Yohei Ito

Inhibitory effects of ursolic and oleanolic ancid on skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

Ursolic acid (UA) and oleanolic acid (OA), which had been isolated from Glechoma hederacea as inhibitors of Epstein-Barr virus (EBV) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), were tested against inhibitory effect on tumor promotion by TPA in vivo. They inhibited effectively the tumor promotion in mouse skin and the activities were comparable to that of a known inhibitor of tumor promotion, retinoic acid (RA). Interestingly, UA was more effective on a single application before initial TPA-treatment than on a continuous application before each TPA-treatment, while OA and RA were ineffective in the same treatment. These data suggest that the role of UA for inhibitory action on tumor promotion differs slightly from those of RA and OA.

Search for possible antitumor promoters by inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus activation; Ursolic acid and oleanolic acid from an anti-inflammatory Chinese medicinal plant, Glechoma hederaceae L.

From an anti-inflammatory Chinese medicinal plant, Glechoma hederaceae L., two triterpene carboxylic acids, ursolic acid (UA) and oleanolic acid (OA) have been isolated as inhibitors of 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Barr virus (EBV) activation in Raji cells. Both acids significantly inhibited the activation at a 1000-fold molar ratio to TPA, and also teleocidin B-4. The dose responses of the acids were very similar to those of the antitumor promoters, retinoic acid (RA) and glycyrrhetinic acid (GA). However, a characteristic property that UA and OA possess, far higher cell viability to the Raji cells than RA to the Raji cells, has been pointed out. Furthermore, enhancement of the inhibitory activity was found in 3-keto derivatives of UA and OA, while either loss of oxygen functionality at C-3 position of UA or oxidation at C-3 of GA led to reduction of the activity. Binding assay suggested that the inhibitory activity should be exhibited by some event caused after binding of TPA to the receptor in the cells.

Combined effect of the extracts from Croton tiglium, Euphorbia lathyris or Euphorbia tirucalli and n-butyrate on Epstein-Barr virus expression in human lymphoblastoid P3HR-1 and Raji cells

The combined usage of n-butyrate and 12-O-tetradecanoylphorbol-13-acetate (TPA) or the oily extracts from Croton tiglium, Euphorbia lathyris or Euphorbia tirucalli exerted a marked effect on induction of Epstein-Barr virus (EBV)-associated early (EA) and viral capsid (VCA) antigens in EBV genome-carrying human lymphoblastoid cell lines. In producer P3HR-1 cells, the enhancing effect of the 2 components was additive both for EA and VCA, while in non-producer Raji cells, a synergistic increase of EA was observed. The possible implication of these findings relating to the cause of EBV-associated diseases is discussed.

A new view of the etiology of nasopharyngeal carcinoma

Abstract The epidemiological finding of high geographical similarity seen in the distribution of Croton tiglium , the parental plant for croton oil, as well as other related members of Euphorbiaceae family, and the nasopharyngeal carcinoma (NPC) in southern regions of China, led to the compilation of these data plus our recent laboratory observations on in vitro induction of Epstein-Barr virus (EBV) in human lymphoblastoid cell lines. The EBV genome-carrying P3HR-1 and Raji cells exerted a dramatic increase in EBV antigens when given a combined treatment with n -butyrate and croton oil, in nanogram concentrations. The Fusobacterium and other common microbial inhabitants of the mouth and nasopharynx of man efficiently produce, in laboratory culture fluids, n -butyric acid with EBV-inducer activity. Furthermore, the seeds and plant per se of C. tiglium and other Euphorbiaceae are currently being used as herbal drug in the southern parts of China. We hypothesize that NPC is initiated by persisting EBV as an essential and basic factor plus the combined action of the bacterial fatty acid and traditional intake of promoter substance of plant origin as cofactors, and that the EBV, induced and activated by such mechanisms, initiates the chain of events which ultimately lead to the emergence of the malignant disease.

Identification of HTLV p19 specific natural human antibodies by competition with monoclonal antibody.

Abstract A competitive binding assay using a monoclonal antibody to the human T-cell lymphoma/leukemia virus (HTLV) p19 was developed for use in detecting natural antibodies to the protein in human sera. The specificity of the assay for HTLV p19 was demonstrated using a variety of antisera. While sera known to contain antibodies to HTLV p19 competed in the assay, antisera prepared against purified HTLV p24, the major core protein of the virus, or against other disrupted type-C retroviruses did not. Sera of Japanese patients with adult T-cell leukemia and similar T-cell malignant lymphomas were examined by this technique for the presence of antibodies to HTLV p19. The results were compared with those obtained by a solid-phase radioimmunoassay (RIA) against disrupted HTLV. The majority of Japanese ATL patients possess natural antibodies to HTLV as shown by solid-phase RIA (88%) and also specifically to HTLV p19 (77%). Similarly, 50% of Japanese patients with similar T-cell malignant lymphomas possess HTLV antibodies by solid-phase RIA and nearly as many (42%) possess anti-p19 reactivity. Twelve and eight percent, respectively, of normal Japanese donors from the ATL endemic region possessed HTLV-specific antibody by the solid-phase RIA or competitive binding assay. Normal donors from nonendemic areas lacked antibodies to HTLV. These results extend our previous findings of natural antibodies to HTLV in Japanese patients with ATL. The finding of p19-specific antibodies in these Japanese sera, together with previous reports of natural antibodies to HTLV p24 in sera from this same geographic cluster, strengthens the association of HTLV with Japanese ATL.

