Clinical uses of recombinant human thyrotropin

Recombinant human thyroid-stimulating hormone (rhTSH), used to enhance diagnostic radioiodine whole body scanning and thyroglobulin testing, has dramatically altered the management of patients with thyroid cancer. Withdrawal from thyroid hormone suppression therapy and subsequent hypothyroidism is no longer the only safe and effective method for thyroid cancer surveillance. Currently, rhTSH is only approved for the monitoring of low-risk patients with well-differentiated thyroid cancer and radioactive iodine administration, in selected cases. Additional applications of rhTSH include enhancing the sensitivity of positron emission tomography in thyroid cancer, the management of multinodular goiter, and dynamic testing of thyroid reserve. The diagnostic and therapeutic role of rhTSH in these areas is discussed in this review.     

Antioxidant activity of supercritical extract of Melissa officinalis subsp. officinalis and Melissa officinalis subsp. inodora

The antioxidant activity of Melissa officinalis subsp. officinalis and of Melissa officinalis subsp. inodora extracts, obtained by using carbon dioxide under supercritical conditions was investigated. The samples were prepared in two steps. A preliminary extraction at 90 bar and 50 degrees C eliminated the essential oil, then a further extraction at 300 bar and 50 degrees C obtained the high molecular mass extract. These samples were tested for autoxidation and the iron or EDTA-mediated oxidation of linoleic acid at 37 degrees C in the absence of solvent, in in vitro systems. During linoleic acid autoxidation and its EDTA-mediated oxidation both M. officinalis and M. inodora extracts showed an antioxidant activity, and no significant differences in their efficacy were observed. None showed any prooxidant activity.     

In vitro studies with methylproamine: a potent new radioprotector

New analogues of the minor groove binding ligand Hoechst 33342 have been investigated in an attempt to improve radioprotective activity. The synthesis, DNA binding, and in vitro radioprotective properties of methylproamine, the most potent derivative, are reported. Experiments with V79 cells have shown that methylproamine is approximately 100-fold more potent than the classical aminothiol radioprotector WR1065. The crystal structures of methylproamine and proamine complexes with the dodecamer d(CGCGAATTCGCG)(2) confirm that the new analogues also are minor groove binders. It is proposed that the DNA-bound methylproamine ligand acts as a reducing agent by an electron transfer mechanism, repairing transient radiation-induced oxidizing species on DNA.     

CD4(+) T-cell recognition of mutated B-RAF in melanoma patients harboring the V599E mutation

The potential of antigen-directed cancer immunotherapy has not been fully realized, perhaps because many commonly targeted tumor associated proteins are not essential to maintaining the malignant cell phenotype. A constitutively activating mutation in the signaling molecule BRAF is expressed frequently in melanomas and may play an important role in the biology of this disease. A 29-mer B-Raf peptide incorporating the V599E mutation was used for in vitro stimulation of lymphocytes derived from melanoma patients, generating MHC class II-restricted CD4(+) T cells specific for this peptide as well as for melanoma cells expressing B-Raf V599E. Mutated B-Raf exemplifies targets that may be ideal for immunotherapy.     

Akt stimulates aerobic glycolysis in cancer cells

Cancer cells frequently display high rates of aerobic glycolysis in comparison to their nontransformed counterparts, although the molecular basis of this phenomenon remains poorly understood. Constitutive activity of the serine/threonine kinase Akt is a common perturbation observed in malignant cells. Surprisingly, although Akt activity is sufficient to promote leukemogenesis in nontransformed hematopoietic precursors and maintenance of Akt activity was required for rapid disease progression, the expression of activated Akt did not increase the proliferation of the premalignant or malignant cells in culture. However, Akt stimulated glucose consumption in transformed cells without affecting the rate of oxidative phosphorylation. High rates of aerobic glycolysis were also identified in human glioblastoma cells possessing but not those lacking constitutive Akt activity. Akt-expressing cells were more susceptible than control cells to death after glucose withdrawal. These data suggest that activation of the Akt oncogene is sufficient to stimulate the switch to aerobic glycolysis characteristic of cancer cells and that Akt activity renders cancer cells dependent on aerobic glycolysis for continued growth and survival.     

Prostate carcinoma and green tea: (-)epigallocatechin-3-gallate inhibits inflammation-triggered MMP-2 activation and invasion in murine TRAMP model

Green tea infusion has been shown to inhibit metastatic spreading of the transgenic adenocarcinoma of mouse prostate (TRAMP). Investigation on the molecular mechanisms triggered by the main green tea flavonoid, (-)epigallocatechin-3-gallate (EGCG), shows that EGCG restrains TRAMP-C1 cell proliferation in a dose-dependent manner, at concentrations (IC(50) < 0.2 microM) equivalent to those measured in the plasma of moderate green-tea drinkers. Up to 10 microM, EGCG does not modify the cell-surface immuno-localization of MMP-2, one of the invasion-instrumental proteinases; but while in default culture conditions these cells secrete mainly pro-MMP-2, in the presence of reconstituted basement membrane (Matrigel) they release almost exclusively pro-MMP-9. In contrast, when stimulated to traverse Matrigel toward a chemo-attractant, in addition to pro-MMP-9, they secrete pro-MMP-2. In the presence of 0.2 microM EGCG, only the level of the latter is markedly lowered in the conditioned medium, in parallel with the invasive behavior (>50%). In vivo, s.c. injection of TRAMP-C1 cells dispersed in Matrigel gives origin to a tumor mass, whose growth is not inhibited by green-tea regimen. This growth is contained greater than two-thirds by LPS-triggered polymorpho-nuclear phagocyte (PMN) recruitment but this effect is abolished by green tea. Nevertheless, while tumor-released pro-MMP-2 is activated by co-incubation of TRAMP-C1 cells with PMNs, in the presence of 10 microM EGCG the activation is almost abolished. These results suggest that inflammatory involvement of prostate carcinoma could be efficaciously prevented by green tea with a concomitant lowering of the invasive potential.     

