The Astragalus Membranaceus Herb Attenuates Leukemia by Inhibiting the FLI1 Oncogene and Enhancing Anti-Tumor Immunity

Abstract

Astragalus membranaceus (AM) herb is a component of traditional Chinese medicine used to treat various cancers. Herein, we demonstrate a strong anti-leukemic effect of AM injected (Ai) into the mouse model of erythroleukemia induced by Friend virus. Chemical analysis combined with mass spectrometry of AM/Ai identified the compounds Betulinic acid, Kaempferol, Hederagenin, and formononetin, all major mediators of leukemia inhibition in culture and in vivo. Docking analysis demonstrated binding of these four compounds to FLI1, resulting in downregulation of its targets, induction of apoptosis, differentiation, and suppression of cell proliferation. Chemical composition analysis identified other compounds previously known having anti-tumor activity independent of the FLI1 blockade. Among these, Astragaloside-A (As-A) has marginal effect on cells in culture, but strongly inhibits leukemogenesis in vivo, likely through improvement of anti-tumor immunity. Indeed, both IDO1 and TDO2 were identified as targets of As-A, leading to suppression of tryptophane-mediated Kyn production and leukemia suppression. Moreover, As-A interacts with histamine decarboxylase (HDC), leading to suppression of anti-inflammatory genes TNF, IL1B/IL1A, TNFAIP3, and CXCR2, but not IL6. These results implicate HDC as a novel immune checkpoint mediator, induced in the tumor microenvironment to promote leukemia. Functional analysis of AM components may allow development of combination therapy with optimal anti-leukemia effect.

Keywords: Astragalus membranaceus; DTO2; FLI1; HDC; IDO1; drug–protein interaction; leukemia inhibition; traditional Chinese medicine; tumor microenvironment.

Figure 1

Astragalus injection (Ai) inhibits leukemia survival and proliferation in culture and blocks leukemogenesis in mice. (A) IC₅₀ analysis of Ai treatment for the indicated leukemia cell lines in culture. (B) Proliferation rate of Ai treatment on HEL cells using the indicated concentration of drug. (C) Microscopic images of HEL cells treated with the indicated concentration of Ai. (D) Apoptosis index of HEL cells treated with the indicated concentration of Ai. Average of three experiments is shown in right panel. (E) Cell cycle index of HEL cells treated with the indicated concentration of Ai treatment. Average of three experiments is shown in the right pane. (F) Survival rate of leukemic mice treated with saline or Ai. (G,H) Spleen weight (G) and hematocrit (H) of control and Ai-treated mice at 90 days after virus inoculation. Data are expressed as mean ± SD, n = 3 independent experiments. *** p < 0.001, ** p < 0.01 and * p < 0.05, ns: no significant differences.

Figure 2

Ai treatment inhibits FLI1 function leading to leukemia suppression. (A) Expression of FLI1 protein in HEL cells treated with the indicated concentration of Ai. Relative density (Rd) of the band is indicated. (B) Expression of MiR145 in HEL cells treated with the indicated concentration of Ai, by RT-qPCR. (C) Expression of FLI1 mRNA in HEL cells treated with the indicated concentration of Ai, by RT-qPCR. (D) Expression of erythroid markers CD71 and CD235a in HEL cell treated with Ai or vehicle control, as determined by flow cytometry. Relative percentage of three experiments is shown in the right panel. (E) Knockdown of FLI1 in HEL cells using shFLI1, as determined by Western blotting. (F) Increase IC₅₀ by Ai treatment in shFLI1-HEL versus scrambled-control cells. Data are expressed as mean ± SD, n = 3 independent experiments. *** p < 0.001, ** p < 0.01 and * p < 0.05, ns: no significant differences.

Figure 3

AM contains several compounds with potent anti-FLI1 activity. (A) The diagram of AM extraction toward identification of active compounds from the published TCMSP database list. The active compounds are based on criteria such as oral benefit (OB) [higher than 30%] and drug-like property (DL) [higher than 0.18]. (B) List of 20 active compounds and their anti-FLI1 activity based on their affinity to bind FLI1. The binding energy to FLI1 for each compound (kcal/mol) is provided.

