Junko Mitsuhashi

Inhibition of the mitogen-activated protein kinase pathway results in the down-regulation of P-glycoprotein

The multidrug resistance gene 1 (MDR1) product, P-glycoprotein (P-gp), pumps out a variety of anticancer agents from the cell, including anthracyclines, Vinca alkaloids, and taxanes. The expression of P-gp therefore confers resistance to these anticancer agents. In our present study, we found that FTI-277 (a farnesyltransferase inhibitor), U0126 [an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)], and 17-allylamino-17-demethoxygeldanamycin (an inhibitor of heat shock protein 90) reduced the endogenous expression levels of P-gp in the human colorectal cancer cells, HCT-15 and SW620-14. In contrast, inhibitors of phosphatidylinositol 3-OH kinase, mammalian target of rapamycin, p38 mitogen-activated protein kinase, and c-Jun NH2-terminal kinase did not affect P-gp expression in these cells. We further found that U0126 down-regulated exogenous P-gp expression in the MDR1-transduced human breast cancer cells, MCF-7/MDR and MDA-MB-231/MDR. However, the MDR1 mRNA levels in these cells were unaffected by this treatment. PD98059 (a MEK inhibitor), ERK small interfering RNA, and p90 ribosomal S6 kinase (RSK) small interfering RNA also suppressed P-gp expression. Conversely, epidermal growth factor and basic fibroblast growth factor enhanced P-gp expression, but the MDR1 mRNA levels were unchanged in epidermal growth factor–stimulated cells. Pulse-chase analysis revealed that U0126 promoted P-gp degradation but did not affect the biosynthesis of this gene product. The pretreatment of cells with U0126 enhanced the paclitaxel-induced cleavage of poly(ADP-ribose) polymerase and paclitaxel sensitivity. Furthermore, U0126-treated cells showed high levels of rhodamine123 uptake. Hence, our present data show that inhibition of the MEK-ERK-RSK pathway down-regulates P-gp expression levels and diminishes the cellular multidrug resistance. [Mol Cancer Ther 2007;6(7):2092–2102]

Functions of the breast cancer resistance protein (BCRP/ABCG2) in chemotherapy.

The breast cancer resistance protein, BCRP/ABCG2, is a half-molecule ATP-binding cassette transporter that facilitates the efflux of various anticancer agents from the cell, including 7-ethyl-10-hydroxycamptothecin, topotecan and mitoxantrone. The expression of BCRP can thus confer a multidrug resistance phenotype in cancer cells, and its transporter activity is involved in the in vivo efficacy of chemotherapeutic agents. Thus, the elucidation of the substrate preferences and structural relationships of BCRP is essential to understanding its in vivo functions during chemotherapeutic treatments. Single nucleotide polymorphisms (SNPs) have also been found to be key factors in determining the efficacy of chemotherapeutics, and those therapeutics that inhibit BCRP activity, such as the SNP that results in a C421A mutant, may result in unexpected side effects of the BCRP- anticancer drugs interaction even at normal dosages. In order to modulate the BCRP activity during chemotherapy, various compounds have been tested as inhibitors of this protein. Estrogenic compounds including estrone, several tamoxifen derivatives in addition to phytoestrogens and flavonoids have been shown to reverse BCRP-mediated drug resistance. Intriguingly, recently developed molecular targeted cancer drugs, such as the tyrosine kinase inhibitors imatinib mesylate, gefitinib and others, can also interact with BCRP. Since both functional SNPs and inhibitory agents of BCRP modulate the in vivo pharmacokinetics and pharmacodynamics of its substrate drugs, BCRP activity is an important consideration in the development of molecular targeted chemotherapeutics.

Breast cancer resistance protein: molecular target for anticancer drug resistance and pharmacokinetics/pharmacodynamics.

