Proteomic analysis of changes in the protein composition of MCF-7 human breast cancer cells induced by all-trans retinoic acid, 9-cis retinoic acid,and their combination

Retinoic acid (all-trans and 9-cis) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment.     

Regulation of CYP26A1 expression by selective RAR and RXR agonists in human NB4 promyelocytic leukemia cells

All-trans retinoic acid (ATRA) can induce complete remission in acute promyelocytic leukemia (APL), but resistance to this treatment develops rapidly partly due to increased ATRA metabolism. Among the cytochrome P450s (CYPs) involved in ATRA metabolism, the ATRA-inducible cytochrome P450 26A1 (CYP26A1) is particularly active although the molecular mechanisms involved in its regulation are not well defined in the target leukemia cells. To study CYP26A1 expression and regulation in APL cells, we used the NB4 promyelocytic leukemia cell line. CYP26A1 constitutive expression was barely detectable in NB4 cells, but ATRA could induce high levels of CYP26A1 expression, which reached a maximum at 72h. To further define CYP26A1 induction mechanisms in the NB4 leukemia cells, we used RARs and RXR selective agonists. The RARalpha agonist BMS753 could elicit maturation, as expected, but not CYP26A1 expression. Treatment with the RARbeta agonist BMS641, or the RARbeta/gamma agonist BMS961, could not elicit maturation, as expected, nor induce CYP26A1 expression. Because CYP26A1 expression could not be induced by RAR ligands alone, NB4 cells were then co-treated with the RXR agonist BMS649. The RXR agonist alone could not induce CYP26A1 expression, nor in combination with either the RARbeta agonist or the RARbeta/gamma agonist. However, the combination of the RXR agonist and the RARalpha agonist could elicit a marked induction of CYP26A1 expression. In conclusion, we have shown that CYP26A1 induction is not essential for the granulocytic maturation of NB4 leukemia cells, and that CYP26A1 induction requires the activation of both RARalpha and RXR in these cells.     

Differential enhancement of leukaemia cell differentiation without elevation of intracellular calcium by plant-derived sesquiterpene lactone compounds

Background and purpose:

All-trans retinoic acid (ATRA) induces complete remission in a majority of acute promyelocytic leukaemia patients, but resistance of leukaemic cells to ATRA and its toxicity, such as hypercalcaemia, lead to a limitation of treatment. Therefore, combination therapies with differentiation-enhancing agents at non-toxic concentrations of ATRA may overcome its side effects. Here, we investigated the effect of plant-derived sesquiterpene lactone compounds and their underlying mechanisms in ATRA-induced differentiation of human leukaemia HL-60 cells.

Experimental approach:

HL-60 cells were treated with four sesquiterpene lactones (helenalin, costunolide, parthenolide and sclareolide) and cell differentiation was determined by NBT reduction, Giemsa and cytofluorometric analyses. Signalling pathways were assessed by western blotting, gel-shift assay and kinase activity determinations and intracellular calcium levels were determined using a calcium-specific fluorescent probe.

Key results:

Helenalin, costunolide and parthenolide, but not sclareolide, increased ATRA-induced HL-60 cell differentiation into a granulocytic lineage. Signalling kinases PKC and ERK were involved in the ATRA-induced differentiation enhanced by all of the effective sesquiterpene lactones, but JNK and PI3-K were involved in the ATRA-induced differentiation enhanced by costunolide and parthenolide. Enhancement of cell differentiation closely correlated with inhibition of NF-κB DNA-binding activity by all three effective compounds. Importantly, enhancement of differentiation induced by 50 nM ATRA by the sesquiterpene lactones was not accompanied by elevation of basal intracellular calcium concentrations.

Conclusions and implications:

These results indicate that plant-derived sesquiterpene lactones may enhance ATRA-mediated cell differentiation through distinct pathways.     

13-cis retinoic acid and isomerisation in paediatric oncology--is changing shape the key to success?

