Efficacy of different sequences of radio- and chemotherapy in experimental models of human melanoma.

Although combination chemotherapy and radiotherapy have become the standard of care in numerous tumors, the mechanisms of interaction are often still unclear. The purpose of this study was to analyze the efficacy of radiation treatment and cisplatin sequences and to investigate their mechanisms of interaction. Three melanoma cell lines were used to evaluate in vitro radiation-induced cytotoxicity before and after cisplatin treatment. Expression levels of a panel of genes were determined by real-time RT-PCR. Cytotoxic effect was evaluated by flow cytometry analysis and Comet assay. We also used normal human dermal fibroblasts (HUDE) to evaluate the cytotoxicity of the two treatments by clonogenic assay. Radiation and cisplatin used singly were not particularly effective in reducing proliferation in melanoma cells. Conversely, radiation treatment followed by cisplatin showed a strong synergistic interaction in all cell lines, with a ratio index ranging from 16 to >100. The synergistic effect was accompanied by apoptosis induction (up to 40%) and an increase in the percentage of comet-shaped nucleoids from 85% to 99%. In parallel, our results also showed that radiation treatment of HUDE fibroblasts followed by cisplatin only induced weak cytotoxicity. Our findings highlight the efficacy of the sequence radiation → cisplatin in reducing cell proliferation and in inducing apoptosis in melanoma cell lines. This sequence also modulated a network of proteins involved in DNA damage repair.

Synthesis and Characterization of TiO2 Nanoparticles for the Reduction of Water Pollutants.

The aim of this manuscript was the optimization of the synthesis of TiO2 nanoparticles (TiO2 NPs) with conditions that could be easily reproducible at the industrial level. Several procedures were tested and those with C12H28O4Ti and CO(NH2)2 as precursors seemed the most promising and, consequently, were improved with different molar ratios, lower temperatures and the addition of NH4Cl as a secondary dopant of nitrogen. The obtained samples were studied with analytical techniques such as X-ray powder diffraction (XRPD) and field emission scanning electron microscopy (FESEM). To complete the study, dye degradation and bacteriological tests were also performed. The results indicate that it is possible to obtain TiO2 NPs at lower temperatures with respect to those used in the literature; the best candidate that could satisfy all the requirements was a sample with a molar ratio of C12H28O4Ti:CO(NH2)2 at 2:1 and obtained at 50 °C.

Improving reliability of live/dead cell counting through automated image mosaicing.

Cell counting is one of the basic needs of most biological experiments. Numerous methods and systems have been studied to improve the reliability of counting. However, at present, manual cell counting performed with a hemocytometer still represents the gold standard, despite several problems limiting reproducibility and repeatability of the counts and, at the end, jeopardizing their reliability in general. We present our own approach based on image processing techniques to improve counting reliability. It works in two stages: first building a high-resolution image of the hemocytometer's grid, then counting the live and dead cells by tagging the image with flags of different colours. In particular, we introduce GridMos (http://sourceforge.net/p/gridmos), a fully-automated mosaicing method to obtain a mosaic representing the whole hemocytometer's grid. In addition to offering more significant statistics, the mosaic "freezes" the culture status, thus permitting analysis by more than one operator. Finally, the mosaic achieved can thus be tagged by using an image editor, thus markedly improving counting reliability. The experiments performed confirm the improvements brought about by the proposed counting approach in terms of both reproducibility and repeatability, also suggesting the use of a mosaic of an entire hemocytometer's grid, then labelled trough an image editor, as the best likely candidate for the new gold standard method in cell counting.

A new avenue toward androgen receptor pan-antagonists: C2 sterically hindered substitution of hydroxy-propanamides.

