Abstract
Abstract Colorectal cancer (CRC) is the most common gastrointestinal malignancy, the third most frequently diagnosed cancer, and the second leading cause of cancer-related mortality worldwide. Major risk factors include physical inactivity, unhealthy dietary habits, and obesity, which has been recognized as an independent contributor to CRC development and progression. Epidemiological evidence consistently associates the consumption of cruciferous vegetables with a lower incidence of several cancers. Their protective effects are mainly attributed to isothiocyanates (ITCs), sulfur-containing compounds that exert anticancer activities through multiple mechanisms, including modulation of detoxification enzymes, regulation of cell-cycle progression, induction of apoptosis, antioxidant and anti-inflammatory effects, inhibition of angiogenesis, and epigenetic regulation. Among ITCs, sulforaphane is the best-characterized compound and displays potent cytostatic and cytotoxic activities. We recently developed a novel class of fluorescent ITCs (NITCs) by conjugating sulforaphane with a fluorinated hydrophobic rhodol analogue. These compounds combine intrinsic green fluorescence with therapeutic potential, making them promising theranostic agents capable of integrating cancer diagnosis, treatment, and therapeutic monitoring. Unlike many fluorescent probes currently available, which are often limited by poor solubility, toxicity, or inadequate cellular uptake, NITCs may simultaneously enable intracellular imaging and anticancer activity. The aim of this project is to evaluate the therapeutic potential of NITCs against CRC and characterize the molecular mechanisms underlying their activity. We will employ complementary preclinical models reproducing the pathological, pharmacological, and molecular features of human CRC, allowing investigation of NITC efficacy under both normal-weight and obesity-associated conditions. Particular attention will be devoted to the influence of the tumor microenvironment on treatment response. The experimental strategy will begin with the characterization of four NITCs in two-dimensional (2D) cultures of human CRC cell lines. Cellular viability, proliferation, cell-cycle progression, and activation of cell death pathways will be investigated together with the expression of relevant molecular markers. Since conventional 2D cultures do not adequately reproduce tumor architecture or cell-cell and cell-matrix interactions, the most promising compounds will subsequently be evaluated in three-dimensional (3D) tumor models generated using the U-cup bioreactor. These models better mimic the structural and biological characteristics of human tumors while preserving the flexibility and reproducibility of in vitro systems. The intrinsic fluorescence of NITCs will also allow direct visualization of their intracellular localization and distribution. An important objective of the project is to determine whether NITCs induce immunogenic cell death (ICD), a form of regulated cell death capable of stimulating antitumor immune responses. Several chemotherapeutic agents, including oxaliplatin, promote ICD by inducing the release of damage-associated molecular patterns (DAMPs), thereby increasing tumor immunogenicity. To evaluate this possibility, DAMP release following NITC treatment will be analyzed, and treated tumor cells will be incubated with dendritic cells to assess phagocytosis, expression of activation markers, and secretion of immune mediators. Cell-cycle progression and activation of specific cell death pathways will also be characterized using established molecular and immunological assays. To assess treatment selectivity, all analyses performed in CRC cells will be repeated in normal colorectal epithelial cell lines. The effects of selected NITCs on the tumor microenvironment will then be investigated in 3D cultures, and results will be directly compared with those obtained in 2D models. These experiments will clarify how tissue architecture and microenvironmental factors influence NITC activity and provide a more reliable prediction of therapeutic efficacy. Finally, the anticancer potential of NITCs will be validated in vivo using a rat model of chemically induced colorectal carcinogenesis. Colorectal tumors will be induced by 1,2-dimethylhydrazine, and NITCs will be administered to evaluate their ability to inhibit tumor formation and reduce tumor multiplicity. Since obesity promotes CRC through chronic