Archives of Pharmacal Research

, Volume 42, Issue 8, pp 684–694 | Cite as

Realgar transforming solution-induced differentiation of NB4 cell by the degradation of PML/RARα partially through the ubiquitin–proteasome pathway

  • Yang Hai
  • Xin Wang
  • Peng Song
  • Jian-yin Li
  • Long-he Zhao
  • Fei Xie
  • Xiang-min Tan
  • Qin-jian Xie
  • Lan Yu
  • Yang Li
  • Zheng-rong Wu
  • Hong-yu LiEmail author
Research Article


PML/retinoic acid receptor alpha (RARα), as a hallmark of acute promyeloid leukemia (APL), is directly related to the outcome of clinical APL remedy. It is reported that arsenicals can effectively degrade PML/RARα, such as arsenic trioxide and realgar. However, the high toxicity or insolubility have hampered their clinical applications. Realgar transforming solution (RTS) was produced from realgar by bioleaching process in our lab. Previous studies demonstrated that RTS had a significant anti-cancer ability on chronic myeloid leukemia through oncoprotein degradation. The capacity of RTS on treating APL is what is focused on in this study. The results showed that RTS had a noticeable sensitivity in NB4 cell, and RTS remarkably down-regulated PML/RARα expression and induced cell differentiation. Further, RTS could accumulate PML/RARα into the nuclear bodies and then execute degradation, which could be reversed by proteasome inhibitor MG132. The results also exhibited that the reduction of RTS-induced PML/RARα expression accompanied by the elevation of ubiquitin and SUMO-1 protein expression. Finally, PML and SUMO-1 had been demonstrated to be co-localized after RTS treatment by immunofluorescence co-localization assay and immunoprecipitation assay. In conclusion, these results suggested that RTS-induced cell differentiation may attribute to the PML/RARα degradation partially through the ubiquitin–proteasome pathway.


Realgar Arsenic PML/RARα SUMO Ubiquitin–proteasome pathway 



This work was supported by the National Natural Science Foundation of China (Grant Numbers 81403145, 81560715, 51501080, 81803779), the Sub-Project of National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (Grant Number 2015ZX09501-004-003-008), The Fundamental Research Funds for the Central Universities of China (lzujbky-2018-136, lzujbky-2018-40, lzujbky-2017-206).

Compliance with ethical standards

Conflict of Interest

No potential conflict of interest was reported by the authors.

Supplementary material

12272_2019_1170_MOESM1_ESM.tiff (1 mb)
Supplementary material 1 (TIFF 1073 kb) Supplemental Fig. A Cytotoxicity of NB4 cell in different arsenic agents’ treatments. B The expression level of PML/RAR⍺ after treating with different arsenic agents. C AV/PI analysis for NB4 cell apoptosis of RTS and ATO incubated in or without MG132. D The expression of CD11b after treating with ATO. E Wright–Giemsa staining after treating with RTS and ATO. The solid red arrows indicated the lobulated nuclei, which represented differentiation cells (× 400)


