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2011년 2월
석사학위논문
Identification of MDR1 dual inhibitor from chemical library
조선대학교 대학원
약학과
이 원 영
화합물 라이브러리에서 MDR1 이중 저해제 발굴
Identification of MDR1 dual inhibitor from chemical library
2011년 2월 25일
조선대학교 대학원
약학과
이 원 영
Identification of MDR1 dual inhibitor from chemical library
지도교수 강 건 욱
이 논문을 약물학 석사학위신청 논문으로 제출함
2010년 10월조선대학교 대학원
약학과
이 원 영
이원영의 석사학위논문을 인준함
위원장 조선대학교 교수 최 홍 석 (印) 위 원 동국대학교 교수 한 효 경 (印) 위 원 조선대학교 교수 강 건 욱 (印)
2010년 11월
조선대학교 대학원
CONTENTS
국 문 초 록
… … … 2
A B S T R A C T … … … 4
1. I n t r o d u c t i o n … … … 6
2. M a t e r i a l s a n d M e t h o d s … … … 9
3. R e s u l t s … … … 1 3
4. D i s c u s s i o n … … … 1 6
5. R e f e r e n c e s … … … 2 0
6. F i g u r e L e g e n d s … … … 2 6
(국문 초록)
화합물 라이브러리에서 MDR1 이중 저해제 발굴
이 원 영
지도 교수 : 강 건 욱 조선대학교 대학원 약학과
성공적인 암 치료에 있어서 심각한 장애요인이 되는 다약제 내성 (MDR) 은
MDR1 [P-glycoprotein (P-gp), ABCB1] 같은 막 투과성 단백질의 과다발현에
기인하고 그 결과 약물방출이 유도된다. 안전하고 효과적으로 MDR 을 역전시키는
물질의 개발은 임상적으로 이러한 문제를 다루는데에 필수적인 접근이라고 할 수
있겠다. 우리의 연구목적은 독소루비신 (아드리아마이신; adriamycin)에 저항성을
가지는 인간 유방암 세포에서 P-gp 의 기능과 발현을 조절할 수 있는 가능성을 지닌
약물을 찾아내고 그 작용기전을 밝혀내는 것이다. 우리는 Hif-1 억제제 역할을 하는
일련의 후보물질들을 대상으로 MDR1 억제효능을 rhodamine-123 retention assay
을 통해 조사하였고, 그 중 AC-507 이라는 물질이 가장 효과적이고 강력하게
MDR1 을 역전시키는 약물이라는 것을 알아내었다. 동시에 AC-507 은 P-gp 발현의
억제를 통해 MCF-7/ADR 세포의 내성을 줄여주고, 세포 내 doxorubicin uptake 도
억제시킴을 확인할 수 있었다. AC-507 을 30 분과 24 시간 처치한 Rhodamine-123
retention 실험은 이 약물이 MDR1 의 활성 및 발현을 동시에 억제하는 이중억제제로
작용함을 보여준다. MCF-7/ADR 세포는 독소루비신에 저항성을 보이며, AC-507 의
전처치는 독소루비신의 반응성을 현저하게 회복시켰다. 더하여, AC-507 에
의한 MDR1 발현 억제는 Hif-1α 와 FoxO1 의 억제가 중요한 역할을 한다는 것을
밝혔다. 이러한 결과들은 AC-507 이 Hif-1α 및 FoxO1 의 억제를 통해
MDR1 발현을 억제하고, 동시에 MDR1 transporter 활성을 직접 저해하여 항암제
저항성을 역전시킬 수 있는 물질임을 시사한다.
ABSTRACT
Identification of dual inhibitor from chemical library
Lee Won-Young
Advisor : Prof. Kang Keon-Wook Ph.D Department of Pharmacy,
Graduate School of Chosun University
Multidrug resistance (MDR), which is a serious obstacle to the success of treating cancer disease, is attributed to the overexpression of transmembrane proteins, such as MDR1 (P-glycoprotein, ABCB1) and facilitates drug efflux. The development of secure and effective MDR-reversing substance is an essential approach to dealing with this problem clinically. Our study aimed to find dual inhibitor for its potential ability to regulate the function and expression of MDR1 in doxorubicin (adriamycin)-resistant human breast cancer cell line, MCF-7/ADR cells and identify molecular mechanistic basis for its action. We tested a series of small molecules which functions as Hif-1 inhibitors using a screening of rhodamine-123 retention assay. Among them, we found out that AC-507 is the most potent MDR-reversing agent. In addition, AC-507 leads to reverse resistance of MCF-7/ADR cells through the reduction of P-gp expression. These were confirmed by MDR1 immunoblottings in MCF-7/ADR cells. Rhodamine-123
retention assay with treating the cells with AC-507 for 30 min and 24 h revealed that the compound possessed direct MDR1 inhibitory action and indirect inhibitory activity against MDR1 expression. MTT cell viability assay and doxorubicin uptake assay showed that AC-507 efficiently reversed doxorubicin uptake and doxorubicin responsiveness in MCF-7/ADR cells. Immunoblot analyses using subcellular fractions indicated that nuclear level of Hif-1α and FoxO1 were decreased by AC-507 treatment. These results suggest that AC-507 is a potential therapeutic agent to reverse MDR1-mediated chemoresistance.
