24 research outputs found
Potenziamento dell’efficacia del cetuximab mediante combinazione con cellule T ingegnerizzate con recettori Fc chimerici per il trattamento del carcinoma del colon retto K-ras mutato.
No full textINTRODUZIONE: L’impiego di anticorpi monoclonali (AM) diretti contro il recettore per il fattore di crescita epidermico (EGFR), tra cui il cetuximab, ha rappresentato un significativo avanzamento nel trattamento del carcinoma colorettale metastatico (mCRC). L’effetto anti-tumorale del cetuximab è dovuto a: i) blocco della proliferazione neoplastica; ii) attivazione di processi apoptotici; iii) induzione di citotossicità cellulare dipendente da anticorpo (ADCC) mediante il legame con i recettori Fc. Tuttavia, diversi fattori limitano l’efficacia terapeutica del cetuximab; tra questi la presenza di mutazioni nel gene Kras (Kras-mut) e la scarsa presenza di cellule Natural Killer, capaci di mediare ADCC, nel microambiente tumorale. In questo studio ipotizziamo di superare i limiti terapeutici del cetuximab attraverso la sua combinazione con cellule T ingegnerizzate con recettori chimerici Fc (Fc-CRs).
MATERIALI E METODI: Due Fc-CRs, denominati CD32-CR e CD16-CR, sono stati generati dalla fusione della regione extracellulare del FcRIIA (CD32) o del FcRIIIA (CD16) con il dominio transmembrana del CD8a e con i domini intracitoplasmatici del CD28 e del CD3. Linfociti T attivati sono stati trasdotti con le chimere e la corretta espressione è stata valutata mediante citofluorimetria e western blot. I recettori sono stati caratterizzati funzionalmente in vitro attraverso test di binding e saggi ELISA. L’attività anti-tumorale delle cellule T trasdotte in associazione al cetuximab è stata valutata in test di deplezione in vitro e in topi SCID xenotrapiantati con la linea cellulare di CRC Kras-mut HCT116.
RISULTATI: Entrambi i Fc-CRs sono espressi correttamente sulla superficie delle cellule T. Il CD32-CR, ma non il CD16-CR, lega in maniera specifica il frammento Fc degli AM anti-EGFR cetuximab e panitumumab. La dose massima di cetuximab necessaria per saturare tutti i CD32-CR che è pari a 10g/ml ed il binding è mantenuto anche in presenza di quantità crescenti di plasma umano. Sia il CD32-CR che il CD16-CR inducono rilascio di citochine in risposta a stimolazione con cetuximab o panitumumab. La combinazione del cetuximab con cellule T CD32-CR+: i) induce l’eliminazione di HCT116 in vitro; ii) riduce significativamente la crescita tumorale in vivo.
CONCLUSIONI: In questo studio preclinico dimostriamo che cellule T armate con CD32-CR potenziano l’efficacia del cetuximab contro CRC Kras-mutato
HSP70 production and inhibition of cell proliferation in molt-4 T-cells after cell-to-cell transmission of HTLV-I: Effect of PGA1
No full textInfection with HTLV-I is associated with leukemic transformation of mature CD4(+) T lymphocytes. PGA(1), a powerful inhibitor of tumour cell proliferation, can prevent the clonal expansion of HTLV-I-infected cells following acute infection of cord blood-derived mononuclear cells. Since the antiproliferative effect of PGA(1) on HTLV-I transformed, chronically infected MT-2 cell line was associated with induction of HSP70, we have investigated the effect of PGA(1) on cell cycle progression and HSP70 production in a leukemic T-cell line (Molt-4) shortly after exposure to HTLV-I in a cell-to-cell transmission model. rate of cell proliferation and HSP70 expression were studied within one duplication cycle of Molt-4 cells after exposure to HTLV-I. growth of both control and virus-exposed cultures was inhibited by treatment with PGA(1) (4 mu g/ml) and cell cycling was arrested preferentially at the G(1)/S interphase. synthesis of HSP70 was induced within 3 h by PGA(1) in control and virus-exposed Molt-4 cells and became undetectable from overnight onward, though the protein accumulated in the cells. the arrest of growth was observed from overnight up to 48 h so that treated cells almost missed one cycle. Interestingly, HSP70 transcript and protein persisted at remarkably high levels in molt-4 cells exposed to HTLV-I in the absence of PGA(1), showing that HSP70 expression can be directly activated during primary infection with this human retrovirus. moreover, in these cocultures, treatment with PGA(1) or heat shock was not able to increase further the elevated level of HSP70 found in untreated cocultures, suggesting that during the early period of the virus-transmission phase, HTLV-I could interfere with HSP70 induction by other inducers
Aspirin inhibits cancer stem cells properties and growth of glioblastoma multiforme through Rb1 pathway modulation
