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Keywords:

  • xanthohumol;
  • AML;
  • CML;
  • angiogenesis;
  • invasion;
  • NF-κB;
  • Akt

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. REFERENCES

BACKGROUND.

Leukemias are dependent on Akt/NF-κB activation and angiogenesis.

METHODS.

The antiangiogenic Akt/NF-κB inhibitor xanthohumol (XN) has in vitro activity against acute and chronic myelogenous leukemia cell lines (AML, CML) and fresh samples from patients were investigated.

RESULTS.

Inhibition of cell proliferation is associated with induction of apoptosis and reduced VEGF secretion. Decreased cell invasion, metalloprotease production, and adhesion to endothelial cells observed in the presence of XN could prevent in vivo life-threatening complications of leukostasis and tissue infiltration.

CONCLUSIONS.

As endothelial cells and hematopoietic cells are mutually correlated in their development and growth, targeting both tumor cells and endothelial cells with agents possessing cytotoxic and antiangiogenic activities may lead to synergistic antitumor effects interrupting a reciprocal stimulatory loop between leukemia and endothelial cells. Cancer 2007. © 2007 American Cancer Society.

Angiogenesis has been associated with the growth, dissemination, and metastasis of solid tumors and increased levels of angiogenic molecules have been correlated with poor prognosis in several solid tumors. In recent studies, increased microvessel density in areas of intense neovascularization was a significant and independent prognostic indicator in early-stage breast, prostate, gastrointestinal, ovarian, and melanoma cancers. Increased vascularity was also reported in chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), acute lymphoid leukemia (ALL), and myelodysplastic syndrome (MDS) and appeared to be independent of cellularity and associated with a significant increase in angiogenic factors,1 suggesting that vascularity in hematologic malignancies is an active and controlled process and may play a role in the leukemogenic process. The successful use of arsenic trioxide and thalidomide in the treatment of hematologic malignancies has been, in part, associated with their antiangiogenic properties. Xanthohumol (XN), the principal flavonoid of the hop plant (Humulus lupulus L.), has cancer chemopreventive activities.2, 3 We have demonstrated that XN has antiangiogenic properties in vitro and in vivo4 associated with blockage of Akt/NF-κB activation. As endothelial cells and hematopoietic cells are mutually correlated in their development and growth, here we investigated the effects of XN on leukemia cells. Our results identify new signaling pathways targeted by XN and suggest potential therapeutic utility in leukemia patients.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. REFERENCES

Reagents

XN was purchased from Alexis Biochemicals (San Diego, Calif), enzyme-linked immunosorbent assay (ELISA) assays for NF-κB activity were performed using a commercially available kit (TransAM, Active Motif, Carlsbad, Calif). Vascular endothelial growth factor (VEGF) protein released into the media by cell lines and clinical samples was measured using a commercial ELISA kit (LISTARFISH; CYTImmune Sciences, College Park, Md).

Cells

U937 CML and human umbilical vein endothelial (HUVE) cells were obtained from the American Type Culture Collection (ATCC, Rockville, Md). MM6 AML cells were kindly provided by Prof. Mingari of our institute and cultured under standard conditions. Mononuclear cells from peripheral blood of healthy donors or CML and AML patients at diagnosis after receiving informed consent were isolated following established procedures5 and cultured in RPMI in the presence of 20% heat-inactivated fetal bovine serum (FBS) and 20 ng/mL human granulocyte-macrophage colony-stimulating factor (hGM-CSF). Clonogenic assays were conducted on normal bone marrow progenitors6 to evaluate XN cytotoxicity.

Cell Proliferation, Apoptosis, Invasion, Gelatin Zymography, and Adhesion Assay

The number of viable cells was measured by the MTT test at different timepoints. A cell death detection ELISA kit (Roche, Mannheim, Germany) was used to measure apoptosis after XN treatment. Chemoinvasion was carried out in BioCoat Matrigel invasion chambers (BD Biosciences, Bedford, Mass) following the manufacturer's instructions. Gelatin zymography and adhesion to endothelial cells were carried out as described previously.4, 7 Cell surface aminopeptidase (CD13/APN) activity was measured after incubating the cells with various concentrations of XN for 4, 12, 16, and 24 hours in complete medium. Cells (40,000 per well) were washed thrice withphosphate-buffered saline (PBS) and incubated 1 hour at 37°C with 2 mL of 100 mM L-alanine-4 methyl-7-coumarinylamide trifluoroacetate (Fluka, Milan, Italy) in 10 mM HEPES-buffered PBS (pH 7.2) containing 0.1% bovine serum albumin. The development of the fluorescent product was measured with a fluorimetric plate reader (excitation wavelength, 360 nm; emission wavelength, 465 nm).

