First, we performed a preliminary cytotoxicity test to determine a safe curcumin dose to be employed in the P-gp modulation study. Then, we set-up the culture of Caco-2 cells in the two systems for 21 days, with regular cell monitoring, including TEER and TEEI (Trans Epithelial Electrical Resistance and Impedance respectively). Finally, the P-gp activity in response to treatment with the two different compounds (Curcumin extract and the synthetic compound APD) was assessed through a transport study using a P-gp substrate: rhodamine-123 (Rh-123). Figure 2 schematically illustrates the experiments performed.
Cell culture and subculturing
Caco-2 cells from the American Type Culture Collection (ATCC) were grown in tissue culture flasks in Dulbecco's Modified Eagle's medium (high glucose) supplemented with 10% (v/v) heat-inactivated Fetal Bovine Serum (FBS), 1% (v/v) L-glutamine, 1% (v/v) non-essential amino acids and 1% (v/v) penicillin/streptomycin. All reagents were purchased from Sigma-Aldrich, Italy, unless stated otherwise.
The LB2 bioreactors, purchased from IVTech srl, are composed of an apical and basal chamber, separated by a Polyethylene terephthalate (PET) membrane. Both chambers are closed by two circular glass slides, one at the top and one at the bottom of the system, to allow live imaging. These are connected through fluidic tubing to separate medium reservoirs (the mixing chambers). The flow was generated by a peristaltic pump (LiveFlow, IVTech Srl, Italy).
Caco-2 cells (between passages 30 and 45) were seeded at a density of 25000 cells/cm2 in bioreactors (through their tubing system) and PET 12-well-plate Transwell® (Corning, Italy). After cell adhesion, the apical chambers of the bioreactor were filled with 2 mL of medium and the basolateral ones with 1 mL of medium. In the Transwells, the apical and basolateral compartments were filled with 500 µL and 1 mL of medium, respectively. All the samples were incubated at 37 °C in a humidified atmosphere of 5% CO2 for three weeks. In the static samples the medium was changed every three days.
After one week, the basal and apical circuits were filled up to a total volume of 10 mL of media respectively and kept in dynamic conditions (under flow) at a flow rate of 150 µL/min, which according to the manufacturer should provide an average shear stress of around 6 × 10− 4 Pa on the membrane. In this set-up, the medium was changed once a week: half the volume was removed and replaced by fresh medium.
Curcuma extract and APD compound preparation
The extract of Turmeric was taken from a common encapsulated supplement (Curcumina Santé, from Santé Naturels, Italy) containing 450 mg of pure curcumin, titled at 95%. It is regularly registered as a food supplement notified to the Italian Ministry of Health. A stock solution of curcumin (45 mg/mL) was diluted in DMSO and stored at − 20 °C.
APD (kindly provided by the medicinal chemistry lab of Rapposelli Simona, Dept.of Pharmacy, UNIPI, Italy) was dissolved in Hank’s Balanced Salt Solution (HBSS) and the stock solution (10 mM) was stored at – 20 °C. This compound belongs to a new class of molecules that showed appreciable modulatory activity on P-gp and a high degree of selectivity (17–19). The authors studied P-gp inhibition activity by 3 combined biological assays among which there was the inhibition of P-gp mediated transport of vinblastine. This derivative was shown to compete with radiolabeled vinblastine (a well-known P-gp substrate) for the P-gp binding site, with an IC50 value of 0.19 mM. (17).
Assessment of curcumin toxicity
The toxicity of curcumin was measured through a preliminary assessment of the metabolic activity of the cells, before and after incubation with the compound at different concentrations.
Caco-2 cells were seeded in 24 well flat-bottom plates (Corning, Italy) and cultured for 7 days. At the seventh day the medium was replaced with fresh medium containing different concentrations of curcumin (11.25, 22.5, 45, 90 µg/mL), assuring the DMSO was present at a non-toxic concentration (0.02%). The cells were incubated with the different solutions at 37 °C for 48 hours and the control samples solution contained only 0.02% DMSO in the cell medium.
