Anti-tumor promoting effects of multiflorane-type triterpenoids and cytotoxic activity of karounidiol against human cancer cell lines.

Forty-nine multiflorane-type triterpenoids consisting of 11 compounds isolated from the seeds of Trichosanthes kirilowii (Cucurbitaceae) and 38 of their derivatives have been evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in Raji cells as a primary screening test for anti-tumor promoters. All of the compounds tested showed an inhibitory effect against EBV-EA activation, and among which 43 were revealed to possess remarkable activity with potencies either comparable to or stronger than that of glycyrrhetic acid, a known natural anti-tumor promoter. Their structure-activity relationship is discussed. Evaluation of the cytotoxic activity of karounidiol (27) against human cancer cell lines exhibited cytotoxicity especially against a human renal cancer.

Inhibitory effects of anthraquinones and bianthraquinones on Epstein-Barr virus activation.

Short-term in vitro assays for anti-tumor promoters were carried out for several anthraquinones and bianthraquinones, which were isolated from Cassia siamea and derived from cascaroside A. Anthraquinone monomers showed higher anti-tumor promoting activity than that of bianthraquinones.

Anti-tumor-promoting effects of 8-substituted 7-methoxycoumarins on Epstein-Barr virus activation assay.

In a search for anti-tumor-promoting agents, we carried out a primary screening of twenty-nine 8-substituted and four 6-substituted derivatives of 7-methoxycoumarins isolated from plants of the Murraya and/or Citrus species (Rutaceae), examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. This investigation indicated that the prenyl (3-methyl-2-butenyl) or 2-hydroxy-3-methylbutyl (or butenyl) unit as an isoprenoid moiety at C-8 on the 7-methoxycoumarin nucleus plays an important role in the anti-tumor-promoting activity. Some of the 8-substituted 7-methoxycoumarins isolated from Murraya species, murrangatin (7), minumicrolin (10) and chloticol (18), were found to significantly inhibit EBV-EA activation, and preserved the high viability of Raji cells, suggesting that 7, 10 and 18 might be valuable anti-tumor-promoting agents.

Inhibitory effects of dehydrozingerone and related compounds on 12-O-tetradecanoylphorbol-13-acetate induced Epstein-Barr virus early antigen activation.

Dehydrozingerone, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, is half an analog of curcumin which is known to have anti-tumor activity. The anti-tumor promoting activity of dehydrozingerone was evaluated by determining the inhibitory effect on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). The concentration needed for 50% inhibition of the tumor promotion (IC50) of dehydrozingerone was similar to that of curcumin. To elucidate the structure-activity relationship on the anti-tumor promoting activity, dehydrozingerone, curcumin, isoeugenol, which has no carbonyl group in the side chain, benzalacetone, which is the basic structure of dehydrozingerone, o-dehydrozingerone, which is the ortho-hydroxyl substituted compound of dehydrozingerone, and their related compounds were investigated using the in vitro short-term assay on TPA-induced EBV-EA activation. o-Dehydrozingerone showed the most potent inhibitory effect in a series of tested dehydrozingerone derivatives and their related monosubstituted benzalacetones. This suggests that the occupation at both ortho positions of the hydroxyl group enhances the anti-tumor promoting activity. Isoeugenol inhibited the tumor promoting activity at a concentration of about one-third of the IC50 of dehydrozingerone. This indicates that the carbonyl group in the side chain has a negative impact on the anti-tumor promoting activity. The inhibitory effects of the carbon-carbon bond in the side chain were studied using benzylacetone with a single bond, benzalacetone with a double bond and 4-phenyl-3-butyn-2-one with a triple bond. 4-Phenyl-3-butyn-2-one inhibited the most potent activity followed by benzalacetone and benzylacetone.

Inhibitory effect of flavonoid derivatives on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumors.

To search for possible anti-tumor promoters, ten flavonoid derivatives (1-10) synthesized from morin and quercetin were examined for their inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation by a short-term in vitro assay. Of these compounds, pentaallyl ethers (9, 10) showed significant inhibitory effects on EBV-EA activation induced by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate. Further, quercetin pentaallyl ether (10) exhibited remarkable inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.

Inhibitory effects of 6-O-acylated L-ascorbic acids possessing a straight- or branched-acyl chain on Epstein-Barr virus activation.