Protein Extraction and Western Blot Analysis

To test the activation of the Akt and NF-κB pathways, MM6 and U937 cells were serum-starved for 16 hours and then treated with 5 μM XN for 6 hours in serum-free medium. Five to 30 minutes before the end of incubation cells were stimulated with TNF-α (10 ng/mL). Cell pellets were lysed in RIPA buffer containing protease inhibitors and equal amounts of samples were resolved by SDS-PAGE, transferred to nitrocellulose, and probed at 4°C overnight with the following antihuman antibodies (Cell Signaling Technology, Beverly, Mass): rabbit polyclonal anti-phospho-Akt (Ser473), anti-phospho p65(Ser539), anti-phospho-IκB (Ser32). After washing, the blots were incubated for 1 hour at room temperature with horseradish peroxidase-conjugated secondary antibodies (Amersham, Cologno Monzese, Italy) and specific complexes were revealed by enhanced chemiluminescence solution (Amersham). An anti-GAPDH antibody conjugated to horseradish peroxidase (Novus Biologicals, Littleton, Colo), or a mouse monoclonal anti-β-tubulin antibody (Sigma, Milano, Italy) were used as loading controls for all samples.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. REFERENCES

Micromolar concentrations of XN dose-dependently inhibited in vitro proliferation of MM6 and U937 cell lines and fresh samples from AML and CML patients (Fig. 1A-C). A notable apoptotic activity of XN was also observed (Fig. 1A-C). To assess the safety of XNon human bone marrow progenitors, we tested the compound in in vitro colony-forming-unit (CFU) assay. At concentrations up to 5 μM, XN did not inhibit growth of bone marrow progenitors isolated from healthy volunteers (data not shown). This is consistent with the previously observed lack of apoptosis in endothelial cells treated with XN.4 Expansion of leukemic cell populations residing within the bone marrow microenvironment involves adhesion of leukemic cells to bone marrow extracellular matrix (ECM) and migration/invasion into the surrounding tissues. Kinase-dependent and -independent mechanisms contribute to the abnormal adhesion and migration/invasion of hematopoietic progenitors. Leukemic blast cells secrete MMP-2 and MMP-9, which represent a marker for dissemination in myeloproliferative malignancies.8 XN significantly inhibited invasion of MM6 and U937 cell lines and patient samples in vitro at concentrations as low as 2.5 μM (Fig. 2A-C). Consistent with this, MMPs release was also inhibited by XN (Fig. 2A-C). Another cell surface metalloprotease, CD13/APN, expressed by myeloid progenitors, monocytes, endothelial cells, and tumor cells has been associated with tumor cell invasion and may be important in the regulation and signaling pathways that control myeloid growth and differentiation.9 CD13/APN activity was decreased in both cell lines exposed to increasing concentrations of XN, with an effect detectable after 4 hours (data not shown) and statistically significant at 16 hours (Fig. 2A,B, right panels). Interestingly, the CD13/APN inhibitor bestatin induces growth inhibition and apoptosis in human leukemic cell lines10 and CD13 activity is down-regulated during differentiation of HL60 induced by ATRA.9

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Figure 1. In vitro effects of xanthohumol (XN) on leukemia cells. XN at 5 and 10 μM significantly reduced leukemia cell proliferation after 48 hours in (A, left) MM6, (B, left) U937, and (C, left) fresh samples from acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) patients (AML is shown and identical results were obtained from CML patients) (in all cases ***P < .001 with respect to controls; 2-tailed t-test). Right panels: 24 hours exposure to XN significantly induced cell apoptosis in the same samples and in a dose-dependent manner (**P < .01, ***P < .001 with respect to controls; 2-tailed t-test).

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Figure 2. The antileukemic agent xanthohumol (XN) has matrix metalloproteinase inhibitory activity. (A-C, left panels) MM6, U937, and fresh samples from acute myelogenous leukemia (AML) patients, respectively: significant inhibition of cell invasion by XN (**P < .01, ***P < .001 with respect to controls; 2-tailed t-test). Serum-free medium (SFM) and supernatants of NIH3T3 fibroblasts were used as negative and positive controls, respectively. Insets: zymography detection of secreted gelatinase activity indicated XN inhibited MMP-2 and MMP-9 release. Identical results were obtained in fresh samples from chronic myelogenous leukemia (CML) patients. Right panels (A,B): cell surface CD13/APN activity in MM6 and U937 cells, respectively; an overnight exposure to XN significantly inhibited aminopeptidase (APN) in a dose-dependent manner (*P < .05, **P < .01, ***P < .001 with respect to controls; 2-tailed t-test). (D) Myeloblast-HUVEC adhesion assay. Calcein-labeled leukemia cells (5 × 105 cells/well) showed a significantly decreased adhesion to HUVE cells exposed to XN for 16 hours (*P < .05, **P < .01, ***P < .001 with respect to controls; 2-tailed t-test).

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Important features of AML and CML are the presence of an increased number of circulating progenitors and extramedullary hematopoiesis. This promotes adhesion of leukemia cells to vascular endothelium and generates conditions favoring leukostasis and tissue infiltration.11 Leukemia cells labeled with the fluorescent dye calcein showed a significantly reduced adherence to confluent endothelial cell monolayers exposed to XN for 16 hours (Fig. 2D). The repressive effects of XN on endothelial cell activation4 might further contribute to decrease the inflammatory reactions causing multiple organ failure in leukemia patients.

