Nrf2 knockout enhances intestinal tumorigenesis in Apc(min/+) mice due to attenuation of anti-oxidative stress pathway while potentiates inflammation.

Mutations in adenomatous polyposis coli (APC) gene are found in more than 80% of colorectal cancer (CRC) patients. The nuclear transcription factor Nrf2 plays a central role in the regulation of oxidative stress and inflammation. Previously, we have shown that chronic inflammation in Nrf2(-/-) (Nrf2 knockout; KO) mice resulted in higher expression of inflammatory markers and cytokines, coupled with higher inflammatory damage to the colonic crypt cells, as compared to the Nrf2(+/+) (wild type; WT) mice. Induction of mutation in the colon by administration of carcinogen, AOM prior to DSS-induced inflammation resulted in higher tumor incidence and numbers in Nrf2KO mice. These results indicate that Nrf2-dependent inhibition of inflammation appears to be critical in inhibiting mutation-initiated colorectal carcinogenesis. In this study, we aim to investigate if loss of Nrf2 would dose-dependently promote intestinal tumorigenesis in Apc(min/+) mice. To demonstrate the in vivo mechanisms, we constructed both Apc mutated and Nrf2 deficient strain Apc(min/+) mice with C57BL/6 Nrf2KO mice to obtain F1, Apc(min/+) ;Nrf2(+/-) and F2, Apc(min/+) ;Nrf2(-/-) mice. Nrf2KO decreased the protein expression of antioxidant enzyme NQO1 in Apc(min/+) . In contrast, Nrf2KO enhanced the expression of inflammatory markers such as COX-2, cPLA, LTB4 in Apc(min/+) . Finally, Nrf2KO resulted in higher level of PCNA and c-Myc expression in intestinal tissue, indicating the deficiency of Nrf2 promotes proliferation of intestinal crypt cells in Apc(min/+) . Taken together, our results suggest that Nrf2KO attenuates anti-oxidative stress pathway, induces inflammation, and increases proliferative potential in the intestinal crypts leading to enhanced intestinal carcinogenesis and adenomas in Apc(min/+) .

Cancer prevention by tea: Evidence from laboratory studies.

The cancer preventive activities of tea (Camellia sinensis Theaceae) have been studied extensively. Inhibition of tumorigenesis by green tea extracts and tea polyphenols has been demonstrated in different animal models, including those for cancers of the skin, lung, oral cavity, esophagus, stomach, small intestine, colon, bladder, liver, pancreas, prostate, and mammary glands. Many studies in cell lines have demonstrated the modulation of signal transduction and metabolic pathways by (-)-epigallocatechin-3-gallate (EGCG), the most abundant and active polyphenol in green tea. These molecular events can result in cellular changes, such as enhancement of apoptosis, suppression of cell proliferation, and inhibition of angiogenesis. Nevertheless, the molecular mechanisms of inhibition of carcinogenesis in animals and humans remain to be further investigated. Future research directions in this area are discussed.

δ-tocopherol is more active than α - or γ -tocopherol in inhibiting lung tumorigenesis in vivo.

In contrast to strong epidemiologic, preclinical, and secondary clinical evidence for vitamin E (tocopherols) in reducing cancer risk, large-scale clinical cancer-prevention trials of α-tocopherol have been negative. This vexing contrast helped spur substantial preclinical efforts to better understand and improve the antineoplastic activity of tocopherol through, for example, the study of different tocopherol forms. We previously showed that the γ-tocopherol-rich mixture (γ-TmT) effectively inhibited colon and lung carcinogenesis and the growth of transplanted lung-cancer cells in mice. We designed this study to determine the relative activities of different forms of tocopherol in a xenograft model, comparing the anticancer activities of δ-tocopherol with those of α- and γ-tocopherols. We subcutaneously injected human lung cancer H1299 cells into NCr nu/nu mice, which then received α-, γ-, or δ-tocopherol or γ-TmT in the diet (each at 0.17% and 0.3%) for 49 days. δ-Tocopherol inhibited tumor growth most strongly. γ-Tocopherol and γ-TmT (at 0.3%) also inhibited growth significantly, but α-tocopherol did not. δ-Tocopherol also effectively decreased oxidative DNA damage and nitrotyrosine formation and enhanced apoptosis in tumor cells; again, γ-tocopherol also was active in these regards but less so, and α-tocopherol was not. Each supplemented diet increased serum levels of its tocopherol - up to 45 µmol/L for α-tocopherol, 9.7 µmol/L for γ-tocopherol, and 1.2 µmol/L for δ-tocopherol; dietary γ- or δ-tocopherol, however, decreased serum α-tocopherol levels, and dietary α-tocopherol decreased serum levels of γ-tocopherol. Each dietary tocopherol also increased its corresponding side-chain-degradation metabolites, with concentrations of δ-tocopherol metabolites greater than γ-tocopherol and far greater than α-tocopherol metabolites in serum and tumors. This study is the first in vivo assessment of δ-tocopherol in tumorigenesis and shows that δ-tocopherol is more active than α- or γ-tocopherol in inhibiting tumor growth, possibly through trapping reactive oxygen and nitrogen species and inducing apoptosis; δ-tocopherol metabolites could contribute significantly to these results.

