JP2003063989A - Agent for overcoming tolerance of anticancer medicine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent for overcoming tolerance of anticancer medicine to carcinoma cells by BCRP (Boreal Caribou Research Program) containing the effective ingredients having steroid hormones, compounds having female hormone action or compounds having antagonistic actions against these hormones: and the anticancer medicine containing (A) steroid hormones, compounds having female hormone actions or compounds having antagonistic actions against these hormones and anticancer medicines to which the carcinoma cells are capable of aquire the tolerance. SOLUTION: Pharmacological effects of conventional anticancer medicines could not have been expressed sufficiently, because of the generation of tolerance, while according to the present invention, the effects of anticancer medicines can easily be recovered and administration of the dosage of the anticancer medicines can easily be achieved, as the result that chemotherapy of cancer can possibly be proceeded with which the side-effects thereof are controlled.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anticancer agent effective against cancer that has acquired resistance to an anticancer agent, and cells useful for screening the anticancer agent.

[0002]

[0002] Camptothecins such as irinotecan hydrochloride and anticancer agents such as mitoxantrone are widely used because of their extremely high anti-malignant effect, but there is a case where the anti-cancer effect may be reduced by long-term continuous use. It has been pointed out.

The mechanism of the resistance of cancer cells to acquire resistance to these anti-cancer drugs has been studied recently.
It is known that BCRP, which is one of the ABC transporters, is involved in the acquisition of resistance to these anticancer drugs (Proc. Natl. Acad. Sci. USA, 95 (26), 15665-15670.
(1998)). That is, it was found that BCRP is expressed in cancer cells when an anticancer agent is used for a long period of time, and the BCRP has an action of reducing the intracellular accumulation of the anticancer agent by excreting the anticancer agent to the outside of the cell.

[0004]

[Problems to be Solved by the Invention] However, the B
No leukemia cell line has been found that has acquired anticancer drug resistance only by increasing CRP expression, and since there is no good experimental system, it has been impossible to develop means for overcoming anticancer drug resistance. Therefore, an object of the present invention is to use leukemia cell lines useful for screening anticancer drug resistance overcoming agents,
Another object of the present invention is to provide an anticancer agent effective against cancer that has acquired anticancer agent resistance.

[0005]

Therefore, the present inventor examined the introduction of the BCRP gene using leukemia cells such as K562 and P388, and found that the leukemia cells possessing the exogenous BCRP gene and acquired resistance to anticancer drugs. Was successfully established. When further screening for various substances using the leukemia cells, a steroid hormone, a compound having a female hormone action or a compound having an antagonistic effect on these hormones, the uptake of the anticancer drug into the leukemia cells that acquired the anticancer drug resistance. Increase
As a result, they have found that they have an effect of overcoming resistance to anticancer agents, and completed the present invention.

That is, the present invention comprises a BCR containing a steroid hormone, a compound having a female hormone action or a compound having an antagonistic action on these hormones as an active ingredient.
The present invention provides an agent for overcoming anticancer drug resistance of cancer cells by P. The present invention also provides an anticancer agent containing (A) a steroid hormone, a compound having a female hormone action or a compound having an antagonistic action on these hormones, and (B) an anticancer agent capable of acquiring resistance to a cancer cell. is there. Furthermore, the present invention provides a leukemia cell having an exogenous BCRP gene.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION First, leukemia cells having an exogenous BCRP gene will be described.

The BCRP gene to be introduced into leukemia cells is registered in the accession number AB056867 of DDBJ, and is Doyle, LA, Yang, W., Abruzzo, LV, Krogman.
n, T., Gao, Y., Rishi, AK and Ross, DD Amultid
rug resistance transporter from human MCF-7 breast
cancer cells Proc. Natl. Acad. Sci. USA 95 (2
6), 15665-15670 (1998). The B
Any CRP gene may be used as long as it can express BCRP, for example, a plasmid inserted into a retrovirus vector, pHaBCRP, pHa-BCRP-IRE.
It can be used as S-DHFR and the like. More specifically, a plasmid in which BCRP with Myc epitope is inserted into a retrovirus vector is particularly preferable.

Leukemia cells do not originally express BCRP, are easily cultured, and BCRP
Since it shows high sensitivity to anticancer drugs transported by K562 cells, P388 cells, L1210 cells,
HL-60 cells, CCRF-CEM cells and the like are preferable.

