Nuclear Medicine and Molecular Imaging

The Korean Society of Nuclear Medicine (대한핵의학회)
권호 표지

Increases in Doxorubicin Sensitivity and Radioiodide Uptake by Transfecting shMDR and Sodium/Iodide Symporter Gene in Cancer Cells Expressing Multidrug Resistance

다약제내성 암세포에서 shMDR과 Sodium/Iodide Symporter 유전자의 이입에 의한 Doxorubicin 감수성과 방사성옥소 섭취의 증가

  • Ahn, Sohn-Joo (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Lee, Yong-Jin (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Lee, You-La (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Choi, Chang-Ik (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Lee, Sang-Woo (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Yoo, Jeong-Soo (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Ahn, Byeong-Cheol (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Lee, In-Kyu (Department of Internal Medicine, School of Medicine, Kyungpook National University) ;
  • Lee, Jae-Tae (Department of Nuclear Medicine, School of Medicine, Kyungpook National University)
  • 안손주 (경북대학교 의과대학 핵의학교실) ;
  • 이용진 (경북대학교 의과대학 핵의학교실) ;
  • 이유라 (경북대학교 의과대학 핵의학교실) ;
  • 최창익 (경북대학교 의과대학 핵의학교실) ;
  • 이상우 (경북대학교 의과대학 핵의학교실) ;
  • 유정수 (경북대학교 의과대학 핵의학교실) ;
  • 안병철 (경북대학교 의과대학 핵의학교실) ;
  • 이인규 (경북대학교 의과대학 내분비내과학교실) ;
  • 이재태 (경북대학교 의과대학 핵의학교실)
  • Published : 2007.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Purpose: Multidrug resistance (MDR) of the cancer cells related to mdr1 gene expression can be effectively treated by selective short hairpin RNA for mdr1 gene (shMDR). Sodium/iodide symporter (NIS) gene is well known to have both reporter and therapeutic gene characteristics. We have co-transfected both shMDR and NIS gene into colon cancer cells (HCT15 cell) expressing MDR and Tc-99m sestamibi and I-125 uptake were measured. In addition, cytotoxic effects of doxorubicin and I-131 therapy were also assessed after transfection. Material and Methods: At first, shMDR was transfected with liposome reagent into human embryonic kidney cells (HEK293) and HCT cells. shMDR transfection was confirmed by RT-PCR and western blot analysis. Adenovirus expressing NIS (Ad-NIS) gene and shMDR (Ad-shMDR) were co-transfected with Ad-NIS into HCT15 cells. Forty-eight hours after infection, inhibition of P-gycoprotein (Pgp) function by shMDR was analyzed by a change of Tc-99m sestamibi uptake and doxorubicin cytotoxicity, and functional activity of induced NIS gene expression was assessed with I-125 uptake assay. Results: In HEK293 cells transfected with shMDR, mdr1 mRNA and Pgp protein expressions were down regulated. HCT15 cells infected with 20 MOI of Ad-NIS was higher NIS protein expression than control cells. After transfection of 300 MOI of Ad-shMDR either with or without 10 MOI of Ad-NIS, uptake of Tc-99m sestamibi increased up to 1.5-fold than control cells. HCT15 cells infected with 10 MOI of Ad-NIS showed approximately 25-fold higher I-125 uptake than control cells. Cotransfection of Ad-shMDR and Ad-NIS resulted in enhanced cytotoxic by doxorubicin in HCT15 cells. I-131 treatment on HCT15 cells infected with 20 MOI of Ad-NIS revealed increased cytotoxic effect. Conclusion: Suppression of mdr1 gene expression, retention of Tc-99m sestamibi, enhanced doxorubicin cytotoxicity and increases in I-125 uptake were achieved in MDR expressing cancer cell by co-transfection of shMDR and NIS gene. Dual therapy with doxorubicin and radioiodine after cotransfection shMDR and NIS gene can be used to overcome MDR.