Keywords: MDR1, MCF-7/ADR, Hif-1α, FoxO1
1. Introduction
Cancer cells can take chemoresistance via overexpression of ATP-binding cassette (ABC) transporters such as multidrug resistance 1 (MDR1 ; or P- glycoprotein, ABCB1) and multidrug resistance-associated proteins. These are able to release variety anticancer agents out of cells and have an interruption of cytotoxic drugs accumulation in cancer cells (1). In addition, P-glycoprotein (P-gp) plays a significant role in absorption, distribution and elimination of various xenobiotics (2-5). P-gp has a very wide spectrum of substrates as well as cancer chemotherapeutic agents, cardiovascular drugs, HIV protease inhibitors, immunosuppressants, antibiotics, steroids and cytokines (2-5). MDR1 which is a member of the ABC transporter family, is the best characterized drug efflux pump (6) and a glycosylated membrane protein of a 170 kDa encoded by the ABCB1 (MDR1) gene (7). Its gene expression levels are modulated by posttranscriptional and transcriptional processes (8).
Breast cancer is the most commonplace malignancy in Western women (9).
The increase in MDR1 expression tends to extend cross-resistance to other diverse therapeutic agents and leads to chemoresistance in breast cancer cells (10).
Accordingly, prevention of MDR1 overexpression could improve the chemotherapy effectiveness.
A conserved DNA binding domain termed the Forkhead box is held by Forkhead box-containing protein, O subfamily (FoxO) transcription factors. There
are four proteins; FoxO1, FoxO3, FoxO4 and FoxO6 in mammals. These have been known as members of the O subfamily FoxO, and FoxO transcriptional activity is modulated by a shuttling system running between the nucleus and the cytoplasm. Phosphorylation-dependent ubiquitination and acetylation can regulate this system (11-13). FoxO factors can control diverse cellular destinies such as differentiation, metabolism and proliferation (14). These are frequently deregulated in some cancers (11, 15). We found that FoxO1 is constantly increased in MCF-7/ADR, adriamycin-resistant breast cancer cells, and FoxO1 has a critical role in the MDR1 gene expression (16).
There is common consent that hypoxia in the depth of solid tumors considerably decreases the chemosensitivity of cancer cells and that experimental hypoxia induces drug resistance to anticancer agents in a variety of cell lines (17).
Moreover, it has been revealed that MDR1 gene is hypoxia responsive through HIF-1 binding to its binding site located in the proximal promoter region (18,19,20). Increased expression or amplification of genes conferring multidrug resistance (MDR), e.g., the genes for the P-glycoprotein (P-gp) of MDR transporters, MDR-associated protein (MRPs), or lung resistance-associated protein constitute the main constraint to increased efficiency of chemotherapeutic anticancer agents and these genes may be modulated by existence of HIF-1 (21-22).
In the present study, we tested a series of small molecules which functions as Hif-1 inhibitors using a screening of rhodamine-123 retention assay. Among them, we found out that AC-507 is the most potent MDR-reversing agent and tried
to elucidate its molecular mechanism to inhibit MDR1.
2. Materials and Methods
2-1. Materials
The anti-MDR1 antibody was supplied by Calbiochem (Darmastadt, Germany). The FoxO1, FoxO3a antibodies, Horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG were purchased from Cell Signaling Technology (Beverly, MA). The antibody against enhancer/enhancer-binding protein (C/EBP)α, (C/EBP) β, Y box binding protein 1(YB-1) and NF-kB/p65 antibodies were provided by Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase- conjugated rabbit anti-gout IgG was obtained from Jackson Immunoresearch Laboratories (West Grove, PA).
2-2. Cell culture
The MCF-7 cells and the MCF-7/ADR cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37℃ in a 5% CO2 humidified atmosphere
2-3. Preparation of nuclear extracts
Nuclear extracts were prepared essentially as described by Schreiber et al.
(23). Briefly, cells in dishes were washed with ice-cold PBS, scraped, transferred to microtubes and allowed to swell after adding 100 μl of lysis buffer containing
10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH7.9), 0.5% Nonidet P-40, 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride. Cell membranes were disrupted by vortexing, and the lysates were incubated for 10 min on ice and centrifuged at 7200g for 5 min. Pellets containing crude nuclei were resuspended in 60 μl of extraction buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride and then incubated for 30 min on ice. The samples were then centrifuged at 15 800g for 10 min to obtain supernatants containing nuclear extracts, which were stored at -80℃ until required.