No full textSeveral clinical studies indicated that the daily use of aspirin or acetylsalicylic acid reduces the cancer risk via cyclooxygenases (Cox-1 and Cox-2) inhibition. In addition, aspirin-induced Cox-dependent and -independent antitumor effects have also been described. Here we report, for the first time, that aspirin treatment of human glioblastoma cancer (GBM) stem cells, a small population responsible for tumor progression and recurrence, is associated with reduced cell proliferation and motility. Aspirin did not interfere with cell viability but induced cell-cycle arrest. Exogenous prostaglandin E2 significantly increased cell proliferation but did not abrogate the aspirin-mediated growth inhibition, suggesting a Cox-independent mechanism. These effects appear to be mediated by the increase of p21 waf1 and p27 Kip1, associated with a reduction of Cyclin D1 and Rb1 protein phosphorylation, and involve the downregulation of key molecules responsible for tumor development, that is, Notch1, Sox2, Stat3, and Survivin. Our results support a possible role of aspirin as adjunctive therapy in the clinical management of GBM patients
Genomic and physiological characterization of Bacilli isolated from salt-pans with plant growth promoting features
No full text© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Petrillo, C., Castaldi, S., Lanzilli, M., Selci, M., Cordone, A., Giovannelli, D., & Isticato, R. Genomic and physiological characterization of Bacilli isolated from salt-pans with plant growth promoting features. Frontiers in Microbiology, 12, (2021): 715678, https://doi.org/10.3389/fmicb.2021.715678.Massive application of chemical fertilizers and pesticides has been the main strategy used to cope with the rising crop demands in the last decades. The indiscriminate use of chemicals while providing a temporary solution to food demand has led to a decrease in crop productivity and an increase in the environmental impact of modern agriculture. A sustainable alternative to the use of agrochemicals is the use of microorganisms naturally capable of enhancing plant growth and protecting crops from pests known as Plant-Growth-Promoting Bacteria (PGPB). Aim of the present study was to isolate and characterize PGPB from salt-pans sand samples with activities associated to plant fitness increase. To survive high salinity, salt-tolerant microbes produce a broad range of compounds with heterogeneous biological activities that are potentially beneficial for plant growth. A total of 20 halophilic spore-forming bacteria have been screened in vitro for phyto-beneficial traits and compared with other two members of Bacillus genus recently isolated from the rhizosphere of the same collection site and characterized as potential biocontrol agents. Whole-genome analysis on seven selected strains confirmed the presence of numerous gene clusters with PGP and biocontrol functions and of novel secondary-metabolite biosynthetic genes, which could exert beneficial impacts on plant growth and protection. The predicted biocontrol potential was confirmed in dual culture assays against several phytopathogenic fungi and bacteria. Interestingly, the presence of predicted gene clusters with known biocontrol functions in some of the isolates was not predictive of the in vitro results, supporting the need of combining laboratory assays and genome mining in PGPB identification for future applications
A possible interplay between HR‐HPV and stemness in tumor development: an in vivo investigation of CD133 as a putative marker of cancer stem cell in HPV18‐infected KB cell line
No full textHigh-risk HPVs (HR-HPVs) are DNA viruses considered as primary etiologic factors in malignancies of the low female genital tract. Their presence has also been documented in oropharyngeal and laryngeal cancers. However, HPV infection is considered a necessary but not sufficient cause of tumoral development; meantime, increasing evidences on the tumorigenic role of cancer stem cells (CSCs) have been documented in the literature. CSCs represent a small subpopulation of neoplastic cells with self-renewal potential, capable of maintaining tumor growth and cell differentiation, also involved in metastatic process, recurrence, and resistance to chemotherapeutic agents. In the present study, performed on KB cell lines, we evaluated the tumor forming potential of CSCs, and their relationship with the HPV infection status. We started our study by identifying the most aggressive cell line on the minimal number of cells being able of growth in vivo in a model of athymic nude mice (BALB/c nu/nu). We used an oral-derived KB cell line separated in the KB-CD133+ and KB-CD133- populations, by using immunomagnetic beads and fluorescence-activated cell sorting (FACS). The separated populations were injected in athymic nude mice (BALB/c nu/nu). Xenograft tumors have been analyzed for tumor size, CD133 expression by immunohistochemistry (IHC) and for DNA HR-HPV integration by in situ hybridization (ISH), comparing CD133-enriched xenograft tumors versus the CD133 non-enriched ones. On standard conditions, the KB cell line has a poor population of glycosylated CD133 marker (<5.0%) when investigated with antibodies versus CD133, and more specifically its glycosylated epitope (AC133). Enriched CD133 KB cells possess a higher capacity of tumor growth in xenograft models of nude mice when compared to KB CD133-negative cells. We observed that the AC133 epitope, extensively used to purifying hematopoietic stem cells, is able to select an epithelial subpopulation of cancer stem cells with aggressive behavior. We retain that CD133 may be a useful target in anticancer strategies including pharmacological and immunological therapies
