Everolimus was purchased from Selleck Chemicals. 7-Ethyl-10-hydroxycamptothecin (SN-38) was purchased from LKT Laboratories, Inc. Cisplatin and paclitaxel were purchased from FUJIFILM Wako Pure Chemical Corporation. Rhodamine 123 was purchased from Molecular Probes (Thermo Fisher Scientific, Inc.). WST-1 and 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS) were obtained from Dojindo Laboratories, Inc.

The human colon carcinoma cell line Caco-2 (research resource identifiers:CVCL_0025) was obtained from the American Type Culture Collection. Caco-2 cells were cultured in DMEM (Sigma-Aldrich; Merck KGaA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G, 100 µg/l streptomycin sulfate and 0.1 mM non-essential amino acids (Gibco; Thermo Fisher Scientific, Inc.). Cells were seeded at a density of 2x10⁴ cells/cm² in culture dishes, incubated in a humidified atmosphere of 5% CO₂ at 37˚C and passaged using 0.05% trypsin-0.02% EDTA (Gibco; Thermo Fisher Scientific, Inc.).

The cells were treated with 1 µM (958.22 µg/ml) everolimus. To establish a subline capable of tolerating 1 µM everolimus, cells were cultured at 37˚C in complete DMEM with 1 µM everolimus for ~3 months. Cells displaying stable growth were isolated and cloned using the limiting dilution method, where cells were serially diluted in 24-well plates to achieve an estimated concentration of <1 cell per well. In preliminary experiments, the proliferation activity and drug sensitivity of several clones were assessed and the chosen clone was named Caco/EV. Caco/EV cells were maintained as Caco-2 cells without the addition of 1 µM everolimus to the DMEM.

The proliferation of Caco-2 and Caco/EV cells was assessed through proliferation curves. On day 0, cells (1,000 cells/well) were seeded in 96-well plates using complete DMEM without everolimus and the cell count was monitored from day 0 to 9. Cell count was determined using the WST-1 assay based on the MTT assay (19-22). The culture medium was substituted with DMEM containing WST-1 reagent solution (10 µl WST-1 including 0.2 mM 1-methoxy PMS + 100 µl culture medium) and after 2 h, absorbance was measured at 450 nm using a microplate reader (Spectra Fluor; Tecan Group Ltd.) with a reference wavelength of 630 nm. The relationship between absorbance and cell count was confirmed through preliminary experiments. Specifically, both cell types were seeded in 96-well plates at a starting density of 2x10⁴ cells/100 µl/well, followed by a 10-step, 2-fold serial dilution. Thirty minutes after seeding, the WST-1 assay was performed as described above, resulting in a calibration curve correlating absorbance with cell count. The doubling time of each cell during the logarithmic phase was calculated as follows: Doubling time=(t₂-t₁) x log 2/(logN₂-logN₁) where t₁ and t₂ represent the time of cell counting and N₁ and N₂ represent the cell count at t₁ and t₂, respectively (19).

The growth inhibitory effects of everolimus, paclitaxel, SN-38 and cisplatin were evaluated in Caco-2 and Caco/EV cells using WST-1 assay (19-22). Both cell lines were seeded at a density of 1,000 (everolimus) or 5,000 (other drugs) cells/well in 96-well plates with DMEM without any drugs on day 0. On day 1, the medium was replaced with fresh DMEM containing the test drugs. The maximum concentration used was 10 for everolimus, 4 µM for paclitaxel, 1 µM for SN-38, and 1024 µM for cisplatin. Following 72 h (paclitaxel, SN-38 and cisplatin) or 144 h (everolimus) incubation at 37˚C, cell viability was assessed using the WST-1 assay as aforementioned. The half-maximal inhibitory concentration (IC₅₀) of the test drugs in both cell lines was calculated using the sigmoid inhibitory effect model (19-22): E=E_max x [1-C^γ/(C^γ + IC₅₀^γ)], where E and E_max represent the surviving fraction (% of control) and its maximum, respectively, C is concentration in the medium and γ represents sigmoidicity factor. This calculation was performed using the non-linear least-squares fitting method (Solver, Microsoft^® Excel 2019). The relative resistance (RR) was obtained by dividing IC₅₀ value for Caco/EV cells by that for Caco-2 cells.

