In vitro anti-leukaemia activity of sphingosine kinase inhibitor.

Compelling evidence indicates the role of sphingosine kinase 1 (SPHK1) deregulation in the processes of carcinogenesis and acquisition of drug resistance, providing the rationale for an effective anti-cancer therapy. However, no highly selective inhibitors of SPHK1 are available for in vitro and in vivo studies, except for the newly discovered 'SK inhibitor' (SKI). The present study showed that, in a panel of myeloid leukaemia cell lines, basal level of SPHK1 correlated with the degree of kinase inhibition by SKI. Exposure to SKI caused variable anti-proliferative, cytotoxic effects in all cell lines. In particular, SKI induced an early, significant inhibition of SPHK1 activity, impaired cell cycle progression and triggered apoptosis in K562 cells. Moreover, SKI acted synergistically with imatinib mesylate (IM) to inhibit cell growth and survival. Finally, the inhibitor affected the clonogenic potential and viability of primary cells from chronic myeloid leukaemia (CML) patients, including one harbouring the IM-insensitive Abl kinase domain mutation T315I. Due to the fact that the phenomenon of resistance to IM remains a major issue in the treatment of patients with CML, the identification of alternative targets and new drugs may be of clinical relevance.

Sphingolipid players in the leukemia arena.

Sphingolipids function as bioactive mediators of different cellular processes, mostly proliferation, survival, differentiation and apoptosis, besides being structural components of cellular membranes. Involvement of sphingolipid metabolism in cancerogenesis was demonstrated in solid tumors as well as in hematological malignancies. Herein, we describe the main biological and clinical aspects of leukemias and summarize data regarding sphingolipids as mediators of apoptosis triggered in response to anti-leukemic agents and synthetic analogs as inducers of cell death as well. We also report the contribution of molecules that modulate sphingolipid metabolism to development of encouraging strategies for leukemia treatment. Finally we address how deregulation of sphingolipid metabolism is associated to occurrence of therapy resistance both in vitro and in vivo. Sphingolipids can be considered promising therapeutic tools alone or in combination with other compounds, as well as valid targets in the attempt to eradicate leukemia and overcome drug resistance.

Resveratrol sensitization of DU145 prostate cancer cells to ionizing radiation is associated to ceramide increase.

Radiotherapy is an established therapeutic modality for prostate cancer. Since it is well known that radiotherapy is limited due to its severe toxicity towards normal cells at high dose and minimal effect at low dose, the search for biological compounds that increase the sensitivity of tumors cells to radiation may improve the efficacy of therapy. Resveratrol, a natural antioxidant, was shown to inhibit carcinogenesis in animal models, and to block the process of tumor initiation and progression. The purpose of this study was to examine whether or not resveratrol can sensitize DU145, an androgen-independent human prostate cancer cell line, to ionizing radiation. We report here that DU145 cells are resistant to ionizing radiation-induced cell death, but pretreatment with resveratrol significantly enhances cell death. Resveratrol acts synergistically with ionizing radiation to inhibit cell survival in vitro. Resveratrol also potentiates ionizing radiation-induced ceramide accumulation, by promoting its de novo biosynthesis. This confirms ceramide as an effective mediator of the anticancer potential induced by resveratrol.

Mutation in the ATP-binding pocket of the ABL kinase domain in an STI571-resistant BCR/ABL-positive cell line.

The major mechanism of action of STI571 is a competitive interference with the ATP-binding site of the Bcr/Abl tyrosine kinase. In the BCR/ABL-positive cell line KBM5, we studied cellular events associated with the in vitro acquisition of resistance to STI571. The emergence of the STI571-resistant phenotype was accompanied by only a marginal increase in the number of copies of the BCR/ABL gene and its level of expression. The activity of the Bcr/Abl kinase (level of autophosphorylation) in resistant cells was, however, incompletely inhibited by STI571, and the acquisition of the high degree of resistance was associated with a single-point mutation leading to a substitution of a threonine-to-isoleucine at position 315 of Abl. In the resistant KBM5-STI571(R1.0) cells, 20% of the BCR/ABL transcripts and 10% of BCR/ABL gene copies on the DNA level were mutated. The mutation was present in all 10 STI571-resistant clones derived from low density clonogenic assay, confirming its presence in all colony-forming cells but only in a fraction of the BCR/ABL gene copies in each cell. The contribution of this mutation to STI571-resistant phenotype remains unknown. Preliminary data showing partial reversibility of resistance in these cells suggest that resistance may be multifactorial. No other mutations were identified in the kinase domain of the BCR/ABL gene.

