stilbenes and Leukemia--Lymphocytic--Chronic--B-Cell

Microtubule targeting agents, such as vinblastine, are usually thought to arrest cells in mitosis and subsequently induce apoptosis. However, they can also cause rapid induction of apoptosis in a cell-cycle phase independent manner. BNC105 is a novel vascular and microtubule disrupting drug that also induces apoptosis rapidly but with markedly increased potency compared to vinca alkaloids and combretastatin A4. BNC105 binds to the colchicine-binding site on tubulin resulting in activation of c-Jun N-terminal kinase (JNK), phosphorylation of ATF2, and induction of ATF3 and Noxa leading to acute apoptosis in chronic lymphocytic leukemia (CLL) cells. Apoptosis induced by BNC105 is dependent upon both JNK activation and Noxa induction. Normal leukocytes and one CLL sample also exhibited JNK activation but not Noxa induction and were resistant to BNC105. This study emphasizes the importance of Noxa and JNK for induction of apoptosis in CLL cells by microtubule targeting drugs, and highlights the potential of BNC105 as a potent therapeutic to treat haematopoietic malignancies.

Topics: Anisoles; Apoptosis; Benzofurans; Cell Line, Tumor; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia, Lymphocytic, Chronic, B-Cell; MAP Kinase Signaling System; Microtubules; Stilbenes; Transfection; Tubulin Modulators; Vinblastine

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon interaction of leukemic cells with a supportive microenvironment. Therefore, the mobilization of tissue-resident CLL cells into the circulation is a useful therapeutic strategy to minimize the reservoir of tumor cells within survival niches. Because the exit of normal lymphocytes from lymphoid tissues depends on the presence of sphingosine-1 phosphate (S1P) and the regulated expression of S1P receptor-1 (S1PR1), we investigated whether the expression and function of S1PR1 can be modulated by key microenvironment signals. We found that activation of CLL cells with CXCL12, fibroblast CD40L(+), BCR cross-linking, or autologous nurse-like cells reduces their S1PR1 expression and the migratory response toward S1P. Moreover, we found that S1PR1 expression was reduced in the proliferative/activated subset of leukemic cells compared with the quiescent subset from the same patient. Similarly, bone marrow-resident CLL cells expressing high levels of the activation marker CD38 showed a lower expression of S1PR1 compared with CD38(low) counterparts. Finally, given that treatment with BCR-associated kinase inhibitors induces a transient redistribution of leukemic cells from lymphoid tissues to circulation, we studied the effect of the Syk inhibitors piceatannol and R406 on S1PR1 expression and function. We found that they enhance S1PR1 expression in CLL cells and their migratory response toward S1P. Based on our results, we suggest that the regulated expression of S1PR1 might modulate the egress of the leukemic clone from lymphoid tissues.

Topics: ADP-ribosyl Cyclase 1; Adult; Aged; Aged, 80 and over; Animals; B-Lymphocytes; CD40 Ligand; Cell Movement; Chemokine CXCL12; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Lysophospholipids; Male; Membrane Glycoproteins; Mice; Middle Aged; Oxazines; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcr; Pyridines; Receptors, CXCR4; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Stilbenes; Syk Kinase; Tumor Cells, Cultured; Tumor Microenvironment

Chronic lymphocytic leukemia (CLL), defined by accumulation of pathogenic B cells, has a very complex biology due to various factors such as inherited, host, and environmental factors. Recently, finding new therapeutic agents or development of novel treatment strategies have been paid attention. Resveratrol and quercetin, important phytoalexins found in many plants, have been reported to have cytotoxic effects on various types of cancer. In this study, we examined cytotoxic, cytostatic, and apoptotic effects of these two important phenolic compounds on 232B4 human CLL cells. Cytotoxic effects of resveratrol and quercetin were determined by MTT cell proliferation assay. Changes in caspase-3 enzyme activity were measured using caspase-3 colorimetric assay. Annexin V-FITC/PI double staining was performed to measure apoptotic cell population. Effects of resveratrol and quercetin on cell cycle profiles of CLL cells were investigated by flow cytometry. Treatment of CLL cells with resveratrol and quercetin caused dose dependent inhibition of cell proliferation and increased apoptotic cell population through induction of caspase-3 activity. Cell cycle analysis displayed cell cycle arrest mainly in G0/G1 for both polyphenols. Our data, in total, showed for the first time that resveratrol and quercetin might block CLL growth through inducing apoptosis and cell cycle arrest.