목적: mdr1유전자를 표적으로 한 short hairpin RNA (shMDR)는 다약재내성을 나타내는 암세포에서 효과적으로 mdr1 유전자의 발현을 억제 할 수 있고 sodium iodide symporter (NIS)는 유전자 치료와 리포터로의 기능을 동시에 나타낼 수 있다. 이 연구에서는 사람 대장암세포(HCT15)에 shMDR과 NIS를 동시에 이입하고 Tc-99m sestamibi와 I-125 섭취를 측정하였고 doxorubicin과 I-131 치료효과도 관찰하였다. 대상 및 방법: 사람 태아 신장 세포주(Human Embryonic Kidney cells; HEK293)에 liposome 시약으로 shMDR을 이입하고 RT-PCR과 western blot으로 분석하였다. shMDR와 NIS 유전자가 발현하는 adenovirus를 만들고 HCT15 세포에 이입 후 48시간에 shMDR에 의한 Pgp의 기능 억제를 확인하기위해 Tc-99m sestamibi 섭취와 doxorubicin 세포독성을 측정하였다. 또한 NIS유전자의 기능을 확인 하기위해 I-125 섭취와 I-131 세포독성도 확인하였다. 결과: shMDR이 이입 된 HEK293 세포에서 mdr1의 mRNA와 Pgp의 발현이 각각 75%, 80% 감소하였다. NIS 유전자가 발현하는 adenovirus를 HCT15 세포에 이입하고 NIS 유전자 발현을 확인 한 결과 대조군에 비해 월등히 높게 발현하였다. Ad-shMDR 300 MOI, Ad-shMDR 300 MOI 와 Ad-NIS 10 MOI를 처리한 경우 Tc-99m sestamibi의 섭취가 대조군보다 1.5배 정도 증가하였다. HCT15 세포에 Ad-NIS 10 MOI를 감염시킨 경우 I-125 섭취가 대조군에 비해 25배 이상 증가였다. 또한 Ad-shMDR와 Ad-NIS를 동시 감염 시켰을 경우 doxorubicin의 세포 독성이 증가하여 나타났고 Ad-NIS 20 MOI를 감염시켰을 때 I-131에 의한 세포독성이 대조군보다 증가하였다. 결론: 세포에 shMDR의 이입으로 mdr1 유전자의 발현이 억제되고 Tc-99m sestamibi의 섭취와 doxorubicin의 세포독성이 증가하였으며 NIS 유전자의 이입으로 I-125의 섭취와 I-131의 세포독성이 증가하였다. 다약제내성세포에 shMDR와 NIS 유전자의 동시 이입은 doxorubicin과 방사성 옥소의 이중치료 효과를 높일 수 있을 것으로 본다.

