Efficacy of Bidens pilosa Extract against Herpes Simplex Virus Infection In Vitro and In Vivo.

The development of strains of herpes simplex virus (HSV) resistant to drugs has been reported among the immunocompromised patients. Thus, there is a need to develop new therapeutic agents for HSV infections. We evaluated the anti-HSV activity of Bidens pilosa (B. pilosa), a tropical weed, in tissue culture cells and a mouse model. B. pilosa extract showed potent virucidal activity. It inhibited plaque formation and suppressed virus yield in Vero and RAW 264.7 cells infected with HSV-1 and HSV-2. Both the binding of virus to host cells and penetration of virus into cells were also blocked by B. pilosa. Furthermore, B. pilosa was effective against thymidine kinase-deficient and phosphonoacetate-resistant HSV-1 strains. B. pilosa treatment increased the survival rate of HSV-infected mice and limited the development of skin lesions. Our results indicate that B. pilosa has anti-HSV activity and is thus a potentially useful medical plant for treatment of HSV infection.

Antitumor activity and macrophage nitric oxide producing action of medicinal herb, Crassocephalum crepidioides.

BACKGROUND: Crassocephalum crepidioides, a plant distributed in Okinawa Islands, is known in folk medicine; however, its anticancer activity has not been investigated. The aim of this study was to determine the in vitro and in vivo antitumor activities of C. crepidioides on murine Sarcoma 180 (S-180) and related molecular mechanisms. METHODS: The antitumor effect of C. crepidioides was evaluated in S-180-cell-bearing mice. Cell growth was assessed using a colorimetric assay. Nitrite and nitrate levels were measured by colorimetry. The expression levels of inducible NO synthase (iNOS) in murine RAW264.7 macrophages was assessed by reverse transcriptase-polymerase chain reaction. Activation of iNOS promoter was detected by reporter gene. Activation of nuclear factor-κB (NF-κB) was evaluated by electrophoretic mobility shift assay. The role of NF-κB signaling was analyzed using inhibitors of NF-κB and dominant-negative mutants, and Western blot analysis. RESULTS: C. crepidioides extract delayed tumor growth in S-180-bearing mice. However, it did not inhibit S-180 cell growth in vitro. Supernatant of cultured C. crepidioides-stimulated RAW264.7 macrophages was cytotoxic to S-180 cells. This cytotoxicity was associated with nitric oxide (NO) production. NF-κB signaling pathway was crucial for the transcriptional activation of iNOS gene. Isochlorogenic acid, a component of C. crepidioides, induced NF-κB activation and iNOS expression. CONCLUSIONS: The results highlight the oncolytic and immunopotentiation properties of C. crepidioides mediated through NF-κB-induced release of NO from macrophages.

Activation of rat liver microsomal glutathione transferase by hepsin.

Rat liver microsomal glutathione transferase (MGST1) is activated by limited proteolysis. Recently we purified a protease, hepsin, from rat liver microsomes that activates MGST1. In the present study the mechanism of MGST1 activation by hepsin was investigated. When MGST1 and hepsin were incubated at room temperature, MGST1 activity was markedly increased and the increase was decreased to the control level by further incubation with disulfide bond reducing agent dithiothreitol. MGST1 dimer was detected by electrophoresis after treatment of MGST1 with hepsin, instead of proteolytic product. MGST1 dimer formation accompanied by an increase in MGST1 activity was observed even in the presence of the protease inhibitor benzamidine. Furthermore, prolonged incubation of both enzymes caused the formation of MGST1 dimer and its proteolytic product. These results clearly show that the protease hepsin stimulates disulfide-linked MGST1 dimer formation resulting in activation of MGST1 and preferential degradation of MGST1 dimer. Since hepsin contains disulfide bonds in the scavenger receptor cysteine-rich (SRCR) domain, it was suggested that the SRCR domain interacts with MGST1 leading to thiol/disulfide exchange between the two enzymes followed by disulfide-linked MGST1 dimer formation.

Anti-adult T-cell leukemia effects of a novel synthetic retinoid, Am80 (Tamibarotene).

Clinical trials for treatment of adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) using all-trans-retinoic acid (ATRA) have shown satisfactory therapeutic responses, although efficacies were limited. Recently, many synthetic retinoids have been developed and among them, a novel synthetic retinoid, Am80 (Tamibarotene) is an RARalpha- and RARbeta-specific retinoid expected to overcome ATRA resistance. The present study examined the inhibitory effects of Am80 on HTLV-I-infected T-cell lines and ATL cells. Am80 had negligible growth inhibition of peripheral blood mononuclear cells but marked growth inhibition of both HTLV-I-infected T-cell lines and ATL cells. Am80 arrested cells in the G1 phase of the cell cycle and induced apoptosis in HTLV-I-infected T-cell lines. It inhibited also the phosphorylation of IkappaBalpha and NF-kappaB-DNA binding, in conjunction with reduction of expression of proteins involved in the G1/S cell cycle transition and apoptosis. Am80 also inhibited the expression of JunD, resulting in suppression of AP-1-DNA binding. Furthermore, severe combined immunodeficient mice with tumors induced by subcutaneous inoculation of HTLV-I-infected T cells, responded to Am80 treatment with partial regression of tumors and no side-effects. These findings demonstrate that Am80 is a potential inhibitor of NF-kappaB and AP-1, and is a potentially useful therapeutic agent against ATL.

Anti-adult T-cell leukemia effects of Bidens pilosa.

We evaluated the effects of Bidens pilosa, a plant found in tropical and subtropical regions, and investigated the molecular pathways responsible for the anti-adult T-cell leukemia (ATL) effect. Water extracts of B. pilosa had growth suppressive effects on human T-cell leukemia virus type 1 (HTLV-1)-infected T-cell lines and ATL cells. B. pilosa extracts arrested cells in G1 cell cycle and induced apoptosis of HTLV-1-infected T-cell lines. B. pilosa extracts inhibited also the phosphorylation of IκB kinase ß and IκBα, and NF-κB-DNA binding, in conjunction with reduction of expression of proteins involved in G1/S cell cycle transition and suppression of apoptosis. Reactive oxygen species played a role in B. pilosa-mediated suppression of NF-κB activity. B. pilosa extracts also inhibited the expression of JunB and JunD, resulting in suppression of AP-1-DNA binding. In animals harboring tumors of HTLV-1-infected T-cell origin, treatment with B. pilosa extracts suppressed tumor growth. Our results suggest that B. pilosa is a potentially useful medicinal plant for treatment of ATL.

Purification of liver serine protease which activates microsomal glutathione S-transferase: possible involvement of hepsin.

Rat liver microsomal glutathione S-transferase (MGST1) is known to be activated by trypsin, however, it has not been clarified whether MGST1 is activated by a protease present in liver. In the present study we purified the MGST1 activating protease from liver microsomes and finally identified that the protease is hepsin, a type II transmembrane serine protease. When the protease was incubated with the purified MGST1 or liposomal MGST1 at 4 degrees C, MGST1 activity was increased 3-4.5 fold after 3-6 d. In electrophoretic and immunoblot analyses after the incubation of MGST1 with the protease MGST1 dimer and its degraded fragment were detected. These results suggest that the rat liver microsomal hepsin functions as MGST1 activating/degrading enzyme.