  1. Akagi T, Shih LY, Kato M, Kawamata N, Yamamoto G, Sanada M, Okamoto R, Miller CW, Liang DC, Ogawa S, Koeffler HP (2008) Hidden abnormalities and novel classification of t(15;17) APL based on genomic alterations. Blood. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Avvisati G, ten Cate JW, Sturk A, Lamping R, Petti MG, Mandelli F (1988) Acquired alpha-2-antiplasmin deficiency in acute promyelocytic leukaemia. Br J Haematol 70:43–48CrossRefPubMedGoogle Scholar
  3. C.CP (2010) Pharmacopoeia of the People’s Republic of China. China Medical Science Press, BeijingGoogle Scholar
  4. Chen ZX, Xue YQ, Zhang R, Tao RF, Xia XM, Li C, Wang W, Zu WY, Yao XZ, Ling BJ (1991) A clinical and experimental study on all-trans retinoic acid-treated acute promyelocytic leukemia patients. Blood 78:1413–1419PubMedGoogle Scholar
  5. Cunningham I, Gee TS, Reich LM, Kempin SJ, Naval AN, Clarkson BD (1989) Acute promyelocytic leukemia: treatment results during a decade at Memorial Hospital. Blood 73:1116–1122PubMedGoogle Scholar
  6. de The H, Chen Z (2010) Acute promyelocytic leukaemia: novel insights into the mechanisms of cure. Nat Rev Cancer 10:775–783CrossRefPubMedGoogle Scholar
  7. de The H, Le Bras M, Lallemand-Breitenbach V (2012) The cell biology of disease: acute promyelocytic leukemia, arsenic, and PML bodies. J Cell Biol 198:11–21CrossRefPubMedPubMedCentralGoogle Scholar
  8. de Thé H, Lavau C, Marchio A, Chomienne C, Degos L, Dejean A (1991) The PML-RARα fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell 66:675–684CrossRefPubMedGoogle Scholar
  9. Fasci D, Anania VG, Lill JR, Salvesen GS (2015) SUMO deconjugation is required for arsenic-triggered ubiquitylation of PML. Sci Signal 8:ra56CrossRefPubMedPubMedCentralGoogle Scholar
  10. Glickman MH, Ciechanover A (2002) The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82:373–428CrossRefPubMedGoogle Scholar
  11. Isakson P, Bjoras M, Boe SO, Simonsen A (2010) Autophagy contributes to therapy-induced degradation of the PML/RARA oncoprotein. Blood 116:2324–2331CrossRefPubMedGoogle Scholar
  12. Lallemand-Breitenbach V, Jeanne M, Benhenda S, Nasr R, Lei M, Peres L, Zhou J, Zhu J, Raught B, de The H (2008) Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway. Nat Cell Biol 10:547–555CrossRefPubMedGoogle Scholar
  13. Lallemand-Breitenbach V, Zhu J, Chen Z, de The H (2012) Curing APL through PML/RARA degradation by As2O3. Trends Mol Med 18:36–42CrossRefPubMedGoogle Scholar
  14. Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R (1991) NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood 77:1080–1086PubMedGoogle Scholar
  15. Muller S, Matunis MJ, Dejean A (1998) Conjugation with the ubiquitin-related modifier SUMO-1 regulates the partitioning of PML within the nucleus. EMBO J 17:61–70CrossRefPubMedPubMedCentralGoogle Scholar
  16. Niu C, Yan H, Yu T, Sun HP, Liu JX, Li XS, Wu W, Zhang FQ, Chen Y, Zhou L, Li JM, Zeng XY, Yang RR, Yuan MM, Ren MY, Gu FY, Cao Q, Gu BW, Su XY, Chen GQ, Xiong SM, Zhang TD, Waxman S, Wang ZY, Chen Z, Hu J, Shen ZX, Chen SJ (1999) Studies on treatment of acute promyelocytic leukemia with arsenic trioxide: remission induction, follow-up, and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients. Blood 94:3315–3324PubMedGoogle Scholar
  17. Rabellino A, Carter B, Konstantinidou G, Wu SY, Rimessi A, Byers LA, Heymach JV, Girard L, Chiang CM, Teruya-Feldstein J, Scaglioni PP (2012) The SUMO E3-ligase PIAS1 regulates the tumor suppressor PML and its oncogenic counterpart PML-RARA. Cancer Res 72:2275–2284CrossRefPubMedPubMedCentralGoogle Scholar
  18. Sanz MA, Jarque I, Martin G, Lorenzo I, Martinez J, Rafecas J, Pastor E, Sayas MJ, Sanz G, Gomis F (1988) Acute promyelocytic leukemia. Therapy results and prognostic factors. Cancer 61:7–13CrossRefPubMedGoogle Scholar
  19. Shen ZX, Chen GQ, Ni JH, Li XS, Xiong SM, Qiu QY, Zhu J, Tang W, Sun GL, Yang KQ, Chen Y, Zhou L, Fang ZW, Wang YT, Ma J, Zhang P, Zhang TD, Chen SJ, Chen Z, Wang ZY (1997) Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood 89:3354–3360PubMedGoogle Scholar
  20. Song P, Chen P, Wang D, Wu Z, Gao Q, Wang A, Zhu R, Wang Y, Wang X, Zhao L, Duan Z, Zhu S, Cui P, Li Y, Li H (2017) Realgar transforming solution displays anticancer potential against human hepatocellular carcinoma HepG2 cells by inducing ROS. Int J Oncol 50:660–670CrossRefPubMedGoogle Scholar