Keywords

References

  1. Eytan GD. Mechanism of multidrug resistance in relation to passive membrane permeation. Biomed Pharmacother 2005;59:90-7 https://doi.org/10.1016/j.biopha.2005.01.003
  2. Galina R, Jocelyne P, Sharom FJ. P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains. FEBS Journal 2005;272:4924-37 https://doi.org/10.1111/j.1742-4658.2005.04905.x
  3. Mahadevan D, List AF. Targeting the multidrug resistance-1 transporter inAML: molecular regulation and therapeutic strategies. Blood 2004;104:1940-51 https://doi.org/10.1182/blood-2003-07-2490
  4. Stierle V, Laigle A, Jolle B. Modulation of MDR1 gene expression in multidrug resistant MCF7 cells by low concentrations of small interfering RNAs. Biochemical Pharmacology 2005;70:1424-30 https://doi.org/10.1016/j.bcp.2005.08.007
  5. Matsui Y, Kobayashi N, Nishikawa M, Takakura Y. Sequence-specific suppression of mdr1a/1b expression in mice via RNA interference. Pharmaceutical Research 2005;22:2091-98 https://doi.org/10.1007/s11095-005-8178-8
  6. Schinkel AH, Jonker JW. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Advanced Drug Delivery Reviews 2003;55:3-29 https://doi.org/10.1016/S0169-409X(02)00169-2
  7. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Craig CM. Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature 1998;391:806-11 https://doi.org/10.1038/35888
  8. Hannon GJ. RNA interference. Nature 2002;418:244-51 https://doi.org/10.1038/418244a
  9. Wu H, Hait W, Yang JM. Small interfering RNA-induced suppression of MDR1 (P-Glycoprotein) restores sensitivity to multidrug-resistant cancer cells. Cancer Res 2003;63:1515-19
  10. Filetti S, Bidart JM, Arturi F, Caillou B, Russo D, Schlumberger M. Sodium/iodide symporter: a key transport system in thyroid cancer cell metabolism. Eur J Endocrinol 1999;141:443-57 https://doi.org/10.1530/eje.0.1410443
  11. Chung JK. Sodium Iodide Symporter: Its role in nuclear medicine. J Nucl Med 2002;43:1188-200
  12. Levy O, Vieja AD, Carrasco N. The Na+/I- Symporter (NIS): recent advances. J Bioenerg Biomembr 1998;30:195-206 https://doi.org/10.1023/A:1020577426732
  13. Lee YJ, Chung JK, Shin JH, Kang JH, Jeong JM, Lee DS et al. In vitro and in vivo properties of a human anaplastic thyroid carcinoma cell line transfected with the sodium iodide symporter gene. Thyroid 2004;14;889-95 https://doi.org/10.1089/thy.2004.14.889
  14. Miyagawa M, Anton M, Wagner B, Haubner R, Souvatzoglou M, Gansbacher B, et al. Non-invasive imaging of cardiac transgene expression with PET: comparison of the human sodium/iodide symporter gene and HSV1-tk as the reporter gene. Eur J Nucl Med Mol Imaging 2005;32:1108-14 https://doi.org/10.1007/s00259-005-1854-4
  15. Lee KH, Kim HK, Paik JY, Matsui T, Choe YS, Choi Y, et al. Accuracy of myocardial sodium/iodide symporter gene expression imaging with radioiodide: evaluation with a dual-gene adenovirus vector. J Nucl Med 2005;46:652-7
  16. Spitzweg C, Dietz AB, O'Connor MK, Bergert ER, Tindall DJ, Young CYF, et al. In vivo sodium iodide symporter gene therapy of prostate cancer. Gene Therapy 2001;8:1524-31 https://doi.org/10.1038/sj.gt.3301558
  17. Boland A, Ricard M, Opolon P, Bidart JM, Patrice Y, Filetti S, et al. Adenovirus-mediated transfer of the thyroid sodium/iodide symporter gene into tumors for a targeted radiotherapy. Cancer Res 2000;60:3484-92
  18. Chung JK, Kang JH. Translational research using the sodium/iodide symporter in imaging and therapy. Eur J Nucl Med Mol Imaging 2004;31:799-802 https://doi.org/10.1007/s00259-004-1475-3
  19. Kinuya S, Yokoyama K, Fukuoka M, Mori H, Shib K, Watanabe N, et al. Anti-angiogenic therapy and chemotherapy affect 99mTc sestamibi and 99mTc-HL91 accumulation differently in tumour xenografts. Nucl Med Commun 2005;26:1067-73 https://doi.org/10.1097/00006231-200512000-00004
  20. Rebbaa A, Zheng X, Chou PM, Mirkin BL. Caspase inhibition switches doxorubicin-induced apoptosis to senescence. Oncogene 2003;22:2805-11 https://doi.org/10.1038/sj.onc.1206366
