IL-1 Family Members in Bone Sarcomas.

IL-1 family members have multiple pleiotropic functions affecting various tissues and cells, including the regulation of the immune response, hematopoietic homeostasis, bone remodeling, neuronal physiology, and synaptic plasticity. Many of these activities are involved in various pathological processes and immunological disorders, including tumor initiation and progression. Indeed, IL-1 family members have been described to contribute to shaping the tumor microenvironment (TME), determining immune evasion and drug resistance, and to sustain tumor aggressiveness and metastasis. This review addresses the role of IL-1 family members in bone sarcomas, particularly the highly metastatic osteosarcoma (OS) and Ewing sarcoma (EWS), and discusses the IL-1-family-related mechanisms that play a role in bone metastasis development. We also consider the therapeutic implications of targeting IL-1 family members, which have been proposed as (i) relevant targets for anti-tumor and anti-metastatic drugs; (ii) immune checkpoints for immune suppression; and (iii) potential antigens for immunotherapy.

A 3D Collagen-Based Bioprinted Model to Study Osteosarcoma Invasiveness and Drug Response.

The biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 µg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research.

Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response.

DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor in children and adolescents. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming and the DNMT1 enzyme has been demonstrated to have an important role in both maintaining the epigenome and controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor proliferation through a mechanism that is independent on DNA methylation. Depletion of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 phase, and DNA damage, as revealed by the induction of γH2AX foci. These effects elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 mutated cells, persistent micronuclei and increased DNA instability was observed. Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 as an adjuvant agent in treating EWS.

miR-214-3p Is Commonly Downregulated by EWS-FLI1 and by CD99 and Its Restoration Limits Ewing Sarcoma Aggressiveness.

Ewing's sarcoma (EWS), an aggressive pediatric bone and soft-tissue sarcoma, has a very stable genome with very few genetic alterations. Unlike in most cancers, the progression of EWS appears to depend on epigenetic alterations. EWS-FLI1 and CD99, the two hallmarks of EWS, are reported to severely impact the malignancy of EWS cells, at least partly by regulating the expression of several types of non-coding RNAs. Here, we identify miR-214-3p as a common mediator of either EWS-FLI1 or CD99 by in silico analysis. MiR-214-3p expression was lower in EWS cells and in clinical samples than in bone marrow mesenchymal stem cells, and this miRNA was barely expressed in metastatic lesions. Silencing of EWS-FLI1 or CD99 restored the expression of miR-214-3p, leading to a reduced cell growth and migration. Mechanistically, miR-214-3p restoration inhibits the expression of the high-mobility group AT-hook 1 (HMGA1) protein, a validated target of miR-214-3p and a major regulator of the transcriptional machinery. The decrease in HMGA1 expression reduced the growth and the migration of EWS cells. Taken together, our results support that the miR-214-3p is constitutively repressed by both EWS-FLI1 and CD99 because it acts as an oncosuppressor limiting the dissemination of EWS cells.

A natural product, voacamine, sensitizes paclitaxel-resistant human ovarian cancer cells.

Most women with ovarian cancer are treated with chemotherapy before or after surgery. Unfortunately, chemotherapy treatment can cause negative side effects and the onset of multidrug resistance (MDR). The aim of this study is to evaluate the chemosensitizing effect of a natural compound, voacamine (VOA), in ovarian (A2780 DX) and colon (LoVo DX) cancer drug-resistant cell lines which overexpress P-glycoprotein (P-gp), in combination with paclitaxel (PTX), or doxorubicin (DOX) or 5-fluorouracil (5-FU). VOA, a bisindole alkaloid extracted from Peschiera fuchsiaefolia, has already been shown to be effective in enhancing the effect of doxorubicin, because it interferes with the P-gp function. Ovarian cancer cytotoxicity test shows that single treatments with VOA, DOX and PTX do not modify cell viability, while pretreatment with VOA, and then PTX or DOX for 72 h, induces a decrease. In colon cancer, since 5-FU is not a-substrate for P-gp, VOA has no sensitizing effect while in VOA + DOX there is a decrease in viability. Annexin V/PI test, cell cycle analysis, activation of cleaved PARP1 confirm that VOA plus PTX induce apoptotic cell death. Confocal microscopy observations show the different localization of NF-kB after treatment with VOA + PTX, confirming the inhibition of nuclear translocation induced by VOA pretreatment. Our data show the specific effect of VOA which only works on drugs known to be substrates of P-gp.