As a means for introducing the BCRP gene into leukemia cells, a retrovirus or the like into which the BCRP gene has been introduced can be easily used according to a conventional method.

The leukemia cells having the BCRP gene thus obtained have acquired anticancer drug resistance by expressing BCRP and reducing the uptake of the anticancer drug into cells, and are used as screening cells for anticancer drug resistance overcoming agents. It is useful. Especially in BCRP-expressing leukemia cells gene-transfected using retrovirus, DM was compared to the parent strain.
Since there is no change in other anticancer drug resistance genes such as R1 and MRP and the handling is good, BCRP
It is excellent in research on overcoming anti-cancer drug resistance. The cells may be directly used for screening in vitro,
It can also be used for in vivo screening by transplanting into an animal such as a mouse.

As a result of examination using the cells, when a compound having a steroid hormone, a female hormone action, or a compound having an antagonistic action to these hormones is added to these cells, sensitivity to an anticancer drug, that is, suppression of cancer cell growth by the anticancer drug is observed. It was found that the effect was restored.
Therefore, these drugs are useful as agents for overcoming anticancer drug resistance of cancer cells.

As the steroid hormone, the compound having a female hormone, and the compound having an antagonistic effect on these hormones, a female hormone and its analogs, a female hormone antagonist and its analogs are preferable. Specifically, estrone, estradiol, estradiol benzoate, estradiol dipropionate, estradiol valerate, ethinyl estradiol,
Estrogen and related substances such as estriol, estriol acetate benzoate, estriol tripropionate, conjugated estrogens, mestranol, diethylstilbestrol, diethylstilbestrol dipropionate, phosfestrol, and estramustine phosphate phosphate. Body; progesterone, pregnenerone, pregnanediol, dydrogesterone, hydroxyprogesterone caproate, medroxyprogesterone acetate, chloromadinone acetate, allylestrenol, gestenolone caproate and other progesterone hormones and their analogs: nortestosterone such as norethisterone and allylsterol Kind; Female hormone antagonists such as tamoxifen citrate and toremifene citrate

The anticancer drug which is the target of the anticancer drug resistance overcoming agent is not limited as long as it is an anticancer drug which causes resistance by BCRP, but for example, camptothecins such as irinotecan hydrochloride and topotecan; anthraquinones such as mitoxantrone; 7 -Staurosporines such as hydroxystaurosporine; and anthracyclines such as adriamycin.

The target cancer of the anticancer drug resistance overcoming agent of the present invention is not particularly limited as long as it is a cancer to which the above anticancer drug is applied.

(A) anticancer drug resistance overcoming agent of the present invention and (B)
When the above-mentioned cancer cell is used in combination with an anti-cancer agent capable of acquiring resistance, the therapeutic effect on the cancer having acquired resistance is restored. Therefore, a composition containing these components (A) and (B) is useful as a new anti-cancer agent. Is.

The anti-cancer drug resistance overcoming agent or the new anti-cancer agent of the present invention may be administered by directly using a formulation in which these components are conventionally used, or may be a new formulation containing these components. . The form of these preparations includes oral preparations, injections (including muscle, subcutaneous, and vein),
Examples include suppositories and external preparations (patches, coatings) and the like.

Although the dose of the anticancer drug resistance overcoming agent of the present invention varies depending on the administration method, symptoms, etc., it is 1 mg to 10 mg per day.
g, especially 100 mg to 3 g is preferred. Further, the dose of the anticancer agent (B) capable of acquiring resistance to the cancer cells may be a usual effective dose, for example, 1 mg to 1 g, particularly 2 mg to 300 mg is preferable.