Breast cancer resistance protein (BCRP) is a half‐molecule ATP‐binding cassette transporter that forms a functional homodimer and pumps out various anticancer agents, such as 7‐ethyl‐10‐hydroxycamptothecin, topotecan, mitoxantrone and flavopiridol, from cells. Estrogens, such as estrone and 17β‐estradiol, have been found to restore drug sensitivity levels in BCRP‐transduced cells by increasing the cellular accumulation of such agents. Furthermore, synthetic estrogens, tamoxifen derivatives and phytoestrogens/flavonoids have now been identified that can effectively circumvent BCRP‐mediated drug resistance. Transcellular transport experiments have shown that BCRP transports sulfated estrogens and various sulfated steroidal compounds, but not free estrogens. The kinase inhibitor gefitinib inhibited the transporter function of BCRP and reversed BCRP‐mediated drug resistance both in vitro and in vivo. BCRP‐transduced human epidermoid carcinoma A431 (A431/BCRP) and BCRP‐transduced human non‐small cell lung cancer PC‐9 (PC‐9/BCRP) cells showed gefitinib resistance. Physiological concentrations of estrogens (10–100 pM) reduced BCRP protein expression without affecting its mRNA levels. Two functional polymorphisms of the BCRP gene have been identified. The C376T (Q126Stop) polymorphism has a dramatic phenotype as active BCRP protein cannot be expressed from a C376T allele. The C421A (Q141K) polymorphism is also significant as Q141K‐BCRP‐transfected cells show markedly low protein expression levels and low‐level drug resistance. Hence, individuals with C376T or C421A polymorphisms may express low levels of BCRP or none at all, resulting in hypersensitivity of normal cells to BCRP‐substrate anticancer agents. In summary, both modulators of BCRP and functional single nucleotide polymorphisms within the BCRP gene affect the transporter function of the protein and thus can modulate drug sensitivity and substrate pharmacokinetics and pharmacodynamics in affected cells and individuals. (Cancer Sci 2005; 96: 457–465)

Estrogen-mediated post transcriptional down-regulation of P-glycoprotein in MDR1-transduced human breast cancer cells

The human multidrug resistance gene 1 (MDR1) encodes the plasma membrane P‐glycoprotein (P‐gp/ABCB1) that functions as an efflux pump for various anticancer agents. We recently reported that estrogens down‐regulate the expression of breast cancer resistance protein (BCRP/ABCG2). In our present study we demonstrate that estrogens also down‐regulate P‐gp expression in the MDR1‐transduced, estrogen receptor α (ER‐α)‐positive human breast cancer cells, MCF‐7/MDR and T‐47D/MDR. The P‐gp expression levels in MCF‐7/MDR cells treated with 100 pM estradiol were found to be 10–20‐fold lower than the levels in these same cells that were cultured without estradiol. In contrast, estradiol did not affect the P‐gp expression levels in the ER‐α‐negative cancer cells, MDA‐MB‐231/MDR and NCI/ADR‐RES. Estrone and diethylstilbestrol were also found to down‐regulate P‐gp in MCF‐7/MDR cells, but progesterone treatment did not produce this effect. Tamoxifen reversed the estradiol‐mediated down‐regulation of P‐gp in MCF‐7/MDR cells, suggesting that ER‐α activity is necessary for the effects of estradiol upon P‐gp. However, estradiol was found not to alter the MDR1 transcript levels in either MCF‐7/MDR and T‐47D/MDR cells, suggesting that post‐transcriptional mechanisms underlie its effects upon P‐gp down‐regulation. MCF‐7/MDR cells also showed eight‐fold higher sensitivity to vincristine when treated with 100 pM estradiol, than when treated with 1 pM estradiol. These results may serve to provide a better understanding of the expression control of ABC transporters, and possibly allow for the establishment of new cancer chemotherapy strategies that would control P‐gp expression in breast cancer cells and thereby increase their sensitivity to MDR1‐related anticancer agents. (Cancer Sci 2006; 97: 1198–1204)

Reduced Activity of Anabolizing Enzymes in 5-Fluorouracil-resistant Human Stomach Cancer Cells

The mechanism of resistance to 5‐fluorouracil (5‐FU) was studied with NUGC‐3/5FU/L, a human stomach cancer cell line which had acquired resistance as a consequence of repeated 5‐day exposures to stepwise‐increasing concentrations of 5‐FU in vitro. NUGC‐3/5FU/L was 200‐fold and over 16‐fold resistant to 96‐h and 1‐h exposures to 5‐FU, respectively. NUGC‐3/5FU/L incorporated less 5‐FU into RNA, indicating resistance to the RNA‐directed action of 5‐FU. On the other hand, NUGC‐3/5FU/L also showed resistance to in situ thymidylate synthase (TS) inhibition by 5‐FU. Polymerase chain reaction‐single‐strand conformation polymorphism analysis of TS cDNA and a FdUMP ligand binding assay showed that quantitative and qualitative alterations of TS are not responsible for this resistance. In contrast, the ability to metabolize 5‐FU to its active metabolites, FUTP and FdUMP, was reduced in NUGC‐3/5FU/L. We found that not only the activities of uridine phosphorylase/kinase and orotate phosphoribosyl‐transferase (OPRT), but also the level of phosphoribosyl pyrophosphate, a cosubstrate for OPRT, were significantly lower in NUGC‐3/5FU/L than in the parent NUGC‐3. These results indicated that resistance to 5‐FU in NUGC‐3/5FU/L is due to reduced activities of 5‐FU‐anabolizing enzymes, but not to an alteration of TS. 2′‐Deoxyinosine effectively enhanced TS inhibition by 5‐FU in the resistant cells, thus markedly sensitizing them to 5‐FU.