Genomics and Therapeutic Vulnerabilities of Primary Bone Tumors.

Osteosarcoma, Ewing sarcoma and chondrosarcoma are rare diseases but the most common primary tumors of bone. The genes directly involved in the sarcomagenesis, tumor progression and treatment responsiveness are not completely defined for these tumors, and the powerful discovery of genetic analysis is highly warranted in the view of improving the therapy and cure of patients. The review summarizes recent advances concerning the molecular and genetic background of these three neoplasms and, of their most common variants, highlights the putative therapeutic targets and the clinical trials that are presently active, and notes the fundamental issues that remain unanswered. In the era of personalized medicine, the rarity of sarcomas may not be the major obstacle, provided that each patient is studied extensively according to a road map that combines emerging genomic and functional approaches toward the selection of novel therapeutic strategies.

Voacamine: Alkaloid with its essential dimeric units to reverse tumor multidrug resistance.

Search for natural substances in association with conventional chemotherapeutic drugs with a chemiosensitizing action easily accessible to the tumor mass has encouraged our studies on voacamine (VOA) and its monomeric units, voacangine and vobasine. Our previous results showed that VOA sensitized multidrug resistant (MDR) osteosarcoma cells (U-2 OS/DX) to doxorubicin (DOX) cytotoxicity. VOA, extracted by Peschiera fuchsiaefolia plant, is a bisindole alkaloid consisting of an Iboga skeleton (voacangine) directly linked to a 2-acyl indole unit (vobasine). High-performance thin-layer chromatography densitometry demonstrated the purity of VOA, voacangine and vobasine samples. Flow cytometry analysis showed that VOA, voacangine and vobasine enhanced DOX accumulation of U-2 OS/DX cells, in equally way, whereas VOA reduced more efficiently DOX efflux. Optical microscopy and clonogenic assay confirmed that VOA was more effective than voacangine and vobasine in enhancing DOX cytotoxic effect. These results showed that monomers linked together are necessary to modulate resistant phenotype of osteosarcoma cells. To complete the study, we evaluated the effect of three compounds on microtubules by confocal microscopy, suggesting that only the whole molecule depolymerizes the microtubules blocking so DOX efflux-mediated by vesicles.

In vitro toxicity assessment of hydrogel patches obtained by cation-induced cross-linking of rod-like cellulose nanocrystals.

With the purpose of designing active patches for photodynamic therapy of melanoma, transparent and soft hydrogel membranes (HMs) have been fabricated by cation-induced gelation of rod-like cellulose nanocrystals (CNCs) bearing negatively charged carboxylic groups. Na+ , Ca2+ , Mg2+ have been used as cross-linkers of cellulose nanocrystal (CNC). The biosafety of this material and of its precursors has been evaluated in vitro in cell cultures. Morphological changes, cell organelles integrity, and cell survival with the tetrazolium salt reduction (MTT) assay were utilized as tests of cytotoxicity. Preliminary investigation was performed by addition of the hydrogel components to the cell culture medium and by incubations of the CNC-HM in direct and indirect contact with a confluent monolayer of A375 melanoma cells. Direct contact assays suffered from interference of physical stress. Careful evaluation of cytotoxicity was obtained considering the overall picture provided by microscopy and biochemical tests performed with the CNC-HM in indirect contact with two melanoma cell lines (A375, M14) and human fibroblasts. CNCs have been demonstrated to be a safe precursor material and CNC-HMs have a good biocompatibility provided that the excess of cations, in particular of Ca2+ is removed. These results indicate that CNC and can be safely used to fabricate biomedical devices such as transparent hydrogel patches, although attention must be paid to the fabrication procedure.

Anticancer activity of "Trigno M", extract of Prunus spinosa drupes, against in vitro 3D and in vivo colon cancer models.