Rapid induction of colon carcinogenesis in CYP1A-humanized mice by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and dextran sodium sulfate.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine produced during the cooking of meats and fish, is suspected to be a human carcinogen. Metabolic activation of PhIP is primarily mediated by the enzyme cytochrome P450 (CYP) 1A2. Metabolism of PhIP by CYP1A2 differs considerably between humans and rodents, with more N(2)-hydroxylation (activation) and less 4'-hydroxylation (detoxication) in humans. Transgenic CYP1A-humanized mice (hCYP1A-mice), which have the human CYP1A1 and CYP1A2 genes but lack the murine orthologs Cyp1a1 and Cyp1a2, provide an excellent opportunity to develop a relevant model to study dietary-induced colon carcinogenesis. The treatment with 200 mg/kg PhIP by oral gavage, followed by 1.5% dextran sodium sulfate (DSS) in the drinking water for 7 days, was found to be an effective combination to induce colon carcinogenesis in hCYP1A-mice. Tumor multiplicity at week 6 was calculated to be 3.75 ± 0.70 and for week 10 was 3.90 ± 0.61 with 80-95% of the tumors being adenocarcinomas. No tumors were found in the similarly treated wild-type mice. Western blots revealed overexpression of ß-catenin, c-Myc, cyclin D1, inducible nitric oxide synthase and cyclooxygenase-2 in colon tumor samples. Strong nuclear localization of ß-catenin was observed in tumors. These results illustrate that PhIP and DSS combination produces rapid colon carcinogenesis in hCYP1A-mice and this is an effective model to mimic human colon carcinogenesis.

Inhibition of inflammation and carcinogenesis in the lung and colon by tocopherols.

Tocopherols, which exist in alpha, beta, gamma, and delta forms, are antioxidative nutrients also known as vitamin E. Although alpha-tocopherol (alpha-T) is the major form of vitamin E found in the blood and tissues, gamma- and delta-T have been suggested to have stronger anti-inflammatory activities. In the present study, using a tocopherol mixture that is rich in gamma-T (gamma-TmT, which contains 57%gamma-T), we demonstrated the inhibition of inflammation as well as of cancer formation and growth in the lung and colon in animal models. When given in the diet at 0.3%, gamma-TmT inhibited chemically induced lung tumorigenesis in the A/J mice as well as the growth of human lung cancer cell H1299 xenograft tumors. gamma-TmT also decreased the levels of 8-hydroxydeoxyguanosine, gamma-H2AX, and nitrotyrosine in tumors. More evident anti-inflammatory and cancer preventive activities of dietary gamma-TmT were demonstrated in mice treated with azoxymethane and dextran sulfate sodium. These results demonstrate the antioxidative, anti-inflammatory, and anticarcinogenic activities of tocopherols.

Pro-oxidative activities and dose-response relationship of (-)-epigallocatechin-3-gallate in the inhibition of lung cancer cell growth: a comparative study in vivo and in vitro.

(-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. Nevertheless, the dose-response relationship of the inhibitory activity in vivo has not been systematically characterized. The present studies were conducted to address these issues, as well as the involvement of reactive oxygen species (ROS), in the inhibitory action of EGCG in vivo and in vitro. We characterized the inhibitory actions of EGCG against human lung cancer H1299 cells in culture and in xenograft tumors. The growth of tumors was dose dependently inhibited by EGCG at doses of 0.1, 0.3 and 0.5% in the diet. Tumor cell apoptosis and oxidative DNA damage, assessed by the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and phosphorylated histone 2A variant X (gamma-H2AX), were dose dependently increased by EGCG treatment. However, the levels of 8-OHdG and gamma-H2AX were not changed by the EGCG treatment in host organs. In culture, the growth of viable H1299 cells was dose dependently reduced by EGCG; the estimated concentration that causes 50% inhibition (IC(50)) (20 microM) was much higher than the IC(50) (0.15 microM) observed in vivo. The action of EGCG was mostly abolished by the presence of superoxide dismutase (SOD) and catalase, which decompose the ROS formed in the culture medium. Treatment with EGCG also caused the generation of intracellular ROS and mitochondrial ROS. Although EGCG is generally considered to be an antioxidant, the present study demonstrates the pro-oxidative activities of EGCG in vivo and in vitro in the described experimental system.

