Inhibition of DNA topoisomerases I and II of compounds from Reynoutria japonica.

Three anthraquinones (1, 2 and 4), three stilbenes (5, 6 and 7) and 3,5-dihydroxybenzyl alcohol (3) were isolated from Reynoutria japonica. Their structures were identified as emodin (1), emodin-8-O-ß-D-glucoside (2), 3,5-dihydroxybenzyl alcohol (3), citreorosein (4), cis-resveratrol (5), trans-resveratrol (6) and trans-resveratrol-5-O-ß-D-glucopyranoside (7) by comparing their physicochemical and spectral data with published data. Compound 3 was isolated for the first time from the Polygonaceae family. Among the purified compounds, 3 showed more potent inhibitory activity against topoisomerase I (IC50: 4 µM) than camptothecin, as the positive control (IC50: 18 µM). Compounds 3, 4, 5, 6 and 7 showed stronger inhibitory activities toward DNA topoisomerase II (IC50: 0.54, 14, 15, 0.77 and 3 µM, respectively) than the positive control, etoposide (IC50: 44 µM). Compounds 1 and 4 displayed weak cytotoxicities against human lung cancer (A549), ovarian cancer (SK-OV-3), human liver hepatoblastoma (HepG2) and colon adenocarcinoma (HT-29) cell lines.

Plant-derived EpCAM antigen induces protective anti-cancer response.

Immunotherapy holds great promise for treatment of infectious and malignant diseases and might help to prevent the occurrence and recurrence of cancer. We produced a plant-derived tumor-associated colorectal cancer antigen EpCAM (pGA733) at high yields using two modern plant expression systems. The full antigenic domain of EpCAM was efficiently purified to confirm its antigenic and immunogenic properties as compared to those of the antigen expressed in the baculovirus system (bGA733). Recombinant plant-derived antigen induced a humoral immune response in BALB/c mice. Sera from those mice efficiently inhibited the growth of SW948 colorectal carcinoma cells xenografted in nude mice, as compared to the EpCAM-specific mAb CO17-1A. Our results support the feasibility of producing anti-cancer recombinant vaccines using plant expression systems.

Immunization with an antigen identified by cytokine tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo.

We have reported previously that s.c. immunization of rats with IL-4 transduced 9L gliosarcoma cells (9L-IL-4) induced a potent antitumor immunity against intracranial, parental 9L tumors. Subcutaneous implantation of 9L-IL-4 influenced the systemic humoral response, which was demonstrated by Th2-type isotype-switching and the induction of cellular immune responses, which played a critical role in the rejection of tumors. Serological analyses of recombinant cDNA expression libraries (SEREX), has recently emerged as a powerful method for serological identification of tumor-associated antigens (TAAs) and/or tumor rejection antigens (TRAs). Because IL-4 is known to activate B cells and to promote humoral responses, and inasmuch as induction of humoral responses by central nervous system tumors has been reported to be minimal, we investigated whether the induction of a potent humoral immune response against 9L TAAs or TRAs in rats immunized s.c. with 9L-IL4 could be demonstrated. Screening of 5 x 10(5) independent clones of 9L-expression cDNA library for the presence of reactive antibodies in the serum from a 91-IL-4 immunized rat led to the identification of three different TAAs. One 9L TAA (clone 29) was demonstrated to be calcyclin, a member of the S-100 family of calcium-binding proteins. The second 9L TAA (clone 37) was demonstrated to be the rat homologue of the J6B7 mouse immunomodulatory molecule. The third TAA (clones 158 and 171) was determined to be the rat homologue of the mouse Id-associated protein 1 (MIDA1), a DNA-binding, protein-associated protein. Northern blotting demonstrated that message for calcyclin was overexpressed in 9L cells. Message encoding MIDA1 was highly expressed in parental 9L cells and thymus and, to a lesser degree, in testis, suggesting that MIDA1 was comparable with the cancer/testis category of TAAs. Sera obtained from animals bearing 9L-IL-4 were found to have a higher a frequency and titer of antibodies to these antigens when compared with sera obtained from rats bearing sham-transduced 9L (9L-neo) cells. To determine whether immunization with these TAAs induced antitumor immunity, animals were immunized by intradermal injection with expression plasmids encoding calcyclin or MIDA1. Subsequent challenge of rats with parental 9L resulted in significant suppression of tumor growth in animals immunized with MIDA1, but not with calcyclin. These results indicate that MIDA1 is an effective 9L TRA and will be useful for the investigation of specific antitumor immunity in this glioma model. Furthermore, these results suggest that this approach, termed "cytokine-assisted SEREX (CAS)," may serve as an effective strategy for identification of TRAs for in animal-glioma models of cytokine gene therapy, and potentially in humans undergoing cytokine gene therapy protocols as well.

