PB1708 THERAPEUTIC POTENCIAL OF GAMBOGIC ACID AND SILIBININ IN ACUTE MYELOBLASTIC LEUKEMIA – IN VITRO STUDIES

Abstract

Background:Acute myeloblastic leukemia (AML) is a malignant hematological disease characterized by an uncontrolled proliferation of multipotent hematopoietic progenitors with subsequent accumulation of immature myeloid cells in the bone marrow. Despite the development of new therapies, standard treatment for AML relies in intensive chemotherapy with combination of several drugs. Natural compounds such as gambogic acid (GA), extracted from Garcinia hanburyi, and silibinin (SLB), from Silybum marianum, showed an antitumoral effect in many neoplasms, by induction of apoptosis and differentiation and proliferation inhibition. SLB display several targets including anti‐apoptotic molecules (survivin), apoptotic proteins (caspases), cell proliferation mediators (NF‐KB), cell cycle regulators (cyclin D1), tumor suppressors (p53), and chaperones (HSP90). On the other hand, GA induces cell death through the interaction with several extrinsic and intrinsic apoptotic proteins, autophagy induction as well as telomerase and proteasome inhibition. However, the effect of these two compounds on AML is still unclear.Aims:The aim of this study was to evaluate the therapeutic potential of gambogic acid and silibinin in monotherapy, in single and daily fractioned administration scheme, and in therapeutic combination, in acute myeloid leukemia cell lines.Methods:We used two in vitro AML models, KG‐1 and THP‐1 cells, incubated in absence and presence of increasing concentrations of GA (5 – 1000 nM) and SLB (0.5 – 250 μM), in monotherapy at single dose and at daily administration (e.g. GA 150 nM in single dose vs GA 50 nM/day during 3 days) and in combination therapy (GA 50 nM + SLB 15 μM). Metabolic activity was evaluated by resazurin assay. Cell death was assessed by optic microscopy (May‐Grünwald‐Giemsa staining) and flow cytometry (FC), using the Annexin V and 7‐AAD double staining. Cell cycle using IP/RNase and caspase‐3 activation were evaluated by FC. Combination Index (CI) was calculated using CompuSyn software. All the adequate statistical analysis was performed considering a level of significance of 95% (p < 0.05).Results:Results show that GA and SLB decrease the metabolic activity in a time, dose and cell line dependent manner, being KG‐1 the most sensitive cells to SLB. After 72 h of incubation, IC50 of GA was 338 nM in KG‐1 cells and 453 nM in THP‐1, while IC50 for SLB was 33 μM and 75 μM in KG‐1 and THP‐1, respectively. Daily administration was more effective in KG‐1 cells, whereas combination therapy have a synergistic effect (CI < 1) in both cell lines (CI=0.17 in KG1; CI=0.16 in THP‐1 cells). These compounds induced cell death by apoptosis, with increased caspase‐3 activation, and typical morphological features of apoptosis, namely blebbing and DNA fragmentation. Cell cycle analysis demonstrated that SLB induced cell cycle arrest in G0/G1, mainly in THP‐1 cell line, while GA had no cytostatic effect.Summary/Conclusion:Our results suggest that GA and SLB may be potential treatment options in acute myeloblastic leukemia, particularly in therapeutic combination. However, their efficacy is depending on cellular characteristics and/or on drug administration scheme.