Isolation and identification of a cytotoxic principle from Chrysosplenium grayanum Maxim. (Saxifragaceae) and its antitumor activities.

A cytotoxic principle was newly isolated from Chrysosplenium grayanum Maxim. (Saxifragaceae) and identified as beta-peltoboykinolic acid (1) on the basis of spectral data. Cytotoxicity of compound 1 was tested against various human cancer cell lines in vitro, and antitumor effect of this compound was demonstrated on Meth.A mouse fibrosarcoma. The experiment of combined treatment with compound 1, mitomycin C, and OK-432 resulted in enhancing the antitumor effect against B16-BL6 mouse melanoma in C57BL/6 mice.

Phenoxazinone synthesis by human hemoglobin.

We found that 2-amino-5-methylphenol was converted to the dihydrophenoxazinone with a reddish brown color by purified human hemoglobin, lysates of human erythrocytes, and human erythrocytes. The reddish brown compound was identified as 2-amino-4,4 alpha-dihydro-4 alpha,7-dimethyl-3H-phenoxazin-3-one by the measurement of NMR spectra, IR spectra, EI mass spectra, and absorption spectra. The changes in this phenoxazinone were studied under various conditions after mixing 2-amino-5-methylphenol with purified oxy- or methemoglobin, or with human erythrocytes. The production of 2-amino-4,4 alpha-dihydro-4 alpha,7-dimethyl-3H-phenoxazine-3-one from 2-amino-5-methylphenol was found to be tightly coupled with the oxidation of ferrous hemoglobin and reduction of ferric hemoglobin under aerobic conditions. By studying the production rates of the dihydrophenoxazinone and the oxido-reduction rates of ferrous and ferric hemoglobins during the reactions of ferrous or ferric hemoglobin with 2-amino-5-methylphenol under aerobic and anaerobic conditions, the reaction mechanism was extensively proposed.

Oxidative condensation of 2-amino-4-methylphenol to dihydrophenoxazinone compound by human hemoglobin.

2-Amino-4-methylphenol was converted to a brownish yellow material by the lysates of human erythrocytes or purified human hemoglobin. The reaction proceeded oxidatively, coupled with the oxidation of hemoglobin. The major component of the brownish yellow material produced by oxidative condensation of 2-amino-4-methylphenol was identified as 3-amino-1,4 alpha-dihydro-4 alpha, 8-dimethyl-2H-phenoxazin-2-one on the basis of its spectral data including NMR spectra, IR spectra, EI mass spectra, and absorption spectra. The changes in 3-amino-1,4 alpha-dihydro-4 alpha,8-dimethyl-2H-phenoxazin-2-one during incubation of purified human hemoglobin and 2-amino-4-methylphenol were analyzed spectrophotometrically and by using HPLC. The reaction mechanism involved may be similar to that of actinomycin synthase, which oxidizes 2-amino-5-methylphenol to the dihydrophenoxazinone.

Inhibition of HIV-reverse transcriptase activity by some phloroglucinol derivatives.

Four phloroglucinol derivatives, named mallotophenone (5-methylene-bis-2,6-dihydroxy-3-methyl-4-methoxyacetophenone), mallotochromene (8-acetyl-5,7-dihydroxy-6-(3-acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-2,2-dimethylchromene), mallotojaponin (3-(3,3(dimethylallyl)5-(3(acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-phloracetophenone) and mallotolerin (3-(3-methyl-2-hydroxybut-3-enyl)-5(3-acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-phloracetophenone), have been tested for their ability to inhibit the activity of human immunodeficiency virus (HIV)-reverse transcriptase. Under the reaction conditions with (rA)n.(dT)12-18 as the template.primer, the enzyme activity was inhibited by approximately 70% in the presence of 10 micrograms/ml mallotochromene or mallotojaponin, whereas mallotophenone and mallotolerin were much less inhibitory to the enzyme. The enzyme activity was also inhibited, though to lesser extent, by these compounds under similar conditions with initiated MS-2 phage RNA as the template.primer. The mode of inhibition was, as analyzed with mallotojaponin, competivite with respect to the template.primer, (rA)n.(dT)12-18, and non-competitive with respect to the triphosphate substrate, dTTP. The Ki value of mallotojaponin for HIV-reverse transcriptase was determined to be 6.1 microM.

Antibody production useful for cytotoxicity against mouse MM2 tumor cells by peritoneal macrophages.