A gamma-tocopherol-rich mixture of tocopherols inhibits chemically induced lung tumorigenesis in A/J mice and xenograft tumor growth.

The present study investigated the effects of a preparation of a gamma-tocopherol-rich mixture of tocopherols (gamma-TmT) on chemically induced lung tumorigenesis in female A/J mice and the growth of H1299 human lung cancer cell xenograft tumors. In the A/J mouse model, the lung tumors were induced by either 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; intraperitoneal injections with 100 and 75 mg/kg on Week 1 and 2, respectively) or NNK plus benzo[a]pyrene (B[a]P) (8 weekly gavages of 2 mumole each from Week 1 to 8). The NNK plus B[a]P treatment induced 21 tumors per lung on Week 19; dietary 0.3% gamma-TmT treatment during the entire experimental period significantly lowered tumor multiplicity, tumor volume and tumor burden (by 30, 50 and 55%, respectively; P < 0.05). For three groups of mice treated with NNK alone, the gamma-TmT diet was given during the initiation stage (Week 0 to 3), post-initiation stage (Week 3 to 19) or the entire experimental period, and the tumor multiplicity was reduced by 17.8, 19.7 or 29.3%, respectively (P < 0.05). gamma-TmT treatment during the tumor initiation stage or throughout the entire period of the experiment also significantly reduced tumor burden (by 36 or 43%, respectively). In the xenograft tumor model of human lung cancer H1299 cells in NCr-nu/nu mice, 0.3% dietary gamma-TmT treatment significantly reduced tumor volume and tumor weight by 56 and 47%, respectively (P < 0.05). In both the carcinogenesis and tumor growth models, the inhibitory action of gamma-TmT was associated with enhanced apoptosis and lowered levels of 8-hydroxydeoxyguanine, gamma-H2AX and nitrotyrosine in the tumors of the gamma-TmT-treated mice. In cell culture, the growth of H1299 cells was inhibited by tocopherols with their effectiveness following the order of delta-T > gamma-TmT > gamma-T, whereas alpha-T was not effective. These results demonstrate the inhibitory effect of gamma-TmT against lung tumorigenesis and the growth of xenograft tumors of human lung cancer cells. The inhibitory activity may be due mainly to the actions of delta-T and gamma-T.

Methyl-selenium compounds inhibit prostate carcinogenesis in the transgenic adenocarcinoma of mouse prostate model with survival benefit.

Chemoprevention of prostate cancer by second-generation selenium compounds in reference to selenomethionine holds strong promise to deal with the disease at the root. Here we used the transgenic adenocarcinoma mouse prostate (TRAMP) model to establish the efficacy of methylseleninic acid (MSeA) and methylselenocysteine (MSeC) against prostate carcinogenesis and to characterize potential mechanisms. Eight-week-old male TRAMP mice (C57B/6 background) were given a daily oral dose of water, MSeA, or MSeC at 3 mg Se/kg body weight and were euthanized at either 18 or 26 weeks of age. By 18 weeks of age, the genitourinary tract and dorsolateral prostate weights for the MSeA- and MSeC-treated groups were lower than for the control (P < 0.01). At 26 weeks, 4 of 10 control mice had genitourinary weight >2 g, and only 1 of 10 in each of the Se groups did. The efficacy was accompanied by delayed lesion progression, increased apoptosis, and decreased proliferation without appreciable changes of T-antigen expression in the dorsolateral prostate of Se-treated mice and decreased serum insulin-like growth factor I when compared with control mice. In another experiment, giving MSeA to TRAMP mice from 10 or 16 weeks of age increased their survival to 50 weeks of age, and delayed the death due to synaptophysin-positive neuroendocrine carcinomas and synaptophysin-negative prostate lesions and seminal vesicle hypertrophy. Wild-type mice receiving MSeA from 10 weeks did not exhibit decreased body weight or genitourinary weight or increased serum alanine aminotransferase compared with the control mice. Therefore, these selenium compounds may effectively inhibit this model of prostate cancer carcinogenesis.

Benzene-induced hematopoietic toxicity transmitted by AhR in wild-type mouse and nullified by repopulation with AhR-deficient bone marrow cells: time after benzene treatment and recovery.