low-grade inflammation, the in vivo model will also allow assessment of NITC efficacy in a physiologically relevant context. We expect a high tumor incidence in carcinogen-treated animals and a significant reduction in animals receiving NITCs during the post-initiation stage of carcinogenesis. Overall, the integration of conventional cell cultures, advanced 3D tumor models, and in vivo studies provides a comprehensive preclinical platform for evaluating the biological activity and therapeutic potential of NITCs. This multidisciplinary approach will identify the molecular mechanisms responsible for their anticancer effects, assess their selectivity and immunogenic properties, and establish their potential as innovative theranostic agents for the treatment of colorectal cancer. Key Results Achieved: This project was supported by two research units with extensive experience in conducting innovative, high-impact research that has been widely disseminated in the past and will continue to be so in the future. The project has generated high-quality publications in peer-reviewed journals (see below), ensuring broad dissemination of its findings. Furthermore, the results have underscored the excellence of Italian scientific research and highlighted the importance of establishing a strong national consortium that brings together bedside and bench scientists. Such multidisciplinary collaboration is essential for achieving long-term advances in oncology, a field that continues to face significant challenges due to the high global burden of cancer-related mortality. Milelli A, Catanzaro E, Greco G, Calcabrini C, Turrini E, Maffei F, Burattini S, Guardigni M, Sissi C, Schnekenburger M, Diederich M, Sestili P, Fimognari C. New rhodol-sulforaphane conjugates as innovative isothiocyanate-based cytotoxic agents for cancer cells. Eur J Med Chem. 2024; 280:116936. doi: 10.1016/j.ejmech.2024.116936. Einaudi G, Oberto A, Bertocchi I, Aimaretti E, Ferreira Alves G, Porchietto E, Cifani C, Micioni Di Bonaventura MV, Collino M, Chiazza F. Effects of Npy1r limbic conditional knock-out on adipose tissue metabolism. Neuropharmacology 2026; 292:110941. doi: 10.1016/j.neuropharm.2026.110941. Casile A, Bonaldo B, Bettarelli M, Papadopoulos P, Micioni Di Bonaventura MV, Nasini S, Comai S, Sut S, Dall'Acqua S, Marraudino M, Gotti S, Cifani C. Central and peripheral monoamine changes in a rat model of Gaming Disorder. Neuropharmacology 2026; 282:110703. doi: 10.1016/j.neuropharm.2025.110703. de Ceglia M, Botticelli L, Micioni Di Bonaventura E, Vargas Fuentes A, Micioni Di Bonaventura MV, Rodriguez de Fonseca F, Cifani C. Targeting the endocannabinoid/paracannabinoid systems in binge eating behavior: Efficacy of dual ligands in a preclinical model. Pharmacological Research 2025; 222:108005. doi: 10.1016/j.phrs.2025.108005. Botticelli L, Micioni Di Bonaventura E, Einaudi G, Provensi G, Costa A, D'Addario C, Cifani C, Micioni Di Bonaventura MV. Is there a role for cannabidiol in obesity, metabolic syndrome and binge eating? British Journal of Pharmacology 2025. doi: 10.1111/bph.70196. Costa A, Micioni Di Bonaventura E, Botticelli L, Eramo B, Gaetani S, Passani MB, Cifani C, Micioni Di Bonaventura MV, Provensi G. Brain histaminergic system: An emerging target for the treatment of feeding and eating-related disorders. Pharmacological Research 2025; 221:107949. doi: 10.1016/j.phrs.2025.107949. de Ceglia M, Romano A, Micioni Di Bonaventura MV, Gavito A, Botticelli L, Micioni Di Bonaventura E, Friuli M, Cifani C, de Fonseca FR, Gaetani S. Cafeteria Diet Abstinence Induces Depressive Behavior and Disrupts Endocannabinoid Signaling in Dopaminergic Areas: A Preclinical Study. Current Neuropharmacology 2025; 23(4):458-474. doi: 10.2174/1570159X23666241107160840. Mercante F, Micioni Di Bonaventura E, Pucci M, Botticelli L, Cifani C, D'Addario C, Micioni Di Bonaventura MV. Repeated binge-like eating episodes in female rats alter adenosine A2A and dopamine D2 receptor genes regulation in the brain reward system. International Journal of Eating Disorder 2024; 57(7):1433-1446. doi: 10.1002/eat.24216.
Dettagli del progetto
Responsabile scientifico: Carmela Fimognari
Strutture Unibo coinvolte:
Dipartimento di Scienze per la Qualità della Vita
Coordinatore:
ALMA MATER STUDIORUM - Università di Bologna(Italy)
Contributo totale di progetto: Euro (EUR) 221.637,00
Contributo totale Unibo: Euro (EUR) 111.043,00
Durata del progetto in mesi: 24
Data di inizio
16/10/2023
Data di fine:
28/02/2026
