Matrine Synergistically Promotes Non-Toxic-Dose RLS3 Induced Ferroptosis in Acute Myeloid Leukemic Cells with MLL Translocation
Authors: Hong Yaonan , Yu Fanhua , Li Man , Dong Jingjie , Shen Yingying , Yang Xiawan , Shao Keding , Liu Qi , Wang Peicheng , Zhou Yuhong , Shen Yiping , Wu Dijiong 
Published: December 2024

MLL-rearrangement (MLL-r), one of the most common forms of chromosomal aberrations in acute myeloid leukemia (AML), was characterized by its high aggressiveness, low remission rates, propensity for recurrence, and shortened survival periods. Therefore, there was an urgent need to identify effective therapeutic targets and develop novel treatments to address this pressing issue. Ferroptosis, a novel form of cell death triggered by iron-dependent oxidative damage, held inherent advantages in inducing ferroptosis of leukemic cells due to their pathological basis of elevated intracellular iron load, which was reported to be triggered by p53 activation. The induction of ferroptosis in MLL-r leukemic cells by ferroptosis activators (such as RSL3, one of the GPX4 inhibitor) may provide novel therapeutic strategies for leukemia. However, its clinical development and application had been significantly constrained due to its toxicity and side effects, including liver and kidney dysfunction.

Matrine (MAT) is an effective quinoline alkaloid component extracted from Sophora flavescens, which exhibits a diverse range of pharmacological activities including anti-tumor, anti-inflammatory, and immunosuppression. Current research has reported that the mechanisms of MAT in anti-leukemia are complex and diverse, and there was a report indicating that MAT can exert an anti-cervical cancer activity by inducing ferroptosis. Our team had previously explored the multiple potential effects of MAT in treating hematologic malignancies, including leukemia, lymphoma, as well as multiple myeloma, while the mechanisms were not fully elaborated.

In this study, we found that MAT alone could not induce ferroptosis in MLL-r leukemic cells (MOLM-13 and MV4-11), but can synergistically promote ferroptosis with a combination of non-toxic dosage of RSL3 (1μM RLS3 (resulting in 96.17% cell viability) and 0.25 μM MAT for MOLM-13 cells, and 0.125 μM RLS3 (resulting in 95.03% cell viability) and 0.25 μM MAT for MV4-11 cells). Results revealed a decrease in mitochondrial membrane potential and glutathione (GSH) levels was observed in the combination group (P &lt;0.05). Additionally, neither MAT nor RSL3 treatment alone induced an increase in intracellular ferrous ion (IFI). However, the combination of MAT and RSL3 effectively promoted the accumulation of IFI in both cell lines (P &lt;0.01). To further confirm the combined effect on regulating the ferroptosis pathway, we assessed the expression of ferroptosis-related proteins. The results indicated that MAT + RSL3 significantly downregulated the protein and gene expression of GPX4 and SLC7A11 in MOLM-13 shCtrl cells, and effect reversed by p53 knockdown. Similarly, this phenomenon was observed in MV4-11 cells (P &lt;0.05). Moreover, the necrosis rates of the Ctrl, MAT, RSL3, and MAT + RSL3 groups were 10.79%, 15.58%, 16.94%, and 82.39%, respectively, in MOLM-13 shCtrl cells; and 11.71%, 7.61%, 16.44%, and 54.85%, respectively, in MV4-11 shCtrl cells. In MOLM-13 shp53 and MV4-11 shp53 cells, these rates were 3.70%, 2.03%, 3.27%, and 2.95%; and 5.92%, 10.22%, 6.62%, and 16.53%, respectively. The combination of MAT and RSL3 effectively promoted the expression of intracellular lipid peroxidation in both shCtrl cell lines (P &lt;0.05). However, there was no significant difference in lipid peroxidation levels compared to the Ctrl group in both shp53 cell lines.

In vivo imaging results demonstrated a reduction in fluorescence intensity in mice treated with the combination of MAT and RSL3 at day 14 compared to the Vehicle group, whereas no significant difference was observed in the MAT or RSL3 solely treated groups. The proportion of CD45+ cells in the bone marrow was significantly decreased in the RSL3 group, MAT + RLS3 group, and the Positive control group (P &lt;0.05), with the lowest proportion (10%) observed in the Positive control group. There was no statistically significant difference in mouse weight among the groups during the treatment period.

In summary, the combination of MAT with the induction of ferroptosis using a non-toxic dosage of RSL3 demonstrated a synergistic effect on promoting cell death, potentially through regulation of the p53/SLC7A11/GPX4 pathway. Further investigation is needed to assess the potential translation of this strategy into clinical applications.