Previously, we found an aryl hydrocarbon receptor (AhR)-transmitted benzene-induced hematotoxicity; that is, AhR-knockout (KO) mice did not show any hematotoxicity after benzene exposure [Yoon, B.I., Hirabayashi, Y., Kawasaki, Y., Kodama, Y., Kaneko, T., Kanno, J., Kim, D.Y., Fujii-Kuriyama, Y., Inoue, T., 2002. Aryl hydrocarbon receptor mediates benzene-induced hematotoxicity. Toxicol. Sci. 70, 150-156]. Furthermore, our preliminary study showed a significant attenuation of benzene-induced hematopoietic toxicity by AhR expression, when the bone marrow (BM) of mice was repopulated with AhR-KO BM cells [Hirabayashi, Y., Yoon, B.I., Li, G., Fujii-Kuriyama, Y., Kaneko, T., Kanno, J., Inoue, T., 2005a. Benzene-induced hematopoietic toxicity transmitted by AhR in the wild-type mouse was negated by repopulation of AhR deficient bone marrow cells. Organohalogen Comp. 67, 2280-2283]. In this study, benzene-induced hematotoxicity and its nullification by AhR-KO BM cells were further precisely reevaluated including the duration of the effect after benzene treatment and recovery after the cessation of exposure. Exposure routes, namely, intraperitoneal (i.p.) injection used in our previous study and intragastric (i.g.) administration used in this study, were also compared in terms of their toxicologic outcomes. From the results of this study, mice that had been lethally irradiated and repopulated with BM cells from AhR-KO mice essentially did not show any benzene-induced hematotoxicity. The AhR-KO BM cells nullified benzene-induced toxicities in notably different hematopoietic endpoints between the i.p. treatment and the i.g. treatment; however, the number of granulo-macrophage colony-forming unit in vitro (CFU-GM) was a common target parameter, the benzene-induced toxicity of which was nullified by the AhR-KO BM cells.

Superior in vivo inhibitory efficacy of methylseleninic acid against human prostate cancer over selenomethionine or selenite.

Methylselenol has been implicated as an active anticancer selenium (Se) metabolite. However, its in vivo efficacy against prostate cancer (PCa) has yet to be established. Here, we evaluated the growth inhibitory effects of two presumed methylselenol precursors methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC) in comparison with selenomethionine (SeMet) and selenite in DU145 and PC-3 human PCa xenografts in athymic nude mice. Each Se was given by a daily single oral dose regimen starting the day after the subcutaneous inoculation of cancer cells. We analyzed serum, liver and tumor Se content to confirm supplementation status and apoptosis indices and tumor microvessel density for association with antitumor efficacy. Furthermore, we analyzed lymphocyte DNA integrity to detect genotoxic effect of Se treatments. The data show that MSeA and MSeC exerted a dose-dependent inhibition of DU145 xenograft growth and both were more potent than SeMet and selenite, in spite of less tumor Se retention than in the SeMet-treated mice. Selenite treatment increased DNA single-strand breaks in peripheral lymphocytes, whereas the other Se forms did not. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and cleaved caspase-3 indices (apoptosis) from MSeC-treated tumors were higher than tumors from control mice or MSeA-treated mice, whereas the microvessel density index was lower in tumors from MSeA-treated mice. In the PC-3 xenograft model, only MSeA was growth inhibitory at a dose of 3 mg/kg body wt. In summary, our data demonstrated superior in vivo growth inhibitory efficacy of MSeA over SeMet and selenite, against two human PCa xenograft models without the genotoxic property of selenite.

Methylseleninic acid enhances taxane drug efficacy against human prostate cancer and down-regulates antiapoptotic proteins Bcl-XL and survivin.

PURPOSE: Our previous work has shown that methylseleninic acid (MSeA) sensitized hormone refractory prostate cancer (HRPCa) cells to apoptosis induced by paclitaxel (Taxol) through enhancing multiple caspases. This study aimed to (a) determine the general applicability of the sensitization effect for taxane drugs in vitro, (b) establish the enhancement of paclitaxel efficacy by MSeA in vivo, and (c) investigate Bcl-XL and survivin as molecular targets of MSeA to augment apoptosis. EXPERIMENTAL DESIGN: DU145 and PC-3 HRPCa cell lines were used to evaluate the in vitro apoptosis effects of paclitaxel, docetaxel and their combination with MSeA, and the molecular mechanisms. DU145 xenograft growth in athymic nude mice was used to evaluate the in vivo efficacy of paclitaxel and its combination with MSeA. The tumor samples were used to examine Bcl-XL and survivin protein abundance. RESULTS: MSeA combination with paclitaxel or docetaxel exerted a greater than additive apoptosis effect on DU145 and PC-3 cells. In nude mice, paclitaxel and MSeA combination inhibited growth of DU145 subcutaneous xenograft with the equivalent efficacy of a four-time higher dose of paclitaxel alone. MSeA decreased the basal and paclitaxel-induced expression of Bcl-XL and survivin in vitro and in vivo. Ectopic expression of Bcl-XL or survivin attenuated MSeA/paclitaxel-induced apoptosis. CONCLUSIONS: MSeA enhanced the efficacy of paclitaxel against HRPCa in vitro and in vivo, at least in part, by down-regulating the basal and paclitaxel-induced expression of both Bcl-XL and survivin to increase caspase-mediated apoptosis. MSeA may be a novel agent to improve taxane combination therapy.