Retinoic acid isomers have been used with some success as chemotherapeutic agents, most recently with 13-cis retinoic acid showing impressive clinical efficacy in the paediatric malignancy neuroblastoma. The aim of this commentary is to review the evidence that 13-cis retinoic acid is a pro-drug, and consider the implications of retinoid metabolism and isomerisation for the further development of retinoic acid for cancer therapy. The low binding affinity of 13-cis retinoic acid for retinoic acid receptors, low activity in gene expression assays and the accumulation of the all-trans isomer in cells treated with 13-cis retinoic acid, coupled with the more-favourable pharmacokinetic profile of 13-cis retinoic acid compared to other isomers, suggest that intracellular isomerisation to all-trans retinoic acid is the key process underlying the biological activity of 13-cis retinoic acid. Intracellular metabolism of all-trans retinoic acid by a positive auto-regulatory loop may result in clinical resistance to retinoic acid. Agents that block or reduce the metabolism of all-trans retinoic acid are therefore attractive targets for drug development. Devising strategies to deliver 13-cis retinoic acid to tumour cells and facilitate the intracellular isomerisation of 13-cis retinoic acid, while limiting metabolism of all-trans retinoic acid, may have a major impact on the efficacy of 13-cis retinoic acid in paediatric oncology.     

Matrix and serine protease expression during leukemic cell differentiation induced by aclacinomycin and all-trans-retinoic acid

In myeloid leukemia, immature leukemic cells are able to egress into peripheral blood to infiltrate extra-medullary organs. We therefore analyzed the migrating and invasive potential of human HL-60 and NB4 cell lines, representative of acute myelogenous leukemia, their ability to express matrix metalloproteases (MMPs), tissue inhibitors of metalloproteases (TIMPs) and urokinase plasminogen activator (uPA) in response to differentiating agents. Granulocytic differentiation by all-trans-retinoic acid (ATRA) and aclacinomycin (ACLA) strongly increased HL-60 and NB4 cell migration and invasion. At mRNA and protein levels, these cell lines produced significant amounts of MMP-9 (HL-60相似文献   

Effects of FK228, a novel histone deacetylase inhibitor,on human lymphoma U-937 cells in vitro and in vivo

FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide] is a novel histone deacetylase inhibitor that shows therapeutic efficacy in Phase I trials of patients with malignant lymphoma. However, its mechanism of action has not been characterized. In this study, we examined the in vitro and in vivo effects of FK228 on human lymphoma U-937 cells. FK228 very strongly inhibited the growth of U-937 cells with an IC(50) value of 5.92 nM. In a scid mouse lymphoma model, mice treated with FK228 once or twice a week survived longer than control mice, with median survival times of 30.5 (0.56 mg/kg) and 33 days (0.32 mg/kg), respectively (vs. 20 days in control mice). Remarkably, 2 out of 12 mice treated with FK228 (0.56 mg/kg once or twice a week) survived past the observation period of 60 days. The apoptotic population of U-937 cells time-dependently increased to 37.7% after 48 hr of treatment with FK228. In addition, FK228 induced G1 and G2/M arrest and the differentiation of U-937 cells to the CD11b(+)/CD14(+) phenotype. Expression of p21(WAF1/Cip1) and gelsolin mRNA increased up to 654- and 152-fold, respectively, after 24hr of treatment with FK228. FK228 caused histone acetylation in p21(WAF1/Cip1) promoter regions, including the Sp1-binding sites. In conclusion, (i) FK228 prolonged the survival time of scid mice in a lymphoma model, and (ii) the beneficial effects of FK228 on human lymphoma may be exerted through the induction of apoptosis, cell cycle arrest, and differentiation via the modulation of gene expression by histone acetylation.     

Differential allosteric modulation by amiloride analogues of agonist and antagonist binding at A(1) and A(3) adenosine receptors