We applied an antibody-dependent macrophage-mediated cytotoxicity (ADMC) test in order to analyze the effector mechanism of the host-mediated antitumor effects induced by OK-432. Adherent peritoneal exudate (PE) cells were obtained from each of high (C3H/He) and low (B10) responder mice treated with OK-432, and 51Cr-labeled MM2 tumor cells were used as target cells. The cytotoxic activity in vitro coincided well with the results obtained in in vivo antitumor experiments. When adherent PE cells from C3H/He mice reacted with anti-MM2 serum from B10 mice, the degree of ADMC was significantly lower than that obtained with the anti-MM2 serum from C3H/He mice. The removal of IgG1 from the anti-MM2 serum induced in B10 mice resulted in the enhancement of ADMC activity. Then mean level of IgG2 in each of anti-MM2 sera from C3H/He and B10 mice was higher than in normal serum, and the IgG1 level in the antiserum from B10 mice was also higher than that in the serum from normal B10 mice. The present work suggested that the active component(s) in anti-MM2 serum participating in ADMC was a specific antibody of the IgG2 subclass, and that the inhibiting factor(s) was the IgG1 subclass.

Inhibition of HIV-reverse transcriptase activity by asterriquinone and its analogues.

Asterriquinone (ARQ; 2,5-bis-[1'-(1", 1"-dimethyl-2"-propenyl)- indol-3'-yl]-3,6-dihydroxy-1,4-benzoquinone) and its three analogues [i.e., 3,6-dihydroxy-2-[2'-(1", 1"-dimethyl-2"-propenyl)-indol-3'-yl]-5-[1', 7'- (1",1"-dimethylpropano)-indol-3'-yl]-1,4-benzoquinone (B1-4), 3,6-dihydroxy-2-[2'-(1", 1"-dimethyl-2"-propenyl)-indol-3'-yl]-5-indol-3'-yl-1,4-benzoquinone (C1-1) and 3,6-dihydroxy-2,5-diindol-3'-yl-1,4-benzoquinone (D-1)] were found to be strong inhibitors of the activity of reverse transcriptase from human immunodeficiency virus type-1. Under the reaction conditions employed, the enzyme activity was inhibited by more than 70% in the presence of 10 microM each of these compounds. The mode of inhibition by these compounds was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive with respect to the triphosphate substrate, dTTP. The Ki values of HIV-1 reverse transcriptase were determined to be 2.3, 1.5, 0.1 and 0.3 microM for ARQ, B1-4, C1-1 and D-1, respectively.

Immune reactivity in bronchogenic carcinoma and its relation to 5-year survival rate.

We performed a prospective study on the correlation of various parameters of the immune response with the 5-year survival rate in patients with bronchogenic carcinoma. Parameters were initially examined before starting treatment. Delayed hypersensitivity skin tests, lymphoblastogenesis, natural killer (NK) cell activity, and interleukin-2 (IL-2) production were employed to assess immune competence. Each reaction was classified into four or five grades in accordance with intensity; the 5-year survival rate of the patients showing each grade of the immune response was calculated. A correlation between response before treatment and the survival rate was most clearly noted for lymphoblastogenesis. The skin tests and the NK cell activity showed poorer correlations, and no exact correlation was noted between the IL-2 production and the immune response.

Interaction of asterriquinone with deoxyribonucleic acid in vitro.

The interaction of asterriquinone (ARQ), a novel antitumor agent isolated from Aspergillus fungi, with deoxyribonucleic acid (DNA), has been studied. The binding of ARQ in vitro with DNA (calf thymus) was ascertained by its behavior in gel filtration using a Sephadex G-25 column at pH 5.4. Some ARQ analogs having no, or less, antitumor activity did not exhibit any evidence of interaction with DNA under the same condition. From the results obtained in this work, the pKa value of ARQs seemed to be critical between 6 and 7 for their binding to DNA and for exhibition of antitumor activity. Also, ARQ showed serious membrane deformations and an inhibitory effect on the membranous adenosine triphosphatase of Ehrlich carcinoma cells.

Cytotoxic and antitumor constituents in pericarps of Mallotus japonicus.

A variety of phloroglucinol derivatives isolated from the pericarps of Mallotus japonicus were assessed for growth inhibiting activity against human larynx (HEp-2) and lung (PC-13) carcinoma cells as well as mouse B16 melanoma, leukemia P388, and L5178Y cells. Most of these derivatives were proved to be significantly cytotoxic in culture. One of them, 3-(3,3-dimethylallyl)-5-(3-acetyl-2,4-dihydroxy-5-methyl-6-methoxybenzyl )- phloracetophenone showed an excellent cytotoxicity against all target tumor cells and a marked prolongation of life-span in mice bearing L5178Y leukemia.