Differential involvement of reactive oxygen species in apoptosis induced by two classes of selenium compounds in human prostate cancer cells.

Selenium is a promising chemopreventive agent for prostate cancer, possibly via an induction of apoptosis. Earlier studies have shown that selenite induces DNA single strand breaks (SSBs), reactive oxygen species (ROS), p53 Ser-15 phosphorylation and caspase-dependent and -independent apoptosis, whereas a methylselenol precursor methylseleninic acid (MSeA) induces caspase-mediated apoptosis regardless of p53 status. Here we address three main questions: What types of ROS are induced by selenite vs. MSeA in LNCaP (p53 wild type, androgen-responsive) and DU145 (mutant p53, androgen-independent) prostate cancer cells? Does ROS generation depend on androgen signaling? What are the relationships among ROS, DNA SSBs, p53 and caspases? We show that selenite (5 microM) induced superoxide and hydrogen peroxide in LNCaP cells much more than in DU145 cells and the ROS generation was not affected by physiological androgen stimulation. MSeA (10 microM) induced apoptosis without either type of ROS in both cell lines. In LNCaP cells, we established superoxide as a primary mediator for selenite-induced DNA SSBs, p53 activation and caspase-mediated apoptosis. Furthermore a p53-dominant negative mutant attenuated selenite-induced ROS, leading to a proportionate protection against apoptosis. The results support the p53-mitochondria axis in a feedback loop for sustaining superoxide production to lead to efficient caspase-mediated apoptosis by selenite. In contrast, caspase-mediated apoptosis induced by MSeA does not involve ROS induction. Since p53 is frequently mutated or deleted in prostate cancer and many other cancers, our results suggest that genotoxic vs. nongenotoxic classes of selenium may exert differential apoptosis efficacy depending on the p53 status of the cancer cells.

Thioredoxin overexpression in mice, model of attenuation of oxidative stress, prevents benzene-induced hemato-lymphoid toxicity and thymic lymphoma.

OBJECTIVE: Reactive oxygen species (ROS), generated following benzene exposure, are considered to trigger the development of hematopoietic neoplasms, although little supporting evidence has been found. In this study, we examined whether the experimental elimination of ROS generated following benzene exposure prevents the development of benzene-induced hematopoietic disorders to clarify the mechanism underlying the development of benzene-induced hematopoietic disorders. METHODS: C57BL/6 mice, overexpressing human thioredoxin (h-Trx-Tg), were used to examine the possible nullification of ROS induction following benzene exposure. The experimental group was exposed to 300 ppm benzene 6 hours/day, 5 days/week, for 26 weeks, and lifetime observation followed by molecular and histopathological examinations were carried out. RESULTS: The present study using h-Trx-Tg mice showed a complete suppression of the development of thymic lymphoma induced by benzene inhalation (0% in h-Trx-Tg vs 30% in wild-type (Wt) mice). This was associated with a 48% decrease in the incidence of clastogenic micronucleated reticulocyte induction in the h-Trx-Tg mice compared with the Wt control after 2 weeks of inhalation. As underlying mechanisms, the attenuation of oxidative stress was accompanied by a complete abrogation of hemato-lymphoid toxicity, as shown by the upregulation of the activity of superoxide-dismutase, and a consequently stable ROS level, as determined by cell sorting using 2', 7'-dichlorodihydrofluorescein diacetate, along with a significant attenuation of the overexpression of a cell cycle-dependent kinase inhibitor, p21. CONCLUSION: The attenuation of benzene-induced oxidative stress and that of the consequent lymphomagenesis were observed for the first time, and these indicate a role of oxidative stress in benzene-induced clastogenesis and lymphomagenesis. (These attenuations were not seen in nonthymic lymphomas, and no leukemias developed in C57BL/6 used in this study.) During the constitutive overexpression of h-Trx, the expression of aryl-hydrocarbon receptor in h-Trx-Tg mice was downregulated, which may also contribute to the attenuation.

