[go: up one dir, main page]
More Web Proxy on the site http://driver.im/ Skip to main content

Advertisement

Springer Nature Link
Log in

Hydralazine inhibits human cervical cancer cell growth in vitro in association with APC demethylation and re-expression

  • Original Article
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript

Abstract

Purpose

The tumor suppressor adenomatous polyposis coli (APC) is frequently silenced by promoter hypermethylation in human cervical cancer. Clinically, it has been approved that DNA methylation inhibitors, such as 5-aza-2′-deoxycytidine (5-Aza-dC), can reverse APC promoter methylation, but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. Hydralazine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. The purpose of this study was to evaluate the effects of hydralazine on APC reactivation and the inhibition of human cervical cancer cells in vitro.

Methods

Expression of APC gene, and methylation status were analyzed by RT-PCR, quantitative real time RT-PCR, and methylation-specific PCR methods. β-Catenin protein that correlates closely with APC was detected by immunohistochemistry method after treatment with hydralazine. MTT and FCM assays were used to observe the changes of proliferation activity, cell cycle, and apoptosis of the cells.

Results

Methylated APC was not expressed in HeLa cell, hemimethylated APC was expressed in CaSki cells, and unmethylated APC was expressed normally in SiHa cells. Hydralazine induces APC expression and promotes demethylation in HeLa and CaSki cells. After treatment with 40 μmol/L hydralazine for 72 h, growth inhibitive rates (%) of HeLa, CaSki, and SiHa cell lines were 52.12 ± 3.78, 44.31 ± 2.59, and 47.73 ± 4.73, respectively. On the contrary, the normal cell ECV304 growth inhibitory rate was only 27.18 ± 0.79. The expression of APC mRNA in HeLa, CaSki, and SiHa cell lines increased 10.35-, 11.40-, and 0.73-fold, respectively. HeLa and CaSki cells were arrested in S phase of the cell cycle by hydralazine, and the percentage of apoptotic cells in the two cell lines treated with hydralazine was increased significantly compared to the untreated cells (P < 0.01). The expression of β-catenin protein in the cell membrane was observed after the treatment with hydralazine.

Conclusions

Hydralazine, an effective inhibitor of APC methylation and promoter of APC re-expression, can inhibit cell growth in human cervical cancer in vitro and be potentially used for the clinical treatment of human cervical cancer.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
£29.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (United Kingdom)

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

5-Aza-C:

5-Azacytidine

5-Aza-dC:

5-Aza-2′-deoxycytidine

APC:

Adenomatous polyposis coli

FAP:

Familial adenomatous polyposis

FCM:

Flow cytometry

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

IHC:

Immunohistochemistry

MSP:

Methylation-specific PCR

References

  1. Kinzler KW, Vogelstein B (1996) Lessons from hereditary colorectal cancer. Cell 87(2):159–170

    Article  PubMed  CAS  Google Scholar 

  2. Papkoff J, Rubinfeld B, Schryver B, Polakis P (1996) Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes. Mol Cell Biol 16(5):2128–2134

    PubMed  CAS  Google Scholar 

  3. Orford K, Crockett C, Jensen JP, Weissman AM, Byers SW (1997) Serine phosphorylation-regulated ubiquitination and degradation of beta-catenin. J Biol Chem 272(40):24735–24738

    Article  PubMed  CAS  Google Scholar 

  4. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW (1997) Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275(5307):1787–1790

    Article  PubMed  CAS  Google Scholar 

  5. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B, Clevers H (1997) Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma. Science 275(5307):1784–1787

    Article  PubMed  CAS  Google Scholar 

  6. Spark AB, Morin PJ, Vogelstein B, Kinzler KW (1998) Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectal cancer. Cancer Res 58(6):1130–1134

    Google Scholar 

  7. Toyota M, Issa JP (2000) The role of DNA hypermethylation in human neoplasia. Electrophoresis 21(2):329–333

    Article  PubMed  CAS  Google Scholar 

  8. Esteller M (2002) CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene 21(35):5427–5440

    Article  PubMed  CAS  Google Scholar 

  9. Tsuchiya T, Tamura G, Sato K, Endoh Y, Sakata K, Jin Z, Motoyama T, Usuba O, Kimura W, Nishizuka S, Wilson KT, James SP, Yin J, Fleisher AS, Zou T, Silverberg SG, Kong D, Meltzer SJ (2000) Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia. Oncogene 19(32):3642–3646

