Yoshikazu Sugimoto

Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization

Breast cancer resistance protein (BCRP) is a half‐molecule ABC transporter highly expressed in mitoxantrone‐resistant cells. In our study we established PA317 transfectants expressing Myc‐tagged BCRP (MycBCRP) or HA‐tagged BCRP (HABCRP). The exogenous BCRP protein migrated as a 70‐kDa protein in SDS‐PAGE under reducing condition, but migrated as a 140‐kDa complex in the absence of reducing agents. The 140‐kDa BCRP complex was heat‐stable but dissociated into 70‐kDa BCRP with the addition of 2‐mercaptoethanol. The 140‐kDa BCRP complex was immunoprecipitated with anti‐Myc antibody from the lysates of PA317 cells double‐transfected with MycBCRP and HABCRP. The 140‐kDa complex reacted with anti‐HA and anti‐BCRP antibodies and after the addition of reducing agents, a 70‐kDa protein reacting with anti‐Myc, anti‐HA and anti‐BCRP antibodies was detected. These results clearly indicate that BCRP forms a homodimer bridged by disulfide bonds. To assess the possible dominant‐negative inhibition of BCRP drug efflux pump, various mutant BCRP cDNAs were isolated by PCR mutagenesis. First, mutant BCRP cDNAs were introduced to parental PA317 cells and tested for their function as drug‐resistance genes. Next, inactive BCRP cDNA clones were introduced to MycBCRP‐transfected cells and tested for the ability to lower drug resistance. Among the 8 inactive mutant cDNA clones tested, HABCRP cDNA clone 15 with an amino acid change from Leu to Pro at residue 554 in the fifth transmembrane domain of BCRP partially reversed the drug resistance of MycBCRP‐transfected cells. These results suggest that homodimer formation is essential for BCRP drug resistance, implicating this dominant‐negative inhibition as a new strategy to circumvent drug resistance.

Phytoestrogens/Flavonoids Reverse Breast Cancer Resistance Protein/ABCG2-Mediated Multidrug Resistance

Breast cancer resistance protein (BCRP), also called ABCG2, confers resistance to anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan. We found previously that sulfated estrogens are physiologic substrates of BCRP. Flavonoids with weak estrogenic activities are called phytoestrogens. In this study, we show that phytoestrogens/flavonoids, such as genistein, naringenin, acacetin, and kaempferol, potentiated the cytotoxicity of SN-38 and mitoxantrone in BCRP-transduced K562 (K562/BCRP) cells. Some glycosylated flavonoids, such as naringenin-7-glucoside, also effectively inhibited BCRP. These flavonoids showed marginal effect on the drug sensitivity of K562 cells. Genistein and naringenin reversed neither P-glycoprotein-mediated vincristine resistance nor multidrug resistance-related protein 1-mediated VP-16 resistance. Genistein and naringenin increased cellular accumulation of topotecan in K562/BCRP cells. K562/BCRP cells also accumulated less [3H]genistein than K562 cells. [3H]genistein transport in the basal-to-apical direction was greater in BCRP-transduced LLC-PK1 (LLC/BCRP) cells, which express exogenous BCRP in the apical membrane, than in parental cells. Fumitremorgin C abolished the increased transport of [3H]genistein in LLC/BCRP cells compared with parental cells. TLC analysis revealed that genistein was transported in its native form but not in its metabolized form. These results suggest that genistein is among the natural substrates of BCRP and competitively inhibits BCRP-mediated drug efflux. The results have two important clinical implications: (a) flavonoids and glycosylated flavonoids may be useful in overcoming BCRP-mediated drug resistance in tumor cells; and (b) coadministration of flavonoids with BCRP-substrate antitumor agents may alter the pharmacokinetics and consequently increase the toxicity of specific antitumor agents in cancer patients.

Estrone and 17β‐Estradiol Reverse Breast Cancer Resistance Protein‐mediated Multidrug Resistance

Breast cancer resistance protein (BCRP), an adenosine triphosphate‐binding cassette transporter, confers resistance to a series of anticancer reagents, including mitoxantrone, SN‐38 and topotecan. In the present study, we found that estrone and l7β‐estradiol potentiated the cytotoxicity of mitoxantrone, SN‐38 and topotecan in BCRP‐transduced K562 cells (K562/BCRP). These estrogens showed only a marginal effect, or none, in parental K562 cells. Estrone and 17β‐estradiol increased the cellular accumulation of topotecan in K562/BCRP cells, but not in K562 cells, suggesting that these estrogens inhibit the BCRP‐mediated drug efflux and overcome drug resistance.

Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells.

STI571, an Abl‐specific tyrosine kinase inhibitor, selectively kills Bcr‐Abl‐containing cells in vitro and in vivo. However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571. We evaluated whether STI571 interacts with P‐glycopro‐tein (P‐gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells. STI571 inhibited the [125l]azidoagosterol A‐photolabeling of P‐gp, but not that of MRP1. K562/MDR cells that overexpress P‐gp were 3.67 times more resistant to STI571 than the parental Philadelphia‐chromosome‐positive (Ph+) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents. The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference. In KB‐G2 cells that overexpress P‐gp, but not Bcr‐Abl, 2.5 μM STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP‐16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine. STI571 increased the accumulation of VCR, but not that of ADM in KB‐G2 cells. STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1. These findings suggest that STI571 is a substrate for P‐gp, but is less efficiently transported by P‐gp than VCR, and STI571 is not a substrate for MRP1. Among the tested reversing agents that interact with P‐gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/ MDR cells. Furthermore, STI571 itself was a potent reversing agent for MDR in P‐gp‐expressing KB‐G2 cells.

