Skip to main content

Advertisement

SpringerLink
Log in

Knockdown of antizyme inhibitor decreases prostate tumor growth in vivo

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

Abstract

The endogenous protein antizyme inhibitor (AZI) is a potential oncogene which promotes cell growth by both inhibiting antizyme (AZ) activity and releasing ornithine decarboxylase (ODC) from AZ-mediated degradation. High levels of ODC and polyamines are associated with numerous types of neoplastic transformation, and the genomic region including AZI is frequently amplified in tumors of the ovary and prostate. To determine whether AZI functionally promotes prostate tumor growth, we made PC3M-LN4 (human) and AT6.1 (rat) cancer cell lines stably expressing shRNA to knockdown antizyme inhibitor 1 (AZI). AZI knockdown was confirmed by western blot, quantitative real-time PCR, and immunofluorescence. To examine the ability of these cells to form tumors in vivo, 1 × 106 cells were injected subcutaneously into nude mice either with (PC3M-LN4) or without (AT6.1) Matrigel. Tumor growth was measured two times per week by caliper. We found that cells in which AZI levels had been knocked down by shRNA formed significantly smaller tumors in vivo in both human and rat prostate cancer cell lines. These results suggest that not only does AZI promote tumor growth, but also that AZI may be a valid therapeutic target for cancer treatment.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

ANOVA:

One-way analysis of variance

AT6.1:

Rat prostate cancer cells

AZ:

Antizyme

AZI:

Antizyme inhibitor

BE-4-4-4-4:

1,19-di-(ethylamino)-5,10,15-triazononadecane

DAPI:

4,6-Diamidino-2-phenylindole

DENSPM:

N1,N11-di(ethyl)norspermine

DFMO:

Difluoromethylornithine

GFP:

Green flourescent protein

HBSS:

Hank’s balanced salt solution

ODC:

Ornithine decarboxylase

PC3M-LN4:

Human prostate cancer cells

qRT-PCR:

Quantitative real-time PCR

SAGE:

Serial analysis of gene expression

shRNA:

Short hairpin RNA

References

  • Agostinelli E, Marques MP, Calheiros R, Gil FP, Tempera G, Viceconte N, Battaglia V, Grancara S, Toninello A (2010) Polyamines: fundamental characters in chemistry and biology. Amino Acids 38(2):393–403. doi:10.1007/s00726-009-0396-7

    Article  PubMed  CAS  Google Scholar 

  • Auvinen M, Paasinen A, Andersson LC, Holtta E (1992) Ornithine decarboxylase activity is critical for cell transformation. Nature 360(6402):355–358. doi:10.1038/360355a0

    Article  PubMed  CAS  Google Scholar 

  • Bachrach U (2004) Polyamines and cancer: minireview article. Amino Acids 26(4):307–309. doi:10.1007/s00726-004-0076-6

    Article  PubMed  CAS  Google Scholar 

  • Bailey HH, Kim K, Verma AK, Sielaff K, Larson PO, Snow S, Lenaghan T, Viner JL, Douglas J, Dreckschmidt NE, Hamielec M, Pomplun M, Sharata HH, Puchalsky D, Berg ER, Havighurst TC, Carbone PP (2010) A randomized, double-blind, placebo-controlled phase 3 skin cancer prevention study of {alpha}-difluoromethylornithine in subjects with previous history of skin cancer. Cancer Prev Res 3(1):35–47. doi:10.1158/1940-6207.capr-09-0096

    Article  CAS  Google Scholar 

  • Basuroy UK, Gerner EW (2006) Emerging concepts in targeting the polyamine metabolic pathway in epithelial cancer chemoprevention and chemotherapy. J Biochem 139(1):27–33. doi:10.1093/jb/mvj022

    Article  PubMed  CAS  Google Scholar 

  • Choi KS, Suh YH, Kim WH, Lee TH, Jung MH (2005) Stable siRNA-mediated silencing of antizyme inhibitor: regulation of ornithine decarboxylase activity. Biochem Biophys Res Commun 328(1):206–212. doi:10.1016/j.bbrc.2004.11.172

    Article  PubMed  CAS  Google Scholar 

  • Coffino P (2001a) Antizyme, a mediator of ubiquitin-independent proteasomal degradation. Biochimie 83(3–4):319–323

    Article  PubMed  CAS  Google Scholar 

  • Coffino P (2001b) Regulation of cellular polyamines by antizyme. Nat Rev Mol Cell Biol 2(3):188–194. doi:10.1038/35056508

    Article  PubMed  CAS  Google Scholar 

  • Gerner EW, Meyskens FL Jr (2004) Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer 4(10):781–792. doi:10.1038/nrc1454