The combined effect against colon-26 cells of heat treatment and immunization with heat treated colon-26 tumour cell extract.

Cancer vaccines represent a promising new strategy for immunotherapy against cancer, but their effects are insufficiently understood. The effect of heat treatment against mouse colon adenocarcinoma cell line (colon-26), and combined effects of heat treatment and immunizing host animals with heat treated colon-26 cell extracts were investigated. Heat treatment of colon-26 cells induced heat-shock protein 70 (HSP70), but not other HSP. Immunization of BALB/cJ mice with heat treated colon-26 cell extract, which was enriched in HSP70, elicited antitumour immunity against subcutaneously injected colon-26 cells. Furthermore, combination therapy of heat treatment and immunization with heat treated colon-26 cell extract significantly reduced tumour volumes compared with heat treatment alone. Similar immunization enhanced the cytotoxic activity of mouse splenic lymphocytes against untreated and heat treated colon-26 cells in an in vitro assay, as well as against heat treated allogenic mouse lymphoma cell line (YAC-1). These findings suggest possible usefulness of heat treated cancer cell extract as a cancer vaccine, especially if given in combination with hyperthermia.

2B1 antigen characteristically expressed on extracellular matrices of human malignant tumors is a large chondroitin sulfate proteoglycan, PG-M/versican.

2B1 is a monoclonal antibody against a large proteoglycan isolated from human yolk sac tumor (M. Sobue et al., Histochem. J., 21: 455-460, 1989). The antigen is expressed in a variety of embryonal tissues as well as most if not all malignant tumor tissues. However, the expression in normal adult tissues is limited to some tissues, such as the smooth muscle layers of the aorta. We characterized the 2B1 antigen isolated from the conditioned medium of human malignant fibrous histiocytoma and found that immunological and biochemical properties are identical to those of a large chondroitin sulfate proteoglycan, PG-M/versican. Partial amino acid sequences of peptides obtained from the core protein by V8 protease digestion and subsequent SDS-PAGE were detected in the reported amino acid sequence of human PG-M/versican with a complete identity. Furthermore, 2B1 was distinctly reactive to the expressed protein by transfection of the cDNA for the shortest form into mouse cells. The results indicate that the antigen is the PG-M core protein, and the epitope may be in one of the globular domains. It is thus likely that PG-M/versican is one of the extracellular matrix components characteristic of human malignant tumors.

Antitumor immunity in strain 2 guinea pigs immunized with potassium chloride extracts of L2C tumor cells.

Extracts of L2C tumor cells stimulated in vitro production of macrophage migration inhibitory factor (MIF) in peritoneal exudate cells from guinea pigs immunized with L2C tumor cells. Guinea pigs immunized with extracts of L2C tumor cells that were active in vitro (in the MIF assay) were completely resistant to challenge with viable tumor cells given 2 weeks later. Furthermore, guinea pigs immunized with extracts of L2C tumor cells within 1 hour after challenge with viable L2C tumor cells survived substantially longer than did nonimmunized controls. The immunoprotective and immunotherapeutic effects seen in guinea pigs given injections of viable L2C tumor cells were obtained with extracts of L2C tumor cells but not with extracts of another guinea pig tumor (line 10 hepatoma) or with extracts of normal guinea pig lymphoid cells.

Influence of autologous serum on in vitro reactivity of peripheral lymphocytes of patients with breast cancer.

The influence of autologous serum on DNA synthesis and phytohemagglutinin (PHA) or pokeweed mitogen (PWM) stimulation of peripheral blood lymphocytes from mammary tumor-bearing women has been studied. Blood samples were collected from patients at the 4 stages of the TNM classification who had not undergone any therapeutic treatment. Lymphocyte functions were significantly reduced only in patients with the largest tumors and in the latest stage of disease. Autologous cancer sera inhibited spontaneous DNA synthesis of lymphocytes and did not inhibit lymphocyte stimulation by mitogens. It is possible that unrecognized inhibiting and stimulating factors, possibly acting in synergism with mitogens, are present in mammary cancer sera.