Intest Res.  2015 Jul;13(3):233-241. 10.5217/ir.2015.13.3.233.

Balsalazide Potentiates Parthenolide-Mediated Inhibition of Nuclear Factor-kappaB Signaling in HCT116 Human Colorectal Cancer Cells

• Kim HY
• Kim SL
• Park YR
• Liu YC
• Seo SY
• Kim SH
• Kim IH
• Lee SO
• Lee ST
• Kim SW

• KMID: 2174471
• DOI: http://doi.org/10.5217/ir.2015.13.3.233

Abstract

BACKGROUND/AIMS
Balsalazide is an anti-inflammatory drug used in the treatment of inflammatory bowel disease. Balsalazide can reduce inflammatory responses via several mechanisms, including inhibition of nuclear factor-kappaB (NF-kappaB) activity. Parthenolide (PT) inhibits NF-kappaB and exerts promising anticancer effects by promoting apoptosis. The present investigated the antitumor effects of balsalazide, combined with PT, on NF-kappaB in a representative human colorectal carcinoma cell line, HCT116.
METHODS
We counted cells and conducted annexin-V assays and cell cycle analysis to measure apoptotic cell death. Western blotting was used investigate the levels of proteins involved in apoptosis.
RESULTS
PT and balsalazide produced synergistic anti-proliferative effects and induced apoptotic cell death. The combination of balsalazide and PT markedly suppressed nuclear translocation of the NF-kappaB p65 subunit and the phosphorylation of inhibitor of NF-kappaB. Moreover, PT and balsalazide dramatically enhanced NF-kappaB p65 phosphorylation. Apoptosis, through the mitochondrial pathway, was confirmed by detecting effects on Bcl-2 family members, cytochrome c release, and activation of caspase-3 and -8.
CONCLUSIONS
Combination treatment with PT and balsalazide may offer an effective strategy for the induction of apoptosis in HCT116 cells.

Keyword

Balsalazide; Parthenolide; NF-kappaB; Apoptosis; Colorectal neoplasms

MeSH Terms

Apoptosis
Blotting, Western
Caspase 3
Cell Cycle
Cell Death
Cell Line
Colorectal Neoplasms*
Cytochromes c
HCT116 Cells
Humans
Inflammatory Bowel Diseases
NF-kappa B
Phosphorylation
Caspase 3
Cytochromes c
NF-kappa B

Figure

• 

  Fig. 1 Inhibitory effects of parthenolide (PT) and balsalazide on cell growth. HCT116 cells were exposed to the indicated concentrations of (A) balsalazide, (B) PT, or (C) 20 mM balsalazide and 10 or 20 µM PT for 24 h. The data represent the mean cell counts ± the standard error of the mean of three independent experiments. *P<0.05, †P<0.001, as compared to control and ‡P<0.05, as compared to PT-treated cells.

• 

  Fig. 2 Apoptotic cell death was induced by parthenolide (PT) and balsalazide. (A) After treatment with PT and/or balsalazide for 24 h, cells were harvested and stained with annexin-V FITC/propidium iodide (PI). (B) Cell cycle modification induced by combination treatment. After treatment with PT and/or balsalazide for 24 h, cells were harvested, fixed, and stained with PI. The percentage of the population in sub-G1 is reported in each histogram and the total number of events analyzed for each condition was 10,000.

• 

  Fig. 3 Down-regulation of nuclear factor-kappa B (NF-κB) signaling by parthenolide (PT) and balsalazide. (A) Nuclear and cytosolic protein extracts were prepared from HCT116 cells treated with PT, balsalazide, or PT and balsalazide for 24 h. Combined balsalazide and PT markedly suppressed nuclear translocation of the NF-κB p65 subunit and inhibitor of NF-κB alpha (IκB-α) phosphorylation. (B) Total protein extracts were prepared from HCT116 cells treated with PT (10, 20 µM), balsalazide (20, 40 mM), or both (10 µM PT plus 20 mM balsalazide) for 24 h. Combined balsalazide and PT dramatically suppressed p65 phosphorylation. Actin was used as the loading control.

• 

  Fig. 4 Down-regulation of caspases by parthenolide (PT) and balsalazide. HCT116 cell lysates were prepared after treatment with PT, balsalazide, or PT and balsalazide for 24 h and then analyzed by western blotting with caspase-3 or caspase-8 antibodies. Combined treatment reduced the levels of full length caspase-3 and caspase-8. However, caspase-3 and caspase-8 did not change when cells were pre-treated with a pan-caspase inhibitor (Z-VAD-FMK). Actin was used as the loading control.

• 

  Fig. 5 Levels of apoptosis-associated proteins following treatment with parthenolide (PT) and balsalazide. HCT116 cell lysates were prepared after treatment with PT, balsalazide, or PT and balsalazide for 24 h and analyzed by western blotting using B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), Bid, Bcl-2-associated X protein (Bax), and cytochrome c antibodies. Actin was used as the loading control.

Reference

1. Sandborn WJ, Hanauer SB. Systematic review: the pharmacokinetic profiles of oral mesalazine formulations and mesalazine pro-drugs used in the management of ulcerative colitis. Aliment Pharmacol Ther. 2003; 17:29–42. PMID: 12492730.

