Hsp90 Inhibitor, 17-AAG, Affects Early Embryonic Development and Apoptosis of Bovine Embryos

Hsp90의 저해제인 17-AAG의 처리에 따른 소 수정란의 배발달 및 세포사멸 양상

  • Hong, Joo-Hee (Department of Biotechnology, College of Engineering, Daegu University) ;
  • Min, Sung-Hun (Department of Biotechnology, College of Engineering, Daegu University) ;
  • Lee, E-Nok (Department of Biotechnology, College of Engineering, Daegu University) ;
  • Son, Hyeong-Hoon (Department of Biotechnology, College of Engineering, Daegu University) ;
  • Park, Hum-Dai (Department of Biotechnology, College of Engineering, Daegu University) ;
  • Koo, Deog-Bon (Department of Biotechnology, College of Engineering, Daegu University)
  • 홍주희 (대구대학교 공과대학 생명공학과) ;
  • 민성훈 (대구대학교 공과대학 생명공학과) ;
  • 이에녹 (대구대학교 공과대학 생명공학과) ;
  • 손형훈 (대구대학교 공과대학 생명공학과) ;
  • 박흠대 (대구대학교 공과대학 생명공학과) ;
  • 구덕본 (대구대학교 공과대학 생명공학과)
  • Received : 2011.08.31
  • Accepted : 2011.09.07
  • Published : 2011.09.30

Abstract

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of several cells. In our previous study, inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the pig embryonic and primary cells was reported. However, its role during early bovine embryonic development is not sufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on early bovine embryonic development. We also investigated several indicators of developmental potential, including structural integrity, gene expression (apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Bovine embryos were cultured in the CR1-aa medium with or without 17-AAG for 7 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG ($33.1{\pm}9.6$ vs $21.7{\pm}8.3%$). The structural integrity of the blastocysts was examined by differential staining. Blastocysts from the dbcAMP-treated group had higher numbers of ICM, TE, and total cells than those from the untreated group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (11.2 vs 3.9, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation bovine blastocysts. The mRNA expression of the pro-apoptotic gene (Bax) increased in 17-AAG treated group, whereas expression of the antiapoptotic gene (Bcl-XL) decreased. In conclusion, Hsp90 also appears to play a direct role in bovine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with apoptosis-related genes expression in developing bovine embryos.

Keywords

References

  1. Basso AD, Solit DB, Munster PN, Rosen N (2002): Ansamycin antibiotics inhibit Akt activation and cyclin D expression in breast cancer cells that overexpress HER2. Oncogene 21(8):1159-1166. https://doi.org/10.1038/sj.onc.1205184
  2. Betts DH, King WA (2001): Genetic regulation of embryo death and senescence. Theriogenology 55(1):171- 191. https://doi.org/10.1016/S0093-691X(00)00453-2
  3. Burrows F, Zhang H, Kamal A (2004): Hsp90 activation and cell cycle regulation. Cell Cycle 3(12): 1530-1536. https://doi.org/10.4161/cc.3.12.1277
  4. Datta SR, Brunet A, Greenberg ME (1999): Cellular survival: a play in three Akts. Genes Dev 13(22): 2905-2927. https://doi.org/10.1101/gad.13.22.2905
  5. Edwards MJ (1998): Apoptosis, the heat shock response, hyperthermia, birth defects, disease and cancer. Where are the common links? Cell Stress Chaperones 3(4):213-220. https://doi.org/10.1379/1466-1268(1998)003<0213:ATHSRH>2.3.CO;2
  6. Hao Y, Lai L, Mao J, Im GS, Bonk A, Prather RS (2004): Apoptosis in parthenogenetic preimplantation porcine embryos. Biol Reprod 70(6):1644-1649. https://doi.org/10.1095/biolreprod.103.026005
  7. Machaty Z, Day BN, Prather RS (1998): Development of early porcine embryos in vitro and in vivo. Biol Reprod 59(2):451-455. https://doi.org/10.1095/biolreprod59.2.451
  8. Neckers L (2002): Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol Med 8(4 Suppl): S55-61.
  9. Pearl LH, Prodromou C, Workman P (2008): The Hsp- 90 molecular chaperone: an open and shut case for treatment. Biochem J 410(3):439-453. https://doi.org/10.1042/BJ20071640
  10. Schulte TW, Blagosklonny MV, Ingui C, Neckers L (1995): Disruption of the Raf-1-Hsp90 molecular complex results in destabilization of Raf-1 and loss of Raf-1-Ras association. J Biol Chem 270(41):24585- 24588. https://doi.org/10.1074/jbc.270.41.24585
  11. Senju M, Sueoka N, Sato A, Iwanaga K, Sakao Y, Tomimitsu S, Tominaga M, Irie K, Hayashi S, Sueoka E (2006): Hsp90 inhibitors cause G2/M arrest associated with the reduction of Cdc25C and Cdc2 in lung cancer cell lines. J Cancer Res Clin Oncol 132: 150-158. https://doi.org/10.1007/s00432-005-0047-7
  12. Solit DB, Chiosis G (2008): Development and application of Hsp90 inhibitors. Drug Discov Today 13 (1-2):38-43. https://doi.org/10.1016/j.drudis.2007.10.007
  13. Son MJ, Park JM, Min SH, Hong JH, Park H, Koo DB (2011): Hsp90 inhibitor induces cell cycle arrest and apoptosis of early embryos and primary cells in pigs. Reprod Dev Biol 35(1):33-45.
  14. Supko JG, Hickman RL, Grever MR, Malspeis L (1995): Preclinical pharmacologic evaluation of geldanamycin as an antitumor agent. Cancer Chemother Pharmacol 36(4):305-315. https://doi.org/10.1007/BF00689048
  15. Taldone T, Gozman A, Maharaj R, Chiosis G (2008): Targeting Hsp90: small-molecule inhibitors and their clinical development. Current Opinion in Pharmacology 8(4):370-374. https://doi.org/10.1016/j.coph.2008.06.015
  16. Whitesell L, Shifrin SD, Schwab G, Neckers LM (1992): Benzoquinonoid ansamycins possess selective tumoricidal activity unrelated to src kinase inhibition. Cancer Res 52(7):1721-1728.
  17. Workman P (2004): Combinatorial attack on multistep oncogenesis by inhibiting the Hsp90 molecular chaperone. Cancer Lett 206(2):149-157. https://doi.org/10.1016/j.canlet.2003.08.032
  18. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, Wang X (1997): Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275(5303):1129-1132. https://doi.org/10.1126/science.275.5303.1129