Inorganic selenium sensitizes prostate cancer cells to TRAIL-induced apoptosis through superoxide/p53/Bax-mediated activation of mitochondrial pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in prostate cancer cells through DR4 and DR5 death receptors, but not in normal prostate cells, which do not express these receptors. Therefore, TRAIL has excellent potential to be a selective prostate cancer therapeutic agent with minimal toxic side effects. However, prostate cancer cells, as many other cancer types, develop resistance to TRAIL, and the underlying molecular mechanisms require further investigation. We hypothesize that selenium may sensitize TRAIL-resistant cells to undergo caspase-mediated apoptosis and increase therapeutic efficacy. Here, we report that TRAIL signaling in LNCaP prostate cancer cells stalled at downstream of caspase-8 and BID cleavage, as indicated by the lack of Bax translocation into mitochondria, and no subsequent activation of the caspase-9 cascade. Selenite induced a rapid generation of superoxide and p53 Ser(15) phosphorylation and increased Bax abundance and translocation into the mitochondria. Selenite and TRAIL combined treatment led to synergistic increases of Bax abundance and translocation into mitochondria, loss of mitochondrial membrane potential, cytochrome c release, and cleavage activation of caspase-9 and caspase-3. Inactivating p53 with a dominant-negative mutant abolished apoptosis without affecting superoxide generation, whereas a superoxide dismutase mimetic agent blocked p53 activation, Bax translocation to mitochondria, cytochrome c release, and apoptosis induced by selenite/TRAIL. In support of Bax as a crucial target for cross-talk between selenite and TRAIL pathways, introduction of Bax into p53 mutant DU145 cells enabled selenite to sensitize these cells for TRAIL-induced apoptosis. Taken together, the results indicate that selenite induces a rapid superoxide burst and p53 activation, leading to Bax up-regulation and translocation into mitochondria, which restores the cross-talk with stalled TRAIL signaling for a synergistic caspase-9/3 cascade-mediated apoptosis execution.

An oriental herbal cocktail, ka-mi-kae-kyuk-tang, exerts anti-cancer activities by targeting angiogenesis, apoptosis and metastasis.

Rigorous and systematic pre-clinical studies are necessary and essential to establish the efficacy and safety of Oriental herbs and formulas in order to transform traditional herbal practices into evidence-based medicine. Here we evaluated the anti-cancer activities of the ethanol extract of Ka-mi-kae-kyuk-tang (KMKKT), a formula of ten Oriental herbs, with a battery of in vitro and in vivo mechanism-based biomarkers involving angiogenesis, apoptosis and metastasis. The results show that KMKKT suppressed the vascular endothelial responses by inhibiting basic fibroblast growth factor (bFGF)-induced ERK1/2 phosphorylation, cell migration as well as tube formation in the human umbilical vein endothelial cell model, and decreased the hypoxia-induced HIF1alpha and vascular epithelial growth factor (VEGF) expression in the mouse Lewis lung carcinoma (LLC) cells in vitro, and inhibited the bFGF-induced angiogenesis in chick chorioallantoic membrane model, and in the Matrigel plugs in mice. Intraperitoneal delivery of KMKKT potently inhibited the growth of the subcutaneously inoculated LLC cells in syngenic mice. In addition, KMKKT inhibited the invasion ability of the mouse colon 26-L5 cancer cells in vitro and decreased their formation of liver metastasis when intraportally inoculated in syngenic mice. Furthermore, KMKKT suppressed the growth of the human PC-3 prostate cancer xenografts in athymic nude mice and averted the cancer-related body weight loss. The in vivo cancer growth suppression was associated with a decreased microvessel density and VEGF abundance as well as an increased PARP cleavage and the TUNEL-positive apoptosis. Together, our data support broad-spectra in vivo anti-cancer activities of KMKKT targeting angiogenesis, apoptosis and metastasis without any adverse effect on the body weight. This formula merits serious consideration for further evaluation for the chemoprevention and treatment of cancers of multiple organ sites.

Potent antiandrogen and androgen receptor activities of an Angelica gigas-containing herbal formulation: identification of decursin as a novel and active compound with implications for prevention and treatment of prostate cancer.