The androgen receptor (AR) represents the primary target for prostate cancer (PC) treatment even when the disease progresses toward androgen-independent (AIPC) or castration-resistant (CRPC) forms. Because small chemical changes in the structure of nonsteroidal AR ligands determine the pharmacological responses of AR, we developed a novel stereoselective synthetic strategy that allows sterically hindered C2-substituted bicalutamide analogues to be obtained. Biological and theoretical evaluations demonstrate that C2-substitution with benzyl and phenyl moieties is a new, valuable option toward improving pan-antagonist behavior. Among the synthesized compounds, (R)-16m, when compared to casodex, (R)-bicalutamide, and enzalutamide, displayed very promising in vitro activity toward five different prostate cancer cell lines, all representative of CPRC and AIPC typical mutations. Despite being less active than (R)-bicalutamide, (R)-16m also displayed marked in vivo antitumor activity on VCaP xenografts and thus it may serve as starting point for developing novel AR pan-antagonists.

Effect of small molecules modulating androgen receptor (SARMs) in human prostate cancer models.

The management of hormone-refractory prostate cancer represents a major challenge in the therapy of this tumor, and identification of novel androgen receptor antagonists is needed to render treatment more effective. We analyzed the activity of two novel androgen receptor antagonists, (S)-11 and (R)-9, in in vitro and in vivo experimental models of hormone-sensitive or castration-resistant prostate cancer (CRPC). In vitro experiments were performed on LNCaP, LNCaP-AR, LNCaP-Rbic and VCaP human prostate cancer cells. Cytotoxic activity was assessed by SRB and BrdU uptake, AR transactivation by luciferase reporter assay and PSA levels by Real Time RT-PCR and ELISA assays. Cell cycle progression-related markers were evaluated by western blot. In vivo experiments were performed on SCID mice xenografted with cells with different sensitivity to hormonal treatment. In hormone-sensitive LNCaP and LNCaP-AR cells, the latter expressing high androgen receptor levels, (R)-9 and (S)-11 exhibited a higher cytotoxic effect compared to that of the reference compound ((R)-bicalutamide), also in the presence of the synthetic androgen R1881. Furthermore, the cytotoxic effect produced by (R)-9 was higher than that of (S)-11 in the two hormone-resistant LNCaP-AR and VCaP cells. A significant reduction in PSA levels was observed after exposure to both molecules. Moreover, (S)-11 and (R)-9 inhibited DNA synthesis by blocking the androgen-induced increase in cyclin D1 protein levels. In vivo studies on the toxicological profile of (R)-9 did not reveal the presence of adverse events. Furthermore, (R)-9 inhibited tumor growth in various in vivo models, especially LNCaP-Rbic xenografts, representative of recurrent disease. Our in vitro results highlight the antitumor activity of the two novel molecules (R)-9 and (S)-11, making them a potentially attractive option for the treatment of CRPC.

Specific Biomarkers Are Associated with Docetaxeland Gemcitabine-Resistant NSCLC Cell Lines.

Five-year survival rate for lung cancer is limited to 10% to 15%. Therefore, the identification of novel therapeutic prognostic factors is an urgent requirement. The aim of this study is thus to highlight specific biomarkers in chemoresistant non-small cell lung cancer cell lines. Therefore, we checked-in the control condition as well as after short-term pharmacological treatment with either docetaxel or gemcitabine-the expression of genes such as tumor suppressor genes (CDKN2A, DAPK, FHIT, GSTP1, MGMT, RARß2, RASSF1A, and TIMP3), genes associated with drug resistance (BRCA1, COX2, ERCC1, IGFBP3, RRM1, and TUBB3), and stemness-related genes (CD133, OCT4, and SLUG) in two cellular models of squamous carcinoma (CAEP) and adenocarcinoma (RAL) of the lung originally established. Their promoter methylation profile was also evaluated. Drug-related genes were upregulated. Cisplatin resistance matched with high levels of BRCA1 and ERCC1 in both cell lines; docetaxel sensitivity of CAEP cells was associated to levels of TUBB3 lower than RAL cells. Although CAEP cells were more sensitive to gemcitabine, both cell lines showed high levels of RRM1. Stemness-related genes were downregulated in the control condition but became upregulated in docetaxel-resistant cells, indicating the selection of a population with stemness features. We did not find an unequivocal correspondence between gene expression and respective DNA promoter methylation status, suggesting the involvement of additional mechanisms of gene expression regulation. These results highlight specific biomarkers consistent with the different responses of the two cell lines to standard pharmacological treatments and indicate specific molecular traits for their chemoresistance.