Fluorescent peptide dH3w: A sensor for environmental monitoring of mercury (II)
No full textHeavy metals are hazardous environmental contaminants, often highly toxic even at extremely low concentrations. Monitoring their presence in environmental samples is an important but complex task that has attracted the attention of many research groups. We have previously developed a fluorescent peptidyl sensor, dH3w, for monitoring Zn2+ in living cells. This probe, designed on the base on the internal repeats of the human histidine rich glycoprotein, shows a turn on response to Zn2+ and a turn off response to Cu2+. Other heavy metals (Mn2+, Fe2+, Ni2+, Co2+, Pb2+ and Cd2+) do not interfere with the detection of Zn2+ and Cu2+. Here we report that dH3w has an affinity for Hg2+ considerably higher than that for Zn2+ or Cu2+, therefore the strong fluorescence of the Zn2+/dH3w complex is quenched when it is exposed to aqueous solutions of Hg2+, allowing the detection of sub-micromolar levels of Hg2+. Fluorescence of the Zn2+/dH3w complex is also quenched by Cu2+ whereas other heavy metals (Mn2+, Fe2+, Ni2+, Co2+, Cd2+, Pb2+, Sn2+ and Cr3+) have no effect. The high affinity and selectivity suggest that dH3w and the Zn2+/dH3w complex are suited as fluorescent sensor for the detection of Hg2+ and Cu2+ in environmental as well as biological samples. © 2018 Siepi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
FCγ Chimeric Receptor-Engineered T Cells: Methodology, Advantages, Limitations, and Clinical Relevance
No full textFor many years, disappointing results have been generated by many investigations, which have utilized a variety of immunologic strategies to enhance the ability of a patient’s immune system to recognize and eliminate malignant cells. However, in recent years, immunotherapy has been used successfully for the treatment of hematologic and solid malignancies. The impressive clinical responses observed in many types of cancer have convinced even the most skeptical clinical oncologists that a patient’s immune system can recognize and reject his tumor if appropriate strategies are implemented. The success immunotherapy is due to the development of at least three therapeutic strategies. They include tumor-associated antigen (TAA)-specific monoclonal antibodies (mAbs), T cell checkpoint blockade, and TAA-specific chimeric antigen receptors (CARs) T cell-based immunotherapy. However, the full realization of the therapeutic potential of these approaches requires the development of strategies to counteract and overcome some limitations. They include off-target toxicity and mechanisms of cancer immune evasion, which obstacle the successful clinical application of mAbs and CAR T cell-based immunotherapies. Thus, we and others have developed the Fc gamma chimeric receptors (Fcγ-CRs)-based strategy. Like CARs, Fcγ-CRs are composed of an intracellular tail resulting from the fusion of a co-stimulatory molecule with the T cell receptor ζ chain. In contrast, the extracellular CAR single-chain variable fragment (scFv), which recognizes the targeted TAA, has been replaced with the extracellular portion of the FcγRIIIA (CD16). Fcγ-CR T cells have a few intriguing features. First, given in combination with mAbs, Fcγ-CR T cells mediate anticancer activity in vitro and in vivo by an antibody-mediated cellular cytotoxicity mechanism. Second, CD16-CR T cells can target multiple cancer types provided that TAA-specific mAbs with the appropriate specificity are available. Third, the off-target effect of CD16-CR T cells may be controlled by withdrawing the mAb administration. The goal of this manuscript was threefold. First, we review the current state-of-the-art of preclinical CD16-CR T cell technology. Second, we describe its in vitro and in vivo antitumor activity. Finally, we compare the advantages and limitations of the CD16-CR T cell technology with those of CAR T cell methodology
Recent perspective on CAR and Fcγ-CR T cell immunotherapy for cancers: Preclinical evidence versus clinical outcomes.
No full textThe chimeric antigen receptor Tcell (CAR-T cell) immunotherapy currently represents a hot research trend and it is expected to revolutionize the field of cancer therapy. Promising outcomes have been achieved using CAR-T cell therapy for haematological malignancies. Despite encouraging results, several challenges still pose eminent hurdles before being fully recognized. Directing CAR-T cells to target a single tumour associated antigen (TAA) as the case in haematological malignancies might be much simpler than targeting the extensive inhibitory microenvironments associated with solid tumours. This review focuses on the basic principles involved in development of CAR-T cells, emphasizing the differences between humoral IgG, T-cell receptors, CAR and Fcγ-CR constructs. It also highlights the complex inhibitory network that is usually associated with solid tumours, and tackles recent advances in the clinical studies that have provided great hope for the future use of CAR-T cell immunotherapy. While current Fcγ-CR T cell immunotherapy is in pre-clinical stage, is expected to provide a sound therapeutic approach to add to existing classical chemo- and radio-therapeutic modalities