Androgen and androgen receptor (AR)-mediated signaling are crucial for the development of prostate cancer. Identification of novel and naturally occurring phytochemicals that target androgen and AR signaling from Oriental medicinal herbs holds exciting promises for the chemoprevention of this disease. In this article, we report the discovery of strong and long-lasting antiandrogen and AR activities of the ethanol extract of a herbal formula (termed KMKKT) containing Korean Angelica gigas Nakai (AGN) root and nine other Oriental herbs in the androgen-dependent LNCaP human prostate cancer cell model. The functional biomarkers evaluated included a suppression of the expression of prostate-specific antigen (PSA) mRNA and protein (IC50, approximately 7 microg/mL, 48-hour exposure) and an inhibition of androgen-induced cell proliferation through G1 arrest and of the ability of androgen to suppress neuroendocrine differentiation at exposure concentrations that did not cause apoptosis. Through activity-guided fractionation, we identified decursin from AGN as a novel antiandrogen and AR compound with an IC50 of approximately 0.4 microg/mL (1.3 micromol/L, 48-hour exposure) for suppressing PSA expression. Decursin also recapitulated the neuroendocrine differentiation induction and G1 arrest actions of the AGN and KMKKT extracts. Mechanistically, decursin in its neat form or as a component of AGN or KMKKT extracts inhibited androgen-stimulated AR translocation to the nucleus and down-regulated AR protein abundance without affecting the AR mRNA level. The novel antiandrogen and AR activities of decursin and decursin-containing herbal extracts have significant implications for the chemoprevention and treatment of prostate cancer and other androgen-dependent diseases.

Mechanism of benzene-induced hematotoxicity and leukemogenicity: current review with implication of microarray analyses.

Benzene is a potent human leukemogen but the mechanism underlying benzene-induced leukemia remains an enigma due to a number of questions regarding the requirement of extraordinarily long exposure, a relatively low incidence of leukemia for genotoxicity of metabolites and a narrow dose range for leukemogenicity over marrow aplasia (overdoses tend to result in marrow aplasia). Moreover, there were previous controversies as to whether the cell cycle is upregulated or suppressed by the benzene exposure. Subsequently, it was found that the cell cycle is suppressed, but how leukemia develops under such suppression of hemopoiesis remains to be clarified. These questions were fortunately resolved with much effort. Benzene exposure was found to induce the expression of p21, an interlocking counterdevice for cell cycle: due to p53 upregulation, thereby inducing the immediate suppression of the kinetics of hemopoietic progenitors followed by the prominent suppression of hemopoiesis. Intermittent benzene exposure (i.e., cessation of exposure during weekends, for example) allowed an immediate recovery from marrow suppression after terminating exposure, which induced continuous oscillatory changes in marrow hemopoiesis. Benzene-induced leukemia was chiefly due to such an oscillatory change in hemopoiesis, which epigenetically developed leukemia more than 1 year later. The mechanisms of benzene-induced leukemogenicity seem to differ between wild-type mice and mice lacking p53. For p53 knockout mice, DNA damage such as weak mutagenicity or chromosomal damage was retained, and such damage induced consequent activation of proto-oncogenes and related genes, which led cells to undergo further neoplastic changes. In contrast, for wild-type mice carrying the p53 gene, a marked oscillatory change in the cell cycle of the stem cell compartment seems to be important. Compatible and discriminative gene expression profiling between the p53 knockout mice and wild-type mice was observed after benzene exposure by microarray analyses.

Senescent B lymphopoiesis is balanced in suppressive homeostasis: decrease in interleukin-7 and transforming growth factor-beta levels in stromal cells of senescence-accelerated mice.

The suppression of the B cell population during senescence has been considered to be due to the suppression of interleukin-7 (IL-7) production and responsiveness to IL-7; however, the upregulation of transforming growth factor-beta (TGF-beta) was found to contribute to B cell suppression. To investigate the mechanism of this suppression based on the interrelationship between IL-7 and TGF-beta during senescence, senescence-accelerated mice (SAMs), the mouse model of aging, were used in this study to elucidate the mechanisms of B lymphopoietic suppression during aging. Similar to regular senescent mice, SAMs showed a decrease in the number of IL-7-responding B cell progenitors (i.e., colony-forming unit pre-B [CFU-pre-B] cells in the femoral bone marrow [BM]). A co-culture system of B lymphocytes and stromal cells that the authors established showed a significantly lower number of CFU-pre-B cells harvested when BM cells were co-cultured with senescent stromal cells than when they were co-cultured with young stromal cells. Interestingly, cells harvested from a senescent stroma and those from the control culture without stromal cells were higher in number than those harvested from a young stroma, thereby implying that an altered senescent stromal cell is unable to maintain self-renewal of the stem cell compartment. Because TGF-beta is supposed to suppress the proliferative capacity of pro-B/pre-B cells, we added a neutralizing anti-TGF-beta antibody to the co-culture system with a pro-B/pre-B cell-rich population to determine whether such suppression may be rescued. However, unexpectedly, any rescue was not observed and the number of CFU-pre-B cells remained unchanged when BM cells were co-cultured with senescent stromal cells compared with the co-culture with young stromal cells, which essentially showed an increase in the number of CFU-pre-B cells (P < 0.001 in 5 microg/ml). Furthermore, TGF-beta protein level in the supernatant of cultured senescent stroma cells was evaluated by enzyme-linked immunoabsorbent assay, but surprisingly, it was found that TGF-beta concentration was significantly lower than that of cultured young stromal cells. Thus, TGF-beta activity was assumed to decline particularly in a senescent stroma, which means a distinct difference between the senescent suppression of B lymphopoiesis and secondary B lymphocytopenia. Concerning proliferative signaling, on the other hand, the level of IL-7 gene expression in cells from freshly isolated BM decreased significantly with age. Therefore, the acceleration of proliferative signaling and the deceleration of suppressive signaling may both be altered and weakened in a senescent stroma (i.e., homeosuppression).