[0019]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) BCRP gene In the present invention, human BCRP cDNA isolated from human placenta mRNA by the PCR method was used. In this PCR, Human placenta Marathon-ready cDNA from Clontech
As a template and human BCRP cDNA as a primer
5'side primer 1S (CCT GAG ATC CTG AGC CTT
TGG TT) (SEQ ID NO: 1) and 3'primer 5A
Two oligonucleotides of S (GAT GGC AAG GGA ACA GAA AAC AAC A) (SEQ ID NO: 2) were used, and an advantage cDNA PCR kit of Clontech was used to perform a reaction once at 94 ° C. for 1 minute. Then, the reaction at 94 ° C. for 30 seconds and 68 ° C. for 3 minutes was repeated 35 times, and finally the reaction at 94 ° C. for 30 seconds and 68 ° C. for 15 minutes was performed once to obtain an amplified cDNA of about 2150 bp. . This was subcloned into pCR2.1 plasmid to obtain ABI PRISM3 from Applied Biosystems.
The nucleotide sequence was determined using a 77 DNA sequencer. The nucleotide sequences of four independent clones were determined, and the nucleotide sequence of the coding region of this gene and the amino acid sequence predicted from the nucleotide sequence were deduced except for the portion presumed to be a mutation by PCR.
This base sequence is shown in SEQ ID NO: 3, and the deduced amino acid sequence is shown in SEQ ID NO: 4. In the present invention, this sequence is designated as wild-type BC.
It is an array of RPs. The sequence of BCRP of the present invention is DDBJ acc
Registered as ession number AB056867.

(2) Preparation of BCRP expression plasmid Next, a second PCR was carried out for the purpose of modifying the ends in order to insert the Myc epitope into this BCRP cDNA and insert it into a retrovirus vector. For the PCR for adding the Myc epitope tag, the PCR described above is used.
Using the human BCRP cDNA obtained by the reaction as a template, M
5'primer 5Myc containing yc epitope tag
-204S (CCC CGC GGC ATG GAA CAA AAA CTC ATC TC
A GAA GAGGAT CTG TCT TCC AGT AAT GTC GAA GTT TTT A
TC CCA GTG TC) (SEQ ID NO: 5) and 3'side primer 8AS (CGC CTC GTG GAT GGC AAG GGA ACA GAA AA
Two oligonucleotides of C AAC A) (SEQ ID NO: 6) were used as primers. PCR is Adva from Clontech
Using the ntage cDNA PCR kit, perform 1 minute reaction at 94 ° C.
After the reaction was repeated, the reaction was performed at 94 ° C for 30 seconds and 68 ° C for 3 minutes for 2 minutes.
The reaction was repeated 0 times, and finally, the reaction was carried out once at 94 ° C. for 30 seconds and 68 ° C. for 15 minutes, and the amplified cDNA of about 2200 bp was amplified.
I got A. The amplified cDNA was subcloned to determine the nucleotide sequence, and it was confirmed that mutation by PCR did not occur. P for adding HA epitope tag
For CR, human BCRP obtained by the above PCR reaction
5'primer 5HA-204S (CCC CGC GGC ATG TAC containing the HA epitope tag using cDNA as a template)
CCA TAC GAC GTC CCA GAC TAC GCT ATG TCT TCC AGT AA
TGTC GAA GTT TTT ATC CCA GTG TC) (SEQ ID NO: 7) and 3'side primer 8AS (CGC CTC GTG GAT GGC
Two oligonucleotides of AAG GGA ACA GAA AAC AAC A) (SEQ ID NO: 6) were used as primers. PCR
Clontech's Advantage cDNA PCR kit was used to perform 1 minute reaction at 94 ° C, then 30 minutes at 94 ° C.
Second, the reaction at 68 ° C. for 3 minutes was repeated 20 times, and finally 94
The reaction is performed once for 30 seconds at 68 ° C and 15 minutes at 68 ° C for about 22
A 00 bp amplified cDNA was obtained. Amplified cd
Subcloning NA to determine the nucleotide sequence and PCR
It was confirmed that there was no mutation caused by. These c
Both ends of the DNA were cleaved with two restriction enzymes SstII and XhoI, and then ligated with a pHa plasmid vector cleaved with SstII and XhoI and T4 DNA ligase. This ligation reaction solution was transformed into E. co
BCR between the SstII site and the XhoI site of the pHa plasmid vector introduced into liDH5alpha.
Clone pHaMycBCR with P cDNA inserted
P and pHaHABCRP were obtained.

(3) Preparation of BCRP retrovirus First, mouse amphotropic retrovirus packaging c
ell line PA317 cells using the calcium phosphate method for pHaMycBCRP and pHaHABCRP.
Was introduced. The cells after gene transfer were selected with 1 ng / mL mitoxantrone to obtain gene-transferred cells. The culture supernatant of these cells was collected and filtered through a 0.45 μm filter to give a retrovirus solution.