Substrate-dependent bidirectional modulation of P-glycoprotein-mediated drug resistance by erlotinib

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) inhibit the function of certain adenosine triphosphate (ATP)‐binding cassette transporters, including P‐glycoprotein/ABCB1 and breast cancer resistance protein (BCRP)/ABCG2. We previously reported an antagonistic activity of gefitinib towards BCRP. We have now analyzed the effects of erlotinib, another EGFR‐TKI, on P‐glycoprotein and BCRP. As with gefitinib, erlotinib effectively reversed BCRP‐mediated resistance to SN‐38 (7‐ethyl‐10‐hydroxycamptothecin) and mitoxantrone. In contrast, we found that erlotinib effectively suppressed P‐glycoprotein‐mediated resistance to vincristine and paclitaxel, but did not suppress resistance to mitoxantrone and doxorubicin. Conversely, erlotinib appeared to enhance P‐glycoprotein‐mediated resistance to mitoxantrone in K562/MDR cells. This bidirectional activity of erlotinib was not observed with verapamil, a typical P‐glycoprotein inhibitor. Flow cytometric analysis showed that erlotinib co‐treatment restored intracellular accumulation of mitoxantrone in K562 cells expressing BCRP, but not in cells expressing P‐glycoprotein. Consistently, erlotinib did not inhibit mitoxantrone efflux in K562/MDR cells although it did vincristine efflux in K562/MDR cells and mitoxantrone efflux in K562/BCRP cells. Intravesicular transport assay showed that erlotinib inhibited both P‐glycoprotein‐mediated vincristine transport and BCRP‐mediated estrone 3‐sulfate transport. Intriguingly, Lineweaver‐Burk plot suggested that the inhibitory mode of erlotinib was a mixed type for P‐glycoprotein‐mediated vincristine transport whereas it was a competitive type for BCRP‐mediated estrone 3‐sulfate transport. Collectively, these observations indicate that the pharmacological activity of erlotinib on P‐glycoprotein‐mediated drug resistance is dependent upon the transporter substrate. These findings will be useful in understanding the pharmacological interactions of erlotinib used in combinational chemotherapy. (Cancer Sci 2009; 100: 1701–1707)

Kinetic Analysis of 5‐Fluorouracil Action against Various Cancer Cells

Based on our recent kinetic analysis, which made it possible to distinguish between the cell‐killing actions of cell cycle phase‐specific and non‐specific agents, we attempted to elucidate the actions of 5‐fluorouracil (FUra) on three different cancer cell lines. By colony‐forming assay, the concentrations of fluorouridine (FUrd), fluorodeoxyuridine (FdUrd) or FUra giving 90% cell kill (IC90) at various exposure times (texps) were obtained. With P388 cells, the curve of texps‐IC90 for FUrd on a log‐log scale was linear with a slope of — 1, which is typical for cell cycle phase‐nonspecific agents. In contrast, the curve for FdUrd showed a much steeper slope than —1, which is characteristic for cell cycle phase‐specific agents. We found that the curve for FUra was exactly the same as that for FUrd, indicating that the mode of FUra action on P388 leukemia is analogous to that of FUrd. Similar results were observed with human colon and renal cancer cell lines, HT‐29 and KU‐2, although when the cells were exposed to relatively low concentrations of FUra for a long time, a cell cycle phase‐specific action became evident.

The identification of two germ-line mutations in the human breast cancer resistance protein gene that result in the expression of a low/non-functional protein

PurposeWe examined the effects of the nine nonsynonymous germ-line mutations/SNPs in the breast cancer resistance protein (BCRP/ABCG2) gene on the expression and function of the protein.Materials and MethodsWe generated cDNAs for each of these mutants (G151T, C458T, C496G, A616C, T623C, T742C, T1291C, A1768T, and G1858A BCRP) and compared the effects of their exogenous expression in PA317 cells with a wild-type control.ResultsPA/F208S cells (T623C BCRP-transfectants) expressed marginal levels of a BCRP protein species (65kDa), which is slightly smaller than wild-type (70kDa), but this mutant did not appear on the cell surface or confer drug resistance. PA/F431L cells (T1291C BCRP-transfectants) were found to express both 70kDa and 65kDa BCRP protein products. In addition, although PA/F431L cells expressed 70kDa BCRP at comparable levels to PA/WT cells, they showed only marginal resistance to SN-38. PA/T153M cells (C458T BCRP-transfectants) and PA/D620N cells (G1858A BCRP-transfectants) expressed lower amounts of BCRP and showed lower levels of resistance to SN-38 compared with PA/WT cells.ConclusionsWe have shown that T623C BCRP encodes a non-functional BCRP and that T1291C BCRP encodes a low-functional BCRP. Hence, these mutations may affect the pharmacokinetics of BCRP substrates in patients harboring these alleles.

