Figure 1. Regulation of MRP2 by FoxO1. (A) Putative binding sites to FoxO in the proximal promoter region of human MRP2 gene. The predicted binding sites for transactivation factors are demonstrated. The numbering is relative to the major transcription initiation site, which is marked by an arrow. The asterisk (∗, ∗∗) indicates putative FoxO binding sites based on highly conserved sequence of Forkhead protein.
(B) Induction of MRP2 proteins by FoxO overexpression. MCF-7 cells were cultured in transfection optimized medium with pcDNA3-FoxO1 or pcDNA3-FoxO3 for 6 h and in serum deprived medium for 18 h.
Cell lysates were subjected to immunoblotting analysis with anti-MRP2 antibody. Equal loading of proteins was verified by actin immunoblotting. (C) FoxO1 up-regulates MRP2 transcriptional activity in MCF-7 cells. MCF-7 cells were co-transfected with a p2635-human MRP2 reporter construct and pcDNA3-FoxO1 in a concentration dependent manner (3-30 ng), followed by reporter assays. The data represents the mean ± S.D. of 3 separate samples (significant versus the control, ∗p<0.05; ∗∗p<0.01; control level = 1)
Figure 2. Activation of MRP2 in TAMR-MCF-7 cells. (A) Immunoblot analysis of MRP2, FoxO1 and FoxO3 in the total cell lysates or nuclear fractions. A representative immunoblot shows MRP2 protein in both MCF-7 and TAMR-MCF-7 cells serum-deprived for 24 h (upper panel). Nuclear fractions and total cell lysates were also obtained from both MCF-7 and TAMR-MCF-7 cells in same conditions, and FoxO1 (middle panel) and FoxO3 (lower panel) in each fraction were immunoblotted with the respective antibody. (B) Reporter activities of deletion mutant human MRP2 promoters in MCF-7 and TAMR-MCF-7 cells. Each cell type was transiently transfected with p2635-MRP2-Luc (left panel) or p491-MRP2-Luc (right panel) plasmid.
Dual luciferase reporter assays were performed on the lysed cells co-transfected with pMRP2-Luc plasmid (firefly lucifefase) and phRL-SV (hRenilla luciferase) (a ratio of 100:1) 18 h after transfection.
Reporter gene activation was calculated as a relative ratio of firefly luciferase to hRenilla luciferase activity. Data represent means ± SD with 6 different samples (significant versus the control, ∗∗p<0.01;
control level = 1). (C) Inhibition of MRP2 protein expression and transcriptional activity by FoxO1 suppression. MRP2 and FoxO1 levels were determined by immunoblotting in TAMR-MCF-7 cells transfected
with FoxO1 siRNA (60 pmole) or control siRNA (60 pmole) (left panel). MCF-7 and TAMR-MCF-7 cells were co-transfected with p2635-MRP2-Luc in combination with FoxO1 siRNA (20 pmole) or control siRNA (20 pmole) (right panel). Data represent the means ± SD with 6 separate samples (significant when compared with MCF-7 cells transfected with con siRNA, ∗∗p<0.01; significant when compared with TAMR-MCF-7 cells transfected with con siRNA, ##p<0.01;
control level = 1).
Figure 3. The factors related with FoxO1 regulation mechanism. (A) Akt and phosphorylated Akt levels were detected in TAMR-MCF-7 cells (left panel) and MCF-7/ADR cells (right panel) compared with MCF-7 cells by immunoblotting. (B) SIRT1 protein expression in TAMR-MCF-7 cells (upper, left panel) and MCF-7/ADR cells (upper, right panel). SIRT1 activities were measured by Fluor de Lys Fluorescence assay. Cell lysates of TAMR-MCF-7 cells (lower, left panel) and MCF-7/ADR cells (lower, right panel) were obtained from the cells cultured in serum deprived medium for 24 h. Instead of SIRT1 enzyme, cell lysates were used to detect the relative activity of SIRT1.
Data represents means ± SD with 3 different samples (significant versus the control MCF-7 cells, ∗∗p<0.01; control level = 1) (C) The effects of PI3-kinase inhibitor and SIRT1 inhibitor on the expression of FoxO1 and MRP2 in TAMR-MCF-7 cells. Expression of FoxO1 and MRP2 was measured in TMAR-MCF-7 cells treated with PI3-kinase inhibitor (LY; LY294002 20 µM) and SIRT1 inhibitor (Nam;
nicotinamide 1 mM) for 24 h.
Figure 4. Requirements of SIRT1 to induce FoxO1-mediated multidrug resistant proteins. (A) Inhibition of p2635-MRP2 and p491-MRP2 reporter gene activation in TAMR-MCF-7 cells by SIRT1 suppressions.
TAMR-MCF-7 cells were treated Nam (nicotinamide; 1 mM), 10 min after transfection of cells with two types of MRP2 reporter plasmids and then the luciferase activity was measured 18 h after inhibitors treatment. Data represent the means ± SD of 3 different samples (significant when compared with control treated cells, ∗∗p<0.01;
control level = 1). (B) Transactivation of MRP2 and MDR1 by SIRT1.
