Controlled Decoration of [60]Fullerene with Polymannan Analogues and Amino Acid Derivatives through Malondiamide-Based Linkers.

In the last few years, nanomaterials based on fullerene have begun to be considered promising tools in the development of efficient adjuvant/delivery systems for vaccination, thanks to their several advantages such as biocompatibility, size, and easy preparation and modification. In this work we reported the chemoenzymatic synthesis of natural polymannan analogues (di- and tri-mannan oligosaccharides characterized by α1,6man and/or α1,2man motifs) endowed with an anomeric propargyl group. These sugar derivatives were submitted to 1,3 Huisgen dipolar cycloaddition with a malondiamide-based chain equipped with two azido terminal groups. The obtained sugar-modified malondiamide derivatives were used to functionalize the surface of Buckminster fullerene (C60) in a highly controlled fashion, and yields (11-41%) higher than those so far reported by employing analogue linkers. The same strategy has been exploited to obtain C60 endowed with natural and unnatural amino acid derivatives. Finally, the first double functionalization of fullerene with both sugar- and amino acid-modified malondiamide chains was successfully performed, paving the way to the possible derivatization of fullerenes with immunogenic sugars and more complex antigenic peptides.

Long-Term Follow-up of a Randomized Study of Oral Etoposide versus Viscum album Fermentatum Pini as Maintenance Therapy in Osteosarcoma Patients in Complete Surgical Remission after Second Relapse.

BACKGROUND: In relapsed osteosarcoma, the 5-yr postrelapse disease-free survival (PRDFS) rate after the second relapse is <20%. In June 2007, a randomized study was started comparing oral etoposide vs Viscum album fermentatum Pini (an extract derived from the parasitic plant Viscum album L., European mistletoe) as maintenance therapy in patients with metastatic osteosarcoma in complete surgical remission after the second relapse. The primary endpoint was the PRDFS rate at 12 months (compared to the historical control rate). This is a long-term updated result. Patients and Methods. 10 patients received oral etoposide 50 mg/m2 daily for 21 days every 28 days for 6 months, and 9 patients received Viscum album fermentatum Pini 3 times/wk subcutaneously for 1 year. The study closed early in July 2011 due to insufficient recruitment. Lymphocyte subpopulations were analyzed at T0, T3, T6, T9, and T12 months. RESULTS: On 30 June 2019, at a median follow-up ITT of 83 months (range 3-144 ms), a median PRDFS of 106 ms (2-144) was observed in the Viscum arm with 5/9 patients who never relapse vs a PRDFS of 7 months (3-134) in the etoposide arm (all patients in the Etoposide arm relapsed) (hazard ratio HR = 0.287, 95% CI: 0.076-0.884, p=0.03). Model forecast 10-yr overall survival rates as 64% in the Viscum arm and 33% in the etoposide arm. Lymphocyte subpopulation counts (CD3, CD4, and CD56) showed an increase in the Viscum arm while a decrease was observed in the etoposide arm during treatment. CONCLUSIONS: After 12 years from the start of the trial, the patients in the Viscum arm continue to show a considerably longer PRDFS compared to oral etoposide, and a trend for an advantage in OS is evident even if the number of treated patients is too small to draw conclusions. Viscum as maintenance treatment after complete surgical remission in relapsed osteosarcoma should be further investigated and compared with other drugs.

Agave negatively regulates YAP and TAZ transcriptionally and post-translationally in osteosarcoma cell lines.

Osteosarcoma (OS) is the most aggressive type of primary solid tumor that develops in bone. Whilst conventional chemotherapy can improve survival rates, the outcome for patients with metastatic or recurrent OS remains poor, so novel treatment agents and strategies are required. Research into new anticancer therapies has paved the way for the utilisation of natural compounds as they are typically less expensive and less toxic compared to conventional chemotherapeutics. Previously published works indicate that Agave exhibits anticancer properties, however potential molecular mechanisms remain poorly understood. In the present study, we investigate the anticancer effects of Agave leaf extract in OS cells suggesting that Agave inhibits cell viability, colony formation, and cell migration, and can induce apoptosis in OS cell lines. Moreover, Agave sensitizes OS cells to cisplatin (CDDP) and radiation, to overcome chemo- and radio-resistance. We demonstrate that Agave extract induces a marked decrease of Yes Associated Protein (YAP) and Tafazzin (TAZ) mRNA and protein expression upon treatment. We propose an initial mechanism of action in which Agave induces YAP/TAZ protein degradation, followed by a secondary event whereby Agave inhibits YAP/TAZ transcription, effectively deregulating the Nuclear Factor kappa B (NF-κB) p65:p50 heterodimers responsible for transcriptional induction of YAP and TAZ.

Anti-EGFR antibody cetuximab enhances the cytolytic activity of natural killer cells toward osteosarcoma.

PURPOSE: Osteosarcoma and Ewing's sarcoma are the most common bone tumors in children and adolescents. Despite intensive chemotherapy, patients with advanced disease have a poor prognosis, illustrating the need for alternative therapies. Sarcoma cells are susceptible to the cytolytic activity of resting natural killer (NK) cells which can be improved by interleukin (IL)-15 stimulation. In this study, we explored whether the cytolytic function of resting NK cells can be augmented and specifically directed toward sarcoma cells by antibody-dependent cellular cytotoxicity (ADCC). EXPERIMENTAL DESIGN: Epidermal growth factor receptor (EGFR) expression was examined on osteosarcoma and Ewing's sarcoma cell lines by flow cytometry and in osteosarcoma biopsy and resection specimens by immunohistochemistry. Cetuximab-mediated ADCC by NK cells from osteosarcoma patients and healthy controls was measured with 4-hour (51)Cr release assays. RESULTS: EGFR surface expression was shown on chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cells (12/12), most primary osteosarcoma cultures (4/5), and few Ewing's sarcoma cell lines (2/7). In the presence of cetuximab, the cytolytic activity of resting NK cells against all EGFR-expressing sarcoma cells was substantially increased and comparable with that of IL-15-activated NK cells. Surface EGFR expression on primary osteosarcoma cultures correlated with EGFR expression in the original tumor. The cytolytic activity of osteosarcoma patient-derived NK cells against autologous tumor cells was as efficient as that of NK cells from healthy donors. CONCLUSION: Our data show that the cytolytic potential of resting NK cells can be potentiated and directed toward osteosarcoma cells with cetuximab. Therefore, cetuximab-mediated immunotherapy may be considered a novel treatment modality in the management of advanced osteosarcoma.

Screening of fibrillogenesis inhibitors of ß2-microglobulin: integrated strategies by mass spectrometry capillary electrophoresis and in silico simulations.

The challenging search of ligands for the amyloidogenic protein ß(2)-microglobulin led us to set up an integrated strategy that combines analytical techniques and molecular modelling. Using a chemical library composed of 90 sulphonated molecules and a novel MS screening approach, we initially single out a few new binders. To check for anti-amyloid activity, the best hit obtained was thoroughly studied by docking analysis, affinity and refolding experiments by capillary electrophoresis and in vitro fibrillogenesis Thioflavin T test. Correlative analysis of the overall results obtained from the MS screening led to develop an equation able to identify the key factors of the affinity for ß(2)-microglobulin and to predict the affinity for novel derivatives. The proposed equation was then used for a virtual screening of a large compound database. Studies on the new hit thus retrieved confirm the predictive potential of both the equation on affinity and of docking analysis on anti-amyloid activity.

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques.

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.