    Article  PubMed  CAS  Google Scholar 

  • Gruendler C, Lin Y, Farley J, Wang T (2001) Proteasomal degradation of Smad1 induced by bone morphogenetic proteins. J Biol Chem 276(49):46533–46543. doi:10.1074/jbc.M105500200

    Article  PubMed  CAS  Google Scholar 

  • Jung MH, Kim SC, Jeon GA, Kim SH, Kim Y, Choi KS, Park SI, Joe MK, Kimm K (2000) Identification of differentially expressed genes in normal and tumor human gastric tissue. Genomics 69(3):281–286. doi:10.1006/geno.2000.6338

    Article  PubMed  CAS  Google Scholar 

  • Kahana C, Asher G, Shaul Y (2005) Mechanisms of protein degradation: an odyssey with ODC. Cell Cycle 4(11):1461–1464

    Article  PubMed  CAS  Google Scholar 

  • Keren-Paz A, Bercovich Z, Kahana C (2007) Antizyme inhibitor: a defective ornithine decarboxylase or a physiological regulator of polyamine biosynthesis and cellular proliferation. Biochem Soc Trans 35(Pt 2):311–313. doi:10.1042/bst0350311

    PubMed  CAS  Google Scholar 

  • Keren-Paz A, Bercovich Z, Porat Z, Erez O, Brener O, Kahana C (2006) Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions. Oncogene 25(37):5163–5172. doi:10.1038/sj.onc.1209521

    PubMed  CAS  Google Scholar 

  • Kim SW, Mangold U, Waghorne C, Mobascher A, Shantz L, Banyard J, Zetter BR (2006) Regulation of cell proliferation by the antizyme inhibitor: evidence for an antizyme-independent mechanism. J Cell Sci 119(Pt 12):2583–2591. doi:10.1242/jcs.02966

    Article  PubMed  CAS  Google Scholar 

  • Lan L, Trempus C, Gilmour SK (2000) Inhibition of ornithine decarboxylase (ODC) decreases tumor vascularization and reverses spontaneous tumors in ODC/Ras transgenic mice. Cancer Res 60(20):5696–5703

    PubMed  CAS  Google Scholar 

  • Levin VA, Hess KR, Choucair A, Flynn PJ, Jaeckle KA, Kyritsis AP, Yung WK, Prados MD, Bruner JM, Ictech S, Gleason MJ, Kim HW (2003) Phase III randomized study of postradiotherapy chemotherapy with combination alpha-difluoromethylornithine-PCV versus PCV for anaplastic gliomas. Clin Cancer Res 9(3):981–990

    PubMed  CAS  Google Scholar 

  • Li X, Coffino P (1992) Regulated degradation of ornithine decarboxylase requires interaction with the polyamine-inducible protein antizyme. Mol Cell Biol 12(8):3556–3562

    PubMed  CAS  Google Scholar 

  • Lim SK, Gopalan G (2007) Antizyme1 mediates AURKAIP1-dependent degradation of Aurora-A. Oncogene 26(46):6593–6603. doi:10.1038/sj.onc.1210482

    Article  PubMed  CAS  Google Scholar 

  • Mangold U (2006) Antizyme inhibitor: mysterious modulator of cell proliferation. Cell Mol Life Sci 63(18):2095–2101. doi:10.1007/s00018-005-5583-4

    Article  PubMed  CAS  Google Scholar 

  • Mangold U, Hayakawa H, Coughlin M, Munger K, Zetter BR (2008) Antizyme, a mediator of ubiquitin-independent proteasomal degradation and its inhibitor localize to centrosomes and modulate centriole amplification. Oncogene 27(5):604–613. doi:10.1038/sj.onc.1210685

    Article  PubMed  CAS  Google Scholar 

  • Matsufuji S, Matsufuji T, Miyazaki Y, Murakami Y, Atkins JF, Gesteland RF, Hayashi S (1995) Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell 80(1):51–60

    Article  PubMed  CAS  Google Scholar 

  • Megosh L, Gilmour SK, Rosson D, Soler AP, Blessing M, Sawicki JA, O’Brien TG (1995) Increased frequency of spontaneous skin tumors in transgenic mice which overexpress ornithine decarboxylase. Cancer Res 55(19):4205–4209

    PubMed  CAS  Google Scholar 

  • Meyskens FL Jr, Gerner EW (1999) Development of difluoromethylornithine (DFMO) as a chemoprevention agent. Clin Cancer Res 5(5):945–951

