188Rhenium Treatment Induces DACT2 Expression in Hepatocellular Carcinoma Cells

Document Type : Original Article


1 Cellular and Molecular Research Center, Research Institute for Prevention of Non-Communicable Diseases, Qazvin University of Medical Sciences, Qazvin, Iran

2 Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

3 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

4 Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5 Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

6 Department of Organic Chemistry, Karaj Branch, Islamic Azad University, Karaj, Iran

7 Department of Physical Chemistry, Faculty of Science, University of Tehran, Tehran, Iran

8 Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow, Russia

9 World-Class Research Center "Digital biodesign and personalized healthcare", Sechenov First Moscow State Medical University, Moscow, Russia

10 Chemistry Department, Lomonosov Moscow State University, Moscow, Russia

11 Department of Regenerative Biomedicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran


Objectives: Epigenetic alterations, including any change in DNA methylation pattern, could be the missing link of understanding radiation-induced genomic instability. Dapper, Dishevelled-associated antagonist of β-catenin homolog 2 (DACT2) is a tumor suppressor gene regulating Wnt/β-catenin. In hepatocellular carcinoma (HCC), DACT2 is hypermethylated, while methylation status of its promoter regulates the corresponding expression. Radionuclides have been used to reduce proliferation and induce apoptosis in cancerous cells. Epigenetic impact of radionuclides as therapeutic agents for treatment of HCC is still unknown. The aim of this study was to evaluate epigenetic impact of 188Rhenium perrhenate (188ReO4) on HCC cells.
Material and Methods: In this in vitro experimental study, HepG2 and Huh7 cells were treated with 188ReO4, receiving 55 and 73 Mega Becquerel (MBq) exposures, respectively. For cell viability measurement, live/dead staining was carried out 18, 24, and 48 hours post-exposure. mRNA expression level of β-Catenin, Wnt1, DNMT1, DACT2 and WIF-1 genes were quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Then, possible regulatory impact of DACT2 upregulation was investigated through evaluating methylation-specific PCR (MS-PCR).
Results: Results showed that viability of both cells was reduced after treatment with 188ReO4 at three time points postexposure compared to the control groups. The qRT-PCR results showed that DACT2 mRNA level was significantly increased at 24, and 48 hours post-exposure in HepG2 cells compared to the control group, while, no significant change was observed in Huh7 cells. Methylation pattern of DACT2 promoter remained unchanged in HepG2 and Huh7 cells.
Conclusion: Treatment with 188ReO4 reduced viability of HepG2 and Huh7 cells. Although DACT2 expression was increased after 188ReO4 exposure in HepG2 cells, methylation pattern of its promoter remained unchanged. This study assessed impacts of the 188 ReO4 β-irradiation on expression and induction of DACT2 epigenetic aberrations as well as the correlation of this agent with viability of cells.