2-4. Immunoblot analysis
After washing with sterile PBS, MCF-7 or MCF-7/ADR cells were lysed in EBC lysis buffer containing 20 mM Tris-HC1 (pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25mM β-glycerophosphate, 2mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride and 1 μg/ml leupeptin. The cell lysates were centrifuged at 10 000g for 10 min to remove the debris, and the proteins were fractionated using a 10% separating gel. The fractionated proteins were then transferred electrophoretically to nitrocellulose paper, and the proteins were immunoblotted with specific antibodies. Horseradish peroxidase- or alkaline phosphatase-conjugated anti-IgG antibodies were used as the secondary antibodies.
The nitrocellulose papers were developed using 5-bromo-4-chloro-3- indolyl phosphate/4-nitroblue tetrazolium or an enhanced chemiluminescence system. For chemiluminescence detection, the LAS3000-mini (Fujifilm, Tokyo, Japan) was used.
2-5. Rhodamine-123 retention assay
The MCF-7 and MCF-7/ADR cells were seeded in 24-well plates. At 80%
confluence, the cells were incubated in fetal bovine serum-free Dulbecco’s modified Eagle’s medium for 18 h. The culture medium was changed with Hanks’
balanced salt solution and the cells were incubated at 37℃ for 30 min. After incubation of the cells with 20 μM rhodamine-123 (R-123) in the presence or absence of verapamil (100 μM) for 90 min, the medium was completely removed.
The cells were then washed three times with an ice-cold phosphate buffer (pH 7.0) and lysed in EBC lysis buffer. The R-123 fluorescence in the cell lysates was measured using excitation and emission wavelengths of 480 and 540 nm, respectively. Fluorescence values were normalized to the total protein content of each sample and presented as the ratio to controls.
2-6. Cellular uptake of doxorubicin.
Doxorubicin uptake was quantified in MCF-7 and MCF-7/ADR cells.
Cells (3x106 cells) were incubated with 30 μM doxorubicin for 60 min, washed with PBS three times, and lysed in EBC lysis buffer containing 20 mM Tris·Cl (pH
7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM β-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM PMSF, and 1 μg/ml leupeptin. After centrifugation of the samples at 10,000 g for 10 min, change in fluorescent absorbance of doxorubicin in the supernatant was determined at excitation and emission wavelengths of 470 nm and 590 nm, respectively. Uptake intensity was expressed as a relative ratio to the fluorescence value for the doxorubicin-treated group.
2-7. MTT cell viability assay.
To determine cell viability, cells were plated at 10^4 cells/well in 96-well plates. For determination of the cytotoxicity of amurensin G, MCF-7/ADR cells were incubated in FBS-free medium with or without amurensin G (0.1–3 μg/ml) for 24 h. Viable adherent cells were stained with 3-(4,5-dimethylthiazol-2-yl)- 2,5- diphenyl-tetrazolium bromide (MTT; 2 mg/ml) for 4 h. The media were then removed and the formazan crystals produced were dissolved by adding 200 μl dimethylsulfoxide/well. Absorbance was assayed at 540 nm. Cell viability was expressed as relative ratios to untreated control cells.
3. Results
3-1. Screening library using a rhodamine-123 retention assay.
We acquired a series of compounds from Dr. Lee K (Dongguk University) as HIF-1a inhibitors (AC301-AC540). In order to screen drug candidates inhibiting drug resistance, we examined the MDR1 transport activity using a rhodamine-123 (R-123, a substrate of MDR1) retention assay (Table 1). Higher level indicated that the R-123 was retained MCF-7/ADR cells and presumably contributed to overcome the acquired chemoresistance. The increased accumulation of R-123 in MCF-7/ADR was revealed in some compounds (10 mM used). We first chose 17 chemicals which had almost 2-fold higher level of the intracellular accumulation ratio of R-123 than control group.
3-2. Inhibitory effect of AC-507 on MDR1 expression.
MDR1 expression changes by 17 chosen chemicals in MCF-7/ADR cells were determined by Western blot analyses. As shown in Fig. 1A, 10 mM AC-507 potently inhibited the protein expression of MDR1, while other chemicals marginally affected MDR1 expression in MCF-7/ADR cells. Thus, we selected AC-507 for further studies. We next determined the concentration-dependent inhibitory activity on MDR1 expression. Treatment of MCF-7/ADR cells with AC-507 (0.3-10 mM) significantly suppressed protein levels of MDR1 in a concentration-dependent manner and maximal inhibition was found at 10 mM (Fig.
1B).
3-3. Role of AC-507 as a dual inhibitor.
To clarify the role of MDR1 down-regulation effect of AC-507 in chemoresistance reversal, MCF-7/ADR cells were treated with same concentrations of AC-507 for 30 min and 24 h, respectively and then determined the accumulation of R-123. As shown in Fig. 2, 30 min incubation of AC-507 caused 1.4 fold increase in R-123 fluorescence, however, 24 h incubation of AC- 507 resulted in 1.9 fold increase in R-123 accumulation. This result indicates that AC-507 plays dual inhibitory action in MDR1 activity; by direct inhibition on transporter activity and indirect MDR1 down-regulation effect.
