A novel anticancer quinolone, (R)-WAC-224, has anti-leukemia activities against acute myeloid leukemia.

Approximately 60%-80% of patients who achieve complete remission eventually relapse after conventional chemotherapy and have poor prognoses despite the recent advances of novel anticancer agents. Continuing development of more effective novel treatments for acute myeloid leukemia (AML) is necessary. We developed (R)-WAC-224 (R-WAC), which is an anticancer quinolone, targeting topoisomerase II. This study evaluated the anti-leukemia potential of R-WAC or racemic WAC-224 (WAC) in vitro and in vivo. R-WAC significantly inhibited the human AML cell line proliferation (MV4-11, HL60, and KG1a), which was comparable to daunorubicin and cytarabine, not affected by P-glycoprotein overexpression. WAC did neither increase serum troponin-T nor decrease the crypt numbers in the small intestine, indicating WAC was less toxic than doxorubicin. R-WAC monotherapy demonstrated prolonged survival in the AML mice model and inhibited tumor growth in the MV4-11 xenograft mice model. Moreover, the combination of R-WAC and cytarabine demonstrated more active anti-leukemia effects than daunorubicin and cytarabine. Finally, R-WAC inhibited the colony-forming abilities using primary AML cells. These results indicate that R-WAC is a promising therapeutic agent for AML.

Effects of a water-soluble extract of Ganoderma lucidum mycelia on aberrant crypt foci induced by azoxymethane and small-intestinal injury by 5-FU in F344 rats.

The present study investigated whether a water-soluble extract from the culture medium of Ganoderma lucidum mycelia (Japanese: Reishi or Mannentake) (designated as MAK) exerted a protective effect against induction of aberrant crypt foci (ACF) by azoxymethane (AOM) and small-intestinal damage induced by the anticancer drug 5-FU. Six-week-old male F344 rats were fed a basic diet (MF), either alone or containing 2.5 % MAK, beginning 1 week before treatment with AOM. The rats were then given subcutaneous injections of AOM (15 mg/kg body weight) once in a week for 3 weeks. Next, beginning 1 day after the final AOM treatment, 25 or 80 mg/kg 5-FU was injected intraperitoneally three times at 5-day intervals. Finally, the rats were killed 3.5 days after the last injection of 5-FU. The large and small intestines were removed, and tissue specimens were examined for both ACF in the large intestine and regeneration of small-intestinal crypts. The number of ACF was significantly decreased by treatment with 25 mg 5-FU and further decreased by 25 mg 5-FU + MAK in comparison with 5-FU alone. Moreover, there was a greater degree of recovery from small-intestinal damage in the 5-FU + MAK groups than in rats that had received 5-FU alone. The present results indicate that MAK ameliorates the colon precancerous lesions induced by AOM and the small-intestinal injury caused by 5-FU, suggesting that MAK could have potential as a preventive agent against colonic precancer, which is a common adverse effect of chemotherapy.

A water-soluble extract from cultured medium of Ganoderma lucidum (Reishi) mycelia attenuates the small intestinal injury induced by anti-cancer drugs.

The present study investigated whether a water-soluble extract from the culture medium of Ganoderma lucidum (Reishi) mycelia (MAK) is able to protect the small intestine against damage induced by anti-cancer drugs. Six-week-old male B6C3F1/Crlj mice were fed a basal diet (MF) alone or with various doses of MAK or Agarics blazei Murrill (AGA) beginning one week before treatment with the anti-cancer drugs. Mice were sacrificed 3.5 days after injection of the anti-cancer drug, the small intestine was removed and tissue specimens were examined for the regeneration of small intestinal crypts. In experiment 1, the number of regenerative crypts after the administration of 5-fluorouracil (5FU) intravenously (250 mg/kg) or intraperitoneally (250 or 500 mg/kg) was compared after treatment with MAK or AGA. MAK protected against 5FU-induced small intestinal injury whereas AGA did not. In experiment 2, we investigated the protective effect of MAK against small intestinal injury induced by the anti-cancer drugs: UFT (tegafur with uracil; 1,000 mg/kg, orally), cisplatin (CDDP; 12.5 and 25 mg/kg, intraperitoneally), cyclophosphamide (CPA; 250 mg/kg, orally) and gefitinib (Iressa; 2,000 and 4,000 mg/kg, orally). UFT and CDDP decreased the number of regenerative crypts, but treatment with MAK attenuated the extent of UFT- or CDDP-induced small intestinal injury. CPA or Iressa plus MAK up-regulated crypt regeneration. The present results indicate that MAK ameliorates the small intestinal injury caused by several anti-cancer drugs, suggesting that MAK is a potential preventive agent against this common adverse effect of chemotherapy.