  1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019; 69(1): 7-34.
  2. Dhanasekaran R, Bandoh S, Roberts LRJF. Molecular pathogenesis of hepatocellular carcinoma and impact of therapeutic advances. F1000Res. 2016; 5.
  3. Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017; 36(11): 1461-1473.
  4. Tsukamoto AS, Grosschedl R, Guzman RC, Parslow T, Varmus HEJC. Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice. Cell. 1988; 55(4): 619-625.
  5. Khalaf AM, Fuentes D, Morshid AI, Burke MR, Kaseb AO, Hassan M, et al. Role of Wnt/β-catenin signaling in hepatocellular carcinoma, pathogenesis, and clinical significance. J Hepatocell Carcinoma. 2018; 5: 61-73.
  6. Feinberg AP, Vogelstein BJN. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983; 301(5895): 89-92.
  7. Dawson MA, Kouzarides T. Cancer epigenetics: from mechanism to therapy. Cell. 2012; 150(1): 12-27.
  8. Tsang DP, Wu WK, Kang W, Lee YY, Wu F, Yu Z, et al. Yin Yang 1‐ mediated epigenetic silencing of tumour‐suppressive microRNAs activates nuclear factor‐κB in hepatocellular carcinoma. J Pathol. 2016; 238(5): 651-664.
  9. Jia Y, Yang Y, Brock MV, Zhan Q, Herman JG, Guo M. Epigenetic regulation of DACT2, a key component of the Wnt signalling pathway in human lung cancer. J Pathol. 2013; 230(2): 194-204.
  10. Cheyette BN, Waxman JS, Miller JR, Takemaru KI, Sheldahl LC, Khlebtsova N, et al. Dapper, a Dishevelled-associated antagonist of β-catenin and JNK signaling, is required for notochord formation. Dev Cell. 2002; 2(4): 449-461.
  11. Wang S, Dong Y, Zhang Y, Wang X, Xu L, Yang S, et al. DACT2 is a functional tumor suppressor through inhibiting Wnt/β-catenin pathway and associated with poor survival in colon cancer. Oncogene. 2015; 34(20): 2575-2585.
  12. Xiang T, Fan Y, Li C, Li L, Ying Y, Mu J, et al. DACT2 silencing by promoter CpG methylation disrupts its regulation of epithelial-tomesenchymal transition and cytoskeleton reorganization in breast cancer cells. Oncotarget. 2016;7(43):70924-70935.
  13. Gao S, Yang Z, Zheng ZY, Yao J, Zhang F, Wu LM, et al. Reduced expression of DACT2 promotes hepatocellular carcinoma progression: involvement of methylation-mediated gene silencing. World J Surg Oncol. 2013; 11: 57.
  14. Yu Y, Yan W, Liu X, Jia Y, Cao B, Yu Y, et al. DACT2 is frequently methylated in human gastric cancer and methylation of DACT2 activated Wnt signaling. Am J Cancer Res. 2014; 4(6): 710-724.
  15. Zhang X, Yang Y, Liu X, Herman JG, Brock MV, Licchesi JD, et al. Epigenetic regulation of the Wnt signaling inhibitor DACT2 in human hepatocellular carcinoma. Epigenetics. 2013; 8(4): 373-382.
  16. Deng Y, Yu B, Cheng Q, Jin J, You H, Ke R, et al. Epigenetic silencing of WIF-1 in hepatocellular carcinomas. J Cancer Res Clin Oncol. 2010; 136(8): 1161-1167.
  17. Liu YL, Yang HP, Gong L, Tang CL, Wang HJJM. Hypomethylation effects of curcumin, demethoxycurcumin and bisdemethoxycurcumin on WIF-1 promoter in non-small cell lung cancer cell lines. Mol Med Rep. 2011; 4(4): 675-679.
  18. Guidoccio F, Boni G, Volterrani D, Mariani G. Radionuclide therapy for tumors of the liver and biliary tract. Nuclear Medicine Textbook. Springer: 2019; 859-879.
  19. Asadian S, Mirzaei H, Kalantari BA, Davarpanah MR, Mohamadi M, Shpichka A, et al. β-radiating radionuclides in cancer treatment, novel insight into promising approach. Pharmacol Res. 2020; 105070.
  20. Bozkurt MF, Salanci BV, Uğur Ö. Intra-arterial radionuclide therapies for liver tumors. Semin Nucl Med. 2016; 46(4): 324-339.
