A Plant Triterpenoid Avicin D Stimulates Adipocytic Differentiation of Bone Marrow Stromal Cells and Promotes Their Pro-Survival Effects On Acute Monoblastic Leukemia Cells
Authors: Tabe Yoko , Jin Linhua , Kazuno Saiko , Fujimura Tsutomu , Matsushita Hiromichi , Ueno Takashi , Andreeff Michael , Gutterman Jordan U. , Konopleva Marina 
Published: October 2019

Abstract
Abstract 4315
A natural triterpene Avicin D induces apoptosis in various tumor cells and regulates cellular metabolism via Glucocorticoid receptor (GR) signalling, known to be involved in adipocyte differentiation (Tang, Annu Rev Biochem. 2012;81:715). Since adipocytes represent an essential component of the aging bone marrow (BM) microenvironment (Tabe, Blood 2004;103:1815), and promote monocytic differentiation and survival of monoblastic leukemia cells (Tabe, ASH, 2011), we examined the potential of Avicin D to modulate BM adipocytes and its effects on the survival of the leukemic cells.
In mature adipocytes derived from BM mesenchymal stem cells (MSCs), Avicin D (1ƒÊM, 48 hrs) significantly increased the number of lipid vesicles without apoptosis induction or significant cell cycle arrest. This was associated with upregulation of adipose-specific genes, PPARG and fatty acid binding protein 4 (FABP4), and inflammation-related haptoglobin (HP) gene (p<0.005, Figure 1). Avicin D further enhanced leptin release from adipocytes and inflammatory cytokines production, IL-6 and IL-8, from both MSCs and adipocytes (Figure 2). Of note, Avicin D increased pro-survival Bcl-2 mRNA expression in adipocytes (fold increase compared to MSCs: MSCs+Avicin D 1.0±0, adipocytes 1.8±0.1, adipocytes+Avicin D 4.4±0.6, p=0.02).
We next investigated the effects of MSCs and adipocytes pre-treated with 1mM Avicn D for 24 hours on co-cultured monoblastic leukemia cell line U937. Avicin D (1ƒÊM) moderately (26±5 %) inhibited cell proliferation of U937 cells cultured alone as detected by MTT assay. Serum-starvation induced cell death in U937 cells was inhibited by MCS or adipocyte co-culture, and further blocked by Avicin D-pretreated adipocytes, which was accompanied with cell cycle arrest (sub-G1 fraction; control U937 36.1±7.1%, co-culture with MSC 17.9±4.1%, MSC+Avicin D 20.9±3.9%, adipocyte 16.9±2.9, adipocyte+Avicin D 11.5±0.8%, p=0.03, G0/G1 phase; control 39.1±5.9%, with MSC 44.3±1.0%, MSC+Avicin D 40.3±1.3%, adipocyte 40.6±1.3, adipocyte+Avicin D 50.4±2.3%, p=0.03). Co-culture of U937 cells with Avicin D-pretreated adipocytes induced upregulation of PPARG and CD36, markers of monocytes/macrophages maturation in U937 cells (fold increase compared to control U937: PPARG: co-culture with adipocytes 2.1±0.4, adipocytes+Avicin D 13.8±6.2; CD36: adipocytes 2.2±0.4, adipocytes+Avicin D 4.7±2.9). Co-culture with Avicin D-pretreated adipocytes also caused 3.1 ±0.4 fold increase of BCL2 mRNA in U937 cells compared to the one of co-cultured with control adipocytes (p=0.02). The purity of U937 cells separated from adipocytes was confirmed by lack of CD90 mRNA expression by PCR. Utilization of the proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ) with two-dimensional-liquid chromatography-tandem mass spectrometry allowed the identification of 1,634 proteins. We found changes in 14 proteins in U937 cells co-cultured with Avicin D-pretreated adipocytes compared to untreated adipocytes. Among 13 down-regulated proteins, three are involved in pyruvate metabolism (Glyoxalase I; p=0.01, Lactate dehydrogenase B; p=0.02, Pyruvate kinase; p=0.03), and four proteins participate in cell cycle progression (DNA replication licensing factor MCM4, MCM5; p=0.02, p=0.03, DNA-dependent protein kinase; p=0.02, Interleukin enhancer-binding factor 3; p=0.005). Voltage-dependent anion-selective channel protein 2, which is involved in the mitochondrial apoptotic pathway via regulation of Bcl-2, and an enzymatic antioxidant Superoxide dismutase were up-regulated (p=0.04, p=0.03). These data indicate that U937 cells co-cultured with Avicin D pretreated adipocytes were induced to undergo cell cycle arrest, escape apoptosis with down-regulation of glucose metabolism.
Altogether, these results suggest that Avicin D induces adipocytic differentiation of BM-derived stromal cells, promotes production of leptin and inflammatory cytokines, which in turn supports survival of monocytic leukemia cells. These findings suggest a contributory role of the aging “pro-inflammatory” bone marrow microenvironment in the reduced efficacy of cytotoxic chemotherapy which is prevalent in elderly AML patients.

Disclosures:
No relevant conflicts of interest to declare.