6-O-Acylated L-ascorbic acids possessing a straight- or branched-acyl chain of varying length from C(4) to C(18) have been synthesized and evaluated their anti-tumor promoting effects on the activation of the Epstein-Barr virus early antigen. The derivatives having a straight- or branched-acyl chain of C(6) to C(11) carbon atoms exhibited marked effects.

Inhibitory effect of Epstein-Barr virus activation by Citrus fruits, a cancer chemopreventor.

To search useful compounds in Citrus fruit for cancer chemoprevention, we carried out a primary screening of extracts of fruit peels and seeds from 78 species of the genus Citrus and those from two Fortunella and one Poncirus species, which were closely related to the genus Citrus. These Citrus extracts inhibited the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) as a useful screening method for anti-tumor promoters. Our results indicated that Citrus containing substances may be inhibit susceptibility factors involved in the events leading to the development of cancer.

Anti-tumor-promoting effects of phenylpropanoids on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis.

In our joint project in the search for anti-tumor promoters from natural plant sources, we carried out a primary screening of 12 phenylpropanoids isolated from Boronia pinnata Sm. (Rutaceae) by examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. All tested compounds in this study showed inhibitory activity against the EBV activation even at 1 x 10 mol ratio without any cytotoxicity. Among 12 phenylpropanoids tested, boropinal-C (1), boropinol-A (5), boropinol-C (9) and 3-(3'-methoxy-4'-prenyloxy)phenyl-1-propene (10), all having a 4'-(3-methylbut-2-enyloxy) group, a so-called prenyloxy group, showed more potent activities. Furthermore, 3-(3'-methoxy-4'-prenyloxy)phenyl-1-propene (10) also exhibited remarkable inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. This investigation indicated that certain phenylpropanoids might be valuable anti-tumor promoters.

Inhibitory effects of monoacylated 2-O-beta-galactosylglycerols on Epstein-Barr virus activation: the significant role of the hexanoyl chain.

Three series of monoacyl-2-O-beta-D-galactosylglycerols bearing an acyl chain of varying length, from C4 to C10, were studied due to their antitumor promoting effects on the activation of the Epstein-Barr virus early antigen (EBV-EA), such activation being induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). This study indicates that it is more the length of the acyl chain that is important for the activity, six carbon atoms resulting in maximum effect, rather than the position of the ester function and the nature of the sugar (galactose or glucose).

Anti-tumor-promoting effects of isoflavonoids on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis.

As a part of screening studies for anti-tumor promoters, fifteen isoflavonoids isolated from plants of the genus Millettia (Leguminosae) were evaluated by examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. All of the compounds tested in this study showed inhibitory activity against EBV, without showing any cytotoxicity. Auriculasin (11) and millepurone (13), which is an oxidized isoflavone analogue, both having one or more prenyl side-chains and a 3',4'-dihydroxyphenyl group in the molecule, showed more potent activity than any of the other compounds tested. Furthermore, millepurone (13) exhibited a marked inhibitory effect on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. The results of the present investigation indicate that some of these isoflavonoids might be valuable anti-tumor promoters.

Cancer chemopreventive activity of 3,5,6,7,8,3',4'-heptamethoxyflavone from the peel of citrus plants.

Nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone (HPT), isolated from the peel of Citrus plants, were examined for the anti-tumor-initiating activity on two-stage carcinogenesis of mouse skin tumors induced by a nitric oxide donor, (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide, as an initiator and 12-O-tetradecanoylphorbol-13-acetate as a promoter. HPT exhibited the remarkable anti-tumor-initiating effect on mouse skin and it suggested the possibility of HPT being a chemopreventive agent against nitric oxide (NO) carcinogenesis.

Anti-tumor promoting activity of polyphenols from Cowania mexicana and Coleogyne ramosissima.

Chemical investigation on polyphenol-rich fractions of Cowania mexicana and Coleogyne ramosissima (Rosaceae) which showed significant inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), has led to the characterization of 10 compounds including C-glucosidic ellagitannin monomers and dimers from the former plant, and 17 polyphenols including flavonoid glycosides from the latter. The effects of individual components and their analogues with related structures on the TPA-induced EBV-EA activation were then evaluated. Among the compounds isolated from C. mexicana, two C-glucosidic ellagitannins, alienanin B and stenophyllanin A and a nitrile glucoside (lithospermoside), and among the constituents from C. ramosissima, two flavonoid glycosides, isorhamnetin 3-0-beta-D-glucoside and narcissin were revealed to possess strong inhibitory effects on EVB-EA activation, the potencies of which were either comparable to or stronger than that of a green tea polyphenol, (-)-epigallocatechin gallate. These polyphenols except for nitrile glucoside, which was not tested owing to an insufficient amount, were also found to exhibit anti-tumor promoting activity in two-stage mouse skin carcinogenesis using 7,12-dimethylbenz[a]anthracene (DMBA) and TPA.

