Single-cell microinjection assay indicates that 7-hydroxycoumarin induces rapid activation of caspase-3 in A549 cancer cells

Soto‑Nuñez,Maribel; Díaz‑Morales,Karen  Azucena; Cuautle‑Rodríguez,Patricia; Torres‑Flores,Víctor; López‑González,José  Sullivan; Mandoki‑Weitzner,Juan  José; Molina‑Guarneros,Juan  Arcadio

doi:10.3892/etm.2015.2765

Single-cell microinjection assay indicates that 7-hydroxycoumarin induces rapid activation of caspase-3 in A549 cancer cells

Authors:
- Maribel Soto‑Nuñez
- Karen Azucena Díaz‑Morales
- Patricia Cuautle‑Rodríguez
- Víctor Torres‑Flores
- José Sullivan López‑González
- Juan José Mandoki‑Weitzner
- Juan Arcadio Molina‑Guarneros
View Affiliations

Affiliations: Department of Pharmacology, School of Medicine, National University of Mexico, Mexico City 04510, Mexico, Research Division, School of Medicine, National University of Mexico, Mexico City 04510, Mexico, Department of Chronic‑Degenerative Diseases, National Institute of Respiratory Diseases ‘Ismael Cosio Villegas’, Mexican Ministry of Health, Mexico City 14080, Mexico
Published online on: September 23, 2015 https://doi.org/10.3892/etm.2015.2765
Pages: 1789-1795
Copyright: © Soto‑Nuñez et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Coumarins have attracted intense interest in recent years due to their apoptogenic effects. The aim of the present study was to determine whether 7-hydroxycoumarin (7-HC) induces changes in caspase-3 (C‑3) activity in A549 human lung carcinoma cells. A range of analytical techniques, including colorimetric and fluorometric assays, western blotting, single‑cell microinjection, fluorescence microscopy and image analysis were conducted to elucidate the effects of 7‑HC. A 24‑h exposure to 1.85 mM 7‑HC induced a 65% increase in C‑3 activity, and a notable conversion of procaspase‑3 to C‑3, in addition to poly(ADP‑ribose)polymerase cleavage. Furthermore, morphological changes associated with apoptosis were observed. Exposure of the cells to 7‑HC for 3 or 6 h increased calcium conductance by 27%. By performing the single‑cell microinjection of a specific fluorescent substrate of C‑3 into previously 7‑HC‑exposed cells, a typical enzymatic kinetic profile of C‑3 activation was identified a number of hours prior to the morphological and biochemical changes associated with apoptosis being observed. These results suggest that the rapid in vivo activation of C‑3 is induced by 7‑HC, the most relevant biotransformation product of coumarin in humans.

