Abstract
Purpose
17-(demethoxy), 17-allylamino geldanamycin (17-AAG) suppresses growth in some cancers by inhibiting Heat shock protein 90 (Hsp90). We examined the effects of 17-AAG-mediated Hsp90 inhibition on human hepatocellular carcinoma (HCC) growth in vitro and in vivo.
Methods
Human HCC cell lines, Hep3B and HuH7, were exposed to 17-AAG and cell viabilities and apoptosis were determined. Cell cycle profiles were analyzed and the G2/M cell cycle checkpoint proteins cdc2 and cyclin B1 were examined. Studies were performed to determine whether 17-AAG-mediated cdc2 decrease was due to altered gene expression, transcription, or protein degradation. The effects of 17-AAG on Hep3B and HuH7 xenograft growth in athymic nude mice were also examined.
Results
Hep3B and HuH7 treated with 17-AAG versus untreated controls showed decreased cell viability and increased apoptosis. Cells treated with 17-AAG also showed an increased fraction in G2/M phase and an associated decrease in cdc2 through protein degradation rather than through other mechanisms. Hsp90 inhibition by 17-AAG also decreased HCC xenograft growth in association with decreased cdc2 expression.
Conclusions
17-AAG-mediated inhibition of Hsp90 abrogates human HCC cell growth in vitro and in vivo through cdc2 decrease, which in turn induces G2/M cell cycle arrest and apoptosis. Hsp90 is a mediator of HCC growth and survival and its inhibition may serve as a potential treatment.
Similar content being viewed by others
References
Bosch FX, Ribes J, Diaz M et al (2004) Primary liver cancer: worldwide incidence and trends. Gastroenterology 127:S5–S16
Young JC, Moarefi I, Hartl FU (2001) Hsp90: a specialized but essential protein-folding tool. J Cell Biol 154:267–273
Welch WJ, Feramisco JR (1982) Purification of the major mammalian heat shock proteins. J Biol Chem 257:14949–14959
Luk JM, Lam CT, Siu AF et al (2006) Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values. Proteomics 6:1049–1057
Calvisi DF, Pascale RM, Feo F (2007) Dissection of signal transduction pathways as a tool for the development of targeted therapies of hepatocellular carcinoma. Rev Recent Clin Trials 2:217–236
Scheufler C, Brinker A, Bourenkov G et al (2000) Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101:199–210
Pratt WB (1998) The hsp90-based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors. Proc Soc Exp Biol Med 217:420–434
Bagatell R, Whitesell L (2004) Altered Hsp90 function in cancer: a unique therapeutic opportunity. Mol Cancer Ther 3:1021–1030
Takayama S, Reed JC, Homma S (2003) Heat-shock proteins as regulators of apoptosis. Oncogene 22:9041–9047
Whitesell L, Lindquist SL (2005) HSP90 and the chaperoning of cancer. Nat Rev Cancer 5:761–772
Garcia-Morales P, Carrasco-Garcia E, Ruiz-Rico P et al (2007) Inhibition of Hsp90 function by ansamycins causes downregulation of cdc2 and cdc25c and G(2)/M arrest in glioblastoma cell lines. Oncogene 26(51):7185–7193
Senju M, Sueoka N, Sato A et al (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
Beliakoff J, Whitesell L (2004) Hsp90: an emerging target for breast cancer therapy. Anticancer Drugs 15:651–662
Lim SO, Park SG, Yoo JH et al (2005) Expression of heat shock proteins (HSP27, HSP60, HSP70, HSP90, GRP78, GRP94) in hepatitis B virus-related hepatocellular carcinomas and dysplastic nodules. World J Gastroenterol 11:2072–2079
Pascale RM, Simile MM, Calvisi DF et al (2005) Role of HSP90, CDC37, and CRM1 as modulators of P16(INK4A) activity in rat liver carcinogenesis and human liver cancer. Hepatology 42:1310–1319
Whitesell L, Mimnaugh EG, De Costa B et al (1994) Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci USA 91:8324–8328
Schulte TW, Neckers LM (1998) The benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin binds to HSP90 and shares important biologic activities with geldanamycin. Cancer Chemother Pharmacol 42:273–279
Clarke PA, Hostein I, Banerji U et al (2000) Gene expression profiling of human colon cancer cells following inhibition of signal transduction by 17-allylamino-17-demethoxygeldanamycin, an inhibitor of the hsp90 molecular chaperone. Oncogene 19:4125–4133
Chiosis G, Huezo H, Rosen N et al (2003) 17AAG: low target binding affinity and potent cell activity––finding an explanation. Mol Cancer Ther 2:123–129
Workman P (2004) Altered states: selectively drugging the Hsp90 cancer chaperone. Trends Mol Med 10:47–51
Seglen PO, Gordon PB (1982) 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci USA 79:1889–1892
Meijer L, Borgne A, Mulner O et al (1997) Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem 243:527–536
Gomez-Flores R, Gupta S, Tamez-Guerra R et al (1995) Determination of MICs for Mycobacterium avium-M. Intracellulare complex in liquid medium by a colorimetric method. J Clin Microbiol 33:1842–1846
Le Gac G, Esteve PO, Ferec C et al (2006) DNA damage-induced down-regulation of human Cdc25C and Cdc2 is mediated by cooperation between p53 and maintenance DNA (cytosine–5) methyltransferase 1. J Biol Chem 281:24161–24170