The diuretic drug amiloride and its analogues were found previously to be allosteric modulators of antagonist binding to A(2A) adenosine receptors. In this study, the possibility of the allosteric modulation by amiloride analogues of antagonist binding at A(1) and A(3) receptors, as well as agonist binding at A(1), A(2A), and A(3) receptors, was explored. Amiloride analogues increased the dissociation rates of two antagonist radioligands, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one ([3H]PSB-11), from A(1) and A(3) receptors, respectively. Amiloride and 5-(N,N-dimethyl)amiloride (DMA) were more potent at A(1) receptors than at A(3) receptors, while 5-(N,N-hexamethylene)amiloride (HMA) was more potent at A(3) receptors. Thus, amiloride analogues are allosteric inhibitors of antagonist binding at A(1), A(2A), and A(3) adenosine receptor subtypes. In contrast to their effects on antagonist-occupied receptors, amiloride analogues did not affect the dissociation rates of the A(1) agonist [3H]N(6)-[(R)-phenylisopropyl]adenosine ([3H]R-PIA) from A(1) receptors or the A(2A) agonist [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5'-N-ethylcarboxamidoadenosine ([3H]CGS21680) from A(2A) receptors. The dissociation rate of the A(3) agonist radioligand [125I]N(6)-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide ([125I]I-AB-MECA) from A(3) receptors was decreased significantly by amiloride analogues. The binding modes of amiloride analogues at agonist-occupied and antagonist-occupied receptors differed markedly, which was demonstrated in all three subtypes of adenosine receptors tested in this study. The effects of the amiloride analogues on the action of the A(3) receptor agonist were explored further using a cyclic AMP functional assay in intact CHO cells expressing the human A(3) receptor. Both binding and functional assays support the allosteric interactions of amiloride analogues with A(3) receptors.     

Relative developmental toxicity potencies of retinoids in the embryonic stem cell test compared with their relative potencies in in vivo and two other in vitro assays for developmental toxicity

The present study determines the relative developmental toxicity potencies of retinoids in the embryonic stem (ES)-D3 cell differentiation assay of the embryonic stem cell test, and compares the outcomes with their relative potencies in in vivo and two other in vitro assays for developmental toxicity. The results reveal that the potency ranking obtained in the ES-D3 cell differentiation assay is similar to the reported potency rankings in the two other in vitro assays for developmental toxicity. TTNPB ((E)-4[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid) was the most potent retinoid, whereas etretinate and retinol had the lowest potency. All-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid and acitretin showed an intermediate potency. In vivo potency rankings of the developmental toxicity of retinoids appear to be dependent on the species and/or exposure regimens used. The obtained in vitro potency ranking does not completely correspond with the in vivo potency rankings, although TTNPB is correctly predicted to be the most potent and retinol the least potent congener. The lack of in vivo kinetic processes in the ES-D3 cell differentiation assay might explain the deviating potency predictions of some retinoids. Therefore, knowledge on the species-dependent in vivo kinetics is essential when using in vitro toxicity data for the estimation of in vivo developmental toxicity potencies within series of related compounds.     

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of alpha-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of alpha-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. gamma-tubulin staining showed that cells treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.     

Regulation of genistein-induced differentiation in human acute myeloid leukaemia cells (HL60, NB4): Protein kinase modulation and reactive oxygen species generation

While it has been reported that genistein induces differentiation in multiple tumour cell models, the signalling and regulation of isoflavone-provoked differentiation are poorly known. We here demonstrate that genistein causes G₂/M cycle arrest and expression of differentiation markers in human acute myeloid leukaemia cells (HL60, NB4), and cooperates with all-trans retinoic acid (ATRA) in inducing differentiation, while ATRA attenuates the isoflavone-provoked toxicity. Genistein rapidly stimulates Raf-1, MEK1/2 and ERK1/2 phosphorylation/activation, but does not stimulate and instead causes a late decrease in Akt phosphorylation/activation which is attenuated by ATRA. Both differentiation and G₂/M arrest are attenuated by MEK/ERK inhibitors (PD98059, U0126) and ERK1-/ERK2-directed small interfering RNAs (siRNAs), and by the PI3K inhibitor LY294002, but not by the p38-MAPK inhibitor SB203580. Genistein stimulates p21^waf1/cip1 and cyclin B1 expression, phosphorylation/activation of ATM and Chk2 kinases, and Tyr15-phosphorylation/inactivation of Cdc2 (Cdk1) kinase, and these effects are attenuated by MEK/ERK inhibitors, while LY294002 also attenuates ERK and ATM phosphorylation. Caffeine abrogates the genistein-provoked G₂/M blockade and alterations in cell cycle regulatory proteins, and also suppresses differentiation. Finally, genistein causes reactive oxygen species (ROS) over-accumulation, but the antioxidant N-acetyl-l-cysteine fails to prevent ERK activation, G₂/M arrest, and differentiation induction. By contrast, N-acetyl-l-cysteine and p38-MAPK inhibitor attenuate the apoptosis-sensitizing (pro-apoptotic) action of genistein when combined with the antileukaemic agent arsenic trioxide. In summary, genistein-induced differentiation in acute myeloid leukaemia cells is a ROS-independent, Raf-1/MEK/ERK-mediated and PI3K-dependent response, which is coupled and co-regulated with G₂/M arrest, but uncoupled to the pro-apoptotic action of the drug.     