Anthocyanins induce cell cycle perturbations and apoptosis in different human cell lines

To investigate the mechanistic basis for the biological properties of anthocyanins, two aglycone anthocyanins [delphinidin (DY) and cyanidin (CY)] were used to examine their effects on cell cycle progression and on induction of apoptosis in human cancer cells (uterine carcinoma and colon adenocarcinoma cells) and in normal human fibroblasts. These compounds differ in the number and position of hydroxyl groups on the beta ring in the molecular structure. Cellular uptake of anthocyanins was confirmed by HPLC analysis and no metabolites were detected. The clonogenic assay showed that CY induces a dose-dependent growth inhibitory effect only in fibroblasts. This effect was confirmed by flow cytometric analysis, showing a significant reduction of cells in S phase. In contrast, DP inhibited cell growth in normal and tumour cell lines. This event is accompanied in fibroblasts by an accumulation of cells in the S phase suggesting a block in the transition from S to G2 phase. On the other hand, in tumour cell lines we observed a reduction of cells in G1 phase, paralleled by the appearance of a fraction of cells with a hypodiploid DNA content, thus demonstrating an apoptotic effect by DP. The occurrence of apoptosis induced by DP was confirmed by morphological and biochemical features, including nuclear condensation and fragmentation, annexin V staining, DNA laddering and poly(ADP-ribose) polymerase-1-proteolysis. Furthermore, the mitochondrial membrane potential of apoptotic cells after treatment with DP was significantly lost. The different effects exerted by DP as compared with CY suggest that the presence of the three hydroxyl groups on the beta ring in the molecular structure of DP may be important for its greater biological activity.     

Comparison of DNA adduct levels in nasal mucosa, lymphocytes and bronchial mucosa of cigarette smokers and interaction with metabolic gene polymorphisms

The recent introduction of biomarkers in population studies of lung cancer has improved the traditional epidemiological approach, especially in the detection of high risk groups. Many inhalable carcinogens form DNA adducts, an initial event in lung carcinogenesis, and therefore the identification of easily accessible sources of DNA for population studies is considered a leading priority in the field. In this study we compared the frequency of DNA adducts in samples from nasal brushing, bronchial biopsy and peripheral blood lymphocytes (PBL) in a group of 55 subjects, both smokers and non-smokers, undergoing bronchoscopy for diagnostic purposes. Polymorphisms in the CYP1A1, GSTM1 and GSTT1 genes were also evaluated. The level of DNA adducts measured by (32)P-labelling assay in nasal mucosa (10(8) relative adduct level, mean +/- SD 1.10 +/- 0.66) was higher than in bronchial mucosa (0.82 +/- 0.36) and in PBL (0.54 +/- 0.39, P < 0.01). DNA adducts measured in nasal mucosa and in PBL were correlated with those in bronchial mucosa (P < 0.01 and P < 0.05, respectively). DNA adducts in smokers were significantly increased in both nasal mucosa and PBL, with a significant dose-response linear trend (P < 0.05). No significant effect on DNA adduction of the genetic polymorphisms investigated was found. Nasal mucosa brushing proved to be a suitable procedure for the (32)P-labelling assay and its use in population studies should be further explored.     

AAV-mediated gene transfer of tissue inhibitor of metalloproteinases-1 inhibits vascular tumor growth and angiogenesis in vivo

The activity of matrix metalloproteinases (MMPs) is a universal feature of cellular invasion, tumor angiogenesis and metastasis, which is counterbalanced and regulated by the natural tissue inhibitors of MMPs (Timps). Here we show that Timp1 gene transfer delivered by an adeno-associated virus (AAV) vector inhibits tumor growth in a murine xenotransplant model. A human Kaposi's sarcoma cell line, forming highly vascularized tumors in vivo and having a high natural permissivity to AAV gene transfer, was transduced to express the Timp1 cDNA. AAV-Timp1-transduced cells secreted high levels of Timp1 that inhibited MMP2 and MMP9 gelatinolytic activity. Following subcutaneous inoculation in nude mice, the AAV-Timp1-transduced cells showed significantly reduced tumor growth when compared to control AAV-LacZ-transduced cells. In addition, direct intratumoral injection of AAV-Timp1 into pre-existing tumors significantly impaired the further expansion of the tumor mass. Histological analyses showed that the AAV-Timp1-transduced tumors had limited development of vascular structures and extensive areas of cell death, suggesting that Timp1 overexpression had an antiangiogenic effect. To further support this conclusion, we demonstrated that AAV-Timp1 transduction significantly reduced endothelial cell migration and the invasion of a Matrigel barrier and strongly inhibited angiogenesis in the chick chorioallantoic membrane assay. These results indicate that transfer and overexpression of the Timp1 gene is a promising therapeutic strategy to target tumor-associated angiogenesis in cancer gene therapy.