Study of the modulation of P-gp activity by curcumin preconditioning in a novel dynamic intestinal in vitro model

P-glycoprotein (P-gp) is the most relevant protein studied in the field of multi-drug resistance, the main obstacle of chemotherapy. The first objective of this work was to observe the modulation of P-gp activity by curcuminoids. In fact it can reduce the concentration and effectiveness of numerous substances absorbed, among which there are toxic substances, but also common chemotherapeutic agent. Curcumin has shown good anti-tumor capacity over the years, acting on different targets involved in the appearance and development of cancer cells. Among these, P-glycoprotein was identified: it is a membrane glycoprotein, with the function of pumping out neutral or weakly basic amphipathic substances from the cytoplasm. In the human gastrointestinal tract, it is found expressed in high concentrations on the apical surfaces of superficial columnar epithelial cells of the colon and distal small bowel. In this work intestinal in vitro models of Caco-2 were used. This cell line of colorectal adenocarcinoma is able to form a cell monolayer that resembles the intestinal epithelium and have a high phenotypic expression of P-glycoprotein. The innovative aspect of this work is that the culture of the cells was performed in a bioreactor capable of better reproducing the intestinal conditions of the physiological environment through the application of medium flow. First of all, a cell viability test was performed to determine the concentration of curcumin that did not cause damage to the cells. Caco-2 monolayers were incubated on a 24-well-plate at different concentrations of curcumin (up to 90 µg/mL) for 48 hours. The metabolic activity of these cells was measured before and after incubation. The results showed no signs of significant toxicity on cellular activity. Then, three parallel studies were performed using the Transwell model and static and dynamic bioreactors, on which membranes Caco-2 cells were seeded and grown for 21 days. They were monitored through parameters such as the measure of the TEER, expression of the integrity of the epithelial barrier, and visual inspection under an optical microscope. After 20 days of culture, the preconditioning of cell monolayer with a solution of Curcuminoids (50 µg/mL), for sixteen hours, was performed. Afterwards, the activity of P-gp was studied through the quantification of changes in the concentration of Rhodamine-123, a fluorescent substrate of P-gp. It was added into the basolateral side of the monolayer and it was analyzed if this was retained or effluxed on the apical side. From the results obtained we have measured a greater accumulation of the Rh-123 substrate in the basolateral side of the in the dynamic bioreactor, and therefore a reduced permeability (Papp) of this membrane to the substance possibly due to a down-regulation of the function of P-glycoprotein. These results could be justified by the structural and biochemical differences of the cell barrier formed in different supports. As already demonstrated in the literature, the flow of dynamic bioreactors can stimulate the formation of a more compact cytoskeleton with more solid tight junctions, compared to static culture conditions. The mechanical stimulation of the flow, in addition to enhancing the expression of proteins with a structural function, we can hypothesize that it is able to increase the activity of functional proteins, which are therefore more "sensitive" to biochemical stimuli coming from outside. At the end a characterization of the structure and of the morphology of the cells was performed in order to stain some key structures such as nuclei, actin filaments and occludin, an integral membrane protein. The different biochemical response observed from the membrane grown under flow conditions gives a first validation of the model from a pharmacodynamic point of view. Therefore this new device is promising for future investigations in pharmacological and toxicological research and could open new ways also for studies considered to be unsuccessful in classical in vitro systems