Topics: Annexin A5; Apoptosis; B-Lymphocytes; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytostatic Agents; Cytotoxins; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Quercetin; Resveratrol; Stilbenes

CLL cells are characterized by high levels of proteins that are post-translationally modified by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties, but it is not clear whether O-GlcNAc is a relevant therapeutic target. The neutraceutical resveratrol is cytotoxic to chronic lymphocytic leukemia cells in vitro. In this study, we found that resveratrol has therapeutic activity as a single agent in vivo in both human chronic lymphocytic leukemia patients and mice with erythroleukemia. Blood and splenic O-GlcNAc levels reflected the changes in tumor burden. Resveratrol directly lowered O-GlcNAc levels in leukemia cells through proteasomal activation, but increasing O-GlcNAc levels in vitro did not prevent cell death. These findings suggest that resveratrol has potential as a novel treatment for some forms of chronic and acute leukemia, and the measurement of O-GlcNAc levels could be a surrogate marker for therapeutic responses.

Topics: 3T3 Cells; Acetylglucosamine; Animals; Drug Antagonism; Humans; Interferons; Leukemia, Erythroblastic, Acute; Leukemia, Lymphocytic, Chronic, B-Cell; Mice; Proteins; Resveratrol; Stilbenes

In this study, we attempted to assess the interactions of resveratrol, a natural compound present in various plant species, with the purine analogues fludarabine and cladribine in terms of their effects on DNA damage and apoptosis in chronic lymphocytic leukemia (CLL) cells. The experiments were performed ex vivo using short-term cell cultures of blood and bone marrow cells from newly diagnosed untreated patients. We analyzed the expression of active caspase-3 and the BCL-2/BAX ratio as markers of apoptosis and the expression of phosphorylated histone H2AX (γH2AX) and activated ATM kinase, which are reporters of DNA damage. The results of our study revealed that resveratrol induced apoptosis in CLL cells in a tumor-specific manner but did not affect non-leukemic cells, and apoptosis was associated with a decreased BCL2/BAX ratio. Here, we report for the first time that both resveratrol + fludarabine and resveratrol + cladribine caused a higher rate of apoptosis in comparison to the rate caused by a single drug. The percentage of apoptotic cells induced by resveratrol alone was higher in the group of patients with better prognostic markers than in those with worse prognostic markers. However, the rates of apoptosis caused by resveratrol combined with purine analogues were independent of ZAP-70 and CD38 expression and the clinical state of the disease; they were only dependent on the presence of high-risk cytogenetic abnormalities. We also observed an increase in γH2AX expression together with a rise in activated ATM in most of the analyzed samples. The obtained results indicate that resveratrol might warrant further study as a new therapeutic option for CLL patients. This naturally occurring substance may be used as a single agent, especially in older persons for whom there are some limitations for the use of aggressive treatment. On the other hand, a lower purine analogue dose could potentially be used in combination with resveratrol because of their combined effect. One of the mechanisms of action of resveratrol is the induction of DNA damage, which ultimately leads to apoptosis.