(4) Preparation of K562 / MycBCRP cells MycBCRP retrovirus solution was used for human leukemia K562
Gene transfer was performed in addition to cell culture. The cells to which the retrovirus had been added were selected with 20 ng / mL of SN-38 (7-ethyl-10-hydroxycamptothecin: activator of irinotecan hydrochloride) to obtain transgenic cells. This cell was named K562 / MycBCRP. K562 and K562 / MycBCRP were cultured in RPMI1640 medium supplemented with 7% fetal bovine serum. K562 /
The expression of BCRP protein in MycBCRP cells was confirmed by Western blotting using anti-BCRP antibody (FIG. 1). Although K562 cells are human leukemia cells, they are not originally expressed in BCRP, are easy to culture, and show high sensitivity to anticancer agents transported by BCRP such as mitoxantrone and irinotecan hydrochloride. It is suitable as a parent strain for transgenic cells. Also, K562 cells and K562 / My
It is possible to transplant a cBCRP cell into an immunodeficient mouse and carry out animal experiments such as a BCRP inhibitor.

(5) Preparation of P388 / MycBCRP cells MycBCRP retrovirus solution was added to mouse leukemia P38
Gene transfer was performed in addition to culture of 8 cells. The cells to which the retrovirus was added were selected with 20 ng / mL of SN-38 to obtain gene-transferred cells. This cell is P388 / My
It was named cBCRP. P388 and P388 / My
cBCRP is RPMI1 supplemented with 7% fetal bovine serum
Cultured in 640 medium. The expression of BCRP protein in P388 / MycBCRP cells was confirmed by Western blot using an anti-BCRP antibody. Although P388 cells are mouse leukemia cells, they do not express BCRP originally, are easy to culture, and show high sensitivity to anticancer agents transported by BCRP such as mitoxantrone and irinotecan hydrochloride.
It is suitable as a parent strain for transgenic cells. Also, P38
8 cells and P388 / MycBCRP cells can be transplanted into mice, and in vivo animal experiments such as BCRP inhibitors are possible.

Example 2 (Cell proliferation test) SN- of K562 cells and K562 / MycBCRP cells
The sensitivity to 38 and mitoxantrone was examined in a cell growth inhibition test. Both cells were seeded on a 12-well plate (Iwaki) at 10,000 cells / 1 mL / well, and then 1 mL per well of the drug diluted to each concentration with medium was added. This plate was incubated at 37 ° C. for 5 days in a 5% carbon dioxide incubator. After 5 days, add the cell solution of each well to a beaker containing 9.5 mL of Cell pack diluted solution (Toago Medical Electronics), and count the cells with Sysmex CDA-500 automatic cell counting device (Toago Medical Electronics). did. In FIG. 2, the number of cells at the time of adding each concentration of the drug was divided by the number of cells without the drug, and expressed as (% of control). K562 / Myc
BCRP showed about 20-fold resistance to SN-38 and about 10-fold resistance to mitoxantrone (FIG. 2).

Example 3 (BC with steroid hormone)
Overcoming Resistance of RP) Next, the effects of various steroid hormones on the resistance of BCRP to anticancer agents were examined. K562 cells and K562 / My
Both cells of cBCRP cells were seeded on a 12-well plate (Iwaki) at 10,000 cells / mL / well, and then 0.5 mL of steroid hormone diluted to each concentration with medium was added per well, and SN diluted to each concentration with medium. -38 or mitoxantrone was added at 0.5 mL per well. This plate was incubated at 37 ° C. for 5 days in a 5% carbon dioxide incubator.
Five days later, 9.5 mL of cell pack diluent (Toago Medical Electronics)
Add the cell solution of each well to the beaker containing
The cell number was measured by Sysmex CDA-500 automatic cell number measuring device (Toago Medical Electronics). FIG. 3 shows that BCRP anticancer drug resistance was overcome by estrone at 3 μM or 10 μM. Estrone did not affect the anticancer drug sensitivity of K562 cells that did not express BCRP, but addition of estrone to the culture of K562 / MycBCRP significantly diminished resistance to SN-38 and mitoxantrone (FIG. 3). ). When a similar test was performed using estradiol instead of estrone, the resistance of K562 / MycBCRP to SN-38 and mitoxantrone was also decreased (FIG. 4). This resistance overcoming effect was also seen with other steroid hormones, estriol, progesterone, and pregnenolone. In Table 1,
The change in sensitivity to SN-38 or mitoxantrone due to the addition of these steroid hormones was expressed as the concentration at which cell growth was inhibited by 50%.