    Article  PubMed  CAS  Google Scholar 

  10. Esteller M, Sparks A, Toyota M, Sanchez-Cespedes M, Capella G, Peinado MA, Gonzalez S, Tarafa G, Sidransky D, Meltzer SJ, Baylin SB, Herman JG (2000) Analysis of adenomatous polyposis coli promoter hypermethylation in human cancer. Cancer Res 60(16):4366–4371

    PubMed  CAS  Google Scholar 

  11. Virmani AK, Rathi A, Sathyanarayana UG, Padar A, Huang CX, Cunnigham HT, Farinas AJ, Milchgrub S, Euhus DM, Gilcrease M, Herman J, Minna JD, Gazdar AF (2001) Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in breast and lung carcinomas. Clin Cancer Res 7(7):1998–2004

    PubMed  CAS  Google Scholar 

  12. Zysman M, Saka A, Millar A, Knight J, Chapman W, Bapat B (2002) Methylation of adenomatous polyosis coli in endometrial cancer occurs more frequently in tumors with microsatellite instability phenotype. Cancer Res 62(13):3663–3666

    PubMed  CAS  Google Scholar 

  13. Jin Z, Tamura G, Tsuchiya T, Sakata K, Kashiwaba M, Osakabe M, Motoyama T (2001) Adenomatous polyposis coli (APC) gene promoter hypermethylation in primary breast cancer. Br J Cancer 85(1):69–73

    Article  PubMed  CAS  Google Scholar 

  14. Eads CA, Lord RV, Wickramasinghe K, Long TI, Kurumboor SK, Bernstein L, Peters JH, DeMeester SR, DeMeester TR, Skinner KA, Laird PW (2001) Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res 61(8):3410–3418

    PubMed  CAS  Google Scholar 

  15. Maruyama R, Toyooka S, Toyooka KO, Harada K, Virmani AK, Zöchbauer-Müller S, Farinas AJ, Vakar-Lopez F, Minna JD, Sagalowsky A, Czerniak B, Gazdar AF (2001) Aberrant promoter methylation profile of bladder cancer and its relationship to clinicopathological features. Cancer Res 61(24):8659–8663

    PubMed  CAS  Google Scholar 

  16. Yang Y, Takeuchi S, Tsukasaki K, Yamada Y, Hata T, Mori N, Fukushima A, Seo H, Koeffler HP, Taguchi H (2005) Methylation analysis of the adenomatous polyposis coli (APC) gene in adult T-cell leukemia/lymphoma. Leuk Res 29(1):47–51

    Article  PubMed  CAS  Google Scholar 

  17. Segura-Pacheco B, Trejo-Becerril C, Perez-Cardenas E, Taja-Chayeb L, Mariscal I, Chavez A, Acuna C, Salazar AM, Lizano M, Duenas-Gonzalez A (2003) Reactivation of tumor suppressor genes by the cardiovascular drugs hydralazine and procainamide and their potential use in cancer therapy. Clin Cancer Res 9(5):1596–1603

    PubMed  CAS  Google Scholar 

  18. Angeles E, Vázquez-Valadez VH, Vázquez-Valadez O, Velázquez-Sánchez AM, Ramírez A, Martínez L, Díaz-Barriga S, Romero-Rojas A, Cabrera G, López-Castanares R, Duenas-González A (2005) Computational studies of 1-hydrazinophtalazine (hydralazine) as antineoplastic agent. Docking studies on methyltransferase. Lett Drug Design Discov 2:282–286

    Article  CAS  Google Scholar 

  19. Arce C, Candelaria M, Segura-Pacheco B, Perez-Cardenas E, Taja- Chayeb L, Duenas-Gonzalez A (2006) Hydralazine target: From blood vessels to the epigenoma. J Transl Med 4:10