Augmentation of various immune reactivities of tumor-bearing hosts with an extract of Cordyceps sinensis.

In order to enhance general reactivity of immune system in the tumor-bearing host, we employed extract of Cordyceps sinensis (CSE) as a biological response modifier. Cordyceps sinensis is an interesting material produced by a kind of mushroom parasitic to larval moths and was used to hasten recovery from exhaustion in ancient China. In this experiment, C57BL/6 mice implanted subcutaneously with syngeneic EL-4 lymphoma cells were employed as the host. Oral administration of the extract leads to a reduction of tumor size and prolongation of the host survival time. As judged by plaque-forming cells against T-dependent (sheep erythrocytes) and T-independent (bacterial lipopolysaccharide) antigens, CSE showed to augment the antibody responses. As for the activities of peritoneal macrophages, chemotaxis was dramatically depressed within a few days after EL-4 transplantation up to the end of life, but treatment with CSE at -14, -7, -4, +4, +7 and +10 days after the tumor transplantation augmented the activity about four times stronger than that of control. Phagocytic activity of macrophages was also decreased in tumor-bearing mice treated with cyclophosphamide (100 mg/kg) 3 and 5 days after tumor transplantation. But administration of CSE restored the activity to more than the normal level. The overall efficacy of CSE was tested with protective activity against systemic infection by Salmonella enteritides. The tumor-bearing mice receiving this medicine lived significantly longer than any other groups without CSE.

Changes in immunological parameters in lung cancer patients undergoing immunotherapy with streptococcal preparation OK-432.

We have studied the immunological status of patients treated with streptococcal preparation OK-432. Two KE of OK-432 was injected intramuscularly once every week for more than three years unless the patients died. The natural killer (NK) activity in those patients who underwent curative surgery for lung cancer and had no sign of recurrence was significantly increased (P less than 0.01) during the OK-432 treatment. However, the NK activity in the patients who had persistent disease (non-resected cases, incompletely resected cases or recurrent cases) was not significantly increased in comparison with that before the immunotherapy. Also, in the cases with no clinical symptoms of recurrence, both the lymphoblastogenetic reactions to the mitogens and the IL-2 production were significantly enhanced (P less than 0.01) during the administration of OK-432. Reactions in the SU-PS (polysaccharide taken from the cell-wall fraction of the Streptococcus pyogenes SU strain and containing 7.2% of protein) skin-test appeared to significantly correlate with the immunological status of the patients under OK-432 therapy, but the PHA and PPD skin reactions showed no definitive enhancement. The survival rate of the patients whose SU-PS skin tests were positive during the OK-432 immunotherapy was significantly higher (P less than 0.01) than that of the patients with negative reactions.

Antitumor activity of an extract of Cordyceps sinensis (Berk.) Sacc. against murine tumor cell lines.

A warm water-extract (ECS) prepared from dried Cordyceps sinensis (Berk.) Sacc., a Chinese traditional medicine, was tested for antitumor activity in vivo and in vitro. Ehrlich ascites carcinoma cells (EAC), allogeneic to ICR mice and Meth A fibrosarcoma (Meth A), syngeneic to BALB/c mice were used as the target tumor cell lines. Mice were inoculated i.p. with 1 x 10(6) EAC or 1 x 10(5) Meth A on Day 0, and ECS or saline (control) was injected i.p. to the mice from Day 1 to Day 4. ECS-treatment increased the median survival time of the allogeneic mice inoculated with EAC to 316% of the control. Eight of the 10 ECS-treated mice survived on the 60th day (Day 60) after EAC implantation. ECS-treatment also increased the median survival time of the syngeneic mice inoculated with Meth A to 312% of the control. Half of the ECS-treated mice survived on Day 60. On the other hand, no cytotoxic effect of ECS was found on either EAC or Meth A in vitro. The antitumor effect of ECS seen in the allogeneic mice was significantly reduced when the mice received whole body X-irradiation (5 Gy) before EAC implantation. These results suggest that the antitumor effect of ECS may be mediated through its immunomodulating action.

In vivo localization of radiolabeled monoclonal antibody to carcinoembryonic antigen (CEA) in a CEA-producing tumor--comparison with polyclonal antibody.