Development of gamma-aminobutyric acid (GABA)ergic neurons in cerebral cortical neurons in primary culture.

The developmental patterns of gamma-aminobutyric acid (GABA)ergic neurons in primary culture obtained from the neopallium of 15-day-old fetus of mouse were investigated in terms of morphological features, GABA metabolism and GABA receptor binding. Morphological investigations revealed that these cells possessed typical features of neurons and the formation of synapses was detected at 10 days after the inoculation. During neuronal growth on polylysine surfaces, GABA contents and activity of GABA transaminase (GABA-T) showed a progressive increase in the time of culture. Similarly, L-glutamic acid decarboxylase (GAD) showed a progressive elevation during neuronal development in vitro, which corresponded well with the change in immunoreactivity to anti-GAD examined immunohistochemically. In addition, the high K+-evoked release of [3H]GABA also showed an enhancement during the growth in vitro. The numbers of binding sites (Bmax) for [3H]muscimol and [3H]flunitrazepam (FLN) also showed increases with the time of incubation, although affinity (Kd) to the labeled ligands did not show any noticeable changes. Moreover, it was observed that [3H]FLN binding was enhanced by GABA even in neurons cultured for 7 days. These results indicate that cerebral cortical neurons in primary culture possess GABA biosynthesizing and degrading systems including a high-affinity uptake mechanism for GABA. The present results also indicate that these cells possess synaptic contacts as well as GABAA receptors coupled with benzodiazepine receptor from a relatively early stage of cellular development.

Development of taurine biosynthesizing system in cerebral cortical neurons in primary culture.

Developmental patterns of taurine biosynthesizing system were investigated using primary cultured neurons prepared from the neopallium of 15-day-old fetal mice by a trypsin treatment in comparison with those in cerebral cortices obtained from age-matched fetal and neonatal mice. The morphological observations by phase contrast and scanning electron micrographies indicated that the cells in primary culture used in the present study possessed typical features of neurons. In addition, the immunohistochemical studies using the antibody to glial fibrillary acidic protein (GFAP), a specific marker for astroglia, revealed that the contamination of astroglias was negligible. The contents of taurine and metabolic intermediates in taurine biosynthesis, cysteine sulfinic acid and cysteic acid, in primary cultured neurons showed decreases during their development, especially during the first week after the inoculation. Similar developmental patterns of these amino acids were observed in cerebral cortices in vivo during perinatal stage, which corresponded to the first week of neuronal growth in vitro. On the other hand, the activities of cysteine sulfinic acid decarboxylase and cysteine dioxygenase, both of which are involved in the biosynthesis of taurine, were found to be increased progressively both in primary cultured neurons and in cerebral cortices in vivo during their growth. The immunohistochemical study using antitaurine antibody obtained from rabbit clearly demonstrated that immunoreactive materials were localized in cell bodies and the processes of neurons, and the intensity of the immunoreactivity in primary cultured neurons also showed a reduction with time of culture. These results indicate that primary cultured neurons used in this study possess a similar capacity to synthesize taurine from cysteine as developing brains in vivo. The present results also strongly suggest the well known decrease in cerebral taurine content in vivo during neonatal stages may be predominantly due to the decrease of taurine in neuronal cells.

Mechanisms for pancreatic hypertrophy induced by long-term administration of bethanechol.

Mechanisms for the hypertrophy of rat pancreas induced by long-term administration of bethanechol were investigated. The administration of bethanechol, an acetylcholine receptor agonist, to male Wistar rats for 14 days induced significant increases in the pancreatic weight and contents of protein, amylase and RNA in the pancreas without altering the content of DNA and the incorporation of [3H]thymidine into DNA. Simultaneous administration of atropine with bethanechol suppressed the bethanechol-induced pancreatic hypertrophy. Long-term administration of other acetylcholine receptor agonists also showed similar effects as produced by bethanechol. CR1505 (loxiglumide; D,L-4-(3,4-dichlorobenzoyl-amino)-5-(N-3-methoxypropyl-pentylam ino)-5- oxopentanoic acid), an antagonist of cholecystokinin receptors, inhibited pancreatic growth induced by long-term administation of pentagastrin, whereas pancreatic hypertrophy induced by bethanechol was not inhibited by CR1505. These results suggest that long-term administration of bethanechol induces pancreatic hypertrophy through direct activation of muscarinic receptors in the pancreas.