  21. Gombeau K, Pereira S, Ravanat JL, Camilleri V, Cavalie I, Bourdineaud J-P, et al. Depleted uranium induces sex-and tissue-specific methylation patterns in adult zebrafish. J Environ Radioact. 2016; 154: 25-33.
  22. Kaup S, Grandjean V, Mukherjee R, Kapoor A, Keyes E, Seymour CB, et al. Radiation-induced genomic instability is associated with DNA methylation changes in cultured human keratinocytes. Mutat Res. 2006; 597(1-2): 87-97.
  23. Tamminga J, Kovalchuk O. Role of DNA damage and epigenetic DNA methylation changes in radiation-induced genomic instability and bystander effects in germline in vivo. Curr Mol Pharmacol. 2011; 4(2): 115-125.
  24. Donkena KV, Young CY, Tindall DJ. Oxidative stress and DNA methylation in prostate cancer. Obstet Gynecol Int. 2010; 2010: 302051.
  25. Christman JK, Sheikhnejad G, Marasco CJ, Sufrin JR. 5-Methyl-2’-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation. Proc Natl Acad Sci USA. 1995; 92(16): 7347-7351.
  26. Franco R, Schoneveld O, Georgakilas AG, Panayiotidis MI. Oxidative stress, DNA methylation and carcinogenesis. Cancer Lett. 2008; 266(1): 6-11.
  27. Rust S, Funke H, Assmann G. Mutagenically separated PCR (MS-PCR): a highly specific one step procedure for easy mutation detection. Nucleic Acids Res. 1993; 21(16): 3623-3629.
  28. Denis H, Ndlovu MN, Fuks FJ. Regulation of mammalian DNA methyltransferases: a route to new mechanisms. EMBO Rep. 2011; 12(7): 647-656.
  29. Antwih DA, Gabbara KM, Lancaster WD, Ruden DM, Zielske SP. Radiation-induced epigenetic DNA methylation modification of radiation-response pathways. Epigenetics. 2013; 8(8): 839-848.
  30. Baylin SB, Ohm JE. Epigenetic gene silencing in cancer– a mechanism for early oncogenic pathway addiction? Nat Rev Cancer. 2006; 6(2): 107-116.
  31. Jia Y, Yang Y, Liu S, Liu S, Herman JG, Lu F, et al. SOX17 antagonizes WNT/β-catenin signaling pathway in hepatocellular carcinoma. Epigenetics. 2010; 5(8): 743-749.
  32. Kim DH, Kim EJ, Kim DH, Park SW. Dact2 is involved in the regulation of epithelial-mesenchymal transition. Biochem Biophys Res Commun. 2020; 524(1): 190-197.
  33. Huang L, Li MX, Wang L, Li BK, Chen GH, He LR, et al. Prognostic value of Wnt inhibitory factor-1 expression in hepatocellular carcinoma that is independent of gene methylation. Tumour Biol. 2011; 32(1): 233-240.
  34. Jiang X, Tan J, Li J, Kivimäe S, Yang X, Zhuang L, et al. DACT3 is an epigenetic regulator of Wnt/β-catenin signaling in colorectal cancer and is a therapeutic target of histone modifications. Cancer Cell. 2008; 13(6): 529-541.
  35. Aypar U, Morgan WF, Baulch JE. Radiation-induced epigenetic alterations after low and high LET irradiations. Mutat Res. 2011; 707(1-2): 24-33.
  36. Danielsson A, Barreau K, Kling T, Tisell M, Carén H. Accumulation of DNA methylation alterations in paediatric glioma stem cells following fractionated dose irradiation. Clin Epigenetics. 2020; 12(1): 26.
  37. Wei W, Chua MS, Grepper S, So SK. Blockade of Wnt-1 signaling leads to anti-tumor effects in hepatocellular carcinoma cells. Mol Cancer. 2009; 8: 76.
  38. Hai B, Yang Z, Shangguan L, Zhao Y, Boyer A, Liu F. Concurrent transient activation of Wnt/β-catenin pathway prevents radiation damage to salivary glands. Int J Radiat Oncol Biol Phys. 2012; 83(1): e109-e116.
  39. Albuquerque C, Pebre Pereira L. Wnt signalling-targeted therapy in the CMS2 tumour subtype: a new paradigm in CRC treatment? Adv Exp Med Biol. 2018; 1110: 75-100.
  40. Nguyen P, Lee S, Lorang-Leins D, Trepel J, Smart DK. SIRT2 interacts with β-catenin to inhibit Wnt signaling output in response to radiation-induced stress. Mol Cancer Res. 2014; 12(9): 1244-1253.