Reporter activity of p2635-MRP2-Luc (left panel) and p195-MDR1-Luc (right panel) was measured in MCF-7 cells transiently co-transfected with SIRT1-constitutive active plasmid (30-300 ng) or pCMV5 vector. Data represents means ± SD with 3 different samples (significant versus the control, ∗p<0.05; ∗∗p<0.01;
control level = 1). (C) Increased transcriptional activity of FHRE promoter by SIRT1. MCF-7 cells transiently co-transfected with FHRE reporter plasmid in combination with SIRT1-constitutive active plasmid (30-300 ng) or pCMV5 vector. Data represent the means ± SD of 3 different samples (significant versus the control, ∗p<0.05;
∗∗p<0.01; control level = 1).
Figure 5. The complementary effects of FoxO1 and SIRT1. (A) Up-regulation of MRP2 and MDR1 transactivation by FoxO1 and/or SIRT1. MCF-7 cells were co-transfected with pcDNA3-FoxO1 (30 ng), constitutive active form of SIRT1 (300 ng) plasmid and both of them with p2635-MRP2 (left panel) or p195-MDR1 (right panel) reporter plasmid and then the luciferase activities were measured 18 h after transfection. Data represent the means ± SD of 3 different samples (significant as compared to pCMV5-transfected MCF-7 cells,
∗∗p<0.01; significant as compared to FoxO1-trasnfected MCF-7 cells,
##<0.01; control level = 1). (B) Reversion of SIRT1 induced MRP2
and MDR1 transactivation by FoxO1 suppression. p2635-MRP2 reporter activities were measured in MCF-7 cells co-transfected with pCMV5 or SIRT1 (300 ng) in combination with FoxO1 siRNA (left panel). p195-MDR1 reporter activities were also measured in the same way (right panel). Data represents means ± SD with 3 different samples (significant when compared with control siRNA and pCMV5-transfected MCF-7 cells, ∗∗p<0.01; significant when compared with control siRNA and SIRT1-transfected MCF-7 cells,
#p<0.05; ##p<0.01; control level = 1)
Figure 6. Effects of SIRT1-regulatory natural compounds, CPP342 and CPP343 on FoxO1-mediated MRP2 expressions. (A) Schematic of CPP342 and CPP343 structure. (B) Inhibitory effect of CPP343 on SIRT1-induced FHRE transactivation. MCF-7 cells were transfected with FHRE reporter plasmid together with pCMV5 or SIRT1 (300 ng).
Six hours after transfection, the cells were either maintained as controls or were treated with CPP343 (0.1-1 µg/ml) for further 18 h. Data represent the means ± SD of 3 different samples (significant as compared to pCMV5-transfected MCF-7 cells, ∗∗p<0.01; significant as compared to SIRT1-trasnfected and untreated control MCF-7 cells,
#p<0.05; ##<0.01; control level = 1). (C) Effect of CPP342 on MDR1
and MRP2 proteins. MCF-7/ADR cells (upper panel) and TAMR-MCF-7 cells were incubated with gradually increased concentrations (0.3-3 µg/ml) of CPP342 for 24 h and expression of MDR1 and MRP2 levels were measured by immunoblotting respectively. (D) Effect of CPP343 on MDR1 and MRP2 proteins. In addition, nuclear level of FoxO1 and FoxO3 was also measured. Total cell lysates and nuclear fractions of MCF-7/ADR cells and TAMR-MCF-7 cells were taken after treating CPP343 (0.3-3 µg/ml) for 24 h.
Figure 7. Overcome of the resistance to doxorubicin in MCF-7/ADR cells treated with CPP343. (A) Cell viability after treating with CPP343.
After incubation of TAMR-MCF-7 (left panel) and MCF-7/ADR (right panel) cells with or without CPP343 (0.1-3 µg/ml) for 24 h, cell viabilities were determined using MTT assays. Data represent the means ± SD of 14 different samples (control level = 1). (B) Doxorubicin cellular uptake. After incubation of MCF-7 and MCF-7/ADR cells with or without CPP343 (0.1-3 µg/ml) for 24 h, doxorubicin (30 µM) was treated for 60 min. Fluorescence intensities of doxorubicin retained in lysates of MCF-7 and MCF-7/ADR were measured using the excitation and emission wavelengths of 470 and 590 nm, 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; control level = 1). (C) Cell viability after treating with
doxorubicin with or without CPP343 treatment. Doxorubicin (30 µM) was treated 24 h after exposure of MCF-7 and MCF-7/ADR cells with or without CPP343 (0.1-1 µg/ml). Cell viabilities were determined by crystal violet assays. Data represent the means ± SD of 14 different samples (significant versus the doxorubicin-treated and CPP343 control
MCF-7/ADR cells, ∗∗p<0.01; control level = 1). (D) Representative photographs of TUNEL assays on cells cultured with or without 30 µM doxorubicin for 24 h. MCF-7/ADR cells were pretreated with CPP343 (0.3-1 µg/ml) 24 h before doxorubicin exposure. Results were confirmed by multiple experiments.