View Figures

View References

1	U.S. National Cancer Institute: Surveillance, Epidemiology, and End Results Program. SEER Stat Fact Sheets: Lung and Bronchus Cancer. http://seer.cancer.gov/statfacts/html/lungb.htmlAccessed. April 14–2015
2	U.S. National Cancer Institute: Targeted Cancer Therapies. http://www.cancer.gov/about-cancer/treatment/types/targeted-therapies/targeted-therapies-fact-sheetAccessed. April 12–2015
3	U.S. Food and Drug Administration (FDA): Search Orphan Drug Designations and Approvals. http://www.accessdata.fda.gov/scripts/opdlisting/oopd/OOPD_Results_2.cfmAccessed. April 14–2015
4	Jain PK: Coumarin: Chemical and pharmacological profile. J App Pharm Sci. 2:236–240. 2012.
5	Casley-Smith JR and Casley-Smith JR: The pathophysiology of lymphedema and the action of benzo-pyrones in reducing it. Lymphology. 21:190–194. 1988.PubMed/NCBI
6	Thornes RD, Lynch G and Seehan MV: Cimetidine and coumarin therapy of melanoma. Lancet. 2:3281982. View Article : Google Scholar : PubMed/NCBI
7	Cox D, O'Kennedy R and Thornes RD: The rarity of liver toxicity in patients treated with coumarin (1,2-benzopyrone). Hum Toxicol. 8:501–506. 1989. View Article : Google Scholar : PubMed/NCBI
8	Thornes D, Daly L, Lynch G, Browne H, Tanner A, Keane F, O'Loughlin S, Corrigan T, Daly P and Edwards G: Irish Melanoma Group: Prevention of early recurrence of high risk malignant melanoma by coumarin. Eur J Surg Oncol. 15:431–435. 1989.PubMed/NCBI
9	Dexeus FH, Logothetis CJ, Sella A, Fitz K, Amato R, Reuben JM and Dozier N: Phase II study of coumarin and cimetidine in patients with metastatic renal cell carcinoma. J Clin Oncol. 8:325–329. 1990.PubMed/NCBI
10	Mohler JL, Gomella LG, Crawford D, Glode LM, Zippe CD, Fair WR and Marshal ME: Phase II evaluation of coumarin (1,2-benzopyrone) in metastatic prostatic carcinoma. Prostate. 20:123–131. 1992. View Article : Google Scholar : PubMed/NCBI
11	Chu CY, Tsai YY, Wang CJ, Lin WL and Tseng TH: Induction of apoptosis by esculetin in human leukemia cells. Eur J Pharmacol. 416:25–32. 2001. View Article : Google Scholar : PubMed/NCBI
12	Lopez-Gonzalez JS, Prado-Garcia H, Aguilar-Cazares D, Molina-Guarneros JA, Morales-Fuentes J and Mandoki JJ: Apoptosis and cell cycle disturbances induced by coumarin and 7-hydroxycoumarin on human lung carcinoma cell lines. Lung Cancer. 43:275–283. 2004. View Article : Google Scholar : PubMed/NCBI
13	Yang JY, Della-Fera MA and Baile CA: Esculetin induces mitochondria-mediated apoptosis in 3T3-L1 adipocytes. Apoptosis. 11:1371–1378. 2006. View Article : Google Scholar : PubMed/NCBI
14	Chuang JY, Huang YF, Lu HF, Ho HC, Yang JS, Li TM, Chang NW and Chung JG: Coumarin induces cell cycle arrest and apoptosis in human cervical cancer HeLa cells through a mitochondria- and caspase-3 dependent mechanism and NF-kappaB down-regulation. Vivo. 21:1003–1009. 2007.
15	Alvarez-Delgado C, Reyes-Chilpa R, Estrada Muñiz E, Mendoza-Rodríguez A, Quintero-Ruiz A, Solano J and Cerbón MA: Coumarin A/AA induces apoptosis-like cell death in HeLa cells mediated by the release of apoptosis-inducing factor. J Biochem Mol Toxicol. 23:263–272. 2009. View Article : Google Scholar : PubMed/NCBI
16	Zhang L, Jiang G, Yao F, He Y, Liang G, Zhang Y, Hu B, Wu Y, Li Y and Liu H: Growth inhibition and apoptosis induced by osthole, a natural coumarin, in hepatocellular carcinoma. PLoS One. 7:e378652012. View Article : Google Scholar : PubMed/NCBI
17	Zheng SY, Li Y, Jiang D, Zhao J and Ge JF: Anticancer effect and apoptosis induction by quercetin in the human lung cancer line A-549. Mol Med Rep. 5:822–826. 2012.PubMed/NCBI
18	Shokoohinia Y, Hosseinzadeh L, Alipour M, Mostafaie A and Mohammadi-Motlagh HR: Comparative evaluation of cytotoxic and apoptogenic effects of several coumarins on human cancer cell lines: Osthole induces apoptosis in p53-deficient H1299 cells. Adv Pharmacol Sci. 2014:8475742014.PubMed/NCBI
19	Finn G, Creaven B and Egan D: Modulation of mitogen-activated protein kinases by 6-nitro-7-hydroxycoumarin mediated apoptosis in renal carcinoma cells. Eur J Pharmacol. 481:159–167. 2003. View Article : Google Scholar : PubMed/NCBI
20	Musa MA, Badisa VL, Latinwo LM, Patterson TA and Owens MA: Coumarin-based benzopyranone derivatives induced apoptosis in human lung (A549) cancer cells. Anticancer Res. 32:4271–4276. 2012.PubMed/NCBI
21	Goel A, Prasad AK, Parmar VS, Ghosh B and Saini N: Apoptogenic effect of 7,8-diacetoxy-4-methylcoumarin and 7,8-diacetoxy-4-methylthiocoumarin in human lung adenocarcinoma cell line: Role of NF-kappa B, Akt, ROS and MAP kinase pathway. Chem Biol Interact. 179:363–374. 2009. View Article : Google Scholar : PubMed/NCBI
22	Goel A, Chhabra R, Ahmad S, Prasad AK, Parmar VS, Ghosh B and Saini N: DAMTC regulates cytoskeletal reorganization and cell motility in human lung adenocarcinoma cell line: An integrated proteomics and transcriptomics approach. Cell Death Dis. 3:e4022012. View Article : Google Scholar : PubMed/NCBI
23	Schilling WH, Crampton RF and Longland RC: Metabolism of coumarin in man. Nature. 221:664–665. 1969. View Article : Google Scholar
24	Mossman T: Rapid colorimetric assay for cellular growth and survival: Applications to proliferation and cytotoxicity assay. J Immunol Methods. 65:55–63. 1983. View Article : Google Scholar : PubMed/NCBI
25	Zhang Y: Single-cell microinjection technologies. Single-cell Analysis: Methods and Protocols. Lindström S and Andersson-Svahn H: (Heidelberg). Humana Press. 169–176. 2012. View Article : Google Scholar
26	Elinos-Báez CM, León F and Santos E: Effects of coumarin and 7-OH-coumarin on bcl-2 and Bax expression in two human lung cancer cell lines in vitro. Cell Biol Int. 29:703–708. 2005. View Article : Google Scholar : PubMed/NCBI
27	Zlabinger GJ, Nöhammer C, Böhmig GA and Menzel JE: Mode of action of coumarin in immune cells. J Cancer Res Clin Oncol. 120(Suppl): S17–S18. 1994. View Article : Google Scholar : PubMed/NCBI
28	Marshall ME, Mohler JL, Edmonds K, Williams B, Butler K, Ryles M, Weiss L, Urban D, Bueschen A and Markiewicz M: An updated review of the clinical development of coumarin (1,2-benzopyrone) and 7-hydroxycoumarin. J Cancer Res Clin Oncol. 120(Suppl): S39–S42. 1994. View Article : Google Scholar : PubMed/NCBI
29	Wittgen HG, van den Heuvel JJ, van den Broek PH, Siissalo S, Groothuis GM, de Graaf IA, Koenderink JB and Russel FG: Transport of the coumarin metabolite 7-hydroxycoumarin glucuronide is mediated via multidrug resistance-associated proteins 3 and 4. Drug Metab Dispos. 40:1076–1079. 2012. View Article : Google Scholar : PubMed/NCBI