Cancer-selective cytotoxic Ca2+ overload in acute myeloid leukemia cells and attenuation of disease progression in mice by synergistically acting polyphenols curcumin and carnosic acid.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by extremely heterogeneous molecular and biologic abnormalities that hamper the development of effective targeted treatment modalities. While AML cells are highly sensitive to cytotoxic Ca2+ overload, the feasibility of Ca2+- targeted therapy of this disease remains unclear. Here, we show that apoptotic response of AML cells to the synergistically acting polyphenols curcumin (CUR) and carnosic acid (CA), combined at low, non-cytotoxic doses of each compound was mediated solely by disruption of cellular Ca2+ homeostasis. Specifically, activation of caspase cascade in CUR+CA-treated AML cells resulted from sustained elevation of cytosolic Ca2+ (Ca2+cyt) and was not preceded by endoplasmic reticulum stress or mitochondrial damage. The CUR+CA-induced Ca2+cyt rise did not involve excessive influx of extracellular Ca2+ but, rather, occurred due to massive Ca2+ release from intracellular stores concomitant with inhibition of Ca2+cyt extrusion through the plasma membrane. Notably, the CUR+CA combination did not alter Ca2+ homeostasis and viability in non-neoplastic hematopoietic cells, suggesting its cancer-selective action. Most importantly, co-administration of CUR and CA to AML-bearing mice markedly attenuated disease progression in two animal models. Collectively, our results provide the mechanistic and translational basis for further characterization of this combination as a prototype of novel Ca2+-targeted pharmacological tools for the treatment of AML.

The MAPK ERK5, but not ERK1/2, inhibits the progression of monocytic phenotype to the functioning macrophage.

Intracellular signaling pathways present targets for pharmacological agents with potential for treatment of neoplastic diseases, with some disease remissions already recorded. However, cellular compensatory mechanisms usually negate the initial success. For instance, attempts to interrupt aberrant signaling downstream of the frequently mutated ras by inhibiting ERK1/2 has shown only limited usefulness for cancer therapy. Here, we examined how ERK5, that overlaps the functions of ERK1/2 in cell proliferation and survival, functions in a manner distinct from ERK1/2 in human AML cells induced to differentiate by 1,25D-dihydroxyvitamin D3 (1,25D). Using inhibitors of ERK1/2 and of MEK5/ERK5 at concentrations specific for each kinase in HL60 and U937 cells, we observed that selective inhibition of the kinase activity of ERK5, but not of ERK1/2, in the presence of 1,25D resulted in macrophage-like cell morphology and enhancement of phagocytic activity. Importantly, this was associated with increased expression of the macrophage colony stimulating factor receptor (M-CSFR), but was not seen when M-CSFR expression was knocked down. Interestingly, inhibition of ERK1/2 led to activation of ERK5 in these cells. Our results support the hypothesis that ERK5 negatively regulates the expression of M-CSFR, and thus has a restraining function on macrophage differentiation. The addition of pharmacological inhibitors of ERK5 may influence trials of differentiation therapy of AML.

ERK 5/MAPK pathway has a major role in 1α,25-(OH)2 vitamin D3-induced terminal differentiation of myeloid leukemia cells.

Vitamin D derivatives, including its physiological form 1α,25(OH)2 vitamin D3 (1,25D), have anti-tumor actions demonstrated in cell culture and confirmatory epidemiological associations are frequently reported. However, their promise for use in the cancer clinic is still incompletely fulfilled, suggesting that a better understanding of the molecular events initiated by these compounds is needed for therapeutic advances. While ERK1/2 has been intensely investigated and is known to transmit signals for cell survival, growth, and differentiation, the role of other MAPK pathways has been studied sporadically. Therefore, we utilized acute myeloid leukemia (AML) cells in culture (HL60 and U937), to determine if ERK5 has a role in 1,25D-induced terminal differentiation which is distinct from the previously shown involvement of ERK1/2. We previously found that inhibition of kinase activity of ERK5 by specific pharmacological inhibitors BIX02189 or XMD8-92 results in higher expression of general myeloid marker CD11b, but a lower expression of the monocytic marker CD14. In contrast, the inhibition of the ERK1/2 pathway by PD98059 or U0126 reduced the expression of all differentiation markers studied. We report here for the first time that the differentiation changes induced by ERK5 inhibitors are accompanied by the inhibition of cell proliferation, and this occurs in the both G1 and G2 phases of the cell cycle. Of note, inhibition of ERK5 auto-phosphorylation by XMD8-92 results in a particularly robust cell cycle arrest in G2 phase in AML cells. This study provides a link between the 1,25D-elevated ERK5 pathway and changes in the cell cycle phase transitions in AML cells. Thus, combinations of vitamin D derivatives and ERK5 inhibitors may be more successful in cancer clinics than 1,25D or analogs alone. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

ERK5 pathway regulates transcription factors important for monocytic differentiation of human myeloid leukemia cells.