3-4. Reversal of doxorubicin resistance by AC-507
To confirm whether AC-507 reduces the cellular uptake of doxorubicin, we then performed doxorubicin uptake study. Accumulation of doxorubicin in MCF-7/ADR cells was concentration-dependently increased by AC-507 (Fig. 3A).
We then performed MTT assay to determine whether doxorubicin responsiveness was recovered by AC-507. Doxorubin up to 10 mM did not cause cell death in MCF-7/ADR cells (data not shown). However, pretreatment of MCF- 7/ADR cells with AC-507 for 24 h, concentration-dependently reduced cell viability of MCF-7/ADR cells exposed to 10 mM doxorubicin (Fig. 3B).
3-5. Nuclear levels of diverse proteins.
Several cis-acting elements are located in the proximal promoter region of the MDR1 gene [Y-box, C/EBP, FoxO and HIF-1 binding sites] (16, 24, 25, 26) In particular, we have previously shown that HIF-1 and FoxO transcription factors
are important for the transcriptional activation of MDR1 gene (16). We observed the nuclear levels of these transcription factors (HIF-1α, FoxO1, FoxO3, C/EBPα, C/EBPβ, p65 and YB-1) using Western blot analyses. As shown in Fig 4, nuclear HIF-1α and FoxO1 levels were decreased by AC-507 treatment, while there were no differences in nuclear levels of the other transcription factors. These data suggest that AC-507 down-regulates MDR1 gene expression through modulating Hif-1α and FoxO1.
4. Discussion
Multi-drug resistance is a significant barrier in the treatment of breast cancer (27). Several ABC-transporter groups are acknowledged to be involved in this. Proteins of MDR1 and MRPs contribute to drive this severe resistance (28- 29). Although these efflux systems facilitate removing harmful chemicals and protecting tissues from toxic chemicals, there is much more serious problem to managing patients who are taking therapeutic drugs. To make matters worse, these proteins have cross-tolerance effects on other unconnected anti-cancer agents, and it results in low success of treatment (30). For this reason, extensive compounds have been studied to identify the effective MDR1 inhibitors to reverse uptake of anticancer drugs from now. Actually, pharmaceutical companies have developed many MDR1 inhibitors. Nevertheless, the inhibitors of MDR1 inhibitors frequently lead to have severe side effects or interrupt the kinetics of other drugs (31). It may be an alternative way to consider the decease of MDR1 expression for improving chemotherapy (31). And it should be important to clarify the mechanistic basis of MDR1 expression for recognizing the potential chemotherapeutic target to overcome drug resistance. Therefore, the present study investigated the effective dual inhibitor of MDR1 from compound library and the pharmacological mechanism of it.
In this study, we found the potential and effective dual inhibitor which play an important role of reversing chemoresistance. AC-507 have a dual action ; direct inhibitory of MDR1 and indirect inhibitory of MDR1 expression. As
mentioned before inhibiting MDR1 directly cause side effects. Because of this worry AC-507 we found must be important role in drug resistance as an alternative way. We expect that its action lead to decrease severe side effect and make synergistic effect direct and indirect inhibition each other. We further investigated that AC-507 reversed MDR1-mediated chemoresistance through down regulation of nuclear level of FoxO1 and HIF-1a.
FoxO plays an important role in cell growth, proliferation, differentiation, longevity, metabolism and tumor development (11,14-15,32). In our previous study, we show that FoxO1 binding to the -150 ~ -144 bp MDR1 gene promoter is a key event in the MDR1 gene transactivation in MCF-7/ADR cells and suggested that FoxO1 could be a target for multidrug resistance (16). In this study, AC-507 decreased nuclear levels of FoxO1 and this may be responsible for the compound’s inhibitory activity on MDR1 expression.
Hypoxia is a common feature of various malignant tumors. The physiology and biochemistry of tumor cells changes to adapt hypoxia. HIF-1 plays a key role of altering the biological characteristics of tumors (33-35). Many studies show that hypoxia helped to advance chemotherapy and radiotherapy resistance of tumor (36-39). Enhanced understanding of tumor MDR influence by hypoxia will assist improve the effect of chemotherapy. It has been reported that Hif-1α protein was upregulated in multiple types of human cancer, including breast, lung, gastric, prostate and colon carcinomas, even in preneoplastics and premalignant lesions, such as colonic adenoma, breast ductal carcinomas in situ and prostate
intraepithelial neoplasia (40-43). More significantly, the overexpression of Hif-1α is an important marker in precancerous lesion for example, early-stage cervical cancer, cervical intra-epithelial neoplasia III and earlystage lymph nod-negative breast cancer (44). Hypoxia is highly identified to induce resistance to drug and radiation in solid tumors (45,46) as well as in multicellular tumor spheroids (47,48). It has not yet been established the reason that hypoxia contributes to drug resistance in anticancer therapy. It has been demonstrated that hypoxia decreases the expression of DNA topoisomerase IIα, which makes cells resistant to topoisomeraseII-targeted drugs such as etoposide and doxorubicin (49).