Structure-activity relationship in potentially anti-tumor promoting benzalacetone derivatives, as assayed by the epstein-barr virus early antigen activation.

The in vitro anti-tumor promoting activities of antimutagenic benzalacetone (4-phenyl-3-buten-2-one), its monosubstituted derivatives and related compounds, cinnamaldehydes and cinnamic acids, were evaluated by determining the inhibitory effect on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. In this short-term assay, benzalacetone, which is the basic structure of dehydrozingerone (one-half analog of curcumin) inhibited the EBV-EA activation; the IC(50) value, the molar ratio of benzalacetone to TPA needed for inhibiting 50% of positive cells activated with 32 pmol TPA, was 129. IC(50) values of 2- and 4-methoxybenzalacetones were about one-half of that of benzalacetone and the methoxy compounds were more effective than hydroxybenzalacetones. IC(50) values of chloro- and trifluoromethyl-benzalacetones were higher than that of benzalacetone, indicating that these compounds are weaker inhibitors. In addition, the position of a substituent on the benzene ring affected the inhibitory effect. In benzalacetone derivatives substituted by a hydroxy-, methoxy-, chloro- or trifluoromethyl group, the 2-substituted derivatives exhibited the strongest inhibitory effect, followed by the 3- and the 4-substituents. Cinnamic acid derivatives also decreased the inhibitory effects in the same order. In the side chain of benzalacetone, the terminal group adjacent to the carbon-carbon double bond also affected the inhibitory effect. The conversions of the methylketone to aldehyde and carboxyl groups, i.e., cinnamaldehyde and cinnamic acid, increased the inhibitory effect: the IC(50) values were about one-third of that of benzalacetone. beta-Methyl styrene, which in the side chain has no carbonyl group adjacent to the double bond, inhibited the EBV-EA activation at the concentration of about one-third of that of benzalacetone, indicating that the carbonyl group negatively affects the inhibitory effect. This agreed with the previous observation between isoeugenol and dehydrozingerone, 4-hydroxy-3-methoxy derivatives of beta-methyl styrene and benzalacetone, respectively. The mechanism of the EBV-EA activation inhibition was discussed by being compared with the inhibition of mutagenesis for which the unsaturated bonded-carbonyl system is necessary.

Phagocytosis-enhancing effect of human leukocyte interferon preparation of human peripheral monocytes in vitro.

It was found that the phagocytosis of human peripheral monocytes was enhanced by human leukocyte interferon preparations. This activity showed species-specificity and a positive correlation between interferon titer and phagocytic activity. When human monocytes were treated by interferon, the pahagocytic activity was enhanced. But when interferon was added to the latex, the enhancement was not observed. The interferon preparation did not only increase the number of phagocytic cells, but also enhanced the degree of phagocytosis in individual cells. The factor responsible for the enhancing effect was inactivated by trypsin, but was not inhibited by actinomycin D.

Characteristics of taurine transport in rat hepatocytes in primary culture.

Characteristics of taurine transport in rat hepatocytes maintained in primary culture for 24 h (cultured hepatocytes) have been investigated. The uptake of [3H] taurine by cultured hepatocytes at 2 degrees C was unsaturable, whereas that at 37 degrees C consisted of unsaturable and saturable processes. The saturable transport system was sodium-dependent and consisted of two processes with low and with high affinities. The latter process (Km, 76.9 microM; Vmax, 0.256 nmole/mg protein/min; activation energy (EA), 37.8 kcal mol-1) was competitively inhibited by 2,4-dinitrophenol and ouabain, as well as by taurine analogues such as hypotaurine and guanidinoethyl sulphonate. The Vmax and EA values found in cultured hepatocytes at 37 degrees C were 6.0 and 6.8 times higher than those found in freshly isolated hepatocytes. These results indicate that taurine transport in hepatocytes in primary culture consisted of unsaturable, and saturable, sodium and energy-dependent carrier-mediated transport processes, respectively. The facilitation of the latter transport system by primary culture of hepatocytes is also suggested.