Tumor induction by monoenergetic neutrons in B6C3F1 mice.

This study was undertaken to investigate induction of tumors by monoenergetic neutrons in B6C3F1 mice. Individual groups of 6 week-old animals of both sexes (about 30 mice/group) were exposed to 0.5 Gy of various monoenergetic neutrons (dose rate 0.5 cGy/min) and then observed for 13 months. The incidences of tumors (mainly liver neoplasms) in non-irradiated male and female controls were 11% and 0%, respectively. In the irradiated animals, the incidences were 53%, 50%, 60% and 43% in males, and 75%, 81%, 71%, and 85% in females, after 0.18, 0.32, 0.6 and 1.0 MeV neutron exposure, respectively. There were no significant differences in the tumor induction rate among the different energy groups.

Differentiation of a hepatic phenotype after heterotropic transplantation of heart, kidney, brain, and skin tissues into liver in F344 rats.

While organ-specific stem cells with roles in tissue injury repair have been documented, their pathogenic significance in diseases and the factors potentially responsible for their activation remain largely unclear. In the present study, heart, kidney, brain, and skin samples from F344 transgenic rats carrying the GFP gene were transplanted into normal F344 rat liver one day after an intraperitoneal injection (i.p.) of carbon tetrachloride (CCl(4)) to test their differentiation capacity. The transplantation was carried out by female donors to male recipients, and vice versa. One week after transplantation, GFP antigen-positive cells with phenotypic characteristics of hepatocytes were noted. After two weeks, their extent increased, and at 4 weeks, large areas of strongly GFP-stained cells developed. All recipient livers had GFP antigen-positive hepatocyte cells. PCR analysis coupled with laser capture micro-dissection (LCM) revealed those cells to contain GFP DNA. Thus, our results indicate that tissue stem cells have multipotential ability, differentiating into hepatocytes when transplanted into an injured liver.

Inhibitory effect of a water-soluble extract from the culture medium of Ganoderma lucidum (Rei-shi) mycelia on the development of pulmonary adenocarcinoma induced by N-nitrosobis (2-hydroxypropyl) amine in Wistar rats.

A water-soluble extract from the culture medium of Ganoderma lucidum (Rei-shi) mycelia (MAK) has been shown to exert a potent chemopreventive effect. The present study was designed to investigate the effects of dietary MAK supplementation on the development of lung tumors initiated by N-nitrosobis (2-hydroxypropyl) amine (BHP) in male Slc:Wistar rats. A total of 77 animals, 6 weeks of age, were divided into 5 groups and given BHP (2,000 ppm) in their drinking water for 10 weeks. The normal controls were not supplied with BHP. After treatment with the carcinogen, the rats were fed a normal control MF solid diet, or the same diet containing MAK (1.25%, 2.5% or 5%) for 12 weeks. Macroscopically, all the doses of MAK reduced the number of nodules, and the effect of 5% MAK was found to be especially significant. Microscopically, an increase in the number of proliferating cell nuclear antigen (PCNA)-negative tumors and a decrease in the number of tumors strongly positive for PCNA were observed in the tissue sections from the rats that had received all the doses of MAK. The present results thus indicate that dietary supplementation with MAK inhibits the development of lung tumors, suggesting that MAK may be a potent chemopreventive agent against lung carcinogenesis.

Aged garlic extract ameliorates physical fatigue.