Additionally, glutathione S-transferase pi (GST-pi), which has been confirmed to be involved in MDR phenotype, has recently been revealed to be increased by hypoxia in several cancer cell lines. A relationship between P-gp and GST-pi has been reported that there may be a general mechanism for regulating the expression of drug resistance-related proteins (50). Several factors associated either directly or indirectly with tumor hypoxia contributed to resistance to anticancer agents in a solid tumor in vivo (51). Cells in hypoxic resions of a tumor tend to stop or slow their rate of progression through the cell cycle. This effect is the result of induced expression of specific proteins, such as the tumor suppressor p53 and the cyclin- dependent kinase inhibitor p27Kip1(52-53) which are increased under hypoxic conditions. This slowing of cell proliferation will lead to induced chemoresistance because almost anticancer agents are more effective with rapidly proliferating cells than nonproliferationg cells. We and others revealed that HIF-1a plays an
important role in MDR1 gene expression in several cancer cells. In this study, AC- 507 suppressed nuclear HIF-1a accumulation in MCF-7/ADR cells, which may be another factor for the down-regulation of MDR1 expression by AC-507.
Overall, our data show that an effective agent AC-507 identified from screening a chemical library appears to have therapeutic potential of overcoming chemoresistance through inhibitory expression of HIF-1α and FoxO1.
5. References
[1] Gottesman,M.M. et al. (2002) Multidrug resistance in cancer: role of ATP- dependent transporters. Nat. Rev. Cancer, 2, 48–58.
[2] Leonard,G.D. et al. (2003) The role of ABC transporters in clinical practice.
Oncologist, 8, 411-424.
[3] Dean,M. et al. (2001) Complete characterization of the human ABC gene family. Bioenerg. Biomembr., 33, 475-479.
[4] Thiebaut,F. et al. (1987) Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc. Natl. Acad. Sci., 84, 7735- 7738.
[5] Marchetti,S. et al. (2007) Concise review: Clinical relevance of drug herb drug interactions mediated by the ABC transporter ABCB1 (MDR1, P-glycoprotein).
Oncologist, 12, 927-941.
[6] Leslie,E.M. et al. (2005) Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense. Toxicol. Appl. Pharmacol.,
204, 216–237.
[7] Ambudkar,S.V. et al. (1999) Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu. Rev. Pharmacol. Toxicol., 39, 361–398.
[8] Sukhai,M. et al. (2000) Regulation of the multidrug resistance genes by stress signals. J. Pharm. Sci., 3, 268–280.
[9] Moulder,S. et al. (2008) Advances in the treatment of breast cancer. Clin.
Pharmacol. Ther., 83, 26–36.
[10] Giai,M. et al. (1991) Chemoresistance in breast tumors. Eur. J. Gynaecol.
Oncol., 12, 359–373.
[11] Barthel,A. et al. (2005) FoxO proteins in insulin action and metabolism.
Trends Endocrinol. Metab., 16, 183-189.
[12] Vogt,P.K. et al. (2005) Triple layer control: phosphorylation, acetylation and ubiquitination of FOXO proteins. Cell Cycle, 4, 908-913.
[13] Hoekman,M.F. et al. (2006) Spatial and temporal expression of FoxO transcription factors in the developing and adult murine brain. Gene Expr. Patterns,
6, 134-140.
[14] Accili,D and Arden,K.C. (2004) FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell, 117, 421-426.
[15] Arden,K.C. (2006) Multiple roles of FOXO transcription factors in mammalian cells point to multiple roles in cancer. Exp. Gerontol., 41, 709-717.
[16] Han,C.Y., et al. (2008) Role of FoxO1 activation in MDR1 expression in adriamycin-resistant breast cancer cells. Carcinogenesis, 29, 1837-1844.
[17] Teicher,B.A. (1994) Hypoxia and drug resistance. Cancer Metastasis Rev., 13, 139–168.
[18] Comerford,K.M., et al. (2002) Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res., 62, 3387-3394.
[19] Comerford,K.M., et al. (2004) c-Jun NH2-terminal kinase activation contributes to hypoxia-inducible factor 1alpha-dependent P-glycoprotein
expression in hypoxia. Cancer Res., 64, 9057-9061.
[20] Zhu,H., et al. (2005) Involvement of hypoxia-inducible factor-1-alpha in multidrug resistance induced by hypoxia in HepG2 cells. J. Exp. Clin. Cancer Res.,
24, 565-574.
[21] Hofer,T. et al. (2002) Oxygen sensing, HIF-1alpha stabilization and potential therapeutic strategies. Pflugers Arch., 443, 503–507.
[22] Semenza,G.L. (2001) HIF-1 and mechanisms of hypoxia sensing. Curr. Opin.
Cell Biol., 13, 167–171.
