The proposed mechanism of YZHI.

BackgroundThe unmet needs in treating acute myeloid leukemia(AML) promote us to look for more effective and less toxic therapies. In this study, we discovered that Yinzhihuang injection(YZHI), a traditional Chinese patent medicine for hepatitis treatment, suppressed the growth of AML cells.MethodAnti-proliferative activities of YZHI were measured by CCK-8 assay. Cell cycle arrest was evaluated by PI staining, and apoptosis was evaluated by annexin V/PI staining. To explore the cell cycle arrest and cell death mechanism induced by YZHI, we assessed a series of assays, including measurements of the protein expression and cellular ATP. The anti-tumor activity was further demonstrated in nude mice.ResultsFlow cytometric and biochemical analysis revealed that YZHI caused cell cycle arrest and induced apoptosis in the AML HL-60 cells. Mechanistically, YZHI activated AMPK by promoting phosphorylation of the kinase. The active AMPK negatively regulated the downstream target mTORC1, leading to the inhibition of cell proliferation and induction of apoptosis. Pretreatment with the AMPK inhibitor compound C rescued YZHI induced apoptosis and partially restored cell proliferation of HL-60. Consistent with the data in vitro, YZHI obviously suppressed subcutaneous xenograft growth in nude mice.ConclusionsIn a word, our data suggest that YZHI can be repurposed for the treatment of AML, which is worthy of further clinical evaluation.</div

S3 Fig -

Original data(A) and blot images(B) underlying apoptosis in Fig 3A–3H. (ZIP)</p

AMPK/mTORC1 signaling pathway mediates YZHI-induced apoptosis and growth inhibition in HL-60 cells.

(A) Cellular ATP levels after incubation with YZHI for 24 h. (B) Western blot analysis of proteins in the AMPK/mTORC1 pathway. (C) Cell viability was determined by CCK-8 assay in HL-60 cells incubated with vehicle or YZHI (8 μg/ml) for 72h following pretreated with vehicle or Compound C(5 μm) for 2h. (D) Apoptosis analysis was performed by flow cytometry in HL-60 cells incubated with vehicle or YZHI (72 μg/ml) for 24h following pretreated with vehicle or Compound C (5 μm) for 2h. (E) The statistical analysis result of (D). (F) Western blot analysis of proteins in the cell apoptosis and AMPK/mTORC1 pathway. All data were presented as mean ± SD (n = 3, **p p p < 0.0001).</p

Original data underlying cell viability in Fig 1A–1D.

Original data underlying cell viability in Fig 1A–1D.</p

YZHI suppresses xenograft growth in vivo model.

(A) The average body weight in YZHI(135.3 mg/kg/d, ip) and vehicle(normal saline, ip) groups. (B) xenograft size from nude mice in YZHI and vehicle groups. (C) xenograft excised from nude mice in each group. (D) The average weight of each xenograft. VEH n = 8, YZHI n = 8. The data were shown as the mean ± SD, **p p < 0.0001.</p

Original data underlying cell viability in Table 1.

Original data underlying cell viability in Table 1.</p

S4 Fig -

Original data(A) and blot images(B) underlying AMPK/mTORC1 signaling pathway in Fig 4A–4F. (ZIP)</p

YZHI inhibits AML cell proliferation.

(A-C) HL-60, KG-1 and THP-1 cells were treated with different concentrations of YZHI for 72 h. YZHI was first diluted to the highest concentration of 125 μg/ml and then twice diluted in turn. (D) HL-60 cells were treated with 24 μg/ml YZHI for various times. Cell viability (% of control) was performed by CCK-8 assay. (mean ± SD, n =  3 biologically independent samples).</p

YZHI induces cell cycle arrest in HL-60 cells.

(A) Cell cycle analysis of HL-60 cells treated with YZHI. (B) Analyze the total cell population in the three different phases of cell cycle(G1, S and G2/M) after 24 h drug treatment. (C) Western blot analysis of cell cycle marker Cyclin D1 after YZHI treatment for 24 h. All data were analyzed as mean ± SD (n = 3, **p p p < 0.0001).</p

S2 Fig -

Original data(A) and blot images(B) underlying cell cycle arrest in Fig 2A–2C. (ZIP)</p