Yonsei Med J.  2020 Jan;61(1):20-29. 10.3349/ymj.2020.61.1.20.

Curcumin Inhibits the Proliferation, Migration, Invasion, and Apoptosis of Diffuse Large B-Cell Lymphoma Cell Line by Regulating MiR-21/VHL Axis

• Chen L
• Zhan CZ
• Wang T
• You H
• Yao R

• KMID: 2466332
• DOI: http://doi.org/10.3349/ymj.2020.61.1.20

Abstract

PURPOSE
Curcumin exerts its anti-cancer effects, partly by targeting special microRNAs, in human cancers. MiR-21 is a key oncomir in carcinogenesis of multiple human cancers. Here, we aimed to further explore the mechanistic insight into the link between curcumin and miR-21 on diffuse large B-cell lymphoma (DLBCL).
MATERIALS AND METHODS
Quantitative real-time PCR assays were performed to assess the levels of miR-21 and Von Hippel-Lindau (VHL) mRNA. In situ hybridization assay was used for miR-21 expression visualization in lymphoma tissues. Western blot was used for determination of VHL protein, Ki-67, caspase-3, and cleaved caspase-3 levels. Dual-luciferase reporter assay and RNA immunoprecipitation assay were employed to confirm the direct target of miR-21. MTT assay, flow cytometric analysis, and transwell assay were used to evaluate cell proliferation, apoptosis, and migration and invasion capacities, respectively.
RESULTS
Curcumin repressed the proliferation, migration, and invasion abilities and promoted apoptosis in SU-DHL-8 cells. Curcumin inhibited miR-21 expression and curcumin exerted its anti-proliferation, anti-migration, anti-invasion, and pro-apoptosis effects by miR-21 in SU-DHL-8 cells. VHL was a direct target of miR-21. Moreover, curcumin exerted its regulatory effects on SU-DHL-8 cells by VHL.
CONCLUSION
Curcumin exerted its anti-proliferation, anti-migration, anti-invasion, and pro-apoptosis functions, at least partly, by repressing miR-21 and regulating VHL expression in DLBCL cell line. Our findings provided a possible molecular mechanism of curcumin-mediated anti-cancer effect.

Keyword

Diffuse large B-cell lymphoma (DLBCL); curcumin; miR-21; Von Hippel-Lindau (VHL)

MeSH Terms

Apoptosis*
B-Lymphocytes*
Blotting, Western
Carcinogenesis
Caspase 3
Cell Line*
Cell Proliferation
Curcumin*
Humans
Immunoprecipitation
In Situ Hybridization
Lymphoma
Lymphoma, B-Cell*
MicroRNAs
Real-Time Polymerase Chain Reaction
RNA
RNA, Messenger
Caspase 3
Curcumin
MicroRNAs
RNA
RNA, Messenger

Figure

• 

  Fig. 1 Curcumin inhibited proliferation and promoted apoptosis of SU-DHL-8 cells. SU-DHL-8 cells were treated with different concentrations (0, 5, 10, 20, 40, and 60 µmol/L) of curcumin. (A) At 0, 24, 48, 72, and 96 h treatment, cell proliferation was detected by MTT assay. (B) After 24 h treatment, cell apoptosis rate was measured by flow cytometric analysis. (C) Western blot was performed to determine the levels of Ki-67, caspase-3, and cleaved caspase-3 in curcumin-treated cells. *p<0.05 vs. control.

• 

  Fig. 2 Curcumin repressed the migration and invasion of SU-DHL-8 cells. SU-DHL-8 cells were treated with different concentrations (0, 5, 10, 20, 40, and 60 µmol/L) of curcumin for 24 h, followed by the measurement of cell migration (A) and invasion (B) abilities by transwell assay (×100 magnification). *p<0.05 vs. control.

• 

  Fig. 3 Curcumin inhibited miR-21 expression of SU-DHL-8 cells. (A) MiR-21 expression was detected in 45 cases of DLBCL tissues and 23 cases of reactive lymphoid hyperplasia tissues. (B) Representative ISH of miR-21 in DLBCL tissues and reactive lymphoid hyperplasia tissues (×100 magnification). (C) MiR-21 expression was assessed in DLBCL cell lines (SU-DHL-8, OCI-LY1 and SU-DHL-10) and normal control CD19+ cells by qRT-PCR. (D) SU-DHL-8 cells were treated with different concentrations (0, 5, 10, 20, 40, and 60 µmol/L) of curcumin for 24 h, followed by the determination of miR-21 expression. *p<0.05 vs. corresponding control. DLBCL, diffuse large B-cell lymphoma; ISH, in situ hybridization.