[23] Schreiber,E. et al. (1990) Astrocytes and glioblastoma cells express novel octamer-DNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins. Nucleic Acids Res., 18, 5495–5503.
[24] Ohga,T. et al. (1998) Direct involvement of the Y-box binding protein YB-1 in genotoxic stress-induced activation of the human multidrug resistance 1 gene. J.
Biol. Chem., 273, 5997-6000.
[25] Combates,N.J. et al. (1994) NF-IL6, a member of the C/EBP family of transcription factors, binds and trans-activates the human MDR1 gene promoter. J.
Biol. Chem., 269, 29715-29719.
[26] Comerford,K.M. et al. (2002) Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer. Res., 15, 3387-3394 [27] Liu,F. et al. (2007) The effect of survivin on multidrug resistance mediated by P-glycoprotein in MCF-7 and its adriamycin resistant cells. Biol. Pharm. Bull., 30, 2279-2283.
[28] Ling,V. (1997) Multidrug resistance: molecular mechanisms and clinical relevance. Cancer Chemother. Pharmacol., 40, Suppl, S3-S8.
[29] Sharom,F.J. (2008) ABC multidrug transporters: structure, function and role in chemoresistance. Pharmacogenomics, 9,105-127.
[30] Lage,H. (2003) ABC-transporters: implications on drug resistance from microorganisms to human cancers. Int. J. Antimicrob. Agents, 22, 188-199.
[31] Zhou,J. et al. (2006) Reversal of P-glycoprotein-mediated multidrug resistance in cancer cells by the c-Jun NH2-terminal kinase. Cancer Res., 66, 445–
452.
[32] Reagan-Shaw S and Ahmad N (2007) The role of Forkhead-box Class O (FoxO) transcription factors in cancer: a target for the management of cancer.
Toxicol. Appl. Pharmacol., 224, 360-368.
[33] Mabjeesh,N.J. et al. (2007) Hypoxia-inducible factor (HIF) in human tumorigenesis. Histol. Histopathol., 22, 559–572.
[34] Jensen,R.L. et al. (2006) Inhibition of hypoxia inducible factor-1alpha (HIF- 1alpha) decreases vascular endothelial growth factor (VEGF) secretion and tumor growth in malignant gliomas. J. Neurooncol., 78,233–247.
[35] Nagle,D.G. et al. (2006) Natural product-based inhibitors of hypoxia- inducible factor-1 (HIF-1). Curr. Drug Targets, 7, 355–369.
[36] Magnon,C. et al. (2007) Radiation and inhibition of angiogenesis by canstatin synergize to induce HIF-1alpha-mediated tumor apoptotic switch. J. Clin. Invest.
117, 1844–1855.
[37] O’Donnell,J.L. et al. (2006) Oncological implications of hypoxia inducible factor-1alpha (HIF-1alpha) expression. Cancer Treat. Rev., 32, 407–416.
[38] Schnitzer,S.E. et al. (2006) Hypoxia and HIF- 1alpha protect A549 cells from drug-induced apoptosis. Cell Death DiVer, 13, 1611–1613.
[39] Sullivan,R. et al. (2008) Hypoxia-induced resistance to anticancer drugs is associated with decreased senescence and requires hypoxia-inducible factor-1 activity. Mol. Cancer Ther., 7, 1961–1973.
[40] Giatromanolaki,A. et al. (2001) Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular proWle of tumours and survival. Br. J. Cancer., 85,881–890.
[41] Liu,L. et al. (2008) Hypoxia-inducible factor-1 alpha contributes to hypoxia- induced chemoresistance in gastric cancer. Cancer Sci., 99, 121–128.
[42] Schindl,M. et al. (2002) Overexpression of hypoxia-inducible factor 1alpha is associated with an unfavorable prognosis in lymph node-positive breast cancer.
Clin. Cancer Res., 8, 1831–1837.
[43] Welsh,S.J. et al. (2002) The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis. Cancer Res., 62, 5089–5095.
[44] Birner,P. et al. (2000) Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer.
Cancer Res., 60, 4693–4696.
[45] Piret,J.P. et al. (2006) Hypoxia protects HepG2 cells against etoposide- induced apoptosis via a HIF-1-independent pathway. Exp. Cell Res., 312, 2908–
2920.
[46] Wu,X.A. et al. (2007) Impact of RNA interference targeting hypoxia- inducible factor-1alpha on chemosensitivity in esophageal squamous cell carcinoma cells under hypoxia. Zhonghua Yi Xue Za Zhi, 87, 2640–2644.
[47] Wartenberg,M. et al. (2001a) Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells. Faseb. J., 15, 995–1005.
[48] Wartenberg,M. et al. (2001b) Down-regulation of intrinsic P-glycoprotein expression in multicellular prostate tumor spheroids by reactive oxygen species. J.
Biol. Chem., 276, 17420–17428.
[49] Ogiso,Y. et al. (2000) Proteasome inhibition circumvents solid tumor resistance to topoisomerase II-directed drugs. Cancer Res., 60, 2429–2434.
