The Diterpenoid Lactone Andrographolide (ANDRO) Stimulates Autophagy and Induces Reactive Oxygen Species (ROS)-Dependent Apoptosis in Lymphoma Cell Lines

ANDRO is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an important herbal medicine used in China. It has been reported to have anti-inflammatory, anti-hypertensive, anti-viral and immunostimulant properties. It has also been shown to inhibit cancer cell proliferation and induce apoptosis in HL-60 (leukemia), PC-3 (prostatic adenocarcinoma), MDA-MB-231 (breast cancer), HepG2 (liver cancer), HeLa (cervical cancer) and HCT116 (colorectal cancer) cell lines. The diterpenoids have been found to generate ROS and may increase apoptosis by altering the cellular redox state. We hypothesized that ANDRO would lead to cell death in lymphoma cell lines and that the effect may be related to altered cellular redox state. We studied the Burkitt p53 mutated Ramos cell line, the mantle cell lymphoma line Granta and L428, a resistant EBV-negative Hodgkin lymphoma cell line. We found that after incubation with increasing concentrations of ANDRO, there was dose and time-dependent cell death as measured by MTT. The IC50 (concentration that achieved 50% cell proliferation inhibition) at 48h was 20μM for Ramos, 40μM for Granta, and 50μM for L428. ROS was measured by oxidation of 2’7’dichlorofluorescein diacetate (DCFDA) to dichlorofluorescein (DCF) and analyzed by fluorescence-activated cell sorting (FACS) following incubation at 1hour (h), 2h, 3h, 5h, 38h, and 48h with ANDRO (20–80μM). ANDRO increased ROS production in all lymphoma cell lines, which was abrogated by the antioxidant N-acetyl-L-cysteine (NAC). Maximum ROS generation with ANDRO was seen at 48h for Ramos (1.7 fold), 5h for Granta (1.6 fold), and 38h for L428 (2.4 fold). To determine the mechanism of cell death, we measured apoptosis by Annexin-V/propidium iodide (PI), and detected by flow cytometry (FACS). Cells were treated with ANDRO in the presence or absence of the reduced glutathione (GSH) depleting agent buthionine sulfoximine (BSO) (100μM) for 28h, 48h, and 72h. We found that the AC50 (concentration that achieved 50% apoptosis) was 40μM for Ramos at 72h, 40μM for Granta at 48h and >80μM for L428 at 48h, while in the presence of BSO it was <10μM for Ramos at 72h, between 30–40μM for Granta at 28h and between 30–40μM for L428 at 48h. Apoptosis was completely blocked, by NAC, both in the presence and absence of BSO. Further, ANDRO induced PARP cleavage and activation of caspases 3, 8, and 9 in Granta and Ramos. Next, we explored the relationship of ANDRO and Forkhead transcription factors. ANDRO caused dephosphorylation of FOXO3a or FOXO1, in a dose- and time-dependent manner, and this was reversible by NAC. Downstream proteins of FOXO3a, Bim, p27^kip1 and the isoforms of the autophagy-related protein LC3B were upregulated, and this was reversed by NAC. The LC3B isoform-II, which is cleaved from LC3B-I, is a marker of autophagy activation. To determine the role of autophagy in cell death related to ANDRO, we inhibited autophagy with 3-methyladenine (1–2mM) and found significant enhancement of ANDRO-induced apoptosis in Granta and Ramos. Finally, ANDRO induced apoptosis (>60% Annexin-V⁺/PI⁺) in malignant B-cells from a patient with chronic lymphocytic leukemia/small lymphocytic lymphoma (trisomy 12, peripheral blood absolute lymphocyte count 95.2 K/uL, bulky adenopathy) very low concentrations (5μM at 18h) in vitro, which was also reversible with NAC. We conclude that ANDRO induces ROS-dependent apoptosis in lymphoma cell lines and in a primary tumor sample, which is enhanced by depletion of GSH and inhibited by the antioxidant NAC. These effects appear to proceed through caspase activation and inhibition of autophagy, and are in part dependent on signaling through forkhead transcription factors and altered cellular redox pathways. Further studies of diterpenoids as single agents or in combination with other anti-lymphoma agents are warranted.