verniciflua (lacquer, Urushi in Japanese). To investigate the antitumor effect in vitro, a plant extract mixture was prepared from six medicinal herbs containing lacquer. The plant extract mixture containing lacquer (Rv-PEM) inhibited the proliferation of several mouse and human tumor cell lines. Rv-PEM had more potent inhibitory effect on the proliferation of human leukemia cell lines (MOLT-3, KG-1) than on other tumor cell lines. The IC50 values of Rv-PEM on MOLT-3 and KG-1 cells were 0.208 and 0.293 mg/mL, respectively. After treating Rv-PEM to the tumor cells, DNA fragmentation and Caspase-3 and -9 activity increased in the treated cells. The mechanisms of the inhibitory proliferation activity of Rv-PEM would involve apoptosis of human leukemia cells (MOLT-3, KG-1, K-562) by the mitochondrial pathway.

Enhancement of caffeine-induced locomotor hyperactivity produced by the combination with L-arginine or taurine in mice: Possible involvement of nitric oxide.

Combinations of caffeine with L-arginine or with taurine can enhance the effect of caffeine, but the mechanisms remain elusive. This study was designed to test the hypothesis that stimulant effects of central nervous system nitric oxide (NO) may explain the beneficial effect of caffeine on combinations with amino acid, L-arginine or taurine. Caffeine increased the spontaneous locomotor activity dose-dependently (2-10 mg/kg) in mice. The locomotor activity induced by caffeine at a dose of 2 mg/kg was enhanced by combined administration of L-arginine at a dose of 600 mg/kg, or taurine at a dose of 400 mg/kg, respectively. For both combinations, enhancement was significantly inhibited by pretreatment with N-nitro-L-arginine methyl ester (L-NAME) at a dose of 40 mg/kg. These results suggest that the enhancement induced by combining caffeine with amino acid might be regulated at least in part by NO in the central nervous system.

[Study for an allergic inflammation model using human lungs and its pharmacological application].

The complement system, which plays an important role in inmate immunity, is considered to be important in the pathophysiology of allergic asthma. A patient with allergic asthma shows the reversible characteristic system of bronchoconstriction, increased mucus secretion, and complicated airway inflammation. Various cytokines secreted from Th2 cells contribute to the system. Cysteinyl-leukotrienes (CysLTs) are also considered to be one of the important mediators involved in asthmatic pathophysiology. However, the effects of a drug on humans may not be the same as those on animals due to species differences in complement-related molecules. In this series of experiments, we tried to establish a model in which the effects of a drug on the production of CysLTs from human lung preparations were evaluated following an anaphylactic reaction. CysLT production increased when the passively sensitized lung tissues were stimulated with anti-IgE antibody. The coaddition of anaphylatoxin, C5a, with the anti-IgE antibody potentiated CysLT production. The response to C3a was weaker when compared with that to C5a. In addition, increased production of CysLTs by adding serum at a specific ratio was dose dependently inhibited by nonpeptide C5a receptor antagonist, W-54011, or a novel complementary peptide inhibitor of C5a, acetyl peptide A. From these results, it is suggested that C5a potentiates cysLT production from human lung tissues and contributes to allergic inflammation like asthma, and thus acetylated peptide A and W-54011 are useful for suppressing allergic inflammation in the lungs.

Antagonistic effects of methanolic extract of Polygala telephioides on morphine responses in mice.

The present study was undertaken to investigate the antagonistic effects of the methanolic extract of Polygala telephioides (PT) on morphine responses in mice. Single administration of PT tended to antagonize the morphine-induced analgesia in a hot-plate test. Moreover, PT (300 mg/kg, p.o.) improved the morphine-induced memory impairment in an elevated plus maze test. However, PT alone had no effect on behaviors in the open-field, hot-plate and elevated plus maze tests. We investigated the effects of PT on naloxone-induced jumping (as withdrawal sign) in morphine-dependent mice. To induce dependence, mice were twice daily treated with morphine (10-45 mg/kg, s.c.) for 5 days. Co-administrations of PT (10, 100 and 300 mg/kg, p.o.) during repeated morphine treatments significantly suppressed the naloxone (10 mg/kg, i.p.)-induced jumping. However, the naloxone-induced jumping was not affected by a single large administration of PT on the 5th day. The inhibitory effect of PT on the naloxone-induced jumping was due to the development of dependence rather than expression of withdrawal sign. Moreover, single administration of PT (30 mg/kg, p.o.) decreased the morphine levels in plasma. These results indicate that PT may be useful in facilitating narcotic detoxification.