2. Sutherland L, Macdonald JK. Oral 5-aminosalicylic acid for induction of remission in ulcerative colitis. Cochrane Database Syst Rev. 2006; CD000543. DOI: 10.1002/14651858.CD000543.pub2. Published online 19 April 2006. PMID: 16625536.

3. Feagan BG, Macdonald JK. Oral 5-aminosalicylic acid for maintenance of remission in ulcerative colitis. Cochrane Database Syst Rev. 2012; 10:CD000544. DOI: 10.1002/14651858.CD000544.pub3. Published online 17 October 2012. PMID: 23076890.

4. Shanahan F, Niederlehner A, Carramanzana N, Anton P. Sulfasalazine inhibits the binding of TNF alpha to its receptor. Immunopharmacology. 1990; 20:217–224. PMID: 1981213.

5. Bantel H, Berg C, Vieth M, Stolte M, Kruis W, Schulze-Osthoff K. Mesalazine inhibits activation of transcription factor NF-κB in inflamed mucosa of patients with ulcerative colitis. Am J Gastroenterol. 2000; 95:3452–3457. PMID: 11151876.

6. Bork PM, Schmitz ML, Kuhnt M, Escher C, Heinrich M. Sesquiterpene lactone containing Mexican Indian medicinal plants and pure sesquiterpene lactones as potent inhibitors of transcription factor NF-κB. FEBS Lett. 1997; 402:85–90. PMID: 9013864.

7. Heinrich M, Robles M, West JE, Ortiz de Montellano BR, Rodriguez E. Ethnopharmacology of Mexican asteraceae (Compositae). Annu Rev Pharmacol Toxicol. 1998; 38:539–565. PMID: 9597165.

8. Murphy JJ, Heptinstall S, Mitchell JR. Randomised double-blind placebo-controlled trial of feverfew in migraine prevention. Lancet. 1988; 2:189–192. PMID: 2899663.

9. Schinella GR, Giner RM, Recio MC, Mordujovich de Buschiazzo P, Ríos JL, Máñez S. Anti-inflammatory effects of South American Tanacetum vulgare. J Pharm Pharmacol. 1998; 50:1069–1074. PMID: 9811170.

10. Hehner SP, Heinrich M, Bork PM, et al. Sesquiterpene lactones specifically inhibit activation of NF-κB-α by preventing the degradation of IκB-α and IκB-β. J Biol Chem. 1998; 273:1288–1297. PMID: 9430659.

11. Oka D, Nishimura K, Shiba M, et al. Sesquiterpene lactone parthenolide suppresses tumor growth in a xenograft model of renal cell carcinoma by inhibiting the activation of NF-κB. Int J Cancer. 2007; 120:2576–2581. PMID: 17290398.

12. Kim SL, Trang KT, Jeon BJ, et al. Synergistic effect of parthenolide in combination with 5-fluorouracil in SW480 cells. Intest Res. 2012; 10:357–364.

13. Trang KT, Kim SL, Park SB, et al. Parthenolide sensitizes human colorectal cancer cells to tumor necrosis factor-related apoptosis-inducing ligand through mitochondrial and caspase dependent pathway. Intest Res. 2014; 12:34–41. PMID: 25349561.

14. Zhang S, Ong CN, Shen HM. Critical roles of intracellular thiols and calcium in parthenolide-induced apoptosis in human colorectal cancer cells. Cancer Lett. 2004; 208:143–153. PMID: 15142672.

15. Wen J, You KR, Lee SY, Song CH, Kim DG. Oxidative stress-mediated apoptosis. The anticancer effect of the sesquiterpene lactone parthenolide. J Biol Chem. 2002; 277:38954–38964. PMID: 12151389.

16. Vermeulen L, De Wilde G, Notebaert S, Vanden Berghe W, Haegeman G. Regulation of the transcriptional activity of the nuclear factor-κB p65 subunit. Biochem Pharmacol. 2002; 64:963–970. PMID: 12213593.

17. Slee EA, Adrain C, Martin SJ. Serial killers: ordering caspase activation events in apoptosis. Cell Death Differ. 1999; 6:1067–1074. PMID: 10578175.

18. Yao J, Jiang Z, Duan W, et al. Involvement of mitochondrial pathway in triptolide-induced cytotoxicity in human normal liver L-02 cells. Biol Pharm Bull. 2008; 31:592–597. PMID: 18379047.

19. Rhodes JM, Campbell BJ. Inflammation and colorectal cancer: IBD-associated and sporadic cancer compared. Trends Mol Med. 2002; 8:10–16. PMID: 11796261.

20. Pinczowski D, Ekbom A, Baron J, Yuen J, Adami HO. Risk factors for colorectal cancer in patients with ulcerative colitis: a case-control study. Gastroenterology. 1994; 107:117–120. PMID: 7912678.

21. Stolfi C, Pellegrini R, Franze E, Pallone F, Monteleone G. Molecular basis of the potential of mesalazine to prevent colorectal cancer. World J Gastroenterol. 2008; 14:4434–4439. PMID: 18680220.