Epstein-Barr virus activation by tung oil, extracts of Aleurites fordii and its diterpene ester 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate

During the screening of plant oils for their Epstein-Barr virus (EBV)-activating potency, we found that tung oil possesses an activity comparable to croton oil. Tung oil from various sources and the extracts from its parental plant Aleurites fordii (Chinese tung oil tree), when used in combination with n-butyrate, were shown to efficiently activate EBV persisting in human lymphoblastoid Raji cells (non-producer). The major diterpene ester in the plant extract, 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (HHPA), also exerted a similar activity. In producer P3HR-1 cells, both tung oil and HHPA increased the yield of infectious EBV by approximately five-fold. Since tung oil is used for the manufacture of oil paints, varnishes, waterproof substance, anticorrosives and other products, the implication of using such an agent with EBV-activating potency in our daily life is assessed and discussed.

Chinese and African Euphorbiaceae plant extracts: markedly enhancing effect on Epstein-Barr virus-induced transformation

Chinese and African Euphorbiaceae plant extracts were shown to have a markedly enhancing effect on Epstein-Barr virus (EBV)-induced transformation of human lymphocytes. When 5 X 10(5) cord blood lymphocytes were seeded into the semisolid agar medium immediately after EBV exposure, 3-10 times more colonies developed in the presence of the plant extracts at their optimal doses. When a smaller number of 5 X 10(4) cells were seeded, transformed colonies were also observed in the presence of the plant extracts but not in their absence. All of the colonies picked up from the agar medium were EBV-determined nuclear antigen (EBNA)-positive and showed the typical blastoid morphology. There were no colonies detected in the EBV-uninfected cultures with the extracts, indicating that the virus was required for the promotion by these plant extracts of this lymphocyte transformation. Euphorbiaceae plants are known to be employed as local herbal drugs in southern China and tropical Africa, and the possible role as a co-factor of the plant extracts in the development of nasopharyngeal carcinoma (NPC) and African Burkitts lymphoma (BL) is discussed.

Integration and methylation of shope papilloma virus DNA in the transplantable V×2 and V×7 rabbit carcinomas

The Shope papilloma virus (SPV) DNA present in SPV-induced benign and malignant rabbit tumors, particularly in the transplantable carcinoma Vx2 and Vx7, was examined with regard to physical states and extent of methylation. Vx2 and Vx7 carcinomas contained 10-22 viral genomes per diploid cell, and domestic and cottontail rabbit papillomas 40-400 and 1000-8000, respectively. The digestion of Vx2 and Vx7 DNA with the restriction enzyme KpnI, which does not cleave SPV DNA, yielded a single virus-specific DNA band about nine times larger than the genome length, but EcoRI, which cuts the circular SPV DNA once, cleaved this DNA to the genome-size fragments. However, three or four weak bands which may contain viral segments linked to cellular sequences were also identified, and at least two were shared by both Vx2 and Vx7 carcinomas. The analysis with a set of MspI and HpaII, which discriminates the methylated DNA sequence -CC*GG-, showed that 10-40% of the sites of viral DNA are methylated in papillomas, 30-80% in primary carcinomas, and more than 90% in the transplantable carcinomas.

Epstein-Barr virus-induced polypeptides: a comparative study with superinfected Raji, IUdR-Treated, and N-butyrate-treated P3HR-1 cells.

Abstract Three Epstein-Barr virus (EBV)-induced in vitro systems, EBV-superinfected Raji, IUdR-, and n -butyrate-treated P3HR-1 cells, were studied using a radioimmunoprecipitation method, and the EBV-specific polypeptide profiles were analyzed with anti-EBV human sera. In the three cell systems, 16, 20, and 22 polypeptides were identified, respectively. All 16 polypeptides detectable in the EBV-infected Raji cells belonged to the early products insensitive to phosphonoacetic acid (PAA) and were also shared by the other two systems. Among these common polypeptides, two with molecular weights of 140,000 (140K) and 120K were identified both in the cytoplasm and in the nucleus. However, these are not the major components of EA induced in the EBV-infected Raji cells. All other 14 polypeptides were found exclusively in the cytoplasm and were precipitated by all VCA(+)EA(+) sera, indicating that all of these polypeptides are EA components and that 90K, 54K, and 38K in particular are the major constituents of the EA. The six polypeptides (150K, 145K, 75K, 46K, 34K, 18K), which were detectable in n -butyrate-treated P3HR-1 cells, and four of which are common to those in IUdR-treated P3HR-1 cells, are the late virus-specific products sensitive to PAA. In contrast to the early polypeptides, these late products were found both exclusively in the cytoplasm and the nucleus. Among these products the 150K polypeptide is probably the main component of VCA.

Effect of an aromatic retinoic acid analog (Ro 10-9359) on growth of virus-induced papilloma (Shope) and related neoplasia of rabbits

Abstract An aromatic analog of retinoic acid (Ro 10-9359), a synthetic compound known to arrest development and growth of chemically-induced skin papillomas and carcinomas of mice, exerts a marked inhibitory effect on induction and development of virus-induced papilloma (Shope) of rabbit skin. The intramuscular administration of 12.5, 50 and 200 mg/kg given twice weekly during the induction phase of the neoplasia substantially inhibited the growth of the papilloma, this inhibition being dose-dependent. When the animals bearing well-established tumors were given a relatively large dose (200 mg/kg) of the compound, there was remarkable inhibition of the papillomatous growths and complete regression occurred in about 60%. The transplantable carcinomas Vx2 and Vx7, both of which originated from the Shope virus-induced papilloma, were less sensitive than their original papillomas to this treatment.