[0027]

[Table 1]

Example 4 (BC with steroid hormone)
Increase in Topotecan Uptake in RP-Expressing Cells) It was examined whether or not these steroid hormones affect the uptake of anticancer agents into cells. K562, K562
/ MycBCRP cells were heated with 20 μM topotecan in the presence or absence of 100 μM estradiol, estriol, estrone, progesterone, and pregnenolone at 37 ° C. for 30 minutes, and then extracellular drug was added to ice-cold phosphate buffered saline. It was washed away with water. Fluorescence of topotecan incorporated into cells was determined by FA of Japan Becton.
It was measured by CS Calibur. The result is shown in FIG. These five steroids did not affect the intracellular uptake of topotecan in K562 cells, but K562 /
Increased intracellular uptake of topotecan in MycBCRP cells. Further, the same experiment was carried out for estradiol-3-benzoate and estradiol-17-acetate, which are derivatives of estradiol, and these estradiol derivatives also showed K562 / Myc.
Increased intracellular uptake of topotecan in BCRP cells (FIG. 6).

Example 5 (Increase of uptake of topotecan in BCRP-expressing cells by antihormonal agents) Next, it was examined whether or not the antihormonal agents affect the uptake of anticancer agents into cells. K562, K562 / My
The cBCRP cells were heated with 20 μM topotecan in the presence or absence of 100 μM tamoxifen, diethylstilbestrol, diethylstilbestrol dipropionate, and toremifene at 37 ° C. for 30 minutes, and then the extracellular drug was ice-cooled. It was washed away with phosphate buffered saline. The fluorescence of topotecan incorporated into cells was measured by FACSCalibur manufactured by Becton Japan. The result is shown in FIG. 7. Similar to steroids, these four antihormonal agents did not affect the uptake of topotecan in K562 cells, but increased the uptake of topotecan in K562 / MycBCRP cells. Therefore, it is considered that these antihormonal agents are also effective in overcoming BCRP resistance.

From the above, it was shown that these steroid hormones and antihormonal agents overcome the anticancer agent resistance by BCRP by increasing (restoring) the intracellular uptake of the anticancer agent which is decreased by the expression of BCRP.

[0031]

EFFECTS OF THE INVENTION According to the present invention, the effect of an anticancer drug, which has not been sufficiently effective due to the development of resistance, can be recovered well, so that the dosage of the anticancer drug can be easily controlled and, as a result, side effects-suppressed cancer chemotherapy Is possible.

[0032]