    Article  PubMed  CAS  Google Scholar 

  20. Zambrano P, Segura-Pacheco B, Pérez-Cárdenas E, Cetina L, Revilla-Vázquez A, Taja-Chayeb L, Chávez-Blanco A, Angeles E, Cabrera G, Sandoval K, Trejo-Becerril C, Chanona-Vilchis J, Duenas-González A (2005) A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes. BMC Cancer 5(1):44–55

    Article  PubMed  CAS  Google Scholar 

  21. Liu HS, Chen CY, Lee CH, Chou YI (1998) Selective activation of oncogenic Ha-ras-induced apoptosis in NIH/3T3 cells. Br J Cancer 77(11):1777–1789

    PubMed  CAS  Google Scholar 

  22. Dong SM, Kim HS, Rha SH, Sidransky D (2001) Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix. Clin Carcinoma Res 7(7):1982–1986

    CAS  Google Scholar 

  23. Issa JP (2003) Decitabine. Curr Opin Oncol 15(6):446–451

    Article  PubMed  CAS  Google Scholar 

  24. Gore SD (2005) Combination therapy with DNA methyltransferase inhibitors in hematologic malignancies. Nat Clin Pract Oncol 2(suppl 1):S30–S35

    Article  PubMed  CAS  Google Scholar 

  25. Cheng JC, Weisenberger DJ, Gonzales FA, Liang G, Xu GL, Hu YG, Marquez VE, Jones PA (2004) Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol Cell Biol 24(3):1270–1278

    Article  PubMed  CAS  Google Scholar 

  26. Cheng JC, Matsen CB, Gonzales FA, Ye W, Greer S, Marquez VE, Jones PA, Selker EU (2003) Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J Natl Cancer Inst 95(5):399–409

    Article  PubMed  CAS  Google Scholar 

  27. Kirsten R, Nelson K, Kirsten D, Heintz B (1998) Clinical pharmacokinetics of vasodilators. Part II. Clin Pharmacokinet 35(1):9–36

    Article  PubMed  CAS  Google Scholar 

  28. Magee LA, Cham C, Waterman EJ, Ohlsson A, von Dadelszen P (2003) Hydralazine for treatment of severe hypertension in pregnancy: meta-analysis. BMJ 327(7421):955–960

    Article  PubMed  CAS  Google Scholar 

  29. Klein L, O’Connor CM, Gattis WA, Gattis WA, Zampino M, de Luca L, Vitarelli A, Fedele F, Gheorghiade M (2003) Pharmacologic therapy for patients with chronic heart failure and reduced systolic function: review of trials and practical considerations. Am J Cardiol 91(9A):18F–40F

    Article  PubMed  CAS  Google Scholar 

  30. Yung R, Chang S, Hemati N, Johnson K, Richardson B (1997) Mechanisms of drug-induced lupus. IV. Comparison of procainamide and hydralazine with analogs in vitro and in vivo. Arthritis Rheum 40(8):1436–1443

    Article  PubMed  CAS  Google Scholar 

  31. Chavez-Blanco A, Perez-Plasencia C, Perez-Cardenas E, Carrasco-Legleu C, Rangel-Lopez E, Segura-Pacheco B, Taja-Chayeb L, Trejo-Becerril C, Gonzalez-Fierro A, Candelaria M, Cabrera G, Duenas-Gonzalez A (2006) Antineoplastic effects of the DNA methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in carcinoma cell lines. Carcinoma Cell Int 6:2

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Immunology, Medical College of Three Gorges University, 443002, Yichang, Hubei, People’s Republic of China

    Yinhong Song & Changju Zhang

  2. Department of Microbiology and Immunology, China Three Gorges University School of Medicine, Yichang, People’s Republic of China

    Changju Zhang

Authors
  1. Yinhong Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Changju Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Changju Zhang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Song, Y., Zhang, C. Hydralazine inhibits human cervical cancer cell growth in vitro in association with APC demethylation and re-expression. Cancer Chemother Pharmacol 63, 605–613 (2009). https://doi.org/10.1007/s00280-008-0773-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00280-008-0773-z

Keywords

Search

Navigation