• 

  Fig. 4 The effects of curcumin on proliferation and apoptosis were mediated by miR-21 in SU-DHL-8 cells. SU-DHL-8 cells were transfected with NC mimics or miR-21 mimics, and then were treated with or without curcumin (20 µmol/L). (A) At 0, 24, 48, 72, and 96 h treatment, MTT assay was performed to detect cell proliferation ability. (B) At 24 h treatment, flow cytometric analysis was used to assess cell apoptosis. (C) Western blot was performed to determine the levels of Ki-67, caspase-3, and cleaved caspase-3 in treated cells. *p<0.05 vs. NC mimics or NC mimics+curcumin.

• 

  Fig. 5 Curcumin exerted its anti-migration and anti-invasion effects by miR-21 in SU-DHL-8 cells. SU-DHL-8 cells were transfected with NC mimics or miR-21 mimics, and then were treated with or without curcumin (20 µmol/L) for 24 h, followed by the determination of cell migration ability (A) and invasion capacity (B) by transwell assay. *p<0.05 vs. NC mimics or NC mimics+curcumin.

• 

  Fig. 6 VHL was a direct target of miR-21. (A) Schematic illustration of the putative binding sites between miR-21 and VHL, and mutation in binding sequence in 3′-UTR of VHL. (B) VHL wild-type or mutant-type reporter plasmids (VHL-WT or VHL-MUT) were constructed and transfected into SU-DHL-8 cells together with NC mimics, miR-21 mimics, anti-NC, or anti-miR-21. Relative luciferase activities were detected. (C) SU-DHL-8 cells were transfected with miR-21 mimics, followed by the detection of VHL mRNA enrichment with anti-Ago2 or anti-IgG. SU-DHL-8 cells were transfected with NC mimics, miR-21 mimics, anti-NC, or anti-miR-21, followed by the measurement of VHL mRNA by qRT-PCR assay (D) and VHL protein by western blot (E). The expression of VHL protein was detected by western blot in DLBCL tissues and reactive lymphoid hyperplasia tissues (F), and in DLBCL cell lines (SU-DHL-8, OCI-LY1, and SU-DHL-10) and normal control CD19+ cells (G). *p<0.05 vs. NC mimics or anti-NC. VHL, Von Hippel-Lindau; DLBCL, diffuse large Bcell lymphoma.

• 

  Fig. 7 Curcumin exerted its regulatory effects by VHL in SU-DHL-8 cells. (A) SU-DHL-8 cells were treated with different concentrations (0, 5, 10, 20, 40, and 60 µmol/L) of curcumin for 24 h, followed by the measurement of VHL mRNA expression. SU-DHL-8 cells were transfected with si-VHL or si-NC and then were treated with 20 µmol/L of curcumin. (B) After 24 h treatment, VHL protein expression was determined by western blot analysis. (C) At 0, 24, 48, 72, and 96 h treatment, cell proliferation ability was assessed by MTT assay. (D) At 24 h treatment, cell apoptosis was detected by flow cytometric analysis. (E) Western blot was performed to determine the levels of Ki-67, caspase-3, and cleaved caspase-3. At 24 h treatment, cell migration ability was determined by transwell assay (F) and cell invasion capacity was evaluated by transwell assay (G). *p<0.05 vs. control or si-NC+curcumin. VHL, Von Hippel-Lindau.

Reference

1. Martelli M, Ferreri AJ, Agostinelli C, Di Rocco A, Pfreundschuh M, Pileri SA. Diffuse large B-cell lymphoma. Crit Rev Oncol Hematol. 2013; 87:146–171.

2. Shaffer AL 3rd, Young RM, Staudt LM. Pathogenesis of human B cell lymphomas. Annu Rev Immunol. 2012; 30:565–610.

3. Tilly H, Gomes da, Vitolo U, Jack A, Meignan M, Lopez-Guillermo A, et al. Diffuse large B-cell lymphoma (DLBCL): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2015; 26 Suppl 5:v116–v125.

4. Nabavi SF, Thiagarajan R, Rastrelli L, Daglia M, Sobarzo-Sánchez E, Alinezhad H, et al. Curcumin: a natural product for diabetes and its complications. Curr Top Med Chem. 2015; 15:2445–2455.