Involvement of microtubules and mitochondria in the antagonism of arsenic trioxide on paclitaxel-induced apoptosis

Arsenic trioxide (As(2)O(3)) at low concentrations (1-10 microM) is effective in the treatment of acute promyelocytic leukemia (APL) and lymphoma and is in clinical trials for treatment of solid tumors. Paclitaxel, an antimicrotubule agent, is highly efficacious in the treatment of adult tumors and is in clinical evaluation in childhood tumors. This study is the first to investigate the combination of arsenic and paclitaxel in the range of clinically achievable concentrations. We found that the simultaneous combination was antagonistic on proliferation of the neuroblastoma SK-N-SH cell line by using the combination index (CI) method. Moreover, a 40+/-5% decrease in paclitaxel-induced apoptosis in cells co-treated with As(2)O(3) confirmed the antagonism. The mechanism of antagonism was studied at the cellular level with 200 nM paclitaxel, twice the IC(50) value, and with 1 microM As(2)O(3) which administered singly did not affect cell survival or the microtubule network. As(2)O(3) antagonized the effects of paclitaxel on tubulin and microtubules. Paclitaxel-induced mitotic block was decreased by 20+/-2% and bundles induced by 200 nM paclitaxel were less condensed in the presence of 1 microM As(2)O(3). As(2)O(3) (10-200 microM) induced a concentration-dependent inhibition of tubulin polymerization in vitro which was maintained in presence of paclitaxel. Spectrophotometric and spectrofluorometric measurements indicated an interaction of As(2)O(3) with tubulin SH groups, without modification of the stoichiometry of paclitaxel binding to tubulin. Moreover, 4 microM As(2)O(3) inhibited the release of cytochrome c from isolated mitochondria by 78+/-10%. Our results show that As(2)O(3) and paclitaxel act antagonistically on mitochondria and microtubules and illustrate the need for careful evaluation of drug combinations.     

Increased apoptotic efficacy of lonidamine plus arsenic trioxide combination in human leukemia cells. Reactive oxygen species generation and defensive protein kinase (MEK/ERK, Akt/mTOR) modulation

Lonidamine is a safe, clinically useful anti-tumor drug, but its efficacy is generally low when used in monotherapy. We here demonstrate that lonidamine efficaciously cooperates with the anti-leukemic agent arsenic trioxide (ATO, Trisenox™) to induce apoptosis in HL-60 and other human leukemia cell lines, with low toxicity in non-tumor peripheral blood lymphocytes. Apoptosis induction by lonidamine/ATO involves mitochondrial dysfunction, as indicated by early mitochondrial permeability transition pore opening and late mitochondrial transmembrane potential dissipation, as well as activation of the intrinsic apoptotic pathway, as indicated by Bcl-X_L and Mcl-1 down-regulation, Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release to the cytosol, XIAP down-regulation, and caspase-9 and -3 cleavage/activation, with secondary (Bcl-2-inhibitable) activation of the caspase-8/Bid axis. Lonidamine stimulates reactive oxygen species production, and lonidamine/ATO toxicity is attenuated by antioxidants. Lonidamine/ATO stimulates JNK phosphorylation/activation, and apoptosis is attenuated by the JNK inhibitor SP600125. In addition, lonidamine elicits ERK and Akt/mTOR pathway activation, as indicated by increased ERK, Akt, p70S6K and rpS6 phosphorylation, and these effects are reduced by co-treatment with ATO. Importantly, co-treatment with MEK/ERK inhibitor (U0126) and PI3K/Akt (LY294002) or mTOR (rapamycin) inhibitors, instead of ATO, also potentiates lonidamine-provoked apoptosis. These results indicate that: (i) lonidamine efficacy is restrained by drug-provoked activation of MEK/ERK and Akt/mTOR defensive pathways, which therefore represent potential therapeutic targets. (ii) Co-treatment with ATO efficaciously potentiates lonidamine toxicity via defensive pathway inhibition and JNK activation. And (iii) conversely, the pro-oxidant action of lonidamine potentiates the apoptotic efficacy of ATO as an anti-leukemic agent.