Establishment and characterization of resistant cell lines
SN38-resistant T47D/SN120 and T47D/SN150 cell sublines were established from the wild-type T47D cells following long-term exposure (of more than 16 months) to 120 nM and 150 nM SN38, respectively. Microscopic observation showed some distinct features in the SN38-resistant T47D sublines compared to their parenteral cell line. In monolayer, T47D/WT cells were relatively consistent in size and shape, while the resistant cells presented a spindle-shaped morphology and were smaller in size (data not shown). Moreover, the doubling times of T47D/SN120 and T47D/SN150 cells were 35.5 h and 32.8 h, respectively, different from that of T47D/WT cells (69.2 h).
Cross-resistance to other anti-cancer drugs of resistant cell lines
MTT assay showed that T47D/SN120 and T47D/SN150 cells were more resistant to SN38 (14.5 and 59.1 times, respectively), irinotecan (1.5 and 3.7 times, respectively), and topotecan (4.9 and 12 times, respectively) as compared to the wild-type drug-sensitive parental cells. In addition to topoisomerase I inhibitors, both T47D/SN120 and T47D/SN150 sublines were cross-resistant to various anti-cancer drugs that are used in breast cancer treatment, including microtubule inhibitors (paclitaxel and vinblastine), anti-metabolites (5-fluorouracil and methotrexate), topoisomerase II inhibitors (doxorubicin and mitoxantrone), estrogen receptor blockers (tamoxifen and endoxifen), an alkylating agent (cisplatin), and a tyrosine kinase inhibitor (gefitinib) (Table 2).
As shown in Table 2, both T47D/SN120 and T47D/SN150 sublines were highly resistant to microtubule inhibitors, including paclitaxel and vinblastine. On the other hand, compared to the T47D/SN120 subline, the T47D/SN150 subline presented weak resistance to gefitinib, 5-FU, and irinotecan, and was sensitive to methotrexate and endoxifen.
Expression profiles of 24 transporters in the T47D/SN sublines
Expression profiles of resistance-related transporters were examined in T47D/WT, T47D/SN120, and T47D/SN150 cells using a resistance diagnostic kit (Drugsporter®). Drugsporter® assays for 24 transporter genes and one house keeping gene GAPDH (49). Among the 24 transporter genes, only MRP2 mRNA showed a concentration-independent increase, from about two-fold to ten-fold in T47D/SN120 and T47D/SN150 cells treated with 3.5-90 nM SN38. After increasing the concentration to 120 nM and 150 nM, a high level of BCRP, MRP1, MRP2, MRP3, and MRP4 mRNA expression was detected in T47D/SN120 and T47D/SN150 cells. There was no difference in the mRNA expression of other genes between the T47D/WT cells and the SN38-resistant T47D sublines. Real-time RT-PCR confirmed that T47D/SN120 and T47D/SN150 cells overexpressed MRP1 (7-fold and 11-fold, respectively), MRP2 (795-fold and 1,061-fold, respectively), MRP3 (57-fold and 96-fold, respectively), MRP4 (204-fold both), and BCRP (536-fold and 3,083-fold, respectively), compared to T47D/WT cells (Fig. 1).
Expression levels of MRP1, MRP2, MRP3, MRP4, and BCRP were determined by western blot analysis. Fifty micrograms of protein was used to detect MRP1, MRP2, MRP3, and MRP4. MRP1, MRP2, MRP3, and MRP4 expression levels in T47D/SN150 cells were compared with those in T47D/SN120 cells. Compared to T47D/SN120 cells, T47D/SN150 cells overexpressed MRP1 (1.2-fold), MRP2 (1.1-fold), MRP3 (1.1-fold), and MRP4 (2.2-fold) proteins (Fig. 2).
Compared to T47D/WT cells, which expressed a trace amount of BCRP, T47D/SN120 and T47D/SN150 cells displayed 85.3-fold and 327.5-fold higher BCRP protein expression, respectively. T47D/SN150 cells displayed 4-fold higher expression of BCRP, as compared to T47D/SN120 cells (Fig. 2).
Sensitivity of SN38-resistant T47D sublines to SN38 in the presence of various chemosensitizers
The effects of several chemosensitizers, including Pgp [verapamil, PSC833, and 3,4,5-trimethoxyflavone (TMF)], MRP (probenecid), and BCRP (genistein) inhibitors, were assessed on SN38-resistant T47D sublines. Among the Pgp inhibitors, only TMF showed chemosensitizing effects on SN38 in a concentration-dependent manner. Probenecid and genistein also sensitized SN38-resistant T47D sublines to SN38 in a concentration-dependent manner (Fig. 3). The chemosensitizing effects of TMF and genistein to SN38 were similar between T47D/SN120 and T47D/SN150 cells. As shown in Table 3, the chemosensitizing index was calculated by dividing the IC50 value in the absence of the chemosensitizer with that in its presence. Chemosensitizing indices of probenecid (5 and 50 µM) and genistein (1 and 10 µM) were 1.3 - 2.0 and 1.2 - 10.8, respectively, in both the cell lines, whereas those for verapamil (1 and 10 µM) and PSC833 (5 and 50 nM) were less than 1.0, with the exception of PSC833 in the T47D/SN120 cell line, which displayed a chemosensitizing index of 1.3.
Involvement of epigenetic gene silencing of MRP1, MRP2, MRP3, MRP4, and BCRP in T47D/WT cells
Next, we investigated whether MRP1, MRP2, MRP3, MRP4, and BCRP could be epigenetically induced in T47D/WT cells following treatment with 2.5 μM 5-aza-2'-deoxycytidine for 96 h or 100 ng/mL trichostatin A (TSA) for 48 h. RT-PCR indicated that 5-aza-2'-deoxycytidine induced the mRNA expression of MRP2, MRP3, MRP4, and BCRP, while TSA induced the mRNA expression of MRP1, MRP2, MRP4, and BCRP in T47D/WT cells (Fig. 4).
Decreased drug accumulation in T47D/SN120 and T47D/SN150 cells
Drug accumulation was assayed to estimate the functional activity of transporters in T47D/SN120 and T47D/SN150 cells. Calcein AM and mitoxantrone were used as fluorescent substrates for MRP and BCRP, respectively, and detected using flow cytometry. Accumulation of both the substrates decreased in T47D/SN120 and T47D/SN150 cells, compared to that in T47D/WT cells. However, upon treatment with the inhibitors probenecid and genistein, there was an increase in the accumulation of both the substrates (Figs. 5-6).
Contrarily, intracellular levels of rhodamine 123, a substrate of Pgp, were not affected following treatment with PCS833, a Pgp inhibitor in T47D/SN120 and T47D/SN150 cells.