저작물 저작물 저작물
저작물 이용 이용 이용 허락서 이용 허락서 허락서 허락서
학 과 약학과 학 번 20077059 과 정 석사 성 명 한글: 조 경 빈 한문 : 趙 慶 彬 영문 : Cho, Kyoung Bin
주 소 광주광역시 북구 용봉동 모아미래도 아파트 101 동 1102 호 연락처 E-MAIL : ccakduky@hanmail.net
논문제목
한글 : 타목시펜 저항성 유방암 세포에서 MRP2 를 발현 시키는 FoxO1 활성에 대한 SIRT1 의 역할
영어 : Requirement of SIRT1-mediated FoxO1 activation for Multidrug Resistance-Associated Protein 2 expression in Tamoxifen resistant breast cancer cells
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저작자: 조 경 빈 (서명 또는 인)
감사의감사의 감사의감사의 글글글글
아직도 부족한 논문에 자꾸만 손이 가서 읽고, 또 읽고, 또 고치기를 반복하다가 드디어 마음 먹고 이 곳에 다다랐습니다. 섣불리 도전했던 지난 학기에 이어 졸업 준비를 먼저 시작했음에도 불구하고 여전히 모자람을 많이 느끼고 있습니다. 한편으로 생각해 보면 이런 나 자신이 이렇게 구색을 갖추어서나마 논문을 낼 수 있게 되기까지는 정말 많은 사람의 도움이 있었을 것이라는 생각을 다시금 해보게 됩니다. 그런 만큼, 다소 허술한 점이 있더라도 논문에 실려 있는 고마운 사람들의 무게만은 감히 재어 볼 수도 없을 것 같습니다. 그런데도 이런 고마움을 표현하기란 왜 이렇게 쑥스러운지 영어로 쓰는 논문 못지 않게 쉽사리 말을 이어 나가기 벅찹니다. 고마움과 죄송한 마음이 교차하여 잠깐씩 숨을 고르며 천천히 한 분, 한 분을 떠올려야겠습니다. 이 글을 읽어 주시는 모든 사람들이 감사의 대상이 될 것이고, 그 분들이 이 글을 읽으며 제 진심을 알아주는 것에도 의의가 있지만 저 자신 역시 앞으로 그 고마움을 잊지 않고 더욱 열심히 살아가기 위해 이 한 글자, 한 글자를 정성스럽게 채우려 합니다.
격려와 지도를 아끼지 않고 늘 열성적인 모습으로 저를 포함한 많은 학생들의 표본이 되어 주신 약학과 교수님들께 감사합니다. 지도 교수님인 강건욱 교수님께는 가장 먼저 감사의 인사 올립니다. 나태해질 때마다 교수님께서 해 주신 말씀들과 즐겁게 연구하시는 모습을 보고 마음가짐을 다잡을 수 있었습니다. 학부생 시절 약물학 실험 노트의
고찰 밑에 달아주신 잘했다는 교수님의 답글 하나로 흥미가 생겨 실험실에 첫발을 들여 놓게 되었고 연구의 꿈을 키우게 되었습니다.
잘하고 잘못함을 엄격하게 구분하여 적절하게 칭찬과 훈계를 해 주셔서 즐거움과 반성하는 마음으로 제가 조금씩 발전하고 진지하게 실험에 임할 수 있게 된 것 같습니다. 위로와 격려의 말씀 건네며 힘을 주신 한효경 교수님, 정혜광 교수님, 최후균 교수님, 실험에 대한 새로운 지식을 깨닫게 해주고 이 논문에 결정적인 도움을 주신 최홍석 교수님과 오원근 교수님께도 이루 말할 수 없는 감사의 마음을 전합니다.
가끔 아옹다옹 하기도 했지만 무려 2년 동안 함께하며 잘 챙겨주고 많이 알려 주었던 실험실 동기인 상은 오빠와 미라에게 고맙습니다.
또한 이번에 같이 졸업하게 된 Yuba씨와 네팔에 있는 그리운 Sindhu씨, Shreeya에게도 전합니다 ‘I express my heartfelt gratitude to Yuba, Sindhu, Shreeya. I really miss you!’. 헤어질 것을 생각하면 섭섭하지만 모두 하고자 하는 바람 이루어 멋진 모습으로 다시 만나기를 진심으로 기원합니다. 언제나 편안하게 대해주며 실험에 관한 정보는 물론, 귀여운 현민이 이야기와 맛있는 이야기로 활력을 불러준 정우 오빠와 옥이 언니에게도 고마움을 전합니다. 새로운 베트남 학생인 Phuong이 실험실에서 즐겁게 생활하며 열심히 공부하길 바랍니다.
선배로써 많은 것을 알려주고 실험에 대해 많은 지도와 격려 아끼지 않았던 창엽 오빠와 상희 언니, 먼 곳에서 찾아와 준 정용 오빠와 진원 오빠에게도 감사합니다. 미국 학회 & 라스베가스 동지인 생화학실 나연이, 약전실의 온갖 지식인인 영빈이와 여유로움이 부러운 친구