Pharmacological interaction with sunitinib is abolished by a germ-line mutation (1291T>C) of BCRP/ABCG2 gene

Sunitinib malate (Sutent, SU11248) is a small‐molecule multitargeted tyrosine kinase inhibitor (TKI) used for the treatment of renal cell carcinoma and imatinib‐resistant gastrointestinal stromal tumors. Some TKIs can overcome multidrug resistance conferred by ATP‐binding cassette transporter, P‐glycoprotein (P‐gp)/ABCB1, multidrug resistance‐associated protein 1 (MRP1)/ABCC1, and breast cancer resistance protein (BCRP)/ABCG2. Here, we analyzed the effects of sunitinib on P‐gp and on wild‐type and germ‐line mutant BCRPs. Sunitinib remarkably reversed BCRP‐mediated and partially reversed P‐gp‐mediated drug resistance in the respective transfectants. The in vitro vesicle transport assay indicated that sunitinib competitively inhibited BCRP‐mediated estrone 3‐sulfate transport and P‐gp‐mediated vincristine transport. These inhibitory effects of sunitinib were further analyzed in Q141K‐, R482G‐, R482S‐, and F431L‐variant BCRPs. Intriguingly, the F431L‐variant BCRP, which is expressed by a germ‐line mutant allele 1291T>C, was almost insensitive to both sunitinib‐ and fumitremorgin C (FTC)‐mediated inhibition in a cell proliferation assay. Sunitinib and FTC did not inhibit 125I‐iodoarylazidoprazosin‐binding to F431L‐BCRP. Thus, residue Phe‐431 of BCRP is important for the pharmacological interaction with sunitinib and FTC. Collectively, this is the first report showing a differential effect of a germ‐line variation of the BCRP/ABCG2 gene on the pharmacological interaction between small‐molecule TKIs and BCRP. These findings would be useful for improving our understanding of the pharmaceutical effects of sunitinib in personalized chemotherapy. (Cancer Sci 2010; 00: 000–000)

BCRP/ABCG2 confers anticancer drug resistance without covalent dimerization

In previous studies, we demonstrated that the breast cancer resistance protein (BCRP, ABCG2) forms an S–S homodimer. The BCRP‐C603S mutant substituting Ser for Cys‐603 in the third extracellular domain formed both a 70–75‐kDa monomer and 140–150‐kDa dimer, suggesting that Cys‐603 is an important residue in the covalent bridge. These results also suggested the involvement of other Cys residues in dimer formation. In the present study, we examined the possible involvement of the other extracellular Cys residues, Cys‐592 and Cys‐608, in the dimerization and transporter functions of BCRP using double and triple Cys‐mutant BCRP transfectants. In SDS–PAGE under non‐reducing conditions, BCRP‐C592S·C603S and BCRP‐C592S·C608S were detected as dimers whereas BCRP‐C603S·C608S and BCRP‐C592S·C603S·C608S were found only as monomers. This finding indicated that no Cys residues other than the three extracellular Cys are responsible for the dimer formation. The formation of BCRP‐C592S·C603S dimer suggested the involvement of Cys‐608 in the covalent linkage of this mutant BCRP. PA/C592S·C603S·C608S‐cl.7 cells showed a significant level of multiple drug resistance and low‐level accumulation of mitoxantrone. These results clearly demonstrate that BCRP functions as a drug resistance protein without covalent dimerization. Among drug‐resistant Cys‐mutant BCRP transfectants, PA/C603S, PA/C592S·C608S, and PA/C592S·C603S·C608S were found to be more resistant to the reversal effects of fumitremorgin C than PA/WT, suggesting some alteration in the substrate recognition in Cys‐mutant BCRPs. In conclusion, Cys‐mediated covalent dimerization is not required for BCRP to function as a transporter. In addition to Cys‐603, Cys‐608 may also be involved in BCRP dimer formation. (Cancer Sci 2010)