  21. Kamiya H, Tsuchiya H, Yamazaki J, Harashima H. Intracellular trafficking and transgene expression of viral and non-viral gene vectors. Adv Drug Deliv Rev 2001;52:153-64 https://doi.org/10.1016/S0169-409X(01)00216-2
  22. Young LS, Searle PF, Onion D, Mautner V. Viral gene therapy strategies: from basic science to clinical application. J Pathol 2006;208:299-318 https://doi.org/10.1002/path.1896
  23. Takara K, Obata Y, Yoshikawa E, Kitada N, Sakaeda T, Ohnishi N, et al. Molecular changes to HeLa cells on continuous exposure to cisplatin or paclitaxel. Cancer Chemother Pharmacol 2006;22
  24. Burak Z, Moretti JL, Ersoy O, San U, Kantar M, Tamgac F, et al. 99mTc-MIBI imaging as a predictor of therapy response in osteosarcoma compared with multidrug resistance.associated protein and P-glycoprotein expression. J Nucl Med 2003;44: 1394-401
  25. Jekerle V, Wang JH, Scollard DA, Reilly RM, Wiese M, Micheline PM. 99mTc-Sestamibi, A sensitive probe for in vivo imaging of P-glycoprotein inhibition by modulators and mdr1 antisense oligodeoxynucleotides. Mol Imaging Biol 2006;13:60-6
  26. Lee J, Ahn BC. Detection of multidrug resistance using molecular nuclear technique. Korean J Nucl Med 2004;38:180-9
  27. Yoo JA, Chung SY, Seo MR, Kwak DS, Ahn BC, Lee KB, Lee J. Comparison of the uptakes of $^{99m}$Tc-sestamibi and $^{99m}$Tc-tetrofosmin in cancer cell lines expressiong multidrug resistance. Korean J Nucl Med 2003;37:178-89
  28. Liu Z, Stevenson GD, Barrett HH, Kastis GA, Bettan M, Furenlid LR, et al. 99mTc glucarate high-resolution imaging of drug sensitive and drug resistant human breast cancer xenografts in SCID mice. Nucl Med Commun 2004;25:711-20 https://doi.org/10.1097/01.mnm.0000130243.06821.90
  29. Morettil JL, Hauet1 N, Caglar M, Rebillard O, Burak Z. To use MIBI or not to use MIBI? That is the question when assessing tumour cells. Eur J Nucl Med Mol Imaging 2005;32:836-42 https://doi.org/10.1007/s00259-005-1840-x
  30. Dwyer RM, Bergert ER, O'Connor MK, Gendler SJ, Morris JC. Sodium iodide symporter-mediated radioiodide imaging and therapy of ovarian tumor xenografts in mice. Gene Therapy 2006;13:60-6 https://doi.org/10.1038/sj.gt.3302599
  31. Yaghoubi SS, Wu L, Liang Q, Toyokuni T, Barrio JR, Gambhir SS, et al. Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors. Gene Therapy 2001;8:1072-80 https://doi.org/10.1038/sj.gt.3301490
  32. Tjuvajev JG, Joshi A, Callegari J, Lindsley L, Joshi R, Blasberg RG. A general approach to the non-invasive imaging of transgenes using cis-linked herpes simplex virus thymidine kinase. Neoplasia 1999;1:315-20 https://doi.org/10.1038/sj.neo.7900053
  33. Yu Y, Annala AJ, Barrio JR, Toyokuni T, Satyamurthy N, Gambhir SS. Quantification of target gene expression by imaging reporter gene expression in living animals. Nat Med 2000;6:933-7 https://doi.org/10.1038/78704
  34. Baron U, Freundlieb S, Gossen M, Bujard H. Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res 1995;23:3605-6 https://doi.org/10.1093/nar/23.17.3605
  35. Bramson J, Hitt M, Gallichan WS, Rosenthal KL, Gauldie J, Graham FL. Construction of a double recombinant adenovirus vector expressing a heterodimeric cytokine: in vitro and in vivo production of biologically active interleukin-12. Hum Gene Therapy 1996;7:333-42 https://doi.org/10.1089/hum.1996.7.3-333
  36. Wood M, Perrotte P, Onishi E, Harper ME, Dinney C, Pagliaro L, et al. Biodistribution of an adenoviral vector carrying the luciferase reporter gene following intravesical or intravenous administration to a mouse. Cancer Gene Ther 1999;6:367-372 https://doi.org/10.1038/sj.cgt.7700090
  37. Hemminki A, Belousova N, Zinn KR, Liu B, Wang M, Chaudhuri TR, et al. An adenovirus with enhanced infectivity mediates molecular chemotherapy of ovarian cancer cells and allows imagingof gene expression. Mol Ther 2001;4:223-231 https://doi.org/10.1006/mthe.2001.0446
  38. Glasgow JN, Everts M, Curiel DT. Transductional targeting of adenovirus vectors for gene therapy. Cancer Gene Ther 2006; 13:830-844 https://doi.org/10.1038/sj.cgt.7700928