SUMO-1 conjugation to intact DNA topoisomerase I amplifies cleavable complex formation induced by camptothecin

DNA topoisomerase I (Topo1) manages the topological state of DNA. Cleavable complexes, the covalent Topo1–DNA intermediates, become DNA damaged when the catalytic cycles are inhibited by the anti-tumor drug camptothecin (CPT). Intriguingly, Topo1 is modified rapidly and extensively with SUMO-1, a ubiquitin-like protein, in response to CPT. This study shows that the sumoylation enhances the cleavable complex formation and apoptosis induced by CPT. Indeed, substitutions of Lys117 and Lys153, identified as Topo1 sumoylation sites, reduced the CPT-induced cleavable complexes without influencing its in vitro catalytic activity. Consistent with this observation, CPT-induced cleavable complexes of wild-type Topo1 increased in a sumoylation-dependent manner. We also found that Topo1 sumoylation occurred independently of CPT when Topo1 was inactivated by mutation of the catalytic Tyr723. These findings suggested that Topo1 inactivation by CPT treatment can trigger Topo1 sumoylation, leading to enhanced cleavable complex formation.

TAT-59, a new triphenylethylene derivative with antitumor activity against hormone-dependent tumors

The antiestrogenic action of TAT-59 [(E)-4-[1-[4-[2-(dimethylamino)ethoxy]-phenyl]-2-(4-isopropyl) phenyl-1-butenyl]phenyl monophosphate] was characterized and compared with that of Tamoxifen (TAM). Its active metabolite, 4-OH-TAT-59, had a high binding affinity to estrogen receptor (ER), present in the cytosol of the uterus of immature rat, similar to estradiol. TAT-59 and 4-OH-TAT-59 inhibited in vitro estrogen-stimulated proliferation of MCF-7 cells at a lower concentration than TAM. In the absence of estradiol, TAT-59 and 4-OH-TAT-59 were effective at a lower concentration than that of 4-OH-Tamoxifen (4-OH-TAM), the active metabolite of TAM. In uterine growth inhibition, the effective dose of TAT-59 was about 3-6-fold lower than that of TAM, in various administration schedules. The minimum effective dose of TAT-59 against in vivo MCF-7 cells was about 3-fold lower than that of TAM. In DMBA-induced rat mammary tumors, TAT-59 inhibited the growth of existing tumors at about a 10-fold lower dose than TAM. Especially in the tumors with low ER levels (10-20 fmol/mg protein), TAT-59 showed a significantly stronger inhibitory effect than TAM. These experiments showed that TAT-59 was more effective in lower doses than TAM, even against the tumors with low ER content.

Reversal of P-glycoprotein mediated multidrug resistance by a newly synthesized 1,4-benzothiazipine derivative, JTV-519

A newly synthesized 1,4-benzothiazipine derivate, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2,3,4,5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) mediated multidrug resistance (MDR) in K562/MDR and KB/MRP cells, respectively. JTV-519 at 3 microM reversed the resistance of K562/MDR cells to vincristine (VCR), taxol, etoposide (VP16), adriamycin (ADM) and actinomycin D and at 0.5 or 1 microM reversed their resistance to STI571. JTV-519 at 10 microM enhanced the accumulation of ADM in K562/MDR cells to the level in parental K562 cells and inhibited the efflux of ADM from K562/MDR cells. Photoaffinity labeling of P-gp with 3H-azidopine was almost completely inhibited by 500 microM JTV-519. JTV-519 at 3 microM also partially reversed the resistance of KB/MRP cells to VCR and at 500 microM partially inhibited the photoaffinity labeling of MRP1 with (125)I-II-azidophenyl agosterol A (125I-azidoAG-A). These results suggest that JTV-519 reversed the resistance to the anti-cancer agents in P-gp and MRP1 overexpressing multidrug-resistant cells by directly binding to P-gp and MRP1, and competitively inhibiting transport of the anti-cancer agents.

Synthesis of a novel class of cdc25A inhibitors from Vitamin D3

We have developed a novel class of cdc25A inhibitors by drastic modification of the hydrophobic and hydrophilic substructures of dysidiolide. The unsaturated derivative 3b strongly inhibited cdc25A (IC50 = 7.7 microM) and caused GI arrest of HL60 cells.

Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91

Retroviral transduction of human hematopoietic stem cells is an attractive strategy in gene therapy; however, transduction efficiency and duration of transgene expression may not be satisfactory in current protocols. Co‐expression of a human multidrug resistance gene (MDR1) with a therapeutic gene affords selectable growth advantage to genetically modified cells.

Construction of a high efficiency retroviral vector for gene therapy of Hunter's syndrome

As an alternative method to the conventional therapies for Hunters syndrome, which is a lethal lysosomal storage disorder, we have developed gene delivery vehicles using a series of retroviral vectors. The objective of this study was to develop a safe and efficient retroviral vector and to optimize conditions for efficient transduction of human bone marrow CD34+ stem cells using our vector.