Radioimmunotherapy and apoptotic induction on CK19-overexpressing human cervical carcinoma cells with Re-188-mAbCx-99.

BACKGROUND: The overexpression of Ck19 antigen occurs frequently in human carcinomas. The strategy and mechanism of radioimmunotherapy using Re-188-mAbCx-99 to Ck19 on human cervical carcinoma cells was investigated in this study. MATERIALS AND METHODS: Using mAbCx-99, the overexpression of Ck19 protein in lysates of cell lines and tissues from various patients' cervixes were verified by immunobinding and immunoblot analysis. The therapeutic effect of Re-188-mAbCx-99 on ME180 cells was examined in vitro by cell proliferation, apoptosis, DNA fragmentation and intemucleosomal levels. RESULTS: A relatively high expression of Ck19 was found in all human cervical carcinoma cell lines (4- to 44-fold) and in tissue lysates (26.8- to 79-fold) from patients (31 out of 34) with cervical, endometrial or ovarian carcinomas compared with that of benign or normal control samples. The growth inhibition of ME180, CC7T and Hela cells were significantly higher (p < 0.001) in the Re-188-mAbCx-99-treated (60-80%) than in the Re-188-MOPCIgG1-treated lines (8-18%) after 72-h treatment. After 48 h of treatment with Re-188-mAbCx-99, ME180 cells significantly exhibited DNA fragmentation and morphological features of apoptosis. This effect markedly elevated the expression of p21, p53 and Bcl-xS protein, while the Mcl-1 and Caspase-8 proteins were down-regulated. CONCLUSION: We suggest that an elevated Ck19 level is associated with disease stage in most patients with cervical cancer. The therapeutic effect of Re-188-mAbCx-99 was verified through apoptosis on targeting the enriched Ck19 protein of carcinoma cells.

Comparison of different adjuvants of protein and DNA vaccination for the prophylaxis of IgE antibody formation.

A high-molecular-weight mite allergen Der f11 that was hardly purified for immunotherapy was used to develop the DNA vaccine pDf11. We have shown that vaccination of mice with pDf11 induces Th1 responses characterized by suppression of IgE responses. In the present study, effects of different adjuvants on pDf11 were first studied. Mice receiving pDf11 +/- CpG, bestatin, and bupivacaine had better suppression of IgE responses than those receiving pDf11 +/- lipofectin or alum. Bestatin could greatly boost IgG2a responses. Immunomodulating effects of different adjuvants between protein and DNA vaccines were further elucidated. CpG was the best for both protein and DNA vaccines to profoundly suppress IgE responses, but alum, bestatin and lipofectin were useless for rDf11 to induce IgE inhibition. Neither did the combination of rDf11 and pDf11 have further IgE suppression. In conclusion, CpG is the unique adjuvant for the protein vaccine rDf11 to inhibit IgE responses. In contrast, the DNA vaccine pDf11 +/- CpG, bestatin, or bupivacaine induces profound suppression of IgE responses.

Purification and characterization of a novel isoallergen of a major Bermuda grass pollen allergen, Cyn d 1.

A novel immunoreactive isoallergen of a major Bermuda grass pollen allergen, Cyn d 1, was purified by the use of a combination of various chromatographic techniques, including high-performance liquid chromatography. This new isoallergen has a pI value of 9.1 and shows significant N-terminal sequence homology with other isoforms. Carbohydrate composition analysis revealed a 10.4% carbohydrate content consisting of 7 different sugar moieties, including arabinose, fucose, galactose, glucose, mannose, xylose and N-acetylglucosamine, as well as a trace amount of rhamnose. Upon periodate oxidation, the binding activities of the Cyn d 1 isoform to murine monoclonal antibodies and human serum IgE and IgG were reduced, suggesting the importance of the carbohydrate moiety in the immune response. The availability of the purified Cyn d 1 basic isoform will allow for further structural and immunological characterization, and ultimately for the design of an appropriate therapy.

Apoptotic effect of caffeic acid phenethyl ester and its ester and amide analogues in human cervical cancer ME180 cells.

BACKGROUND: Caffeic acid phenylether ester (CAPE) has potent antioxidant, anti-inflammatory, antiviral, anti-proliferative, immunomodulatory and pro-apoptotic activities. The activities of CAPE and its novel synthetic derivatives, caffeic acid octyl ester (CAO) and 1-octyl caffeamide (CAN-8), were investigated in this study. MATERIALS AND METHODS: Cultured human cells were incubated with or without these compounds. The effect of these compounds on cell apoptosis, intracellular level of hydrogen peroxide and mitochondrial potential were analyzed. Western blot analysis was used to study the effect of alterations in protein level of caspases, Bcl-2 family, p21, p53 and c-Jun upon drug treatment. RESULTS: These compounds arrested cell proliferation, triggered cell apoptosis and caused a marked scavenging effect of hydrogen peroxide. Apoptosis induced by CAPE or CAO is associated with increased expression of p53, p21 and c-Jun. While the levels of Bcl-2 and Bcl-xL were relatively unchanged, these compounds induced a marked reduction in Mcl-1 level. The CAPE- or CAO-induced apoptosis was also accompanied by a rapid loss of mitochondrial transmembrane potential and activation of caspase-3 and caspase-8, suggesting a mitochondrial-dependent mechanism. In causing these cellular actions, CAO was shown to be comparable or more potent than CAPE, whereas the amide analogue CAN-8 displayed much weaker activities than both CAPE and CAO. Since these three compounds contain similar antioxidant functionality, the difference in their potency suggests that the octyl moiety in CAO is an important determinant for the enhanced activities. CONCLUSION: We have characterized a novel CAPE structure analogue, CAO, which showed strong antioxidant and proapoptotic activities. In addition, we demonstrated that down-regulation of Mcl-1 gene expression and activation of caspase-8 are associated with CAPE-triggered cell apoptosis.