Different reports demonstrated the constitutive activation of NF-κB in lymphoid and myeloid malignancies, underscoring its implication in malignant transformation.12 Overexpression of NF-κB may lead to chemoresistance and inhibition of this pathway may lead to successful therapy. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is also crucial to many aspects of cell growth, survival, and apoptosis, and its constitutive activation has been implicated in both the pathogenesis and the progression of a variety of neoplasms, including leukemias.13 Evidence accumulated over the recent years has indicated the PI3K/Akt signal transduction pathway as a major one responsible for cancer resistance to conventional therapies. Indeed, pharmacologic inhibitors of PI3K/Akt potentiate the apoptotic action of antileukemic drugs.14 Simultaneous activation of multiple signal transduction pathways confers poor prognosis in AML,15 therefore targeting NF-κB activation, as well as its upstream regulator Akt might constitute an additional strategy to improve conventional therapies. We previously reported that NF-κB and Akt are targets of XN in endothelial cells.4 Because blockade of NF-κB is usually associated with suppression of invasion and metalloproteinase production,16 we examined whether XN could affect Akt/NF-κB activation in leukemia cells. Treatment of MM6 (Fig. 3A) and U937 (Fig. 3B) cells for 5, 15, or 30 minutes with 10 ng/mL TNF-α produced a strong NF-κB activation, as detected by ELISA assay. Preincubation for 6 hours in the presence of 5 μM XN significantly inhibited this TNF-α-induced NF-κB activation (Fig. 3A,C; *P <.05, **P < .01, 2-tailed t-test). Western blot analysis (Fig. 3B,D, MM6 and U937, respectively) confirmed that XN significantly represses the levels of nuclear phosphorylated p65, as well as the levels of cytosolic phosphorylated IκBα, after stimulation with TNF-α. These data indicate an inhibitory activity at or upstream of protein kinase IKK, which phosphorylates IκBα, suggesting that XN is able to interfere with the signaling pathways leading to NF-κB activation. Under the same conditions, XN also inhibited Akt phosphorylation.

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Figure 3. Effects of xanthohumol XN on NF-κB and Akt activation. Enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that 6 hours treatment with 5 μM XN reduced the amount of active NF-κB in TNF-α (10 ng/mL) stimulated (A) MM6 and (C) U937 cells. Means ± SEM are shown (*P < .05, **P < .01 with respect to control, 2-tailed t-test). (B,D) Western blot analysis shows that XN repressed nuclear phosphorylated-p65 as well as -cytosolic IκBα and -Akt levels in MM6 and U937 cells, respectively, after stimulation with TNF-α. As a control for loading, the same blots were reprobed with anti-GAPDH and -β-tubulin antibodies. (E) ELISA analysis for VEGF showed that 16 hours exposure to XN reduced the amount of secreted VEGF. Means ± SEM are shown (*P < .05, **P < .01 with respect to control, 2-tailed t-test). Similar results were obtained after treatment of fresh leukemia from chronic myelogenous leukemia (CML) patients.

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Leukemias are angiogenesis-dependent malignancies1, 17 and angiogenesis is strictly dependent on Akt/NF-κB activation. VEGF release by leukemic blasts may be an important stimulus for angiogenesis in the bone marrow as, in CML, overexpression of VEGF and increased angiogenesis are consistent findings1 and a prognostic significance of VEGF expression in chronic phase CML has been proposed.18 Moreover, in the NOD/SCID mouse model efficiency and speed of engraftment of myeloid and lymphoid human leukemias correlate with VEGF production.19 Because we recently demonstrated an antiangiogenic activity of XN4 and VEGF production correlates with Akt activation,20 we tested whether XN could also affect VEGF secretion. Sixteen hours of exposure to increasing concentrations of XN significantly reduced VEGF levels in both cell lines and clinical samples to about 50% of control values (Fig. 3E, *P < .05, **P < .01, 2-tailed t-test). Because the endothelium is quite sensitive to VEGF exposure, the reduction in VEGF production observed may be physiologically significant. Another intriguing aspect of VEGF-driven angiogenesis in hematologic neoplasia is the finding that VEGF-stimulated endothelial cells produce stem cell factor, granulocyte/macrophage-colony stimulating factor, and interleukin 6. These cytokines, in turn, may act as growth factors for myeloid and lymphoid malignant cells, thus generating a paracrine machinery between hematopoietic malignant cells and newly generated endothelium. In this scenario, XN could inhibit the angiogenic process through decreasing VEGF secretion in leukemic cells and also through inhibiting endothelial cell activities, causing the interruption of a reciprocal stimulatory loop between leukemic and endothelial cells.

Collectively, these findings generate a rationale for investigating the clinical efficacy of XN: 1) It shows direct4 and indirect antiangiogenic properties; and 2) It directly induces tumor cell apoptosis, suggesting that a combination of direct toxicity against leukemia cells with antiangiogenic activity could potently improve therapeutic efficacy and potentially repress acquisition of resistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. REFERENCES
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