[Sequence list]                                SEQUENCE LISTING <110> Japanese Foundation for Cancer Research <120> Reagents reversing anticancer drug resistance <130> P03651308 <140> <141> <160> 7 <170> PatentIn Ver. 2.1 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       primer for human BCRP <400> 1 cctgagatcc tgagcctttg gtt 23 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       primer for human BCRP <400> 2 gatggcaagg gaacagaaaa caaca 25 <210> 3 <211> 1968 <212> DNA <213> Homosapiens <400> 3 atgtcttcca gtaatgtcga agtttttatc ccagtgtcac aaggaaacac caatggcttc 60 cccgcgacag cttccaatga cctgaaggca tttactgaag gagctgtgtt aagttttcat 120 aacatctgct atcgagtaaa actgaagagt ggctttctac cttgtcgaaa accagttgag 180 aaagaaatat tatcgaatat caatgggatc atgaaacctg gtctcaacgc catcctggga 240 cccacaggtg gaggcaaatc ttcgttatta gatgtcttag ctgcaaggaa agatccaagt 300 ggattatctg gagatgttct gataaatgga gcaccgcgac ctgccaattt caaatgtaat 360 tcaggttacg tggtacaaga tgatgttgtg atgggcactc tgacggtgag agaaaactta 420 cagttctcag cagctcttcg gcttgcaaca actatgacga atcatgaaaa aaacgaacgg 480 attaacaggg tcattcaaga gttaggtctg gataaagtgg cagactccaa ggttggaact 540 cagtttatcc gtggtgtgtc tggaggagaa agaaaaagga ctagtatagg aatggagctt 600 atcactgatc cttccatctt gttcttggat gagcctacaa ctggcttaga ctcaagcaca 660 gcaaatgctg tccttttgct cctgaaaagg atgtctaagc agggacgaac aatcatcttc 720 tccattcatc agcctcgata ttccatcttc aagttgtttg atagcctcac cttattggcc 780 tcaggaagac ttatgttcca cgggcctgct caggaggcct tgggatactt tgaatcagct 840 ggttatcact gtgaggccta taataaccct gcagacttct tcttggacat cattaatgga 900 gattccactg ctgtggcatt aaacagagaa gaagacttta aagccacaga gatcatagag 960 ccttccaagc aggataagcc actcatagaa aaattagcgg agatttatgt caactcctcc 1020 ttctacaaag agacaaaagc tgaattacat caactttccg ggggtgagaa gaagaagaag 1080 atcacagtct tcaaggagat cagctacacc acctccttct gtcatcaact cagatgggtt 1140 tccaagcgtt cattcaaaaa cttgctgggt aatccccagg cctctatagc tcagatcatt 1200 gtcacagtcg tactgggact ggttataggt gccatttact ttgggctaaa aaatgattct 1260 actggaatcc agaacagagc tggggttctc ttcttcctga cgaccaacca gtgtttcagc 1320 agtgtttcag ccgtggaact ctttgtggta gagaagaagc tcttcataca tgaatacatc 1380 agcggatact acagagtgtc atcttatttc cttggaaaac tgttatctga tttattaccc 1440 atgaggatgt taccaagtat tatatttacc tgtatagtgt acttcatgtt aggattgaag 1500 ccaaaggcag atgccttctt cgttatgatg tttaccctta tgatggtggc ttattcagcc 1560 agttccatgg cactggccat agcagcaggt cagagtgtgg tttctgtagc aacacttctc 1620 atgaccatct gttttgtgtt tatgatgatt ttttcaggtc tgttggtcaa tctcacaacc 1680 attgcatctt ggctgtcatg gcttcagtac ttcagcattc cacgatatgg atttacggct 1740 ttgcagcata atgaattttt gggacaaaac ttctgcccag gactcaatgc aacaggaaac 1800 aatccttgta actatgcaac atgtactggc gaagaatatt tggtaaagca gggcatcgat 1860 ctctcaccct ggggcttgtg gaagaatcac gtggccttgg cttgtatgat tgttattttc 1920 ctcacaattg cctacctgaa attgttattt cttaaaaaat attcttaa 1968 <210> 4 <211> 655 <212> PRT <213> Homosapiens <400> 4 Met Ser Ser Ser Asn Val Glu Val Phe Ile Pro Val Ser Gln Gly Asn   1 5 10 15 Thr Asn Gly Phe Pro Ala Thr Ala Ser Asn Asp Leu Lys Ala Phe Thr              20 25 30 Glu Gly Ala Val Leu Ser Phe His Asn Ile Cys Tyr Arg Val Lys Leu          35 40 45 Lys Ser Gly Phe Leu Pro Cys Arg Lys Pro Val Glu Lys Glu Ile Leu      50 55 60 Ser Asn Ile Asn Gly Ile Met Lys Pro Gly Leu Asn Ala Ile Leu Gly  65 70 75 80 Pro Thr Gly Gly Gly Lys Ser Ser Leu Leu Asp Val Leu Ala Ala Arg                  85 90 95 Lys Asp Pro Ser Gly Leu Ser Gly Asp Val Leu Ile Asn