5. Liu CH, Huang HY. Antimicrobial activity of curcumin-loaded myristic acid microemulsions against Staphylococcus epidermidis. Chem Pharm Bull (Tokyo). 2012; 60:1118–1124.

6. Meng B, Li J, Cao H. Antioxidant and antiinflammatory activities of curcumin on diabetes mellitus and its complications. Curr Pharm Des. 2013; 19:2101–2113.

7. Lüer S, Troller R, Aebi C. Antibacterial and antiinflammatory kinetics of curcumin as a potential antimucositis agent in cancer patients. Nutr Cancer. 2012; 64:975–981.

8. Hashish EA, Elgaml SA. Hepatoprotective and nephroprotective effect of curcumin against copper toxicity in rats. Indian J Clin Biochem. 2016; 31:270–277.

9. Lin J, Chen A. Curcumin diminishes the impacts of hyperglycemia on the activation of hepatic stellate cells by suppressing membrane translocation and gene expression of glucose transporter-2. Mol Cell Endocrinol. 2011; 333:160–171.

10. Mudduluru G, George-William JN, Muppala S, Asangani IA, Kumarswamy R, Nelson LD, et al. Curcumin regulates miR-21 expression and inhibits invasion and metastasis in colorectal cancer. Biosci Rep. 2011; 31:185–197.

11. Toden S, Okugawa Y, Jascur T, Wodarz D, Komarova NL, Buhrmann C, et al. Curcumin mediates chemosensitization to 5-fluorouracil through miRNA-induced suppression of epithelial-to-mesenchymal transition in chemoresistant colorectal cancer. Carcinogenesis. 2015; 36:355–367.

12. Pandey A, Vishnoi K, Mahata S, Tripathi SC, Misra SP, Misra V, et al. Berberine and curcumin target survivin and STAT3 in gastric cancer cells and synergize actions of standard chemotherapeutic 5-fluorouracil. Nutr Cancer. 2015; 67:1293–1304.

13. Hammond SM. An overview of microRNAs. Adv Drug Deliv Rev. 2015; 87:3–14.

14. Reddy KB. MicroRNA (miRNA) in cancer. Cancer Cell Int. 2015; 15:38.

15. Hayes J, Peruzzi PP, Lawler S. MicroRNAs in cancer: biomarkers, functions and therapy. Trends Mol Med. 2014; 20:460–469.

16. Simonian M, Mosallayi M, Mirzaei H. Circulating miR-21 as novel biomarker in gastric cancer: diagnostic and prognostic biomarker. J Cancer Res Ther. 2018; 14:475.

17. Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A, Lund AH. Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol Chem. 2008; 283:1026–1033.

18. Chan JK, Blansit K, Kiet T, Sherman A, Wong G, Earle C, et al. The inhibition of miR-21 promotes apoptosis and chemosensitivity in ovarian cancer. Gynecol Oncol. 2014; 132:739–744.

19. Cui Q, Vari F, Cristino AS, Salomon C, Rice GE, Sabdia MB, et al. Circulating cell-free miR-494 and miR-21 are disease response biomarkers associated with interim-positron emission tomography response in patients with diffuse large B-cell lymphoma. Oncotarget. 2018; 9:34644–34657.

20. Li J, Fu R, Yang L, Tu W. miR-21 expression predicts prognosis in diffuse large B-cell lymphoma. Int J Clin Exp Pathol. 2015; 8:15019–15024.

21. Go H, Jang JY, Kim PJ, Kim YG, Nam SJ, Paik JH, et al. MicroRNA-21 plays an oncogenic role by targeting FOXO1 and activating the PI3K/AKT pathway in diffuse large B-cell lymphoma. Oncotarget. 2015; 6:15035–15049.

22. Fu R, Li C, Shao Z. Aberrant overexpression and regulatory mechanism of mir-21 in diffuse large B cell lymphoma. Am Soc Hematology. 2014; 124:5171.

23. Ali S, Ahmad A, Banerjee S, Padhye S, Dominiak K, Schaffert JM, et al. Gemcitabine sensitivity can be induced in pancreatic cancer cells through modulation of miR-200 and miR-21 expression by curcumin or its analogue CDF. Cancer Res. 2010; 70:3606–3617.