In 2018 there were over 1.8 million new cases worldwide of colorectal cancer and relapses after clinical treatments. Many studies ascribe the risk of the appearance of this cancer to the Western life style : a sedentary life, obesity, and low -fiber, high -fat diets can promote the onset of disease. Several studies have shown supplement phytochemicals to have an inhibiting effect on the growth of various cancers through the activation of apoptosis. Our goal was to prove the effectiveness of a natural compound in the combined therapy of colorectal cancer. Trigno M supplement was an optimal candidate as anticancer product for its high concentrations of phenolic acids, flavonoids and anthocyanins. Our work showed the antitumor activity of Trigno M, extract of Prunus spinosa drupes combined with the nutraceutical activator complex (NAC), in 2D, 3D and in vivo colorectal cancer models. The cellular model we used both in vitro and in vivo was the HCT116 cell line, particularly suitable for engraftment after inoculation in mice. Trigno M inhibited the growth and colony formation of HCT116 cells (35%) as compared to the chemotherapy treatment with 5-fluorouracil (80%) used in clinical therapy. The reduction of the morphological dimensions in the spheroid cells after Trigno M, was compared with 5-fluorouracil demonstrating the efficacy of the Trigno M compound also in 3D models. Flow cytometric analysis on 3D cells showed a significant increase in the apoptotic cell fraction after Trigno M treatment (44.8%) and a low level of necrotic fraction (6.7%) as compared with control cells. Trigno M and 5-fluorouracil induced the apoptosis in a comparable percentage. Monotherapy with Trigno M in severely immunodeficient mice, carrying colon rectal cancer xenografts, significantly reduced tumor growth. The histopatological analysis of the ectopic tumors showed a lower level of necrosis after Trigno M treatment compared with the control. We conclude that Trigno M is well tolerated by mice, delays colorectal cancer growth in these animals and should be weighed up for integration of the current multi-drug protocols in the treatment of colon carcinoma.

Targeting Autophagy to Overcome Human Diseases.

Autophagy is an evolutionarily conserved cellular process, through which damaged organelles and superfluous proteins are degraded, for maintaining the correct cellular balance during stress insult. It involves formation of double-membrane vesicles, named autophagosomes, that capture cytosolic cargo and deliver it to lysosomes, where the breakdown products are recycled back to cytoplasm. On the basis of degraded cell components, some selective types of autophagy can be identified (mitophagy, ribophagy, reticulophagy, lysophagy, pexophagy, lipophagy, and glycophagy). Dysregulation of autophagy can induce various disease manifestations, such as inflammation, aging, metabolic diseases, neurodegenerative disorders and cancer. The understanding of the molecular mechanism that regulates the different phases of the autophagic process and the role in the development of diseases are only in an early stage. There are still questions that must be answered concerning the functions of the autophagy-related proteins. In this review, we describe the principal cellular and molecular autophagic functions, selective types of autophagy and the main in vitro methods to detect the role of autophagy in the cellular physiology. We also summarize the importance of the autophagic behavior in some diseases to provide a novel insight for target therapies.

Cytotoxic and Apoptotic Activities of Prunus spinosa Trigno Ecotype Extract on Human Cancer Cells.

The aim of this work was to demonstrate that a natural compound, not-toxic to normal cells, has cytotoxic and sensitizing effects on carcinoma cells, with the final goal of combining it with chemotherapeutic drugs to reduce the overall dose. Prunus spinosa Trigno ecotype (PsT) drupe extract with a nutraceutical activator complex (NAC) made of amino acids, vitamins and mineral salt blends, has shown in vitro anticancer activity. The cytotoxic effect of (PsT + NAC)® has been evaluated on human cancer cells, with an initial screening with colorectal, uterine cervical, and bronchoalveolar cells, and a subsequent focus on colon carcinoma cells HCT116 and SW480. The viability reduction of HCT116 and SW480 after treatment with (PsT 10 mg/mL + NAC)® was about 40% (p < 0.05), compared to control cells. The cell's survival reduction was ineffective when the drug vehicle (NAC) was replaced with a phosphate buffer saline (PBS) or physiological solution (PS). The flow cytometry evaluation of cancer cells' mitochondrial membrane potential showed an increase of 20% depolarized mitochondria. Cell cycle analysis showed a sub G1 (Gap 1 phase) peak appearance (HCT116: 35.1%; SW480: 11.6%), indicating apoptotic cell death induction that was confirmed by Annexin V assay (HCT116: 86%; SW480: 96%). Normal cells were not altered by (PsT + NAC)® treatments.