Gly Ala Pro             100 105 110 Arg Pro Ala Asn Phe Lys Cys Asn Ser Gly Tyr Val Val Gln Asp Asp         115 120 125 Val Val Met Gly Thr Leu Thr Val Arg Glu Asn Leu Gln Phe Ser Ala     130 135 140 Ala Leu Arg Leu Ala Thr Thr Met Thr Asn His Glu Lys Asn Glu Arg 145 150 155 160 Ile Asn Arg Val Ile Gln Glu Leu Gly Leu Asp Lys Val Ala Asp Ser                 165 170 175 Lys Val Gly Thr Gln Phe Ile Arg Gly Val Ser Gly Gly Glu Arg Lys             180 185 190 Arg Thr Ser Ile Gly Met Glu Leu Ile Thr Asp Pro Ser Ile Leu Phe         195 200 205 Leu Asp Glu Pro Thr Thr Gly Leu Asp Ser Ser Thr Ala Asn Ala Val     210 215 220 Leu Leu Leu Leu Lys Arg Met Ser Lys Gln Gly Arg Thr Ile Ile Phe 225 230 235 240 Ser Ile His Gln Pro Arg Tyr Ser Ile Phe Lys Leu Phe Asp Ser Leu                 245 250 255 Thr Leu Leu Ala Ser Gly Arg Leu Met Phe His Gly Pro Ala Gln Glu             260 265 270 Ala Leu Gly Tyr Phe Glu Ser Ala Gly Tyr His Cys Glu Ala Tyr Asn         275 280 285 Asn Pro Ala Asp Phe Phe Leu Asp Ile Ile Asn Gly Asp Ser Thr Ala     290 295 300 Val Ala Leu Asn Arg Glu Glu Asp Phe Lys Ala Thr Glu Ile Ile Glu 305 310 315 320 Pro Ser Lys Gln Asp Lys Pro Leu Ile Glu Lys Leu Ala Glu Ile Tyr                 325 330 335 Val Asn Ser Ser Phe Tyr Lys Glu Thr Lys Ala Glu Leu His Gln Leu             340 345 350 Ser Gly Gly Glu Lys Lys Lys Lys Ile Thr Val Phe Lys Glu Ile Ser         355 360 365 Tyr Thr Thr Ser Phe Cys His Gln Leu Arg Trp Val Ser Lys Arg Ser     370 375 380 Phe Lys Asn Leu Leu Gly Asn Pro Gln Ala Ser Ile Ala Gln Ile Ile 385 390 395 400 Val Thr Val Val Leu Gly Leu Val Ile Gly Ala Ile Tyr Phe Gly Leu                 405 410 415 Lys Asn Asp Ser Thr Gly Ile Gln Asn Arg Ala Gly Val Leu Phe Phe             420 425 430 Leu Thr Thr Asn Gln Cys Phe Ser Ser Val Ser Ala Val Glu Leu Phe         435 440 445 Val Val Glu Lys Lys Leu Phe Ile His Glu Tyr Ile Ser Gly Tyr Tyr     450 455 460 Arg Val Ser Ser Tyr Phe Leu Gly Lys Leu Leu Ser Asp Leu Leu Pro 465 470 475 480 Met Arg Met Leu Pro Ser Ile Ile Phe Thr Cys Ile Val Tyr Phe Met                 485 490 495 Leu Gly Leu Lys Pro Lys Ala Asp Ala Phe Phe Val Met Met Phe Thr             500 505 510 Leu Met Met Val Ala Tyr Ser Ala Ser Ser Met Ala Leu Ala Ile Ala         515 520 525 Ala Gly Gln Ser Val Val Ser Val Ala Thr Leu Leu Met Thr Ile Cys     530 535 540 Phe Val Phe Met Met Ile Phe Ser Gly Leu Leu Val Asn Leu Thr Thr 545 550 555 560 Ile Ala Ser Trp Leu Ser Trp Leu Gln Tyr Phe Ser Ile Pro Arg Tyr                 565 570 575 Gly Phe Thr Ala Leu Gln His Asn Glu Phe Leu Gly Gln Asn Phe Cys             580 585 590 Pro Gly Leu Asn Ala Thr Gly Asn Asn Pro Cys Asn Tyr Ala Thr Cys         595 600 605 Thr Gly Glu Glu Tyr Leu Val Lys Gln Gly Ile Asp Leu Ser Pro Trp     610 615 620 Gly Leu Trp Lys Asn His Val Ala Leu Ala Cys Met Ile Val Ile Phe 625 630 635 640 Leu Thr Ile Ala Tyr Leu Lys Leu Leu Phe Leu Lys Lys Tyr Ser                 645 650 655 <210> 5 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       primer for human BCRP <400> 5 ccccgcggca tggaacaaaa actcatctca gaagaggatc tgtcttccag taatgtcgaa 60 gtttttatcc cagtgtc 77 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       primer for human BCRP <400> 6 cgcctcgtgg atggcaaggg aacagaaaac aaca 34 <210> 7 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       primer for human BCRP <400> 7 ccccgcggca tgtacccata cgacgtccca gactacgcta tgtcttccag taatgtcgaa 60 gtttttatcc cagtgtc 77