24. Wilken R, Veena MS, Wang MB, Srivatsan ES. Curcumin: a review of anti-cancer properties and therapeutic activity in head and neck squamous cell carcinoma. Mol Cancer. 2011; 10:12.

25. Ye M, Zhang J, Zhang J, Miao Q, Yao L, Zhang J. Curcumin promotes apoptosis by activating the p53-miR-192-5p/215-XIAP pathway in non-small cell lung cancer. Cancer Lett. 2015; 357:196–205.

26. Epelbaum R, Schaffer M, Vizel B, Badmaev V, Bar-Sela G. Curcumin and gemcitabine in patients with advanced pancreatic cancer. Nutr Cancer. 2010; 62:1137–1141.

27. Bansal SS, Goel M, Aqil F, Vadhanam MV, Gupta RC. Advanced drug delivery systems of curcumin for cancer chemoprevention. Cancer Prev Res (Phila). 2011; 4:1158–1171.

28. Li Y, Zhang T. Targeting cancer stem cells by curcumin and clinical applications. Cancer Lett. 2014; 346:197–205.

29. Bassiouny AR, Atteya MA, El-Rashidy FAH, Neenaa HM. Curcumin and EGCG suppress apurinic/apyrimidinic endonuclease 1 and induce complete remission in B-cell non-Hodgkin’s lymphoma patients. Funct Foods Health Dis. 2011; 1:525–544.

30. Pancione M, Giordano G, Remo A, Febbraro A, Sabatino L, Manfrin E, et al. Immune escape mechanisms in colorectal cancer pathogenesis and liver metastasis. J Immunol Res. 2014; 2014:686879.

31. Saini S, Arora S, Majid S, Shahryari V, Chen Y, Deng G, et al. Curcumin modulates microRNA-203-mediated regulation of the Src-Akt axis in bladder cancer. Cancer Prev Res (Phila). 2011; 4:1698–1709.

32. Taverna S, Giallombardo M, Pucci M, Flugy A, Manno M, Raccosta S, et al. Curcumin inhibits in vitro and in vivo chronic myelogenous leukemia cells growth: a possible role for exosomal disposal of miR-21. Oncotarget. 2015; 6:21918–21933.

33. Chen W, Wang H, Chen H, Liu S, Lu H, Kong D, et al. Clinical significance and detection of microRNA-21 in serum of patients with diffuse large B-cell lymphoma in Chinese population. Eur J Haematol. 2014; 92:407–412.

34. Liu K, Du J, Ruan L. MicroRNA-21 regulates the viability and apoptosis of diffuse large B-cell lymphoma cells by upregulating B cell lymphoma-2. Exp Ther Med. 2017; 14:4489–4496.

35. Moore LE, Nickerson ML, Brennan P, Toro JR, Jaeger E, Rinsky J, et al. Von Hippel-Lindau (VHL) inactivation in sporadic clear cell renal cancer: associations with germline VHL polymorphisms and etiologic risk factors. PLoS Genet. 2011; 7:e1002312.

36. Kong W, He L, Richards EJ, Challa S, Xu CX, Permuth-Wey J, et al. Upregulation of miRNA-155 promotes tumour angiogenesis by targeting VHL and is associated with poor prognosis and triple-negative breast cancer. Oncogene. 2014; 33:679–689.

37. Amara K, Trimeche M, Ziadi S, Laatiri A, Hachana M, Korbi S. Prognostic significance of aberrant promoter hypermethylation of CpG islands in patients with diffuse large B-cell lymphomas. Ann Oncol. 2008; 19:1774–1786.

38. Zhang KL, Han L, Chen LY, Shi ZD, Yang M, Ren Y, et al. Blockage of a miR-21/EGFR regulatory feedback loop augments anti-EGFR therapy in glioblastomas. Cancer Lett. 2014; 342:139–149.

39. Wang Y, Wang S, Wu Y, Ren Y, Li Z, Yao X, et al. Suppression of the growth and invasion of human head and neck squamous cell carcinomas via regulating STAT3 signaling and the miR-21/β-catenin axis with HJC0152. Mol Cancer Ther. 2017; 16:578–590.

40. Sikora E, Bielak-Zmijewska A, Piwocka K, Skierski J, Radziszewska E. Inhibition of proliferation and apoptosis of human and rat T lymphocytes by curcumin, a curry pigment. Biochem Pharmacol. 1997; 54:899–907.