Mechanisms of benzene-induced hematotoxicity and leukemogenicity: cDNA microarray analyses using mouse bone marrow tissue.

Although the mechanisms underlying benzene-induced toxicity and leukemogenicity are not yet fully understood, they are likely to be complicated by various pathways, including those of metabolism, growth factor regulation, oxidative stress, DNA damage, cell cycle regulation, and programmed cell death. With this as a background, we performed cDNA microarray analyses on mouse bone marrow tissue during and after a 2-week benzene exposure by inhalation. Our goal was to clarify the mechanisms underlying the hematotoxicity and leukemogenicity induced by benzene at the level of altered multigene expression. Because a few researchers have postulated that the cell cycle regulation mediated by p53 is a critical event for benzene-induced hematotoxicity, the present study was carried out using p53-knockout (KO) mice and C57BL/6 mice. On the basis of the results of large-scale gene expression studies, we conclude the following: (a) Benzene induces DNA damage in cells at any phase of the cell cycle through myeloperoxidase and in the redox cycle, resulting in p53 expression through Raf-1 and cyclin D-interacting myb-like protein 1. (b) For G1/S cell cycle arrest, the p53-mediated pathway through p21 is involved, as well as the pRb gene-mediated pathway. (c) Alteration of cyclin G1 and Wee-1 kinase genes may be related to the G2/M arrest induced by benzene exposure. (d) DNA repair genes such as Rad50 and Rad51 are markedly downregulated in p53-KO mice. (e) p53-mediated caspase 11 activation, aside from p53-mediated Bax gene induction, may be an important pathway for cellular apoptosis after benzene exposure. Our results strongly suggest that the dysfunction of the p53 gene, possibly caused by strong and repeated genetic and epigenetic effects of benzene on candidate leukemia cells, may induce fatal problems such as those of cell cycle checkpoint, apoptosis, and the DNA repair system, finally resulting in hemopoietic malignancies. Our cDNA microarray data provide valuable information for future investigations of the mechanisms underlying the toxicity and leukemogenicity of benzene.

Decreased sperm number and motile activity on the F1 offspring maternally exposed to butyl p-hydroxybenzoic acid (butyl paraben).

Butyl p-hydroxybenzoic acid (butyl paraben, BP) is widely used as a preservative in food and cosmetic products. Routledge et al showed that BP is weakly estrogenic in both in vitro and in vivo (rat uterotrophic) analyses. We investigated whether maternal exposures to BP during gestation and lactation periods affected the development of the reproductive organs of the F1 offspring. Pregnant Sprague-Dawley rats were injected subcutaneously with 100 or 200 mg/kg of BP from gestation day (GD) 6 to postnatal day (PND) 20. In the group exposed to 200 mg/kg of BP, the proportion of pups born alive and the proportion of pups surviving to weaning were decreased. The body weights of female offspring were significantly decreased at PND 49. The weights of testes, seminal vesicles and prostate glands were significantly decreased in rats exposed to 100 mg/kg of BP on PND 49. In contrast, the weights of female reproductive organs were not affected by BP. The sperm count and the sperm motile activity in the epididymis were significantly decreased at doses of 100 and 200 mg/kg of BP. In accordance with the sperm count in the epididymis, the number of round spermatids and elongated spermatids in the seminiferous tubule (stage VII) were significantly decreased by BP. Testicular expression of estrogen receptor (ER)-alpha and ER-beta mRNA was significantly increased in 200 mg/kg of BP treated group at PND 90. Taken together, these results indicated that maternal exposure of BP might have adverse effects on the F1 male offspring.