[50] Weissenberger,C. et al. (2000) Is there any correlation between MDR1, GST- pi-expression and CEA?. Anticancer Res., 20, 5139–5144.
[51] Graeber,T.G. et al. (1996) Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature, 379, 88–91.
[52] Gardner,L.B. et al. (2001) Hypoxia inhibits G1/S transition through regulation of p27 expression. J. Biol. Chem., 276, 7919–7926.
[53] Wenger,R.H. et al. (1998) Up-regulation of hypoxia-inducible factor-1alpha is not suYcient for hypoxic/anoxic p53 induction. Cancer Res., 58, 5678–5680.
6.Figure Legends
Table1AC-301 1.105±0.042* AC-368 1.034±0.062 AC-435 0.893±0.075 AC-481 1.680±0.036 AC-302 0.924±0.044* AC-369 0.765±0.069 AC-438 0.979±0.036* AC-482 0.976±0.045 AC-303 0.908±0.037* AC-370 2.118±0.060 AC-439 0.955±0.074 AC-483 1.863±0.117 AC-304 0.945±0.042* AC-371 1.166±0.072 AC-440 1.036±0.122 AC-484 0.984±0.027 AC-306 1.391±0.050 AC-372 1.081±0.028* AC-441 1.186±0.046* AC-485 0.872±0.029 AC-307 0.942±0.002** AC-373 1.127±0.060 AC-442 1.761±0.099 AC-486 0.895±0.013 AC-332 0.957±0.017* AC-374 1.014±0.047* AC-443 1.025±0.097 AC-487 1.297±0.036 AC-334 1.081±0.092 AC-375 1.159±0.041* AC-445 0.921±0.031* AC-488 1.062±0.027 AC-335 2.140±0.206 AC-376 1.077±0.032* AC-446 0.852±0.020* AC-506 1.583±0.109 AC-336 0.987±0.077 AC-377 1.135±0.030* AC-447 1.144±0.045* AC-507 2.466±0.200 AC-337 1.091±0.034* AC-378 0.865±0.042* AC-448 0.855±0.012* AC-510 0.901±0.038 AC-338 1.018±0.034* AC-379 1.734±0.032* AC-449 0.978±0.060 AC-513 0.785±0.023 AC-339 1.005±0.025* AC-380 0.882±0.067 AC-450 1.640±0.050 AC-514 1.148±0.036 AC-340 1.174±0.096 AC-381 0.753±0.039* AC-451 0.907±0.021* AC-515 1.595±0.047 AC-341 0.997±0.030* AC-382 1.124±0.047* AC-452 0.930±0.072 AC-516 1.018±0.051 AC-342 1.042±0.056 AC-383 0.889±0.053 AC-453 0.849±0.028* AC-517 1.223±0.032 AC-343 1.023±0.027* AC-384 0.963±0.084 AC-454 1.099±0.036* AC-518 1.060±0.028 AC-344 1.035±0.045* AC-385 0.753±0.028* AC-455 1.049±0.092 AC-519 1.169±0.012 AC-345 1.201±0.164 AC-386 1.075±0.094 AC-456 1.028±0.022* AC-520 1.043±0.055 AC-346 1.004±0.015* AC-387 0.815±0.040* AC-457 2.013±0.095 AC-521 0.727±0.028 AC-347 0.967±0.033* AC-414 0.812±0.052 AC-458 1.230±0.046* AC-522 1.108±0.056 AC-348 1.093±0.087 AC-415 0.891±0.030* AC-459 1.690±0.064 AC-523 1.218±0.046 AC-349 0.942±0.049* AC-416 1.153±0.122 AC-461 0.877±0.021* AC-524 1.121±0.029 AC-351 1.010±0.029* AC-417 1.042±0.145 AC-462 0.757±0.055 AC-525 1.090±0.144 AC-352 1.048±0.083 AC-418 1.174±0.149 AC-463 0.845±0.012* AC-526 0.867±0.058 AC-353 0.916±0.028* AC-419 1.123±0.228 AC-464 0.932±0.036* AC-527 1.082±0.028 AC-354 0.951±0.059 AC-420 1.306±0.142 AC-465 0.999±0.061 AC-529 1.184±0.010 AC-355 1.082±0.045* AC-422 0.921±0.011* AC-466 0.780±0.028* AC-530 1.083±0.029 AC-356 1.386±0.069 AC-423 1.128±0.016* AC-469 0.925±0.033* AC-531 1.091±0.010
AC-357 0.821±0.031* AC-424 0.898±0.002** AC-470 0.846±0.032* AC-532 0.995±0.034 AC-358 2.142±0.618 AC-425 1.085±0.037* AC-471 1.766±0.023* AC-533 0.867±0.040 AC-359 1.038±0.033* AC-426 0.889±0.024 AC-472 1.707±0.074 AC-534 1.198±0.083 AC-360 0.937±0.029* AC-427 1.026±0.048 AC-473 1.253±0.023* AC-535 1.179±0.039*
AC-361 1.057±0.085 AC-428 1.159±0.191 AC-474 2.040±0.048* AC-536 1.078±0.028*
AC-362 0.979±0.064 AC-429 1.188±0.113 AC-475 1.494±0.050 AC-537 1.043±0.052 AC-363 1.126±0.259 AC-430 1.787±0.537 AC-476 1.836±0.007** AC-538 1.527±0.072 AC-364 1.762±0.436 AC-431 1.136±0.143 AC-477 1.479±0.036* AC-539 1.109±0.020*
AC-365 0.999±0.322 AC-432 0.995±0.017 AC-478 1.237±0.065 AC-540 1.132±0.009**
AC-366 0.829±0.064 AC-433 1.115±0.039 AC-479 1.551±0.068 AC-367 1.495±0.202 AC-434 1.778±0.548 AC-480 1.154±0.024*
Table 1.