    PubMed  CAS  Google Scholar 

  • Meyskens FL Jr, McLaren CE, Pelot D, Fujikawa-Brooks S, Carpenter PM, Hawk E, Kelloff G, Lawson MJ, Kidao J, McCracken J, Albers CG, Ahnen DJ, Turgeon DK, Goldschmid S, Lance P, Hagedorn CH, Gillen DL, Gerner EW (2008) Difluoromethylornithine plus sulindac for the prevention of sporadic colorectal adenomas: a randomized placebo-controlled, double-blind trial. Cancer Prev Res 1(1):32–38. doi:10.1158/1940-6207.capr-08-0042

    Article  CAS  Google Scholar 

  • Mi Z, Kramer DL, Miller JT, Bergeron RJ, Bernacki R, Porter CW (1998) Human prostatic carcinoma cell lines display altered regulation of polyamine transport in response to polyamine analogs and inhibitors. Prostate 34(1):51–60

    Article  PubMed  CAS  Google Scholar 

  • Murakami Y, Ichiba T, Matsufuji S, Hayashi S (1996) Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase. J Biol Chem 271(7):3340–3342

    Article  PubMed  CAS  Google Scholar 

  • Murakami Y, Matsufuji S, Kameji T, Hayashi S, Igarashi K, Tamura T, Tanaka K, Ichihara A (1992) Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature 360(6404):597–599. doi:10.1038/360597a0

    Article  PubMed  CAS  Google Scholar 

  • Murakami Y, Suzuki J, Samejima K, Kikuchi K, Hascilowicz T, Murai N, Matsufuji S, Oka T (2009) The change of antizyme inhibitor expression and its possible role during mammalian cell cycle. Exp Cell Res 315(13):2301–2311. doi:10.1016/j.yexcr.2009.04.024

    Article  PubMed  CAS  Google Scholar 

  • Murakami Y, Suzuki J, Samejima K, Oka T (2010) Developmental alterations in expression and subcellular localization of antizyme and antizyme inhibitor and their functional importance in the murine mammary gland. Amino Acids 38(2):591–601. doi:10.1007/s00726-009-0422-9

    Article  PubMed  CAS  Google Scholar 

  • Newman RM, Mobascher A, Mangold U, Koike C, Diah S, Schmidt M, Finley D, Zetter BR (2004) Antizyme targets cyclin D1 for degradation. A novel mechanism for cell growth repression. J Biol Chem 279(40):41504–41511. doi:10.1074/jbc.M407349200

    Article  PubMed  CAS  Google Scholar 

  • Nishioka K, Melgarejo AB, Lyon RR, Mitchell MF (1995) Polyamines as biomarkers of cervical intraepithelial neoplasia. J Cell Biochem Suppl 23:87–95

    Article  PubMed  CAS  Google Scholar 

  • Palmer AJ, Wallace HM (2010) The polyamine transport system as a target for anticancer drug development. Amino Acids 38(2):415–422. doi:10.1007/s00726-009-0400-2

    Article  PubMed  CAS  Google Scholar 

  • Pegg AE (2006) Regulation of ornithine decarboxylase. J Biol Chem 281(21):14529–14532. doi:10.1074/jbc.R500031200

    Article  PubMed  CAS  Google Scholar 

  • Peralta Soler A, Gilliard G, Megosh L, George K, O’Brien TG (1998) Polyamines regulate expression of the neoplastic phenotype in mouse skin. Cancer Res 58(8):1654–1659

    PubMed  CAS  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45

    Article  PubMed  CAS  Google Scholar 

  • Schaner ME, Davidson B, Skrede M, Reich R, Florenes VA, Risberg B, Berner A, Goldberg I, Givant-Horwitz V, Trope CG, Kristensen GB, Nesland JM, Borresen-Dale AL (2005) Variation in gene expression patterns in effusions and primary tumors from serous ovarian cancer patients. Mol Cancer 4:26. doi:10.1186/1476-4598-4-26

    Article  PubMed  Google Scholar 

  • Schaner ME, Ross DT, Ciaravino G, Sorlie T, Troyanskaya O, Diehn M, Wang YC, Duran GE, Sikic TL, Caldeira S, Skomedal H, Tu IP, Hernandez-Boussard T, Johnson SW, O’Dwyer PJ, Fero MJ, Kristensen GB, Borresen-Dale AL, Hastie T, Tibshirani R, van de Rijn M, Teng NN, Longacre TA, Botstein D, Brown PO, Sikic BI (2003) Gene expression patterns in ovarian carcinomas. Mol Biol Cell 14(11):4376–4386. doi:10.1091/mbc.E03-05-0279