22. Bernstein CN, Nugent Z, Blanchard JF. 5-aminosalicylate is not chemoprophylactic for colorectal cancer in IBD: a population based study. Am J Gastroenterol. 2011; 106:731–736. PMID: 21407180.

23. Kim SL, Trang KT, Kim SH, et al. Parthenolide suppresses tumor growth in a xenograft model of colorectal cancer cells by inducing mitochondrial dysfunction and apoptosis. Int J Oncol. 2012; 41:1547–1553. PMID: 22895542.

24. Kim SL, Lee ST, Trang KT, et al. Parthenolide exerts inhibitory effects on angiogenesis through the downregulation of VEGF/VEGFRs in colorectal cancer. Int J Mol Med. 2014; 33:1261–1267. PMID: 24573421.

25. Kim SL, Kim SH, Trang KT, et al. Synergistic antitumor effect of 5-fluorouracil in combination with parthenolide in human colorectal cancer. Cancer Lett. 2013; 335:479–486. PMID: 23507557.

26. Grivennikov S, Karin E, Terzic J, et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell. 2009; 15:103–113. PMID: 19185845.

27. Zhao ZJ, Xiang JY, Liu L, Huang XL, Gan HT. Parthenolide, an inhibitor of the nuclear factor-κB pathway, ameliorates dextran sulfate sodium-induced colitis in mice. Int Immunopharmacol. 2012; 12:169–174. PMID: 22155740.

28. Richmond A. NF-κB, chemokine gene transcription and tumour growth. Nat Rev Immunol. 2002; 2:664–674. PMID: 12209135.

29. Hengartner MO. The biochemistry of apoptosis. Nature. 2000; 407:770–776. PMID: 11048727.

30. Chiarugi V, Magnelli L, Cinelli M, Basi G. Apoptosis and the cell cycle. Cell Mol Biol Res. 1994; 40:603–612. PMID: 7787878.

31. Baker SJ, Reddy EP. Modulation of life and death by the TNF receptor superfamily. Oncogene. 1998; 17:3261–3270. PMID: 9916988.

32. Reed JC. Double identity for proteins of the Bcl-2 family. Nature. 1997; 387:773–776. PMID: 9194558.

33. Estrov Z, Manna SK, Harris D, et al. Phenylarsine oxide blocks interleukin-1β-induced activation of the nuclear transcription factor NF-κB, inhibits proliferation, and induces apoptosis of acute myelogenous leukemia cells. Blood. 1999; 94:2844–2853. PMID: 10515888.

34. Barkett M, Xue D, Horvitz HR, Gilmore TD. Phosphorylation of IκB-α inhibits its cleavage by caspase CPP32 in vitro. J Biol Chem. 1997; 272:29419–29422. PMID: 9367996.

35. Korsmeyer SJ. Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood. 1992; 80:879–886. PMID: 1498330.

36. Li L, Aggarwal BB, Shishodia S, Abbruzzese J, Kurzrock R. Nuclear factor-κB and IκB kinase are constitutively active in human pancreatic cells, and their down-regulation by curcumin (diferuloylmethane) is associated with the suppression of proliferation and the induction of apoptosis. Cancer. 2004; 101:2351–2362. PMID: 15476283.

37. Liptay S, Weber CK, Ludwig L, Wagner M, Adler G, Schmid RM. Mitogenic and antiapoptotic role of constitutive NF-κB/Rel activity in pancreatic cancer. Int J Cancer. 2003; 105:735–746. PMID: 12767057.

38. Kong R, Sun B, Jiang H, et al. Downregulation of nuclear factor-κB p65 subunit by small interfering RNA synergizes with gemcitabine to inhibit the growth of pancreatic cancer. Cancer Lett. 2010; 291:90–98. PMID: 19880242.

39. Lee JU, Hosotani R, Wada M, et al. Role of Bcl-2 family proteins (Bax, Bcl-2 and Bcl-X) on cellular susceptibility to radiation in pancreatic cancer cells. Eur J Cancer. 1999; 35:1374–1380. PMID: 10658530.

40. Lee MA, Park GS, Lee HJ, et al. Survivin expression and its clinical significance in pancreatic cancer. BMC Cancer. 2005; 5:127. PMID: 16202147.

41. Tamm I, Kornblau SM, Segall H, et al. Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias. Clin Cancer Res. 2000; 6:1796–1803. PMID: 10815900.

42. Bai J, Sui J, Demirjian A, Vollmer CM Jr, Marasco W, Callery MP. Predominant Bcl-XL knockdown disables antiapoptotic mechanisms: tumor necrosis factor-related apoptosis-inducing ligand-based triple chemotherapy overcomes chemoresistance in pancreatic cancer cells in vitro. Cancer Res. 2005; 65:2344–2352. PMID: 15781649.

43. El-Rayes BF, Ali S, Ali IF, Philip PA, Abbruzzese J, Sarkar FH. Potentiation of the effect of erlotinib by genistein in pancreatic cancer: the role of Akt and nuclear factor-κB. Cancer Res. 2006; 66:10553–10559. PMID: 17079479.