To compare accumulation of the 125I-labeled antibodies (anti-carcinoembryonic antigen (CEA) monoclonal antibody and polyclonal antibody) to a CEA-producing tumor (SC-2-JCK), an in vivo localization study was performed in nude mice. The tumor-to-blood ratio at 120 hours after injection rose to 4.6 for the monoclonal antibody, but remained at 1.3 for the polyclonal antibody. However, no differences were noted between the antibodies up to 72 hours after injection. In autoradiograms, selective accumulation of the tracer was noted in the tumor for both antibodies. However, no superiority or inferiority of imaging for either of the antibodies could be definitely determined.

Biological active macrocyclic diterpenoids from chinese drug "Fáng Féng Cáo"; II. Derivatives of ovatodiolids and their cytotoxity.

Isolated macrocyclic diterpenoids, ovatodiolide ( 1), 4, 5-epoxyovatodiolide ( 2), anisomelic acid ( 3), 4, 7-oxycycloanisomelic acid ( 4), and 4-methylene-5-oxoanisomelic acid ( 6), from the Chinese crude drug "Fáng Féng Cáordquo;, and the derivatives of the isolated compounds were tested for their growth inhibiting activities in culture of KB cell line. Besides, the effects of 1 on Ehrlich carcinoma in mice are reported.

Studies on cytotoxic constituents in pericarps of Mallotus japonicus, Part II.

Two new phloroglucinol derivatives, mallotolerin (8) and mallotochromanol (9), were isolated from the cytotoxic fraction of the pericarps of Mallotus japonicus. The new derivatives were identified as 3-(3-methyl-2-hydroxybut-3-enyl)-5-(3-acetyl-2,4-dihydroxy-5-me thy l-6- methoxybenxyl)-phlorbutyrophenone (8) and 8-acetyl-5,7-dihydroxy-6-(3-acetyl-2,4-dihydroxy-5-methyl-6- methoxybenxyl)2,2-dimethyl-3-hydroxychroman (9) by their respective chemical and spectral data. These compounds were found to be cytotoxic against KB and L-5178Y cell lines in culture and to be moderately effective in inhibiting the growth of Ehrlich carcinoma in mice. Cytotoxic activities of the isolated phloroglucinol derivatives (1-9) from the pericarps are also discussed.

Production of potent immunoglobulins by group A streptococcus for antibody dependent cell-mediated cytotoxicity in mice.

Antibody-dependent cell-mediated cytotoxicity (ADCC) through serum, which was obtained from B6D2F1 mice pretreated with heated group A streptococci, and spleen cells of normal B6D2F1 mice was examined for different kinds of mouse tumor cell line by using methods to estimate specific 51Cr-release from each 51Cr-labeled target cells. Serum obtained from mice 14 days after the injection with heated streptococci was evidently potent to cause ADCC for all tumor cells tested. When the experiments were carried out under the condition which E/T ratio is 50:1, specific 51Cr-release from each target cells were about 32% for L1210 cells, about 12% for MOPC 31C cells, and about 23% for MH 134 and about 10% for P 388 cells, respectively. Meanwhile, serum obtained from either BCG-injected mice or normal mice was not so much potent for these tumor cells. Experiments to identify immunoglobulin subclasses participating in antibody-dependent cell-mediated cytotoxic reactions clearly showed that only IgG2a immunoglobulin was effective to induce cytotoxic reaction. All classes or subclasses of serum immunoglobulins from mice pretreated with group A streptococci were estimated using single radial immunodiffusion plates which were incorporated with monospecific rabbit antiserum for each classes or subclasses. Results showed that either IgM or IgG2a levels were significantly increased 7 days and 14 days after the injection, but other immunoglobulin classes or subclasses did not. A possible consideration on the production of immunoglobulin to cause ADCC in mice treated with heated streptococci was discussed.

Studies on cytotoxic constituents in pericarps of Mallotus japonicus, Part I.

Two new phloroglucinol derivatives, named mallotophenone and mallotochromene, were isolated from the pericarps of Mallotus japonicus together with two known compounds, 3-(3,3-dimethylallyl)-5-(3-acetyl-2,4-dihydroxy-5-methyl-6- methoxybenzyl)-phloracetophenone and 2,6-dihydroxy-3-methyl-4-methoxyacetophenone. The new derivatives were confirmed to be 5-methylene-bis-2,6-dihydroxy-3-methyl-4-methoxyacetophenone and 8-acetyl-5,7-dihydroxy-6-(3-acetyl-2,4-dihydroxy-5-methyl-6- methoxybenzyl)2,2-dimethylchromene by their respective chemical and spectral data. Several of the phloroglucinol derivatives isolated from this tree were cytotoxic against the KB system and L-5178Y in cell culture.