Mitochondrial-mediated apoptosis in lymphoma cells by the diterpenoid lactone andrographolide, the active component of Andrographis paniculata.
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.
Yang
Shuo
S
Department of Medicine, Northwestern University Feinberg School of Medicine, Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, Illinois, USA.
Evens
Andrew M
AM
Prachand
Sheila
S
Singh
Amareshwar T K
AT
Bhalla
Savita
S
David
Kevin
K
Gordon
Leo I
LI
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
D000970:Antineoplastic Agents; D004224:Diterpenes; D017382:Reactive Oxygen Species; C030419:andrographolide
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