Research on Different Compound Combinations of Realgar-Indigo Naturalis Formula to Reverse APL Arsenic Resistance By Regulating Autophagy through mTOR Pathway

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia, and 90% of APL patients carry positive fusion gene of the promyelocytic leukemia protein (PML)- retinoic acid receptor α (RARα), which drives the development of APL. All-trans retinoic acid and arsenic trioxide is able to cure 90% of APL patients by targeting PML-RARα. However, there are still 5%-10% of patients who will relapse due to drug resistance. Compound realgar-indigo naturalis formula (RIF) was developed based on the characteristics of APL pathogenesis. It contains four Chinese herbs: realgar, natural indigo, danshen root and heterophylly falsestarwort root. Clinical studies have confirmed that RIF has a certain efficacy on APL, which shown non-inferiority compared with arsenic trioxide. In addition, RIF combined with all-trans retinoic acid has become the first-line treatment regimen in APL guidelines in China. This study further clarified the efficacy of RIF on arsenic-resistant APL, explored the mechanism, and laid the foundation for further improving the efficacy of APL.

O bjective: To investigate the mechanism of different combinations of the compound of RIF in reversing arsenic resistance in APL.

M ethods: The arsenic-resistant cell line HL60-PML ^A216V-RARα, which could stably express PML ^A216V-RARα, was established by lentiviral transfection. Among compounds of RIF, the active compound of realgar is tetra-arsenic tetra-sulfide (As ₄S ₄, A), for natural indigo is indirubin (I), for danshen root is tanshinone IIa (T), and the for heterophylly falsestarwort root is total saponins of Radix Pseudostellariae (S). The four compounds were applied on the HL60-PML ^A216V-RARα cell line. Cell viability was detected by CCK-8 method. Hoechst staining was used to observe cell morphology, Annexin V/PI double staining was applied to detect cell apoptosis, JC-1 detection was used to detect mitochondrial membrane potential, flow cytometry was used to detect CD33 expression to evaluate cell differentiation, and Western blotting was used to observe the changes in the levels of PML ^A216V-RARα, apoptosis-related factors, PI3K/AKT/mTOR and autophagy in the control, A, ITS and AITS groups. The PI3K inhibitor LY294002 and the autophagy inhibitor Baf A1 were combined and applied to observe the changes in the levels of PML ^A216V-RARα, mTOR and p62 after blocking the PI3K or autophagy pathway.

Results: The arsenic-resistant cell line HL60-PML ^A216V-RARα was successfully established. Compared with the control group, A, ITS and AITS groups all inhibited the growth of HL60-PML ^A216V-RARα cells in a time-dependent manner ( P < 0.0001). The IC50 value of AITS was significantly lower than the other groups ( P < 0.0001), and AITS group exhibited the strongest inhibitory effect, followed by ITS and A. Hoechst staining and JC-1 detection both showed that AITS had the strongest effect in inducing apoptosis. Annxin V/PI detection showed that the apoptosis rate of the control group was 10.04%, 15.19% for A group, 12.46% for ITS group, and 28.05% for AITS group. The differences between the groups were statistically significant ( P < 0.0001). Detection of CD33 expression showed: control group (79.72%), ITS group (79.19%), A group (28.60%), AITS group (24.46%), and there were significant differences between groups ( P < 0.001 or P < 0.0001). AITS could reduce PML ^A216V-RARα protein, caspase3, Bcl-2 levels more than ITS and A ( P < 0.05 or P < 0.0001), and ITS was better than AITS and A in increasing cleaved PARP-1 levels ( P < 0.0001). AITS decreased the levels of PI3K, mTOR, P-mTOR, AKT, p-AKT and p62 and increased the level of LC3B ( P < 0.001 or P < 0.0001). Electron microscopic detection of autophagy showed that AITS had the highest level of autophagy, AITS combined with LY294002 increased the level of p62 and decreased the level of mTOR ( P < 0.0001) compared to the AITS group; while when AITS was combined with Baf A1, the level of p62 increased ( P < 0.0001) and the level of mTOR also increased ( P < 0.001). Autophagy could induced by serum starvation. As the concentration of fetal bovine serum decreased, the effects of degrading PML ^A216V-RARα and p62, and LC3B level increasing were stronger.

C onclusion: Tanshinone IIa, indirubin and total saponins of Radix Pseudostellariae could enhance the effect of tetra-arsenic tetra-sulfide down-regulating PML ^A216V-RARα, and the mechanism was suggested to be related to inhibiting mTOR pathway to activate autophagy.