Foxo 3a Signaling Mediates Apoptosis in Lymphoma Cells By the Diterpenoid Lactone Andrographolide (Andro), the Active Component of Andrographis Paniculata
Authors: Yang Shuo , Ding Bo , Ying Fei , Svetlichnaya Jana , Tom Austin , Prachand Sheila , Wang Yanzhuang , Gordon Leo I 
Published: October 2019

Abstract
Introduction: 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 lymphoma, leukemia and other solid tumor cell lines. We have shown that Andro caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples that was mediated through mitochondrial pathways and enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK (Yang et al Clin Cancer Res 2010; 16(19):4755). We hypothesized that the tumor suppressor, FOXO3a may be involved in signaling pathways that lead to apoptosis and to test that hypothesis we investigated the role of FOXO3A in Andro induced signaling in lymphoma.
Methods: We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma (MCL) line Granta, the transformed 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 and MCL. We transfected shRNA FOXO3a by electroporation to build stable cells with constant knockdown of FOXO3a in Ramos and SUDHL4 cell lines. We then compared the cell viability (MTT and Golgi fragmentation), apoptosis (Annexin V by flow), c-MYC and Bcl2 expression, death receptors 4 (DR4) expression and cell cycle related proteins in wild type and FOXO3a knockdowns.
Results: We found that Andro resulted in nuclear translocation of FOXO3a in Ramos at early time points. We found that shRNA stable knockdown of FOXO3a in Ramos and SUDHL4 cell lines protected cells (Ramos and SUDHL4) from Andro-induced apoptosis (Figure 1). Moreover, in multiple cell lines, we found that Andro decreased c-MYC expression, which was abrogated in part by FOXO3A knockdown compared with wild type cells. Similarly, reduction in mitochondrial membrane potential by Andro is abrogated in the FOXO 3a knockdown cells. These data suggest that FOXO3a regulates c-MYC stabilization by mitochondrial proteins (for example TFAM and MAD-1). In the Granta cell line, derived from Mantle Cell Lymphoma (MCL) and in an MCL patient sample, Andro reduced c-MYC expression. We also found that Andro induced Death Receptor 4 (DR4) at the mRNA and protein level in Granta cells in a dose-dependent manner. The cell cycle control proteins Aurora, p21, p27 (the latter 2 regulated by FOXO3a), are also increased by Andro. When cell death was measured by Golgi fragmentation and subsequent collapse, we found that Andro induced Golgi fragmentation in Granta and SUDHL4 cells
Conclusion: Andro-induced lymphoma cell apoptosis is mediated through multiple signaling pathways, including FOXO3a, which appears to play a significant role, perhaps by regulating c-MYC stabilization and BCL2 expression and cell cycle proteins. These data suggest that this novel diterpenoid lactone compound deserves further pre-clinical and clinical testing in malignant lymphoma.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.