Chronotherapy with low-dose modified-release prednisone for the management of rheumatoid arthritis: a review.

To date, rheumatoid arthritis (RA) remains a debilitating, life-threatening disease. One major concern is morning symptoms (MS), as they considerably impair the patients' quality of life and ability to work. MS change in a circadian fashion, resembling the fluctuations of inflammatory cytokines such as interleukin-6, whose levels are higher in RA patients compared to healthy donors. Conversely, serum levels of the potent anti-inflammatory glucocorticoid cortisol are similar to that of healthy subjects, suggesting an imbalance that sustains a pro-inflammatory state. From a therapeutic point of view, administering synthetic glucocorticoids (GCs) to RA patients represents an optimal strategy to provide for the inadequate levels of cortisol. Indeed, due to their high efficacy in RA, GCs remain a cornerstone more than 60 years after their first introduction, and despite the development of a wide range of targeted agents. However, to improve safety, low-dose GCs have been introduced, that have demonstrated high efficacy in reducing disease activity, radiological progression, and improving patients' signs and symptoms especially in early RA when added to conventional disease-modifying antirheumatic drugs. A further improvement has been provided by the development of modified-release prednisone, which, by taking advantage of the circadian fluctuations of inflammatory cytokines, cortisol and MS, is given at bedtime to be released approximately 4 hours later. Several studies have already demonstrated the efficacy of this agent on disease activity, MS, and quality of life in the setting of established RA. Moreover, preliminary studies have shown that this new formulation not only has no impact on the adrenal function, but likely improves it. This review is a comprehensive, updated summary of the current evidence on the use of GCs in RA, with focus on the efficacy and safety of low-dose prednisone and modified-release prednisone, the latter representing a rational, cost-effective, and tailored approach to maximize the benefit/risk ratio in RA patients.

Epoetin beta for the treatment of chemotherapy-induced anemia: an update.

Epoetin beta belongs to the class of erythropoiesis-stimulating agents (ESAs) that are currently available to treat anemic patients receiving chemotherapy. Chemotherapy-induced anemia affects a high percentage of cancer patients and, due to its negative effects on disease outcome and the patient's quality of life, should be treated when first diagnosed. Initial trials with ESAs have shown efficacy in improving quality of life and reducing the need for blood transfusions in patients with chemotherapy-induced anemia. However, recent meta-analyses have provided conflicting data on the impact of ESAs on survival and tumor progression. Here we provide an overview of these recent data and review the role of epoetin beta in the treatment of chemotherapy-induced anemia over the past 20 years.

The proteasome inhibitor PS-341 inhibits growth and induces apoptosis in Bcr/Abl-positive cell lines sensitive and resistant to imatinib mesylate.