Cells (2x10⁶ cells/60 mm culture dish) were pre-cultured at 37˚C for 48 h, after which total RNA was extracted using GenElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich; Merck KGaA). mRNA expression levels were quantified using RT-qPCR, employing three independent samples (22-24). RT of 500 ng total RNA was performed using the PrimeScript RT reagent kit (Takara Bio, Inc.) and a thermal cycler (i-Cycler, Bio-Rad Laboratories, Inc.). RT was conducted in 20 µl reaction buffer at 37˚C for 15 min and terminated by heating at 85˚C for 5 sec followed by cooling at 4˚C qPCR was conducted using the 7500 Fast Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, Inc.) and SYBR^® Premix Ex Taq (Takara Bio, Inc.). PCR reaction was performed at 95˚C for 30 sec, followed by 40 cycles of 95˚C for 3 sec and 60˚C for 30 sec; dissociation curve analysis was initiated at 95˚C for 15 sec, followed by 60˚C for 1 min and 95˚C for 15 sec. β-actin served as the internal standard. Primer sequences and were synthesized by GeneDesign, Inc. (Table I). The comparative Cq method was employed to compare relative expression levels of target mRNAs (22-25).

Caco-2 and Caco/EV cells (5x10⁴ cells/well) were seeded in 24-well plates (7,8). The plates were incubated for 10 days in a humidified atmosphere of 5% CO₂ air at 37˚C with the complete DMEM being replaced with fresh medium every 2 days. Cells were washed three times with warmed Hanks' balanced salt solution (HBSS) containing phenol red and HEPES. Fresh HBSS containing 3 µM Rhodamine 123 was added, followed by further incubation for 5, 15, 30, 60, 90 and 120 min at 37˚C. The accumulation process was terminated by removing HBSS from the wells, followed by three washes with ice-cold phosphate-buffered saline. Cells were solubilized with 0.3 M NaOH and aliquots were neutralized using 0.3 M HCl. A total of 100 µl neutralized cell-solubilized solution were transferred to 96-well black plates and the fluorescence intensity of Rhodamine 123 was measured at excitation and emission wavelengths of 485 and 535 nm, respectively, using Spectra Fluor. Protein content was determined using the Lowry method (26), with bovine serum albumin (FUJIFILM Wako Pure Chemical Corporation) serving as the standard.

Data are presented as the mean ± SD of ≥3 independent experimental repeats. Comparisons between groups were conducted using the unpaired Student's t test. Statistical analysis was performed using JMP^® Pro 15.2.0 (SAS Institute Japan Ltd., Tokyo). A two-tailed P<0.05 was considered to indicate a statistically significant difference.

Despite the clinically achievable plasma concentration of everolimus being ~60 ng/ml (equivalent to 0.06 µM) at the clinical dose of 10 mg (27), Caoc-2 cells were culture in DMEM supplemented with 1 µM everolimus, because exposure to lower concentrations did not affect cell proliferation. After 3 months, cells displaying stable growth were cloned and named Caco/EV. Preliminary experiments confirmed a strong correlation between absorbance in the WST assay and cell count (data not shown).

Growth curves of Caco-2 and Caco/EV cells displayed a logarithmic phase starting within 2 days of cell seeding; this phase persisted for ≥7 days (Fig. 1). On day 4, the proliferation rates of both cell types were comparable, with a doubling time of 28.4 h and 32.8 h for Caco-2 and Caco/EV cells, respectively.

IC₅₀ for everolimus in Caco/EV cells was significantly lower than that in Caco-2 cells, showing 14-fold resistance compared with that in Caco-2 cells (Table II). IC₅₀ for paclitaxel, a substrate of ABCB1, were significantly lower in Caco/EV than in Caco-2 cells, indicating enhanced sensitivity to paclitaxel. IC₅₀ value of SN-38, a substrate of ABCG2, was significantly lower in Caco/EV cells than in Caco-2 cells. The IC₅₀ of cisplatin in Caco-2 cells was comparable to that in Caco/EV cells.

mRNA expression of ABCB1 in Caco/EV cells was significantly lower than that in Caco-2 cells, with an estimated 72% decrease (Fig. 2). mRNA expression of ABCG2 was also significantly reduced in Caco/EV cells.

Rhodamine 123 is a substrate of ABCB1 (7,8,20). The cellular accumulation of Rhodamine 123 in both cell lines was time-dependent and reached steady state after 90 min (Fig. 3). The accumulation of Rhodamine 123 in Caco/EV cells was significantly higher than that in Caco-2 cells from 15 min.