Topics: ADP-ribosyl Cyclase 1; Adult; Antineoplastic Agents; Apoptosis; Ataxia Telangiectasia Mutated Proteins; bcl-2-Associated X Protein; Caspase 3; Cell Cycle Proteins; Cladribine; DNA-Binding Proteins; Drug Therapy, Combination; Enzyme Activation; Histones; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Membrane Glycoproteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Purines; Resveratrol; Stilbenes; Tumor Suppressor Proteins; Vidarabine; ZAP-70 Protein-Tyrosine Kinase

The mammalian target of rapamycin (mTOR) is emerging as a promising target for antitumor therapy. However, the mechanism that contributes to its regulation in B lymphomas remains unknown. This study shows that in follicular lymphoma (FL) cells, mTOR is active because the cells displayed rapamycin-sensitive phosphorylation of p70S6 kinase and 4E-BP1. Moreover, immunohistochemistry applied on lymph node tissue sections obtained from patients with FL revealed that, in most cases, p70S6 kinase was highly phosphorylated compared to normal tonsillar tissue. In FL cells, mTOR was under control of both phospholipase D (PLD) and phosphatidylinositol 3-kinase (PI3K). Moreover, we demonstrated that Syk plays a central role in mTOR activation because we found that both expression and activity are elevated compared to normal or chronic lymphocytic leukemia B cells. We also provide evidence that Syk operates through PLD- and PI3K-independent pathways. Finally, Syk inhibition by piceatannol or by siRNA plasmids resulted in a potent inhibition of mTOR activity in FL cells, as well as in mantle cell lymphoma, Burkitt lymphoma, and diffuse large B-cell lymphoma. These findings suggest that the Syk-mTOR pathway has a critical function in FL survival, and therefore, that Syk could be a promising new target for B-lymphoma therapy.

Topics: Burkitt Lymphoma; Cell Line, Tumor; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Lymphoma, B-Cell; Lymphoma, Follicular; Lymphoma, Mantle-Cell; Neoplasm Proteins; Palatine Tonsil; Phosphatidylinositol 3-Kinases; Phospholipase D; Protein Kinases; Protein-Tyrosine Kinases; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Signal Transduction; Stilbenes; Syk Kinase; TOR Serine-Threonine Kinases

We have previously reported that combretastatin-A4 prodrug (CA4P), anantitubulin/antiangiogenic agent isolated from the South African willow tree Combretum caffrum, induced cell death primarily through mitotic catastrophe in a panel of human B-lymphoid tumors. In this study, we investigated the molecular aspects of the mitotic catastrophe and whether or not it shares the same pathways of apoptosis. For this we studied the effect of CA4P on selected markers of apoptosis [caspases 9 and 3, poly(ADP-ribose) polymerase (PARP), bcl-2, and bax] and G2-M protein regulators (p53, MDM2, 14-3-3sigma, GADD45, cdc2, cdc25, chk1, wee1, p21, and cyclin B1). The chronic lymphocytic leukemia cell line WSU-CLL was used for this purpose. Western blot analysis showed that 24 h of CA4P (5 nM) exposure induces caspase 9 activation and PARP cleavage. However, the addition of Z-Val-Ala-Asp-fluoromethylketone (a general caspase inhibitor) or Z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (a caspase 9 inhibitor) before CA4P treatment did not block cell death. No change in bcl-2 or bax protein expression was observed. Exposure of WSU-CLL cells to 4 and 5 nM CA4P was associated with overproduction of total p53 and no dramatic change in MDM2, 14-3-3sigma, GADD45, the cyclin-dependent kinase cdc2, its inhibitory phosphorylation, the cdc2-inhibitory kinase (wee1), chk1, or cdc25 hyperphosphorylation. The overaccumulation of p21 and cyclin B1 protein was obvious at 24 h. Furthermore, CA4P treatment showed an increase in the expression of a marker of mitosis (mitotic protein monoclonal-2 antibody) and an overaccumulation of the cyclin B in the nucleus. Our findings suggest that CA4P induces mitotic catastrophe and arrest of WSU-CLL cells mostly in the M phase independent of p53 and independent of chk1 and cdc2 phosphorylation pathways. Apoptosis is a secondary mechanism of death in a small proportion of cells through activation of caspase 9 and PARP cleavage. The two mechanisms of cell death, i.e., mitotic catastrophe and apoptosis, are independent of each other in our model.