BACKGROUND AND OBJECTIVES: Imatinib mesylate (IM) is the choice treatment for Bcr/Abl-positive malignancies. Emergence of resistance to IM warrants the exploration of novel well-tolerated anticancer agents. We intended to evaluate the effect of PS-341 on proliferation, survival, and cellular events in Bcr/Abl-positive cells sensitive and resistant to IM, and to investigate the effect of PS-341 and IM in conjunction. DESIGN AND METHODS: Bcr/Abl-positive cell lines sensitive (p210Bcr/Abl KBM5, p210Bcr/Abl KBM7, and p190Bcr/Abl Z-119) or resistant (KBM5-R) to IM were treated with PS-341 alone or in combination with IM. The effect on cell growth was determined using the MTT assay. Cell-cycle analysis was performed by propidium iodide staining. Apoptosis was evaluated by measurement of sub-G1 DNA content, annexin V binding, and caspase 3 activity assays. Levels of apoptotic proteins, P-IkBalpha, Bcr/Abl, and phosphorylated Bcr/Abl were determined by western blotting. NF-kappaB activity was evaluated by electromobility gel shift assays. RESULTS: PS-341 exerted growth inhibition effects in IM-sensitive and -resistant cells. This phenomenon correlated with accumulation of cells in the G2/M phase of cell cycle; transient downregulation of NFkappaB DNA binding activity; downregulation of Bcl-xL; activation of caspase 3, induction of apoptosis; inhibition of expression and phosphorylation of Bcr/Abl. Sequential combination of PS-341 followed by IM demonstrated a synergistic pro-apoptotic effect in IM-sensitive cells; concomitant exposure was antagonistic. INTERPRETATION AND CONCLUSIONS: PS-341 suppresses growth and induces apoptosis in Bcr/Abl-positive cells sensitive and resistant to IM. The use of PS-341 should be explored in patients with chronic myelogenous leukemia resistant to IM. Trials of combinations of PS-341 and IM require cautious design.

RAS mutations contribute to evolution of chronic myelomonocytic leukemia to the proliferative variant.

PURPOSE: The biological and clinical heterogeneity of chronic myelomonocytic leukemia features renders its classification difficult. Moreover, because of the limited knowledge of the mechanisms involved in malignant evolution, chronic myelomonocytic leukemia remains a diagnostic and therapeutic challenge and a poor prognosis disease. We aimed to verify the biological and clinical significance of the discrimination, based on the leukocyte count, between myelodysplastic chronic myelomonocytic leukemia (MD-CMML) and myeloproliferative chronic myelomonocytic leukemia (MP-CMML). EXPERIMENTAL DESIGN: Peripheral blood samples from 22 patients classified as MD-CMML and 18 as MP-CMML were collected at different time points during disease course, and patients' clinical characteristics were examined. RAS mutational screening was done by sequencing and, for each substitution identified, a highly selective allele-specific PCR was set up to screen all specimens. RESULTS: MP-CMML patients showed a significantly poorer survival (P = 0.003) and a higher frequency of RAS mutations (P = 0.033) by sequencing compared with MD-CMML. Overall, five MD-CMML patients progressed to myeloproliferative disease: in two, allele-specific PCR unveiled low levels of the RAS mutations predominating in the myeloproliferative phase at the time of myelodysplastic disease, documenting for the first time the expansion of a RAS mutated clone in concomitance with chronic myelomonocytic leukemia evolution. Moreover, one of the progressed patients harbored the FLT3-ITD and two MP-CMML patients presented with the JAK2 V617F substitution. All these lesions were mutually exclusive. CONCLUSIONS: Our results strongly suggest RAS mutations to function as a secondary event that contributes to development of the chronic myelomonocytic leukemia variant with the poorer prognosis (MP-CMML) and therefore advise their detection to be implemented in chronic myelomonocytic leukemia diagnostics and monitoring.

Effect of inositol hexaphosphate (IP(6)) on human normal and leukaemic haematopoietic cells.

Inositol hexaphosphate (IP(6)), a naturally polyphosphorylated carbohydrate, has been reported to have significant in vivo and in vitro anticancer activity against numerous tumours, such as colon, prostate, breast, liver and rhabdomyosarcomas. To confirm this activity in haematological malignancies and to characterize some of the mechanisms of IP(6) action, we analysed its effects on human leukaemic cell lines and fresh chronic myelogenous leukaemia (CML) progenitor cells using a combined cellular and molecular approach. IP(6) had a dose-dependent cytotoxic effect on all of the evaluated cell lines, with accumulation in the G2M phase in two out of five cell lines tested. At the molecular level, cDNA microarray analysis after IP(6) exposure showed an extensive downmodulation of genes involved in transcription and cell cycle regulation and a coherent upregulation of cell cycle inhibitors. Furthermore, IP(6) treatment of fresh leukaemic samples of bone marrow CD34+ CML progenitor cells significantly inhibited granulocyte-macrophage colony-forming unit (CFU-GM) formation (P = 0.0062) in comparison to normal bone marrow specimens, which were not affected. No differentiating effect on HL60 cells was observed. Taken together, our results confirm the antiproliferative activity of IP(6) and suggest that it may have a specific antitumour effect also in chronic myeloid leukaemias, via active gene modulation.

