Active hexose-correlated compound enhances extrinsic-pathway-mediated apoptosis of Acute Myeloid Leukemic cells

<div><p>Active Hexose Correlated Compound (AHCC) has been shown to have many immunostimulatory and anti-cancer activities in mice and in humans. As a natural product, AHCC has potential to create safer adjuvant therapies in cancer patients. Acute Myeloid Leukemia (AML) is the least curable and second-most common leukemia in adults. AML is especially terminal to those over 60 years old, where median survival is only 5 to 10 months, due to inability to receive intensive chemotherapy. Hence, the purpose of this study was to investigate the effects of AHCC on AML cells both <i>in vitro</i> and <i>in vivo</i>. Results showed that AHCC induced Caspase-3-dependent apoptosis in AML cell lines as well as in primary AML leukopheresis samples. Additionally, AHCC induced Caspase-8 cleavage as well as Fas and TRAIL upregulation, suggesting involvement of the extrinsic apoptotic pathway. In contrast, monocytes from healthy donors showed suppressed Caspase-3 cleavage and lower cell death. When tested in a murine engraftment model of AML, AHCC led to significantly increased survival time and decreased blast counts. These results uncover a mechanism by which AHCC leads to AML-cell specific death, and also lend support for the further investigation of AHCC as a potential adjuvant for the treatment of AML.</p></div

AHCC decreases AML-cell proliferation.

<p>MV4-11 cells (1 x 10⁶ cells/ml) were treated with AHCC as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181729#pone.0181729.g001" target="_blank">Fig 1</a>, then incubated on Methocult-media-containing plates for 2 weeks. Colonies were counted in a blinded fashion. (A). Graph of 3 separate experiments. (B). Photographs of representative plates. *** p≤0.001.</p

AHCC decreases survival of AML cells.

<p>The AML cell line MV4-11 (1 x 10⁶ cells/ml) and primary AML-patient leukopheresis samples (3 x 10⁶ cells/ml) were treated with 0, 1, 5 or 10 mg/ml AHCC for 24 or 48 hours. Trypan Blue Exclusion was done with (A) MV4-11 cells (n = 3 separate experiments) and (B) primary AML leukopheresis samples (n = 7 donors). (C-D). MV4-11 (C, n = 3 separate experiments) and patient leukopheresis samples (D, n = 7 donors) were treated as above and then analyzed via flow cytometry following Annexin V and Propidium Iodide (PI) staining. * p≤0.05; ** p≤0.01.</p

AHCC-induced cell death is Caspase-3-dependent.

<p>The AML cell line MV4-11 (1 x 10⁶ cells/ml) and primary AML-patient leukopheresis samples (3 x 10⁶ cells/mL) were treated with 0, 1, 5 or 10 mg/ml AHCC for 24 hours. (A-B). Western blotting was done to measure cleaved Caspase-3, using Calreticulin as a loading control. Representative blots are shown for MV4-11 (A, n = 3 separate experiments) and primary leukopheresis samples (B, n = 3 donors). (C-E). OCI-AML3 (C), MOLM-13 (D) and THP-1 (E) cells were treated with AHCC as above. Western blotting was done to measure cleaved Caspase-3, using Calreticulin as a loading control (n = 3 separate experiments).</p

AHCC is not toxic toward healthy monocytes.

<p>Healthy-donor monocytes (5 x 10⁶ cells/ml, n = 3 donors) were treated with 0, 1, 5 or 10 mg/ml of AHCC for 24 hours. (A). Trypan Blue counts were done to measure viability. (B). Western blotting was done to measure cleaved Caspase-3, using Calreticulin as the loading control. Representative blots shown. ** p≤0.01.</p

MiR-155 induction by microbes/microbial ligands requires NF-kB-dependent de novo protein synthesis

MiR-155 regulates numerous aspects of innate and adaptive immune function. This miR is induced in response to toll-like receptor ligands, cytokines, and microbial infection. We have previously shown that miR-155 is induced in monocytes/macrophages infected with Francisella tularensis and suppresses expression of the inositol phosphatase SHIP to enhance activation of the PI3K/Akt pathway, which in turn promotes favorable responses for the host. Here we examined how miR-155 expression is regulated during infection. First, our data demonstrate that miR-155 can be induced through soluble factors of bacterial origin and not the host. Second, miR-155 induction is not a direct effect of infection and it requires NF-κB signaling to up-regulate fos/jun transcription factors. Finally, we demonstrate that the requirement for NF-κB-dependent de novo protein synthesis is globally shared by microbial ligands and live bacteria. This study provides new insight into the complex regulation of miR-155 during microbial infection

AHCC induces Caspase-8 cleavage and upregulation of Fas and TRAIL.

<p>The AML cell line MV4-11 (1 x 10⁶ cells/ml) was treated with 0, 1, 5 or 10 mg/ml AHCC for 24 hours. (A-B). Western blotting was done to measure cleaved Caspase-8, with Calreticulin as a loading control (A, representative blot shown), and densitometric analysis was performed (B, n = 3 separate experiments). (C-D). Fas (C, n = 3 separate experiments) and TRAIL (D, n = 4 separate experiments) were measured by qPCR. * p≤0.05.</p

AHCC increases apoptosis in most AML cell lines.

<p>The AML cell lines OCI-AML3 (left panels, 1 x 10⁶ cells/ml), MOLM-13 (middle panels, 1 x 10⁶ cells/ml) and THP-1 (right panels, 1 x 10⁶ cells/ml) were treated with 0, 1, 5 or 10 mg/ml of AHCC for 24 or 48 hours. (A). Trypan Blue Exclusion was done to measure viability (n = 3 separate experiments). (B). The 3 respective cell lines were treated as above and Annexin V / Propidium Iodide (PI) staining was measured using flow cytometry (n = 3 separate experiments). * p≤0.05.</p

AHCC increases survival <i>in vivo</i>.

<p>NSG mice (n = 10 per group) were intravenously injected with 0.3 x 10⁶ MV4-11 cells, then monitored for one week. Mice were then treated with either PBS or AHCC (600 mg/kg) twice a week for two weeks via gavage. (A). Survival time was measured and plotted. (B). Peripheral blood was collected and white-blood-cell (WBC) counts done at day 21 and day 27 (n = 3 mice per group). (C). Wright-Giemsa stains of peripheral blood was performed at day 27 to visualize blast count and morphology, for control (top panel) and AHCC-treated (bottom panel) mice. * p≤0.05; ** p≤0.01.</p