Figure 4

Betulinic acid (Ba) binds to FLI1 and inhibits HEL cell proliferation and suppresses leukemogenesis. (A) The chemical structure of Ba. (B) Three-dimensional (left) and two-dimensional (right) interaction of Ba to FLI1 and its binding energy. (C) Ba downregulates FLI1 protein in a dose-dependent manner. (D) Ba induces MiR145 transcription in a dose-dependent manner. (E) Effect of Ba on FLI1 transcription, determined by RT-qPCR. (F) Ba inhibits HEL cell proliferation dose dependently in culture. (G) Ba blocks leukemogenesis in the mouse model of erythroleukemia induced by F-MuLV. (H-I) Spleen weight (H) and hematocrit (I) of leukemic mice treated with Ba. Data are expressed as mean ± SD, n = 3 independent experiments. *** p < 0.001, ** p < 0.01 and * p < 0.05.

Figure 5

Kaempferol (Ka) blocks FLI1 function, causing inhibition of HEL cell proliferation and suppression of erythroleukemogenesis. (A) The chemical structure of Ka. (B) Three-dimensional (top) and two-dimensional (bottom) structure of binding of Ka to FLI1 protein with its binding affinity (kcal/mol). (C) Ka treatment of HEL cells downregulates FLI1 protein expression, by Western blotting. (D) Ka increases the transcription of MiR145 in HEL cells, in a dose-dependent manner. (E) Ka treatment downregulates FLI1 mRNA expression in HEL cells. (F) Ka treatment of HEL cells suppresses cell proliferation in a dose-dependent manner. (G) Ka exhibits a higher IC₅₀ in shFLI1-HEL cells versus scrambled-control cells. (H) Ka significantly suppresses erythroleukemogenesis in vivo. Data are expressed as mean ± SD, n = 3 independent experiments. *** p < 0.001, ** p < 0.01 and * p < 0.05.

Figure 6

Hederagenin (He) and Formononectin (Fn) inhibits FLI1 to block HEL cell proliferation and suppress erythroleukemia cell proliferation. (A,F) Molecular structure of He (A) and Fn (F). (B,G) Three- and two-dimensional structure of interaction of He (B) and Fn (G) to FLI1 with their binding energy (kcal/mol). (C,H) Inhibition of HEL cell proliferation by He (C) and Fn (H) in culture at the indicated doses. (D,I) Downregulation of FLI1 protein by He (D) and Hn (I) at the indicated doses, determined by Western blot. (E,J) Induction of MiR145 by He (E) and Fn (J), by RTqPCR. Data are expressed as mean ± SD, n = 3 independent experiments. *** p < 0.001 and ** p < 0.01.

Figure 7

Astragaloside A (As-A) inhibits leukemia by altering immune checkpoints. (A) Chemical structure of As-A. (B) Three- and two-dimensional structure of As-A with its binding energy to FLI1, determined by molecular docking. (C) Effect of As-A on FLI1 expression, as determined by Western blotting. (D) Effect of As-A on MiR145 expression using the indicated doses. (E) As-A (15 mg/kg) inhibits leukemia in the mouse model of erythroleukemia induced by F-MuLV. (F–H) Expression of the IFN-γ (F), TDO2 (G), and IDO1 (H) genes in HEL cells treated with or without IFN-γ and As-A, as determined by RT-qPCR. (I) Kyn activity in HEL cells treated with or without IFNγ and As-A, as determined by Elisa. (J–L) Expression of the HDC (J), TNF (K), and IL1B (L) in HEL cells treated for 24 h with the indicated doses of As-A, determined by RT-qPCR. Data are expressed as mean ± SD, n = 3 independent experiments. *** p < 0.001, ** p < 0.01 and * p < 0.05 versus Ctrl groups; ^### p < 0.001, ^## p < 0.01 and ^# p < 0.05 versus IFN-γ groups.

Figure 8

Graphical image. The mode of leukemia inhibition by Astragalus membranaceus (AM). AM contains a large number of compounds exhibiting various functions to confer anti-cancer activity. At least four compounds, Ba, Ka, He, and Fn, exert their anti-cancer activity through inhibition of FLI1 function. Other compounds also inhibit leukemia through other mechanisms. While the As-A compound displays a marginal direct effect on leukemia cells in culture, it blocks immunity against leukemia cells by blocking the function of the checkpoint proteins IDO1/DOT2 and pro-inflammatory protein HDC within the tumor microenvironment. The combine effect of these compounds represents the optimal inhibitory response of AM on leukemic cells.