Aged garlic extract (AGE) has recently received attention as a potent anti-fatigue agent. The principal aim of this study was to elucidate the mechanism responsible for the ameliorating effect of AGE on physical fatigue in rats caused by repeated endurance exercise on a mechanical treadmill apparatus. Rats were subjected to endurance exercise 5 times per week for 4 weeks. AGE at a dosage of 2.86 g/kg was administrated to rats 30 min before every exercise. Succinate dehydrogenase (SDH) activity in the gastrocnemius and soleus muscles and superoxide dismutase (SOD) activity, nitric oxide (NO) metabolites, and lactic acid concentration in plasma were evaluated as biomarkers of physical fatigue. SDH activity was increased 2-4-fold by repeated endurance exercise in comparison with unexercised (intact) rats, and AGE further up-regulated this activity by 40%. SOD activity was increased 5-fold, whereas AGE maintained it at a level equivalent to that in intact rats. Levels of NO metabolites were slightly decreased, whereas AGE enhanced them 2-fold. Lactic acid concentration was not changed in any of the groups. These results indicate that AGE may facilitate the turnover of aerobic glucose metabolism, attenuate oxidative stress, and promote oxygen supply based on vasodilation, suggesting that AGE ameliorates the various impairments associated with physical fatigue.

Aged garlic extract inhibits development of putative preneoplastic lesions in rat hepatocarcinogenesis.

A unique garlic preparation, aged garlic extract (AGE), was examined for its modifying effect on diethylnitrosamine (DEN)-induced neoplasia of the liver in male F344 rats, using the medium-term bioassay system based on the 2-step model of hepatocarcinogenesis. Carcinogenic potential was scored by comparing the numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci. GST-P-positive foci were significantly decreased in rats treated with AGE at doses of 2, 5, and 10 mL/kg, i.g., 5 times per week during the promotion phase. In addition, to clarify the mechanism underlying the inhibitory effect of AGE, the effect of AGE on hepatocellular proliferation was evaluated using partially hepatectomized rats as a liver-regeneration model. The bromodeoxyuridine-labeling indices in the livers of the AGE group were significantly lower than those in the control group at 24 h, the maximum proliferation period after partial hepatectomy. These findings indicate that AGE inhibited the development of putative preneoplastic lesions in rat hepatocarcinogenesis, involving a slowing in the proliferation rate of liver cells after partial hepatectomy.

A miso (Japanese soybean paste) diet conferred greater protection against hypertension than a sodium chloride diet in Dahl salt-sensitive rats.

The purpose of this study was to compare the effects of miso and sodium chloride (NaCl) on blood pressure in both sexes of Dahl and SD rats. The systolic and diastolic blood pressures (SBP/DBP) were measured at 2, 4, 8 and 12 weeks of treatment with a miso diet including 2.3% NaCl, a high-sodium diet including 2.3% or 1.9% NaCl, or a normal diet including 0.3% NaCl (MF diet; Oriental Yeast Co., Tokyo, Japan). The rats were autopsied after 12 weeks on a diet. DBP in male Dahl rats was significantly increased by the 2.3% NaCl diet as compared with that in the MF group (p < 0.01) or miso group (p < 0.05) from 4 weeks of treatment. Thereafter, SBP and DBP in both the high NaCl groups were significantly increased when compared with the MF or miso group. SBP in female Dahl rats on 2.3% NaCI was significantly increased from 8 weeks after treatment. Nephropathy was observed in both sexes of Dahl rats but not SD rats. These results show that blood pressure was not increased by the miso diet.

Decrease in size of azoxymethane induced colon carcinoma in F344 rats by 180-day fermented miso.

The present study was designed to investigate the effects of fermented miso (fermented soybean paste) on the induction of colon tumors by azoxymethane (AOM) in male F344 rats. A total of 91 rats, 6 weeks of age, were divided into 5 groups and given weekly subcutaneous injections of AOM (15 mg/kg body wt) for 3 weeks. The animals were placed on diets one week before the first AOM dose: commercial normal control MF diet or a diet containing 10% 2-year, 180-day fermented, or 3-4-day fermented miso. There were no differences in body and organ weights, and no aberrant crypt foci (ACF) among carcinogen-treated groups at week 25. The rates of tumor incidence were 45%, 85%, 75% and 60% with the 2-year, 180-day, and 3-4-day fermented miso and MF, respectively, and those for colon tumors were 34%, 55%, 60% and 55%, respectively. The size of well-differentiated adenocarcinomas and total (well differentiated and signet ring cell) adenocarcinomas in the 180-day fermented miso group was significantly smaller than that in the 2-year fermented miso and MF+AOM groups. Nuclear staining of beta-catenin in colon tumors was increased for the 3-4-day fermented miso compared to the 180-day fermented miso. Cdx2 staining tendency was decreased in colon tumors and adenocarcinomas compared to normal mucosa and ACF, which stained in 100% of cases. In addition, the PCNA index was significantly reduced in the 180-day group compared with those groups receiving the 3-4-day fermented miso and MF diet. The germinal region was also decreased. The present results indicate that dietary supplementation with 180-day fermented dietary miso could act as a chemopreventive agent for colon carcinogenesis.