    Article  PubMed  CAS  Google Scholar 

  • Shantz LM, Levin VA (2007) Regulation of ornithine decarboxylase during oncogenic transformation: mechanisms and therapeutic potential. Amino Acids 33(2):213–223. doi:10.1007/s00726-007-0531-2

    Article  PubMed  CAS  Google Scholar 

  • Smith MK, Trempus CS, Gilmour SK (1998) Co-operation between follicular ornithine decarboxylase and v-Ha-ras induces spontaneous papillomas and malignant conversion in transgenic skin. Carcinogenesis 19(8):1409–1415

    Article  PubMed  CAS  Google Scholar 

  • Svensson KJ, Welch JE, Kucharzewska P, Bengtson P, Bjurberg M, Pahlman S, Ten Dam GB, Persson L, Belting M (2008) Hypoxia-mediated induction of the polyamine system provides opportunities for tumor growth inhibition by combined targeting of vascular endothelial growth factor and ornithine decarboxylase. Cancer Res 68(22):9291–9301. doi:10.1158/0008-5472.can-08-2340

    Article  PubMed  CAS  Google Scholar 

  • Tang H, Ariki K, Ohkido M, Murakami Y, Matsufuji S, Li Z, Yamamura K (2009) Role of ornithine decarboxylase antizyme inhibitor in vivo. Genes Cells 14(1):79–87. doi:10.1111/j.1365-2443.2008.01249.x

    Article  PubMed  CAS  Google Scholar 

  • Thompson PA, Wertheim BC, Zell JA, Chen WP, McLaren CE, LaFleur BJ, Meyskens FL, Gerner EW (2010) Levels of rectal mucosal polyamines and prostaglandin E2 predict ability of DFMO and sulindac to prevent colorectal adenoma. Gastroenterology 139(3):797–805, 805 e791. doi:10.1053/j.gastro.2010.06.005

    Google Scholar 

  • Tsuji T, Todd R, Meyer C, McBride J, Liao PH, Huang MF, Chou MY, Donoff RB, Wong DT (1998) Reduction of ornithine decarboxylase antizyme (ODC-Az) level in the 7, 12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis model. Oncogene 16(26):3379–3385. doi:10.1038/sj.onc.1201887

    Article  PubMed  CAS  Google Scholar 

  • van Duin M, van Marion R, Vissers K, Watson JE, van Weerden WM, Schroder FH, Hop WC, van der Kwast TH, Collins C, van Dekken H (2005) High-resolution array comparative genomic hybridization of chromosome arm 8q: evaluation of genetic progression markers for prostate cancer. Genes Chromosom Cancer 44(4):438–449. doi:10.1002/gcc.20259

    Article  PubMed  Google Scholar 

  • Wan T, Hu Y, Zhang W, Huang A, Yamamura K, Tang H (2010) Changes in liver gene expression of Azin1 knock-out mice. Z Naturforsch C 65(7–8):519–527

    PubMed  CAS  Google Scholar 

  • Zagaja GP, Shrivastav M, Fleig MJ, Marton LJ, Rinker-Schaeffer CW, Dolan ME (1998) Effects of polyamine analogues on prostatic adenocarcinoma cells in vitro and in vivo. Cancer Chemother Pharmacol 41(6):505–512

    Article  PubMed  CAS  Google Scholar 

  • Zell JA, Pelot D, Chen WP, McLaren CE, Gerner EW, Meyskens FL (2009) Risk of cardiovascular events in a randomized placebo-controlled, double-blind trial of difluoromethylornithine plus sulindac for the prevention of sporadic colorectal adenomas. Cancer Prev Res 2(3):209–212. doi:10.1158/1940-6207.capr-08-0203

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Dr. Isaiah Fidler, University of Texas MD Anderson Cancer Center, for providing PC3M-LN4 cells, and Dr. John Isaacs, John Hopkins University, for providing AT6.1 cells. Funding for these studies was provided by grant CA037393 from the National Institutes of Health.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Vascular Biology Program, Department of Surgery, Children’s Hospital Boston, Boston, MA, 02115, USA

    Rachelle R. Olsen & Bruce R. Zetter

  2. Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia

    Ivy Chung

Authors
  1. Rachelle R. Olsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ivy Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bruce R. Zetter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bruce R. Zetter.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Olsen, R.R., Chung, I. & Zetter, B.R. Knockdown of antizyme inhibitor decreases prostate tumor growth in vivo. Amino Acids 42, 549–558 (2012). https://doi.org/10.1007/s00726-011-1032-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-011-1032-x

Keywords

Search

Navigation