hymecromone and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Hyaluronan (HA) is the main glycosaminoglycan of the extracellular matrix. CD44 is the most important HA receptor, and both have been associated with poor prognosis in cancer. Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively activated tyrosine kinase (Breakpoint Cluster Region - Abelson murine leukemia viral oncogene homolog1, BCR-ABL). It is mainly treated with BCR-ABL inhibitors, such as imatinib. However, the selection of resistant cells leads to treatment failure. The aim of this work was to determine the capacity of HA (high molecular weight) to counteract the effect of imatinib in human CML cell lines (K562 and Kv562). We demonstrated that imatinib decreased HA levels and the surface expression of CD44 in both cell lines. Furthermore, HA abrogated the anti-proliferative and pro-senescent effect of Imatinib without modifying the imatinib-induced apoptosis. Moreover, the inhibition of HA synthesis with 4-methylumbelliferone enhanced the anti-proliferative effect of imatinib. These results suggest that Imatinib-induced senescence would depend on the reduction in HA levels, describing, for the first time, the role of HA in the development of resistance to imatinib. These findings show that low levels of HA are crucial for an effective therapy with imatinib in CML.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Fusion Proteins, bcr-abl; Humans; Hyaluronan Receptors; Hyaluronic Acid; Hymecromone; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt

Chronic myeloid leukemia (CML) is a myeloproliferative syndrome characterized by the presence of the Philadelphia chromosome which encodes a constitutively activated tyrosine kinase (BCR-ABL). The first line treatment for CML consists on BCR-ABL inhibitors such as Imatinib. Nevertheless, such treatment may lead to the selection of resistant cells. Therefore, it is of great value to find molecules that enhance the anti-proliferative effect of first-line drugs. Hyaluronan is the main glycosaminglican of the extracellular matrix which is involved in tumor progression and multidrug resistance. We have previously demonstrated that the inhibition of hyaluronan synthesis by 4-methylumbelliferone (4MU) induces senescence and can revert Vincristine resistance in CML cell lines. However, the effect of 4MU on Imatinib therapy remains unknown. The aim of this work was to determine whether the combination of 4MU with Imatinib is able to modulate the proliferation as well as apoptosis and senescence induction in human CML cell lines. For this purpose the ATCC cell line K562, and its multidrug resistant derivate, Kv562 were used. Cells were exposed to 4MU, Imatinib or a combination of both. We demonstrated that 4MU and Imatinib co-treatment abrogated the proliferation of both cell lines. However, such co-treatment did not increase the levels of apoptosis when compared with the treatment with Imatinib alone. For both cell lines the mechanisms of tumor suppression involved was senescence, since the combination of 4MU and Imatinib arrested the cell cycle and increased senescence associated β-galactosidase activity and senescence associated heterochromatin foci presence when compared to each drug alone. Moreover, 4MU, Imatinib and 4MU + Imatinib decreased pAkt/Akt ratio in both cell lines and reduced the pERK/ERK ratio only in K562 cells. These findings highlight the potential use of 4MU together with Imatinib for CML therapy.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Extracellular Signal-Regulated MAP Kinases; Humans; Hymecromone; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt

In the current study we examined the ability of 4-methylumbelliferone (4-MU), which can inhibit hyaluronic acid synthesis, to sensitize K562 chronic myelogenous leukemia (CML) cells to doxorubicin therapy. Exposure of K562 cells to doxorubicin led to increased hyaluronic acid synthase (HAS) gene expression and increased levels of cell surface hyaluronic acid. Furthermore, exposure of K562 cells to exogenous HA caused resistance to doxorubicin-induced cell death. The combination of low dose 4-MU and doxorubicin led to increased apoptosis when compared to higher doses of any agent alone. Additionally, treatment with 4-MU led to a significant reduction in doxorubicin-induced increase in HA cell surface expression. Mechanistically, 4-MU treatment led to an increase in p38 activation and PARP cleavage. The role of p38 in 4-MU/doxorubicin-treated K562 cells was confirmed when p38 inhibitors led to protection from 4-MU/doxorubicin-induced apoptosis. Together, results from this study suggest that treatment with 4-MU increases the sensitivity of CML to chemotherapeutics by decreasing their HA-mediated resistance to apoptosis.

Topics: Antibiotics, Antineoplastic; Cell Death; Cell Proliferation; Dose-Response Relationship, Drug; Doxorubicin; Drug Screening Assays, Antitumor; Humans; Hyaluronan Synthases; Hyaluronic Acid; Hymecromone; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Structure-Activity Relationship; Tumor Cells, Cultured

As an inhibitor of hyaluronic acid (HA) synthesis, 4-methylumbelliferone (4-MU) has been shown to induce apoptosis of various types of cancer cells. However, the potential impact of 4-MU-induced apoptosis on leukemia cells via modulation of HA is not well-known.. We examined apoptosis of chronic myelogenous leukemia (CML) cells after treatment with 4-MU using annexin V/propidium iodide (V/PI) analysis and poly (ADP-ribose) polymerase (PARP) cleavage. We further examined the mechanisms of 4-MU-induced apoptosis by measuring mitochondrial membrane potential and reactive oxygen species generation. Using real-time PCR, and western blot analysis we examined signaling pathways involved in apoptosis.. The current study demonstrated that treatment of CML cells with 4-MU led to induction of apoptosis through PARP cleavage and loss of mitochondria membrane potential. Mechanistically, 4-MU led to increased p53 mRNA expression, elevated cytoplasmic protein levels of cytochrome c and B-cell lymphoma-2-associated X (BAX) and reduced levels of B-cell lymphoma-2 (BCL2) expression. Addition of exogenous soluble HA was able to protect 4-MU-exposed cells from apoptosis.. Our findings suggest that 4-MU induces apoptosis in CML cells by activating components associated with the intrinsic apoptosis pathway.

Topics: Apoptosis; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; Hyaluronic Acid; Hymecromone; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Potential, Mitochondrial; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Signal Transduction