Mitogen-activated protein kinases (MAPKs) are important transducers of external signals for cell growth, survival, and other cellular responses including cell differentiation. Several MAPK cascades are known with the MEK1/2-ERK1/2, JNK, and p38MAPKs receiving most attention, but the role of MEK5-ERK5 in intracellular signaling deserves more scrutiny, as this pathway transmits signals that can complement ERK/2 signaling. We hypothesized that the ERK5 pathway plays a role in the control of monocytic differentiation, which is disturbed in myeloid leukemia. We therefore examined the cellular phenotype and key molecular events which occur when human myeloid leukemia cells, acute (AML) or chronic (CML), are forced to differentiate by vitamin D derivatives (VDDs). This study was performed using established cell lines HL60 and U937, and primary cultures of blasts from 10 patients with ML. We found that ERK5 and its direct downstream target transcription factor MEF2C are upregulated by 1,25D in parallel with monocytic differentiation. Further, inhibition of ERK5 activity by specific pharmacological agents BIX02189 and XMD8-92 alters the phenotype of these cells by reducing the abundance of the VDD-induced surface monocytic marker CD14, and concomitantly increasing surface expression of the general myeloid marker CD11b. Similar results were obtained when the expression of ERK5 was reduced by siRNA or short hairpin (sh) RNA. ERK5 inhibition resulted in an expected decrease in MEF2C activation. We also found that in AML cells the transcription factor C/EBPß is positively regulated, while C/EBPα is negatively regulated by ERK5. These findings provide new understanding of dysregulated differentiation in human myeloid leukemia.

Distinct combinatorial effects of the plant polyphenols curcumin, carnosic acid, and silibinin on proliferation and apoptosis in acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a malignancy without effective treatment for most patients. Here we demonstrate that combinations of the dietary plant polyphenols--curcumin and carnosic acid--at noncytotoxic concentrations of each agent, produced a synergistic antiproliferative effect and a massive apoptotic cell death in HL-60 and KG-1a human AML cells. In contrast, combinations of curcumin and another plant polyphenol silibinin had a predominantly additive cytostatic effect, without pronounced cytotoxicity. Neither polyphenol combination affected viability of normal human fibroblasts or proliferating and nonproliferating blood cells. Early stage of curcumin/carnosic acid-induced apoptosis was associated with cleavage (activation) of caspase-8, caspase-9, and caspase-3 and the proapoptotic protein Bid, but not with oxidative stress or altered levels of other Bcl-2 family proteins (Bcl-2, Bcl-xl, Mcl-1, Bax, and Bak). Inhibitors of caspase-8 and caspase-9 markedly attenuated apoptosis, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. Caspase-8 inhibition abrogated Bid cleavage and strongly reduced caspase-9 activation, suggesting that the cross-talk mechanism mediated by caspase-8-dependent Bid cleavage can contribute to the activation of the intrinsic apoptotic pathway by curcumin + carnosic acid. Collectively, these results suggest a mechanistic basis for the potential use of dietary plant polyphenol combinations in the treatment and prevention of AML.

Effect of hydrogen peroxide production and the Fenton reaction on membrane composition of Streptococcus pneumoniae.

As part of its aerobic metabolism, Streptococcus pneumoniae generates high levels of H(2)O(2) by pyruvate oxidase (SpxB), which can be further reduced to yield the damaging hydroxyl radicals via the Fenton reaction. A universal conserved adaptation response observed among bacteria is the adjustment of the membrane fatty acids to various growth conditions. The aim of the present study was to reveal the effect of endogenous reactive oxygen species (ROS) formation on membrane composition of S. pneumoniae. Blocking carbon aerobic metabolism, by growing the bacteria at anaerobic conditions or by the truncation of the spxB gene, resulted in a significant enhancement in fatty acid unsaturation, mainly cis-vaccenic acid. Moreover, reducing the level of OH(.) by growing the bacteria at acidic pH, or in the presence of an OH(.) scavenger (salicylate), resulted in increased fatty acid unsaturation, similar to that obtained under anaerobic conditions. RT-PCR results demonstrated that this change does not originate from a change in mRNA expression level of the fatty acid synthase II genes. We suggest that endogenous ROS play an important regulatory role in membrane adaptation, allowing the survival of this anaerobic organism at aerobic environments of the host.

Differences in membrane fluidity and fatty acid composition between phenotypic variants of Streptococcus pneumoniae.

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P < or = 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P < or = 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P < or = 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition.