Targeting CD13 (aminopeptidase-N) in turn downregulates ADAM17 by internalization in acute myeloid leukaemia cells

International audienceSecreted matrix metalloproteinases (MMP)-2 and MMP-9 and membrane-anchored aminopeptidase-N/CD13 are abnormally expressed in human acute myeloid leukaemia (AML). We previously showed that CD13 ligation by anti-CD13 monoclonal antibodies can induce apoptosis in AML cells. Here, we assessed ADAM17 expression in primary blood blasts CD13+CD33+ from patients with AML. Primary AML cells expressed ADAM17 transcript and its surface expression was higher in subtype M4 (myelomonocytic) and M5 (monocytic) AML specimens than in M0 and M1/M2 (early and granulocytic) specimens. In AML cell lines defining distinct AML subfamilies (HL-60/M2, NB4/M3, THP-1/M5, U937/M5) and primary AML cells cultured ex vivo, anti-CD13 antibodies downregulated surface CD13 and ADAM17 without affecting MMP-2/-9 release. Knockdown of CD13 by siRNA prevented anti-CD13-mediated ADAM17 downregulation, indicating that CD13 is required for ADAM17 downregulation. Soluble ADAM17 was not detected in the medium of anti-CD13 treated cells, suggesting that ADAM17 was not shed. After ligation by anti-CD13, CD13 and ADAM17 were internalized. Subsequently, we found that ADAM17 interacts with CD13. We postulate that the interaction of ADAM17 with CD13 and its downregulation following CD13 engagement has important implications in AML for the known roles of ADAM17 in tumour-associated cell growth, migration and invasion

The antiangiogenic phloroglucinol hyperforin inhibits the secretion of proMMP-2, proMMP-9 and VEGF-A during apoptosis of primary acute myeloid leukemia cells

Aim: Angiogenesis is observed in acute myeloid leukemia (AML). AML cells abnormally proliferate and are resistant to death. Positive regulators of angiogenesis, VEGF-A and matrix metalloproteinases (MMPs) 2 and 9 are markers of disease status in AML. The natural phloroglucinol hyperforin (HF) displays antitumoral properties of potential pharmacological interest. Herein, we investigated the effects of HF on MMP-2/9 and VEGF-A expression and survival of primary AML cells.Methods: Blood and bone marrow samples were collected in 45 patients with distinct subtypes defined by French American British classification, i.e., M0, M1, M2, M3, M4, and M5. Levels of MMPs and VEGF-A in leukemic blood cells and culture supernatants were determined by RT-PCR, ELISA, and gelatin zymography (MMPs). The balance between cell death and survival was assessed by flow cytometry with analysis of phosphatidylserine externalization and caspase-3 activation.Results: The administration of HF promoted a caspase-associated apoptosis in primary AML blasts (from blood and bone marrow), but not normal blood cells and monocytes. In addition, HF inhibited the levels of secreted proMMP-2, proMMP-9, and VEGF-A without altering transcripts. The induction of apoptosis by HF significantly paralleled the inhibition of MMP-2/9 and VEGF-A release by HF. No differences were seen in response to the deleterious effects of HF between AML cells of distinct subtypes.Conclusion: Our results suggest that HF, through its proapoptotic and potential antiangiogenic properties (by inhibiting MMP-2/9 and VEGF-A) on primary AML cells, might be a useful experimental agent, in combination with existing drugs, for new therapeutic approaches in the treatment of this incurable disease

Hyperforin inhibits Akt1 kinase activity and promotes caspase-mediated apoptosis involving Bad and Noxa activation in human myeloid tumor cells.

BACKGROUND: The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo. METHODOLOGY AND RESULTS: HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser(473)) and Akt1 substrate Bad (at Ser(136)) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells. SIGNIFICANCE: Our data provide new evidence that HF's pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment

A new model of cell dynamics in Acute Myeloid Leukemia involving distributed delays

International audienc

Zosuquidar restores drug sensitivity in P-glycoprotein expressing acute myeloid leukemia (AML)

Abstract Background Chemotherapeutic drug efflux via the P-glycoprotein (P-gp) transporter encoded by the MDR1/ABCB1 gene is a significant cause of drug resistance in numerous malignancies, including acute leukemias, especially in older patients with acute myeloid leukemia (AML). Therefore, the P-gp modulators that block P-gp-mediated drug efflux have been developed, and used in combination with standard chemotherapy. In this paper, the capacity of zosuquidar, a specific P-gp modulator, to reverse chemoresistance was examined in both leukemia cell lines and primary AML blasts. Methods The transporter protein expressions were analyzed by flow cytometry using their specific antibodies. The protein functionalities were assessed by the uptake of their fluorescence substrates in presence or absence their specific modulators. The drug cytotoxicity was evaluated by MTT test. Results Zosuquidar completely or partially restored drug sensitivity in all P-gp-expressing leukemia cell lines tested and enhanced the cytotoxicity of anthracyclines (daunorubicin, idarubicin, mitoxantrone) and gemtuzumab ozogamicin (Mylotarg) in primary AML blasts with active P-gp. In addition, P-gp inhibition by zosuquidar was found to be more potent than cyclosporine A in cells with highly active P-gp. Conclusion These in vitro studies suggest that zosuquidar may be an effective adjunct to cytotoxic chemotherapy for AML patients whose blasts express P-gp, especially for older patients.</p

A subset of bone marrow stromal cells regulate ATP-binding cassette gene expression via insulin-like growth factor-I in a leukemia cell line

International audienceThe importance of the insulin-like growth factor, IGF, as a signaling axis in cancer development, progression and metastasis is highlighted by its effects on cancer cells, notably proliferation and acquired resistance. The role of the microenvironment within which cancer cells evolve and which mediates this effect is far from clear. Here, the involvement of IGF-I in inducing multidrug resistance in a myeloid leukemia cell line, grown in the presence of bone marrow-derived stromal cells called `Hospicells' (BMH), is demonstrated. We found that i) drug sensitive as well as resistant leukemia cells express IGF-I and its receptor IGF-IR. However, the resistant cells were found to secrete high levels of IGF-I. ii) Presence of exogenous IGF-I promoted cell proliferation, which decreased when an inhibitor of IGF-IR (picropodophyllin, PPP) was added. iii) BMH and IGF-I are both involved in the regulation of genes of the ATP binding cassette (ABC) related to resistance development (MDR1, MRP1, MRP2, MRP3 and BCRP). iv) The levels of ABC gene expression by leukemia cells were found to increase in the presence of increasing numbers of BMH. However, these levels decreased when IGF-IR was inhibited by addition of PPP. v) Co-culture of the drug-sensitive leukemia cells with BMH induced protection against the action of daunorubicin. This chemoresistance was amplified by the presence of IGF-I whereas it decreased when IGF-IR was inhibited. Our results underline the role of microenvironment in concert with the IGF-1 pathway in conferring drug resistance to leukemia cells

Analysis of a New Model of Cell Population Dynamics in Acute Myeloid Leukemia

International audienc