Screening library using the rhodamine-123 retention assay. After incubation of MCF-7/ADR cells with 20 μM R-123 for 90 min, the R-123 fluorescence values in cell lysates were measured using the excitation and emission wavelengths of 480 and 540 nm, respectively. 10μM of each compound was added 24 h before rhodamine-123 loading. The values were divided by total protein contents of each sample. Data represents means ± SD of 4 separate samples (significant versus the control MCF-7/ADR cells, **p<0.01; significant versus the vehicle treated MCF-7/ADR cells, *p<0.05).Fig1A
Fig1B
Figure 1A.
Expression of MDR1 protein with a series of compounds devided 3 groups. Western blot analysis was performed in MCF-7/ADR cells treated with 10μM of each compound for 24h.Figure 1B.
Inhibitory effect of AC-507 on MDR1 expression. MCF-7/ADR cells were incubated with AC-507 (0.3-10μM) for 24h and total lysates were subjected to immunoblotting for MDR1.Fig.2
MCF7/ADR 30min 24h
Relative Rhodamine-123 retention (%)
0 50 100 150 200 250
**
**
Figure 2.
Role of AC-507 as a dual inhibitor. Rhodamine-123 retention. After incubation of MCF-7 and MCF-7/ADR cells with 20 μM R-123 for 90 min, the R- 123 fluorescence values in cell lysates were measured using the excitation and emission wavelengths of 480 and 540 nm, respectively. AC-507 10μM was added 30min and 24 h before rhodamine-123 loading. The values were divided by total protein contents of each sample. Data represents means ± SD of 6 separate samples (significant versus the control MCF-7 cells, **p<0.01; significant versus the vehicle treated MCF-7/ADR cells, #p<0.05).Fig3A
Fig3B
MCF7/ADR 0.1 0.3 1 3 10
Doxorubicin uptake (%)
0 20 40 60 80 100 120 140
( Doxorubicin, mM )
MCF7/ADR 0.3uM 1uM 3uM 10uM
Survival (%)
0 20 40 60 80 100
120 *
*
(Doxorubicin)
MCF7/ADR 0.3uM 1uM 3uM 10uM
Survival (%)
0 20 40 60 80 100 120
(Doxorubicin)
*
**
**
Figure 3A.
MCF-7/ADR cells overcame doxorubicin resistance by AC-507.Cellular uptake of doxorubicin. After incubation of MCF-7/ADR cells with AC- 507 (0.1-10μM) for 24h, doxorubicin (30μM) was treated for 60min. Intensities of doxorubicin fluorescence retained in cell lysates of MCF-7/ADR were measured using the excitation and emission wavelengths of 470 and 590nm respectively. The values were divided by total protein content of each sample. Data represent the means ± SD of 3 different samples (significant versus the untreated MCF-7/ADR cells, *p<0.05; **p<0.01).
Figure 3B.
Cell viability of doxorubicin treatment after AC-507 pretreatment.Cell viabilities were determined by MTT assays 24 h after exposure of MCF- 7/ADR cells with Dox (3and 10μM) with AC-507 (0.3-10μM) pretreatment. Data represent means ± SDs of 6 separate samples.
Fig4
Figure 4.
Nuclear levels of diverse proteins. Representative immunoblots illustrate the expression of nucrear fractions in MCF-7/ADR cells incubated with 0.3-10μM AC-507 for 24h.저작물 이용 허락서
학 과 약학과 학 번 20097137 과 정 석사, 박사 성 명 한글: 이 원 영 한문 :李 源 榮 영문 :Lee Won Young 주 소 서울시 송파구 잠실본동 우성 4 차아파트 102-601
연락처 E-MAIL : lee320@hanmail.net
논문제목 한글 : 화합물 라이브러리에서 MDR1 이중 저해제 발굴
영어 : Identification of MDR1 dual inhibitor from chemical library
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동의여부 : 동의( ○ ) 반대( ) 2010 년 10 월 15 일
저작자: 이 원 영 (서명 또는 인)
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