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Purpose: 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. Experimental Design: 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 (MEFBax−/−/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. Clin Cancer Res; 16(19); 4755–68. ©2010 AACR.

Purpose: We investigated the cytotoxicity and mechanisms of cell death of the broad-spectrum histone deacetylase (HDAC) inhibitor PCI-24781, alone and combined with bortezomib in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines and primary lymphoproliferative (CLL/SLL) cells. Experimental Design: Apoptosis, mitochondrial membrane potential, cell cycle analysis, and reactive oxygen species (ROS) were measured by flow cytometry, whereas caspase activation was determined by Western blot. Nuclear factor κB (NF-κB)-related mRNAs were quantified by reverse transcription-PCR, NF-κB–related proteins by Western blotting, and NF-κB DNA-binding activity by electromobility shift assay. Finally, gene expression profiling was analyzed. Results: PCI-24781 induced concentration-dependent apoptosis that was associated with prominent G0/G1 arrest, decreased S-phase, increased p21 protein, and increased ROS in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. Dose-dependent apoptosis with PCI-24781 was also seen among primary CLL/SLL cells. PCI-24781–induced apoptosis was shown to be ROS- and caspase-dependent. Combined PCI-24781/bortezomib treatment resulted in strong synergistic apoptosis in all non-Hodgkin lymphoma lines (combination indices, 0.19-0.6) and was additive in Hodgkin lymphoma and primary CLL/SLL cells. Further, PCI-24781/bortezomib resulted in increased caspase cleavage, mitochondrial depolarization, and histone acetylation compared with either agent alone. Gene expression profiling showed that PCI-24781 alone significantly down-regulated several antioxidant genes, proteasome components, and NF-κB pathway genes, effects that were enhanced further with bortezomib. Reverse transcription-PCR confirmed down-regulation of NF-κB1 (p105), c-Myc, and IκB-kinase subunits, where NF-κB DNA binding activity was decreased. Conclusion: We show that PCI-24781 results in increased ROS and NF-κB inhibition, leading to caspase-dependent apoptosis. We also show that bortezomib is synergistic with PCI-24781. This combination or PCI-24781 alone has potential therapeutic value in lymphoma.