Jurchott K, Bergmann S, Stein U et al (2003) YB–1 as a cell cycle-regulated transcription factor facilitating cyclin A and cyclin B1 gene expression. J Biol Chem 278:27988–27996
Fujii T, Nomoto S, Koshikawa K et al (2006) Overexpression of pituitary tumor transforming gene 1 in HCC is associated with angiogenesis and poor prognosis. Hepatology 43:1267–1275
Yang J, Yang JM, Iannone M et al (2001) Disruption of the EF-2 kinase/Hsp90 protein complex: a possible mechanism to inhibit glioblastoma by geldanamycin. Cancer Res 61:4010–4016
Banerji U, Walton M, Raynaud F et al (2005) Pharmacokinetic-pharmacodynamic relationships for the heat shock protein 90 molecular chaperone inhibitor 17-allylamino, 17-demethoxygeldanamycin in human ovarian cancer xenograft models. Clin Cancer Res 11:7023–7032
Yin X, Zhang H, Burrows F et al (2005) Potent activity of a novel dimeric heat shock protein 90 inhibitor against head and neck squamous cell carcinoma in vitro and in vivo. Clin Cancer Res 11:3889–3896
Gannon JV, Nebreda A, Goodger NM et al (1998) A measure of the mitotic index: studies of the abundance and half-life of p34cdc2 in cultured cells and normal and neoplastic tissues. Genes Cells 3:17–27
Burger AM, Fiebig HH, Stinson SF et al (2004) 17-(Allylamino)-17-demethoxygeldanamycin activity in human melanoma models. Anticancer Drugs 15:377–387
Hostein I, Robertson D, DiStefano F et al (2001) Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis. Cancer Res 61:4003–4009
Giovannetti E, Backus HH, Wouters D et al (2007) Changes in the status of p53 affect drug sensitivity to thymidylate synthase (TS) inhibitors by altering TS levels. Br J Cancer 96:769–775
Bossi G, Sacchi A (2007) Restoration of wild-type p53 function in human cancer: relevance for tumor therapy. Head Neck 29:272–284
Kelland LR, Sharp SY, Rogers PM et al (1999) DT-Diaphorase expression and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock protein 90. J Natl Cancer Inst 91:1940–1949
de Carcer G (2004) Heat shock protein 90 regulates the metaphase-anaphase transition in a polo-like kinase-dependent manner. Cancer Res 64:5106–5112
Georgakis GV, Li Y, Rassidakis GZ et al (2006) Inhibition of heat shock protein 90 function by 17-allylamino-17-demethoxy-geldanamycin in Hodgkin’s lymphoma cells down-regulates Akt kinase, dephosphorylates extracellular signal-regulated kinase, and induces cell cycle arrest and cell death. Clin Cancer Res 12:584–590
Wolf F, Sigl R, Geley S (2007) ‘… The end of the beginning’: cdk1 thresholds and exit from mitosis. Cell Cycle 6:1408–1411
Castedo M, Perfettini JL, Roumier T et al (2002) Cyclin-dependent kinase-1: linking apoptosis to cell cycle and mitotic catastrophe. Cell Death Differ 9:1287–1293
Okamoto J, Mikami I, Tominaga Y et al (2008) Inhibition of Hsp90 leads to cell cycle arrest and apoptosis in human malignant pleural mesothelioma. J Thorac Oncol 3:1089–1095
Nomura M, Nomura N, Newcomb EW et al (2004) Geldanamycin induces mitotic catastrophe and subsequent apoptosis in human glioma cells. J Cell Physiol 201:374–384
Morla AO, Draetta G, Beach D et al (1989) Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis. Cell 58:193–203
Welch PJ, Wang JY (1992) Coordinated synthesis and degradation of cdc2 in the mammalian cell cycle. Proc Natl Acad Sci USA 89:3093–3097
Rubinsztein DC, Gestwicki JE, Murphy LO et al (2007) Potential therapeutic applications of autophagy. Nat Rev Drug Discov 6:304–312
Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717–1721
Nomura N, Nomura M, Newcomb EW et al (2007) Geldanamycin induces G2 arrest in U87MG glioblastoma cells through downregulation of Cdc2 and cyclin B1. Biochem Pharmacol 73:1528–1536
Bagatell R, Khan O, Paine-Murrieta G et al (2001) Destabilization of steroid receptors by heat shock protein 90-binding drugs: a ligand-independent approach to hormonal therapy of breast cancer. Clin Cancer Res 7:2076–2084
Grem JL, Morrison G, Guo XD et al (2005) Phase I and pharmacologic study of 17-(allylamino)-17-demethoxygeldanamycin in adult patients with solid tumors. J Clin Oncol 23:1885–1893
Banerji U, O’Donnell A, Scurr M et al (2005) Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino, 17-demethoxygeldanamycin in patients with advanced malignancies. J Clin Oncol 23:4152–4161
Ronnen EA, Kondagunta GV, Ishill N et al (2006) A phase II trial of 17-(Allylamino)-17-demethoxygeldanamycin in patients with papillary and clear cell renal cell carcinoma. Invest New Drugs 24:543–546
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Watanabe, G., Behrns, K.E., Kim, JS. et al. Heat shock protein 90 inhibition abrogates hepatocellular cancer growth through cdc2-mediated G2/M cell cycle arrest and apoptosis. Cancer Chemother Pharmacol 64, 433–443 (2009). https://doi.org/10.1007/s00280-008-0888-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00280-008-0888-2