Cell barrier monitoring and morphological analysis
Monolayer formation and maintenance were verified every three days with an optical microscope (Olympus AX81, Olympus Italy) both in the transwell and in the bioreactors. Moreover, transepithelial electric resistance (TEER) measurements, which are related to tight junction formations and thus provide an indication of cellular layer tightness, were performed.
Usually, TEER is measured applying a low-frequency (f < 5 kHz) current stimulus across the cellular barrier and recording the resulting voltage (16, 20). In this study, it was monitored at 40 Hz with an integrated cellular impedance-meter (16, 21), in the bioreactors, and at 12.5 Hz with an Epithelial VoltOhmMeter (EVOM, World Precision Instruments, Florida USA) provided with chopsticks electrodes, in the Transwells. As reported in Cacopardo et al (16), the measurements performed by the two instruments are equivalent.
The final TEER values were obtained by subtracting the blank resistance values, related to the membrane and the culture medium, and multiplying by the surface area of the respective membrane (i.e. 1.2 cm2 for the TW and 1.8 cm2 for the bioreactors).
Finally, transepithelial electric impedance (TEEI) spectra were acquired in the bioreactors within a frequency range of 40-10000 Hz, providing a more precise indication regarding the formation of a tight monolayer.
Assessment of P-gp Activity
On the twentieth day of culture, all the samples were rinsed with PBS and then incubated with a curcumin solution (50 µg/mL) that was added to the apical compartments through the mixing chambers in the bioreactors (10 mL) and directly in the apical compartment in the Transwells (0.5 mL). Basolateral compartments were filled fresh medium: 10 mL for the bioreactors and 1 mL for the Transwells. The flow rate was set to 150 µL/min in both circuits. All samples were incubated at 37 °C overnight.
After the treatment, the curcumin solution was removed, and the samples were washed with PBS. The activity of P-gp was then evaluated using a fluorescent compound Rhodamine-123 (Rh-123) (Sigma-Aldrich, Italy), which is a tracer dye used as a substrate in the functional studies of MDR phenotype cells and in particular of the P-glycoprotein (22) (23). HBSS (ThermoFisher Scientific) was added in the apical compartments and the Rh-123 solution (10 µM in HBSS) in the basolateral ones. The same procedure was carried out for the Transwell samples. At the time-points of 0 and 120-minutes, 100 µL samples were collected from the apical sites and their volumes were replaced with pre-warmed HBSS. After two hours the samples were also collected from the basolateral ones. The Rh-123 concentration was determined using a plate reader (PerkinElmer, USA) setting filter wavelengths to 485 nm (excitation) and 535 nm (emission).
Positive and negative control experiments were performed, repeating the protocol. In the positive one APD (100 µM) was added to the apical sites of models instead of curcumin. The aim of this test was to verify the inhibition of P-gp activity by the third-generation drug, together with the lower passage of Rh-123 on the apical side of the cells.
Finally, a negative control to measure the basal activity of P-gp in physiological conditions, without any cell preconditioning was performed. All the experiments were conducted in parallel in the two in vitro models (dynamic and static) for each of the three conditions, with n = 6 experiments per condition. The three different experiments are illustrated in Fig. 3.
Data analysis
The analysis of the effect of curcumin or APD on P-gp activity is based on Rh-123 efflux from the basolateral side to the apical side of planar cell cultures. Mathematically, P-gp activity is expressed as a function of Rh-123 mass (µg) on both apical and basal sides at 120 min (t1apical and t1basal, respectively), as well as Rh-123 initially administered to the experimental set-up (t0basal). All these parameters were normalized with respect to the blanks (i.e., measurements without cells) and combined into the equation as follows:
To appreciate how much the treatment with the substances (curcumin vs APD) inhibited the activity of the protein, the following equation was used:
Statistics based on two-way ANOVA analysis was performed using the Tukey’s test for multiple comparisons and setting statistical significance at p < 0.05, with GraphPad Prism (GraphPad Software, San Diego, CA, USA). Error bars in results section represent standard deviations (n = 6).