Abstract
Colorectal cancer stands as the most common type of gastrointestinal cancer, the third most common cancer type, and the second leading cause of cancer-related mortality worldwide. Rectal cancer alone is responsible for one third of the diagnosed cases, and a trend for an increase in the early-age (under 50 years) diagnostic of rectal cancer has been observed recently, with the age of onset dropping in prospective estimates for the USA and Europe. Known risk factors promoting colorectal cancer development include both the lack of physical exercise and unhealthy eating habits, namely a diet mainly composed of processed foods, red meat, fat from animal sources, and low on vegetable, fruit, fiber, and calcium consumption. In 2016, the International Agency for Research on Cancer (IARC) concluded that obesity was associated with an increased risk of 13 types of cancer, including colorectal cancer. Evidence also suggests that weight gain during adulthood is associated with an elevated risk of developing colorectal cancer when compared with adults whose weight remains stable. Numerous epidemiological studies reported a lower risk of suffering various types of cancers when cruciferous vegetables are consumed. Their beneficial properties are attributed to the presence of sulfurous components, called isothiocyanates. Many in vitro and in vivo studies have reported that isothiocyanates elicit relevant pharmacological effects through multiple mechanisms that include modulation of phases I and II detoxification pathway enzymes, regulation of cell-cycle arrest and control of cell growth, induction of apoptosis, antioxidant activity, anti-inflammatory activity, anti-angiogenic effects, and epigenetic regulation. For this reason, isothiocyanates have been proposed as a possible therapy for the treatment of certain cancers. Among isothiocyanates, the most studied is sulforaphane, which exhibits cytostatic and cytotoxic activities mediated by a plethora of different and partly interdependent molecular mechanisms. Recently, we condensed sulforaphane with a fluorinated hydrophobic rhodol analogue, thus obtaining compounds called NITCs (newly synthesized ITCs). NITCs have intrinsic green fluorescence and can represent a theranostic platform. Theranostics refers to the union of diagnostic and therapeutic applications into a single agent thus leading to a promising therapeutic paradigm involving diagnosis, drug delivery and monitoring of treatment response. In parallel, for diagnosis purposes, particular interest is given to the development of new reactive fluorescent probes involving a minimum perturbation of the biological system. This is essential in order to understand the structure and function of cellular processes. For example, the probe should be able to penetrate the outer lipid/phospholipid membrane at a relatively high speed while preserving integrity and performance at a cellular level, and it should feature an intracellular localization profile while preserving cell viability and proliferation as well as membrane permeability. However, the application of these probes is limited due to several properties, such as: low solubility in water and in culture media, pH range, toxicity, and cell penetration. The project is designed to evaluate whether the NITCs can represent an innovative pharmacological strategy for the treatment of colorectal cancer. Studying how NITCs could impact on key molecular pathways of cancer cells would be of great interest to understand their clinical potential. The integrated preclinical models proposed here, which reproduce the biological features of human tumors by a pathological, pharmacological, and molecular point of view, will be critical to understand the mechanisms through which NITCs exposure could affect tumor development and will give the extent of their activity in comparison to those changes that occurred in normal-weight and obese animals. Moreover, the study on cells cultured in 3D will provide valuable information on the effects of tumor microenvironment in the modulation of their anticancer effects. Experiments will be carried out through an integrated experimental strategy. In particular, we will use experimental models already set-up and validated in our laboratories and characterized by a different degree in predicting clinical outcome. We will perform a preliminary characterization of the cellular and molecular effects of four NITCs in 2D cultures of human colorectal cell lines. 2D monolayer cells have been widely used in cancer cell biology for several decades. However, the so-called “flat biology” suffers from disadvantages associated with the loss of tissue-specific architecture, mechanical and biochemical cues, and cell-to-cell and cell-to-matrix interactions thus making it a relatively poor model to predict drug responses for certain diseases such as cancer. Tumor 3D models have been recently adapted to experimental cancer research and compensate for many of the deficiencies seen in 2D models. They have the advantage of being less experimentally demanding, more high-throughput and of employing cells of human origin. In addition, 3D models much better recapitulate the tumor architecture and biology compared to the widely used 2D models. A bioreactor device (U-cup) will be used to build 3D models. Although classic cytotoxic chemotherapy has been traditionally considered immunosuppressive, various clinical studies demonstrate that it often interacts positively with immunotherapy. Anticancer agents such as oxaliplatin induce cell death accompanied by the release of crucial signals, rendering cancer cells ‘visible’ to the immune system. These signals initiate a cascade of events that can lead to long-term responses in immunocompetent hosts, a mechanism described as immunogenic cell death (ICD). Therefore, it is of major therapeutic relevance to explore the immunogenic potential of both clinically applied anti-cancer drugs and currently developed compounds. In this context, it is of utmost interest to characterize the molecular mechanisms of how cells die upon treatment as this subsequently influences the onset of ICD. The ability of selected NITCs to induce ICD will be tested. Since ICD leads to the release of damage-associated molecular patterns (DAMPs) from dying tumor cells, with ensuing activation of immune responses, we will analyze DAMPs. To identify malignant cells succumbing to a putative ICD inducer, they will be fed to dendritic cells, followed by (1) phagocytosis assays; (2) assessment of activation markers on the dendritic cells surface and functional markers in conditioned media. We will also study the effects of NITCs on cell-cycle progression using propidium iodide and expression of genes involved in the specific cell death pathway using specific antibodies. To explore the potential selectivity of NITCs for cancer cells, their cytotoxic potential will be analyzed on normal colorectal cell lines. All the analyses will be performed as described above. Since U-cup is able to mimic tumor microenvironment, based on the results from 2D models experiments, the most promising NITCs will be studied for their effects on cellular components of tumor microenvironment. The effects of NITCs in the 3D models will be compared with those in 2D models. Of note, taking into account the fluorescence of NITCs, we will also observe their cellular distribution. Obesity is a chronic low-grade inflammation. Inflammation is a mechanism through which obesity increases the risk of developing colorectal cancer and promotes its progression. Additionally, around 11% of colorectal cancer cases have been attributed to overweight and obesity in Europe. Epidemiological data have established an association between obesity and the 30-70% increased risk of colorectal cancer in men. In this project, 1,2-dimethylhydrazine will be used to induce colorectal cancer in rat models. NITC will be administered to test the possible pharmacological arrest of tumor development (inhibition of tumor formation and tumor multiplicity). We expect a high tumor incidence in 1,2-dimethylhydrazine treated rats and a significant reduction of tumor incidence in 1,2-dimethylhydrazine-treated rats that had received NITC at the post-initiation stage of colon carcinogenesis. Taking into account the fluorescence characterizing NITCs, it will be also possible to image tissues that express fluorescence and gain information on the pharmacokinetic profile of NITCs and their specific biodistribution. Moreover, the analysis of the anticancer potential of NITCs on the U-cup and the comparison with the results observed on the in vivo model represent a complex but complementary experimental approach, which will provide an exciting opportunity to predict the cellular mechanisms of the new NITCs and their clinical potential.
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