[Brief description of drawings]

FIG. 1 is a diagram showing the results of Western blotting showing that BCRP protein is expressed in K562 / MycBCRP cells.

FIG. 2 is a diagram showing the degree of acquisition of anticancer drug resistance in K562 / MycBCRP cells.

FIG. 3 shows the effect of estrone on the resistance of K562 / MycBCRP cells to anticancer drugs.

FIG. 4 shows the effect of estradiol on the resistance of K562 / McyBCRP cells to anticancer drugs.

FIG. 5: K562 / My by various steroid hormones
It is a figure which shows the increasing effect of the topotecan uptake | capture of cBCRP cell.

FIG. 6: K562 with various steroid hormone analogs
It is a figure which shows the increase effect of the topotecan uptake | capture of / MycBCRP cell.

FIG. 7: K562 / MycBC with various antihormonal agents
It is a figure which shows the increase effect of topotecan uptake | capture of RP cell.

[Procedure amendment]

[Submission date] September 4, 2001 (2001.9.4)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 2

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction content]

Here, as the steroid hormone, the compound having a female hormone action, and the compound having an antagonistic action on these hormones, a female hormone and its analogs, a female hormone antagonist and its analogs are preferable. Specifically, estrone, estradiol, estradiol benzoate, estradiol dipropionate, estradiol valerate, ethinyl estradiol, estriol, estriol acetate benzoate, estriol tripropionate, conjugated estrogen, mestranol, diethylstilbestrol, diethylstil Bestrol dipropionate, phosfestrol, estrogen sodium phosphate and other estrogen and its analogs; progesterone,
Progesterone and its analogues such as pregnenerone, pregnanediol, dydrogesterone, hydroxyprogesterone caproate, medroxyprogesterone acetate, chlormadinone acetate, allylestrenol, gestanolonecaproate; nortestosterone such as norethisterone, allylsterol; tamoxifensite. Female hormone antagonists such as rate and toremifene citrate are included.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 35/00 A61P 35/00 43/00 121 43/00 121 C12N 5/10 C07J 1/00 // C07J 1/00 17/00 17/00 C12N 5/00 B C12N 15/09 15/00 AF Term (reference) 4B024 AA11 BA80 CA04 DA03 EA02 GA11 4B065 AA93Y AA94X AA97X AB01 AC14 AC20 BA02 CA24 CA44 CA46 4C084 AA17 NA14 ZB26 ZB27 ZC75 4C086 AA01 AA02 DA08 MA02 MA04 NA14 ZB26 ZB27 ZC75 4C091 AA01 BB03 BB05 BB07 CC01 DD01 EE04 FF01 GG01 HH01 JJ01 KK01 LL01 MM01 NN01 PA02 PA09 QQ01

Claims (9)

[Claims] 1. An agent for overcoming anticancer drug resistance of cancer cells by BCRP, which comprises a compound having a steroid hormone, a female hormone action, or a compound having an antagonistic action against these hormones as an active ingredient. 2. The steroid hormone, the compound having a female hormone action or the compound having an antagonistic action on these hormones is a female hormone or the steroid hormone receptor antagonist is a female hormone receptor antagonist.
The anticancer drug resistance overcoming agent described. 3. An anticancer agent containing (A) a steroid hormone, a compound having a female hormone action or a compound having an antagonistic action on these hormones, and (B) an anticancer agent capable of acquiring resistance to a cancer cell. 4. The anticancer agent according to claim 3, wherein the compound having a steroid hormone, a female hormone action, or a compound having an antagonistic action on these hormones is a female hormone or a female hormone receptor antagonist. 5. The anticancer agent according to claim 3 or 4, wherein the anticancer agent capable of acquiring resistance to cancer cells is selected from camptothecins, mitoxantrone, 7-hydroxystaurosporine and adriamycin. 6. The anticancer agent according to any one of claims 3 to 5, wherein the anticancer agent is an anticancer agent for a cancer that can acquire or has acquired resistance to an anticancer agent. 7. A leukemia cell having an exogenous BCRP gene. 8. A P388 having an exogenous BCRP gene
The leukemia cell according to claim 7, which is also a K562 cell. 9. The leukemia cell according to claim 7, which is a cell that has acquired resistance to an anticancer drug.