  21. Unnikrishnan D, Dutcher JP, Varshneya N, Lucariello R, Api M, Garl S, Wiernik PH, Chiaramida S (2001) Torsades de pointes in 3 patients with leukemia treated with arsenic trioxide. Blood 97:1514–1516CrossRefPubMedGoogle Scholar
  22. Vitaliano-Prunier A, Halftermeyer J, Ablain J, de Reynies A, Peres L, Le Bras M, Metzger D, de The H (2014) Clearance of PML/RARA-bound promoters suffice to initiate APL differentiation. Blood 124:3772–3780CrossRefPubMedGoogle Scholar
  23. von Mikecz A (2006) The nuclear ubiquitin–proteasome system. J Cell Sci 119:1977–1984CrossRefGoogle Scholar
  24. Wang ZY (2001) Arsenic compounds as anticancer agents. Cancer Chemother Pharmacol 48(Suppl 1):S72–S76CrossRefPubMedGoogle Scholar
  25. Wang ZY, Chen Z (2000) Differentiation and apoptosis induction therapy in acute promyelocytic leukaemia. Lancet Oncol 2:101–106CrossRefGoogle Scholar
  26. Wang L, Zhou G-B, Liu P, Song J-H, Liang Y, Yan X-J, Xu F, Wang B-S, Mao J-H, Shen Z-X, Chen S-J, Chen Z (2008) Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia. Proc Natl Acad Sci USA 105:4826CrossRefPubMedGoogle Scholar
  27. Wang Z, Cao L, Kang R, Yang M, Liu L, Zhao Y, Yu Y, Xie M, Yin X, Livesey KM, Tang D (2011) Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARalpha oncoprotein. Autophagy 7:401–411CrossRefPubMedPubMedCentralGoogle Scholar
  28. Wang X, Zhang X, Xu Z, Wang Z, Yue X, Li H (2013) Reversal effect of arsenic sensitivity in human leukemia cell line K562 and K562/ADM using realgar transforming solution. Biol Pharm Bull 36:641–648CrossRefPubMedGoogle Scholar
  29. Wang X, Chen B, Zhao L, Zhi D, Hai Y, Song P, Li Y, Xie Q, Inam U, Wu Z, Yu L, Li H (2017) Autophagy enhanced antitumor effect in K562 and K562/ADM cells using realgar transforming solution. Biomed Pharmacother 98:252–264CrossRefPubMedGoogle Scholar
  30. Warrell RP Jr, Frankel SR, Miller WH Jr, Scheinberg DA, Itri LM, Hittelman WN, Vyas R, Andreeff M, Tafuri A, Jakubowski A, Gabrilove J, Gordon MS, Dmitrovsky E (1991) Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid). N Engl J Med 324:1385–1393CrossRefPubMedGoogle Scholar
  31. Xie QJ, Cao XL, Bai L, Wu ZR, Ma YP, Li HY (2014a) Anti-tumor effects and apoptosis induction by Realgar bioleaching solution in Sarcoma-180 cells in vitro and transplanted tumors in mice in vivo. Asian Pac J Cancer Prev 15:2883–2888CrossRefPubMedGoogle Scholar
  32. Xie QJ, Cao XL, Bai L, Wu ZR, Ma YP, Li HY (2014b) Anti-tumor effects and apoptosis induction by Realgar bioleaching solution in Sarcoma-180 cells in vitro and transplanted tumors in mice in vivo. Asian Pac J Cancer Prev 15:2883–2888CrossRefPubMedGoogle Scholar
  33. Zhang P, Wang S, Hu L, Shi F, Qiu F, Hong L, Han X, Yang H, Song Y, Liu Y, Zhou J, Jin Z (1996) Arsenic trioxide treated 72 cases of acute promyelocytic leukemia. Chin J Hematol 17:58–62Google Scholar
  34. Zhang J, Zhang X, Ni Y, Yang X, Li H (2007) Bioleaching of arsenic from medicinal realgar by pure and mixed cultures. Process Biochem 42:1265–1271CrossRefGoogle Scholar
  35. Zhang X, Xie QJ, Wang X, Wang B, Li HY (2010a) Biological extraction of realgar by Acidithiobacillus ferrooxidans and its in vitro and in vivo antitumor activities. Pharm Biol 48:40–47CrossRefPubMedGoogle Scholar
  36. Zhang XW, Yan XJ, Zhou ZR, Yang FF, Wu ZY, Sun HB, Liang WX, Song AX, Lallemand-Breitenbach V, Jeanne M, Zhang QY, Yang HY, Huang QH, Zhou GB, Tong JH, Zhang Y, Wu JH, Hu HY, de The H, Chen SJ, Chen Z (2010b) Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML. Science 328:240–243CrossRefPubMedGoogle Scholar
  37. Zhu J, Koken MH, Quignon F, Chelbialix MK, Degos L, Wang ZY, Chen Z, De The H (1997) Arsenic-induced PML is targeting onto nuclear bodies: implications for the treatment of acute promyelocytic leukemia. Proc Natl Acad Sci USA 94:3978–3983CrossRefPubMedGoogle Scholar
  38. Zhu HH, Wu DP, Jin J, Li JY, Ma J, Wang JX, Jiang H, Chen SJ, Huang XJ (2013) Oral tetra-arsenic tetra-sulfide formula versus intravenous arsenic trioxide as first-line treatment of acute promyelocytic leukemia: a multicenter randomized controlled trial. J Clin Oncol 31:4215–4221CrossRefPubMedGoogle Scholar

Copyright information

© The Pharmaceutical Society of Korea 2019

Authors and Affiliations

  1. 1.School of PharmacyLanzhou UniversityLanzhouChina
  2. 2.Institute of Microbiology, School of Life SciencesLanzhou UniversityLanzhouChina
  3. 3.Gansu Corps Hospital of CAPFLanzhouChina

Personalised recommendations