Topics: 14-3-3 Proteins; Antineoplastic Agents, Phytogenic; bcl-2-Associated X Protein; Biomarkers, Tumor; Blotting, Western; Caspase 3; Caspase 9; Caspases; CDC2 Protein Kinase; Cell Nucleus; Cell Survival; Checkpoint Kinase 1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Activation; Enzyme Inhibitors; Exonucleases; Exoribonucleases; Flow Cytometry; G2 Phase; Genetic Markers; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mitosis; Neoplasm Proteins; Nuclear Proteins; Phosphorylation; Poly(ADP-ribose) Polymerases; Prodrugs; Protein Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Stilbenes; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Trans-resveratrol, its dimer epsilon-viniferin and two preparations of vineatrol (a grape-derived polyphenol fraction isolated from vine-shots extracts) were compared for their effects on the proliferation and survival of normal and leukemic human lymphocytes. The two different batches of vineatrol (vineatrol 10 and 25%) was obtained by HPLC fractionation and contained 10 and 25% trans-resveratrol, respectively. The different polyphenols were added to cultures of leukemic cells from chronic B cell malignancies (B-cell chronic lymphocytic leukemia, B-CLL or hairy cell leukemia, HCL) or normal peripheral blood-derived mononuclear cells (PBMC) as a control. The different polyphenols displayed anti-proliferative effect on the leukemic cells, as estimated by the observed inhibition of tritiated thymidine uptake and the reduction of cell recovery. Vineatrol 10% was the most potent whereas vineatrol 25% and resveratrol displayed comparable activity, epsilon-viniferin only exhibiting slight effets. The same order of potency was observed for their capacity to induce apoptosis in leukemic B cells. In contrast, the survival of normal peripheral blood mononuclear cells (PBMC) was little affected in the presence of these polyphenolic compounds and higher concentrations were required in order to elicit cell death. Polyphenol-driven apoptosis in chronic leukemic B cells was shown to correlate with an activation of caspase 3, a drop in the mitochondrial transmembrane potential, a reduction in the expression of the anti-apoptotic protein bcl-2, as well as a reduction in the expression of the inducible nitric oxide synthase (iNOS). Our data therefore indicate that vine-shoots may be a convenient and natural source of material for the purification of resveratrol and other polyphenolic compounds of putative therapeutic interest.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; B-Lymphocytes; Benzofurans; Caspase 3; Caspases; Cell Division; Flavonoids; Humans; Intracellular Membranes; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocytes, Mononuclear; Membrane Potentials; Mitochondria; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phenols; Plant Extracts; Polymers; Polyphenols; Proto-Oncogene Proteins c-bcl-2; Resveratrol; Stilbenes; Vitis

Trans-resveratrol was analysed for its apoptotic and growth inhibitory activity in human B-cell lines derived from chronic B-cell malignancies (WSU-CLL and ESKOL), and in leukaemic lymphocytes from patients with B-cell chronic lymphocytic leukaemia (B-CLL). Resveratrol displayed antiproliferative activity on both B-cell lines, as estimated by the decrease in cell recovery and inhibition of thymidine uptake. Furthermore, resveratrol induced apoptosis in the two cell lines as well as in B-CLL patients' cells, as evidenced by the increase in annexin V binding, caspase activation, DNA fragmentation and decrease of the mitochondrial transmembrane potential DeltaPsim. We previously reported that nitric oxide (NO), endogenously released by an iNO synthase (iNOS) spontaneously expressed in these leukaemic cells, contributed to their resistance towards apoptosis. We show here that resveratrol inhibited both iNOS protein expression and in situ NO release in WSU-CLL, ESKOL and B-CLL patients'cells. In addition, Bcl-2 expression was also inhibited by resveratrol. Thus, downregulation of the two anti-apoptotic proteins iNOS and Bcl-2 can contribute to the apoptotic effects of resveratrol in leukaemic B cells from chronic leukaemia. Our data suggest that this drug is of potential interest for the therapy of B-CLL.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; B-Lymphocytes; Cell Division; Gene Expression; Genes, bcl-2; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Resveratrol; Stilbenes; Tumor Cells, Cultured