title: Mitochondrial-mediated apoptosis in lymphoma cells by the diterpenoid lactone andrographolide, the active component of Andrographis paniculata.
abstract: OBJECTIVE
  Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, antiviral, and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in leukemia and solid tumor cell lines.
  
  
  METHODS
  We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma (MCL) line Granta, the follicular lymphoma (FL) cell line HF-1, and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL, DLBCL, and MCL.
  
  
  RESULTS
  We found that andrographolide resulted in dose- and time-dependent cell death as measured by MTT. Andrographolide significantly increased reactive oxygen species (ROS) production in all cell lines. To determine mechanism of cell death, we measured apoptosis by Annexin V/propidium iodide in the presence and absence of the antioxidant N-acetyl-l-cysteine (NAC), the glutathione (GSH)-depleting agent buthionine sulfoxamine (BSO), or caspase inhibitors. We found that apoptosis was greatly enhanced by BSO, blocked by NAC, and accompanied by poly(ADP-ribose) polymerase cleavage and activation of caspase-3, caspase-8, and caspase-9. We measured BAX conformational change and mitochondrial membrane potential, and using mouse embryonic fibroblast (MEF) Bax/Bak double knockouts (MEF(Bax-/-/Bak-/-)), we found that apoptosis was mediated through mitochondrial pathways, but dependent on caspases in both cell lines and patient samples.
  
  
  CONCLUSIONS
  Andrographolide caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples, which was enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK. Further studies of diterpenoid lactones in lymphoma are warranted.
authors.affiliation: Department of Medicine, Northwestern University Feinberg School of Medicine, Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, Illinois, USA.
authors.forename: Shuo
authors.initials: S
authors.lastname: Yang
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Evens
authors.forename: Sheila
authors.initials: S
authors.lastname: Prachand
authors.forename: Amareshwar T K
authors.initials: AT
authors.lastname: Singh
authors.forename: Savita
authors.initials: S
authors.lastname: Bhalla
authors.forename: Kevin
authors.initials: K
authors.lastname: David
authors.forename: Leo I
authors.initials: LI
authors.lastname: Gordon
mesh_terms: D035685:Andrographis / Q000737:chemistry*; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004224:Diterpenes / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008223:Lymphoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors
chemical_list: D000970:Antineoplastic Agents; D004224:Diterpenes; D017382:Reactive Oxygen Species; C030419:andrographolide
references.citation: Redox Rep. 2009;14(4):176-84
references.pmid: 19695125
references.citation: Oncogene. 2009 Jul 23;28(29):2690-6
references.pmid: 19503098
references.citation: Br J Pharmacol. 2005 Mar;144(5):680-6
references.pmid: 15678086
references.citation: J Pharm Pharmacol. 2009 Jan;61(1):69-78
references.pmid: 19126299
references.citation: Pak J Pharm Sci. 2007 Jan;20(1):9-15
references.pmid: 17337421
references.citation: J Immunol. 2004 Sep 15;173(6):4207-17
references.pmid: 15356172
references.citation: Blood. 2005 Feb 1;105(3):1265-73
references.pmid: 15388578
references.citation: Int J Toxicol. 2009 Jul-Aug;28(4):308-17
references.pmid: 19636073
references.citation: Breast Cancer Res. 2008;10(6):R104
references.pmid: 19061505
references.citation: Biochem J. 1999 Feb 15;338 ( Pt 1):107-13
references.pmid: 9931305
references.citation: J Ethnopharmacol. 2004 Jun;92(2-3):291-5
references.pmid: 15138014
references.citation: J Asian Nat Prod Res. 2008 May-Jun;10(5-6):467-73
references.pmid: 18464090
references.citation: Science. 2001 Apr 27;292(5517):727-30
references.pmid: 11326099
references.citation: J Pharmacol Exp Ther. 2008 Apr;325(1):226-35
references.pmid: 18174384
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Bioorg Med Chem Lett. 2004 Sep 20;14(18):4711-7
references.pmid: 15324893
references.citation: Zhong Xi Yi Jie He Xue Bao. 2008 Dec;6(12):1238-45
references.pmid: 19063836
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Feb 15;877(5-6):502-6
references.pmid: 19158000
references.citation: BMC Cancer. 2004 Jun 18;4:26
references.pmid: 15207007
references.citation: J Exp Ther Oncol. 2003 May-Jun;3(3):147-58
references.pmid: 14641821
references.citation: Methods. 2008 Mar;44(3):229-34
references.pmid: 18314053
references.citation: Biochem Pharmacol. 2006 Jul 14;72(2):132-44
references.pmid: 16740251
references.citation: In Vivo. 2005 May-Jun;19(3):551-7
references.pmid: 15875775
references.citation: Phytother Res. 2000 Aug;14(5):333-8
references.pmid: 10925397
references.citation: Mol Cancer Ther. 2008 Jul;7(7):2170-80
references.pmid: 18645026
references.citation: J Cell Sci. 2008 Jul 1;121(Pt 13):2186-96
references.pmid: 18544634
references.citation: Planta Med. 2005 Dec;71(12):1106-11
references.pmid: 16395645
references.citation: Phytomedicine. 2000 Oct;7(5):351-64
references.pmid: 11081986
references.citation: Int J Radiat Oncol Biol Phys. 2010 Jul 15;77(4):1232-9
references.pmid: 20610044
references.citation: Nat Rev Cancer. 2002 Sep;2(9):647-56
references.pmid: 12209154
references.citation: Blood. 2006 Mar 15;107(6):2501-6
references.pmid: 16291594
references.citation: Mol Cell. 2005 Feb 18;17(4):525-35
references.pmid: 15721256
references.citation: J Clin Pharm Ther. 2004 Feb;29(1):37-45
references.pmid: 14748896
references.citation: Cancer Lett. 2009 Apr 18;276(2):180-8
references.pmid: 19097688
references.citation: J Nat Prod. 2008 May;71(5):852-5
references.pmid: 18357994
references.citation: Drug Metab Lett. 2008 Dec;2(4):261-8
references.pmid: 19356103
references.citation: J Pharmacol Exp Ther. 2005 Jan;312(1):366-72
references.pmid: 15331658