br Materials and Methods br Results
Materials and Methods
Results All of the studied transporter genes were expressed at detectable levels in all the analyzed samples at mRNA level by means of applied rt-PCR method (CT<35). The lowest Caspase-1, human recombinant proteinase was observed for ABCC4, ABCC1, ABCG2, SLC22A3, and SLC22A18 (less than 10% of mean expression for the house-keeping reference genes), while SLC22A1 mRNA levels were the highest in both study groups. The analysis of mRNA content revealed significant differences in ABCB11, ABCC1, ABCG2, SLC10A1, SLC16A1, SLCO1B1 and SLCO2B1 gene expression between livers from organ donors and patients undergoing surgical resection of metastatic tumors. When median mRNA quantity in liver donors was set as reference (100%), median expression of those genes in metastatic livers was significantly higher (179% for ABCB11, 167% for ABCC1, 442% for ABCG2, 437% for SLC10A1, 278% for SLC16A1, 224% for SLCO1B1, and 172% for SLCO2B1). No significant differences were observed in the case of the other studied transporter genes. The results of mRNA quantification are presented in Fig. 1, Fig. 2, Fig. 3, Fig. 4 and Supplementary Table 2. The protein abundance analysis revealed that MRP4 was below the detection limit in all liver samples, while MRP1 and BCRP were quantified only in single individuals. OCT3 levels were also below detection limit in a significant number of samples from both study groups, while P-gp and MRP3 were absent in some livers (>2), but only in metastatic livers. The protein abundance (mean and median) of most of the transporters was higher in livers from cancer patients than in organ donors, but the difference was significant only in the case of NTCP (SLC10A1) – mean value in metastases vs. donors group: 114.5 ± 177.8 vs. 31.8 ± 9.5 fmol/mg, median value: 88.8 vs. 32.5 fmol/mg, p = 0.001, respectively. That observation was in concordance with mRNA analysis results. In contrast, protein abundance of P-gp (ABCB1) was significantly higher in the donors group (mean value in metastatic vs. donors group: 4.3 ± 8.3 vs. 24.2 ± 9.1 fmol/mg, median value: 0.9 vs. 24.2 fmol/mg, p = 6*10-4), despite similar mRNA expression in both groups. Organ donors were also characterized by numerically higher OATP1B3 (SLCO1B3) protein content, but the difference was not significant (mean value in metastatic vs. donors livers: 60.0 ± 53.1 vs. 116.3 ± 60.8 fmol/mg, median value: 56.0 vs. 114.1 fmol/mg, p = 0.095). The results of protein analysis are presented in Fig. 1, Fig. 2, Fig. 3, Fig. 4 and Supplementary Table 3. Interindividual variability in protein abundance was greater in metastatic livers for all analyzed transporters, as documented by a higher covariance coefficient (Supplementary Table 3). Additionally, there were significant (0.5 < r < 0.8) correlations between mRNA expression and protein abundance for ABCB11/BSEP, SLC22A7/OAT2 and SLCO1B13/OATP1B3 (negative) in donor livers, and SLC22A1/OCT1, SLC22A3/OCT3, SLC22A7/ OAT2 and SLCO1B3/OATP1B3 observed in metastatic livers (Table 2). Protein levels of the plasma membrane marker Na+/K + ATPase levels were similar in both groups (results not shown). Cytokine gene expression was evaluated in liver samples from both groups. Following mean relative mRNA levels of evaluated genes were observed in metastatic liver patients, while compared to mean value in organ donors: 145% for IL1B, 70% for IL6, 262% for IL10, and 242% for TNF. The differences between groups were only significant in case of TNF (p = 0.021, Supplementary Fig. 1).
Discussion In the current study, we compared for the first time drug transporter expression and protein abundance in two sets of liver samples, i.e. non-tumoral liver tissue from patients undergoing resection of colon cancer metastases and liver samples from organ donors (all were also liver donors), which are widely used as the reference, presumably healthy, livers [, , , ]. Moreover, they are also utilized to estimate transporter expression/abundance in general population [, , ]. The present study results show several significant differences between the aforementioned types of liver tissues, which may potentially impact conclusions, depending on the reference tissue used.