Detecting cytokeratin 19 mRNA in the peripheral blood cells of cervical cancer patients and its clinical-pathological correlation.

OBJECTIVE: The aim of this study was to study the presence of cytokeratin 19 (CK19)-expressing cancer cells in the blood of preoperative patients with FIGO stage Ib and IIb cervical cancers who received radical hysterectomy and to investigate the cells' clinical significance. METHODS: CK19 mRNA in the blood cells of the patients was detected preoperatively by a newly designed nested reverse transcriptase-polymerase chain reaction, which excluded pseudogenes a and b, performed on 84 patients with stage Ib and IIb cervical carcinoma. Possible correlations between clinicopathological factors were then analyzed. RESULTS: The sensitivity of this assay was 1 CK19-mRNA-positive cell per 10(6) peripheral blood mononuclear cells. Results showed that 21.4% of the 84 patients with cervical carcinoma had CK19-mRNA-positive cells in the blood, in comparison with 5.7% of the 35 patients with benign gynecological tumors and 0% of the 28 healthy controls (P = 0.037 and 0.006, respectively). The positive tests in the cervical cancer patients were not associated with prognostic factors including stage, pelvic lymph node metastasis, pathological types, bulky tumor size (> or =4 cm), differentiation, parametrial extension, lymphovascular space involvement, deep stromal invasion, or age. CONCLUSIONS: This study revealed the presence of circulating CK19-expressing cancer cells in the blood of patients with untreated early-stage cervical carcinomas, indicating that cervical cancer disseminated early. The survival effect of this phenomenon must be clarified. This detection assay provides an early checkpoint in the multistep process for developing metastasis in cervical cancer patients.

Induction of specific Th1 responses and suppression of IgE antibody formation by vaccination with plasmid DNA encoding Der f 11.

DNA vaccines encoding low-molecular-weight allergens have been used to prevent IgE responses. A high-molecular-weight mite allergen Der f 11 that was hardly to be purified for immunotherapy was used to develop a DNA vaccine here. Vaccination of mice with plasmid DNA encoding Df11 (pDf11) induced Th1 responses characterized by IgG2a responses and spleen cell secretion of IFN-gamma. In contrast, sensitization with recombinant Der f 11 (rDf11) and alum induced Th2 responses characterized by IgE responses and spleen cell secretion of IL-4 and IL-5. Vaccination with pDf11 prevented the induction of IgE responses. Moreover, it could inhibit on-going IgE responses. The debate whether CD4+ or CD8+ T cells were the regulatory cells to inhibit IgE responses by DNA vaccination was also examined. First, sensitization of pDf11-vaccinated mice after depletion of CD8+ T cells still showed suppression of IgE responses. Secondly, adoptive transfer of either CD4- or CD8-depleted spleen cells from pDf11-vaccinated mice suppressed IgE responses. In conclusion, this is the first report to confirm the therapeutic effect of a DNA vaccine encoding a strong allergen on specific IgE responses. Both CD4+ and CD8+ T cells are crucial for the immunomodulation of IgE responses by pDf11.

The concentration of serum transforming growth factor beta-1 (TGF-beta1) is decreased in cervical carcinoma patients.

Transforming growth factor beta-1 (TGF-beta1) is a multifunctional growth factor and known to inhibit the proliferation of epithelial cell. The relationship between serum TGF-beta1 level and the presence of cervical cancer was investigated in this study. The patients with cervical squamous cell carcinoma (SCC) had significantly lower level of serum TGF-beta1 (39.14 +/- 9.03 ng/mL; mean +/- SD) than those with myoma (49.17 +/- 9.38 ng/mL) and normal subjects (49.13 +/- 8.81 ng/mL), (p < 0.007 and p < 0.001, respectively). TGF-beta1 level was also lower in the patients with cervical intraepithelial neoplasia (CIN3) (42.07 +/- 9.38 ng/mL) than in normal controls (49.13 +/- 8.81 ng/mL) (p < 0.04). It suggested that diminution of the production of TGF-beta1 has close association with the neoplastic transformation of cervical epithelium.