Detection of micrometastatic cells in breast cancer by RT-pCR for the mammaglobin gene.

The detection of circulating cancer cells in the bone marrow (BM) and peripheral blood (PB) of patients with solid tumors may be useful for disease staging. To this aim, we evaluated the expression of the mammaglobin gene by reverse transcriptase polymerase chain reaction (RT-PCR) in 60 patients with breast cancer. Moreover, several controls were examined to test the specificity of this marker. The positive cases included 23.6% of the patients with and 9% of those without metastasis. Only 4/60 negative controls analyzed were positive by PCR. Our results show high specificity and a good correlation with disease status.

Changes associated with the development of resistance to imatinib (STI571) in two leukemia cell lines expressing p210 Bcr/Abl protein.

BACKGROUND: Although various mechanisms have been recognized as being associated with the development of resistance to imatinib mesylate in vitro and in clinical situations, their relative significance and contributions remain poorly understood, as is the sequence of events leading to the selection of the resistant phenotype. Experimental in vitro systems involving well defined cell lines and conditions can be used to some advantage to answer specific questions and to develop in vitro models of imatinib resistance that would reflect its potential heterogeneity. METHODS: Two cell lines, KBM5 and KBM7, which expressed p210 Bcr/Abl and which differed in their inherent sensitivity to imatinib, the number of copies of the BCR/ABL fusion gene, and the activation of apoptotic pathways, were grown in vitro in the presence of increasing concentrations of imatinib. The resistant cells were analyzed for cell cycle progression, apoptotic response after exposure to imatinib, expression of Bcr/Abl, tyrosine kinase activity, and the presence of mutations within the adenosine triphosphate (ATP) coding domain of BCR/ABL. At various levels of resistance, the cells were transferred into drug-free media, and the stability of the resistant phenotype was determined in the absence of the drug. RESULTS: In KBM7 cells, the development of resistance was characterized by loss of apoptotic response to the drug, amplification of BCR/ABL, increased levels of expression of p210 Bcr/Abl, and decreased inhibition of Bcr/Abl tyrosine kinase (TK) activity by imatinib. No mutations within the ATP-binding domain of Bcr/Abl were identified, and resistance remained stable in the absence of the drug. In KBM5 cells, which previously were found to be characterized by the acquisition of a single C-T mutation at ABL nucleotide 944 (T315I) at high levels of resistance, this same mutation was detected at an intermediate level, but not at a low level, of resistance. The response of KBM5 cells to imatinib was characterized by a low level of apoptotic response, a marginal increase in BCR/ABL copy number, a modest increase in p210 expression, and a highly imatinib-resistant Bcr/Abl TK. Partial reversal of resistance was observed in highly resistant KBM5-STI571(R1.0) cells, which continued to display the C-T mutation. In KBM5 cells with an intermediate level of resistance, the T315I mutation was no longer detectable upon their reversal to the sensitive phenotype. CONCLUSIONS: BCR/ABL amplification with subsequent overexpression of Bcr/Abl protein, loss of apoptotic response, or point mutation of the ATP-binding site of BCR/ABL was associated alternatively with the acquisition of the resistant phenotype, supporting the notion that multiple mechanisms are involved in the induction of resistance to imatinib. The initial number of BCR/ABL copies itself was not related directly to the degree of resistance. The reversibility of the resistance may be complete, partial, or irreversible, depending on the mechanism(s) involved and the degree of resistance. Both cell lines serve as models for further elucidation of various aspects of imatinib-resistance mechanisms.