Protective effects of a water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia and Agaricus blazei murill against X-irradiation in B6C3F1 mice: Increased small intestinal crypt survival and prolongation of average time to animal death.

Radioprotective effects of a water-soluble extracts from cultured medium of Ganoderma lucidum (Rei-shi) mycelia (designed as MAK) and Agaricus blazei (Agaricus) against the shortening of survival time or the injury of crypt by X-irradiation were investigated in male B6C3F1 mice. MAK and Agaricus at three different doses were mixed into basal diet into biscuits at 5, 2.5 and 1.25% and administered from 1 week before irradiation. MAK (5% group) significantly prolonged animal survival as compared with basal diet group (control group) after 7 Gy of X-ray irradiation at a dose rate of 2 Gy min(-1). At doses of 8, 10 and 12 Gy X-irradiation at a dose rate of 4 Gy min(-1) MAK (5% group) significantly increased crypt survival as compared to other groups. These results suggest that MAK can act as a radioprotective agent.

Damage of the mouse testis by tritiated water and 137Cs-gamma-rays.

Tritiated water at 23.2, 46.3 or 92.5 MBq/animal and 137Cs-gamma-rays at 9.5 Gy (equivalent 370 MBq) or lower doses were administered to 6-week old male C3H/HeNCrj and C57BL/6NCrj mice, as well as F1 Crj: B6C3F1 (C3H x C57BL) progeny. Each set of six to ten animals were autopsied 30 days after the first irradiation. Testis weights were decreased dose dependently, relative values being highest in the C3H and lowest in the C57BL case, with B6C3F1 intermediate. Vacuolization in seminiferous tubules appeared in the 23.2 MBq group and increased with the dose. Focal pyknosis and karyomegaly were found at 46.3 MBq, while primary and secondary spermatocytes and spermatids disappeared with 92.5 MBq. Only a few spermatogonia and Sertoli cells remained after exposure to 9.5 Gy 137Cs-gamma-rays. Sizes of seminiferous tubules were decreased dose dependently, with no strain differences. When male B6C3F1 mice were irradiated with Cs-gamma-rays at 0.119 (equivalent 4.63 MBq tritiated water) or 2.38 Gy (equivalent 92.5 MBq tritiated water), body weights and size of the seminiferous tubules were decreased at both doses, and the larger dose also caused reduction of testis weight and abnormal sperm. However, all changes except for the alteration in weights had disappeared 1 month after the final irradiation. It is considered that the size of seminiferous tubules may be a good parameter for radiation damage in the testis.

Pharmacokinetic study of allixin, a phytoalexin produced by garlic.

The pharmacokinetic behavior of allixin (3-hydroxy-5-methoxy-6-methyl-2-penthyl-4H-pyran-4-one) was investigated in an experimental animal, mice. Allixin was administered using an inclusion compound because the solubility of allixin in aqueous solution is very low. The allixin content in serum and in the organs of administered animals was analyzed by liquid chromatography (LC)-MS. Most of the administered allixin disappeared within 2 h, and the bioavailability of allixin was estimated to be 31% by obtained area under the blood concentration-time curve (AUC). The metabolites of allixin were studied using the metabolic enzyme fraction of liver and liver homogenate. Several new peaks corresponding to allixin metabolites were observed in the HPLC chromatoprofile. The chemical structure of the metabolites was investigated using LC-MS and NMR. Three of them were identified as allixin metabolites having a hydroxylated pentyl group.