Cytotoxic effect, differentiation, inhibition of growth and theoretical calculations of an N,N-donor ligands and its platinum(II), palladium(II) and copper(II) complexes.

The new pyrazole ligand 5-(2-hydroxyphenyl)-3-methyl-1-(2-pyridylo)-1H-pyrazole-4-carboxylic acid methyl ester (2) and the corresponding Pt(II), Pd(II) and Cu(II) complexes 3-5 have been synthesized as potential anticancer compounds, and characterized using IR, and (1)H NMR as well as mass spectrometry. The 3-D structures of the Cu(II) complexes were determined by quantum mechanic calculation DFT methodology (density functional theory). The cytotoxicity assay of the ligand and complexes has been performed on leukemia cell lines. In general, the complexes showed lower cytotoxicity than cisplatin, and the Pt(II) and Cu(II) complexes were found to be more efficient in the induction of leukemia cell death than the Pd(II) complex. Our investigations indicate that the antiproliferating activity of the Pt(II) and Cu(II) complexes was partly due to the modulation of cellular differentiation.

Combined effects of doxorubicin and STI571 on growth, differentiation and apoptosis of CML cell line K562.

STI571 (imatinib mesylate; Gleevec) is an inhibitor that targets the tyrosine kinase activity of Bcr-Abl present in chronic myelogenous leukemia (CML) cells. Some preclinical studies have demonstrated that the combination of STI571 with chemotherapeutic drugs results in enhanced toxicity in Bcr-Abl-positive leukemias. We investigated the potential benefit of using STI571 to down-regulate Bcr-Abl activity for the enhancement of doxorubicin anti-proliferative action in K562 cell line derived from blast crisis of CML. At low concentrations of both drugs (40 nM doxorubicin combined with STI571 in the range of 100-150 nM), the antiproliferative effects were mainly due to cellular differentiation as assessed by benzidine staining for hemoglobin synthesis level and real-time PCR for gamma-globin expression. Higher concentrations of STI571 used in combinations with doxorubicin caused mainly apoptosis as shown by DNA degradation and nuclear fragmentation visualized by fluorescence microscopy after DAPI staining, changes in cell morphology observed after Giemza-May Grünwald staining and cellular membrane organization estimated by flow cytometry after Annexin V staining. As compared with either drug alone, cotreatment with STI571 and DOX induced stronger cellular responses. A low concentration of STI571 in combination with a low concentration of DOX might be tested as an alternative approach to increasing the efficacy of chemotherapy against CML.

Accumulation of gamma-globin mRNA and induction of irreversible erythroid differentiation after treatment of CML cell line K562 with new doxorubicin derivatives.

Human chronic myelogenous leukemia (CML) cell line K562 can be chemically induced to differentiate and express embryonic and fetal globin genes. In this study, the effects of doxorubicin (DOX), an inducer of K562 cell erythroid differentiation, with those of epidoxorubicin (EDOX) as well as newly synthesized derivatives of both drugs (DOXM, DOXH, and EDOXM) on cell growth and differentiation were compared. Our results revealed that DOX, EDOX and their derivatives caused irreversible differentiation of K562 cells into more mature hemoglobin-containing cells. This phenomenon was linked to time-dependent inhibition of cell proliferation. Considering the impact of the structure of newly synthesized anthracyclines on their cellular activity, our data clearly indicated that among tested anthracyclines DOXM, a morpholine derivative of DOX exerted the highest antiproliferative and differentiating activity. An increase of gamma-globin mRNA level caused both by high transcription rate and by mRNA stabilization, as well as an enhancement of expression but not activity of erythroid transcription factor GATA-1 were observed. Therefore, a high level of hemoglobin-containing cells in the presence of DOXM resulted from transcriptional and post-transcriptional events on gamma-globin gene regulation. The same morpholine modification introduced to EDOX did not cause, however, similar effects on cellular level. Characterization of new powerful inducers of erythroid differentiation may contribute to the development of novel compounds for pharmacological approach by differentiation therapy to leukemia or to beta-globin disorder, beta-thalassemia.

Comparative studies on the mechanism of cytotoxic action of novel platinum II complexes with pyrazole ligands.

In search for new platinum-based anticancer drugs, four cisplatin analogues, which contain pyrazole rings as non-leaving ligands, have been synthesized: cis-PtCl(2)(3,5-DM HMPz)(2), cis-PtCl(2)(Pz)(2), cis-PtCl(2)(ClMPz)(2), and cis-PtCl(2)(HMPz)(2), where Pz=pyrazole, H=hydroxyl, M=methyl. We tested their cytotoxicity, apoptosis induction ability, DNA damaging and modification properties comparing them in respect to the parent compound. The cytotoxic activity of these platinum pyrazole complexes toward the murine leukemia cell line was 2.9-3.8 times lower than actvity of cisplatin. The tested compounds varied in their mechanism of action by producing different DNA lesions. The most interesting compound seems to be the complex with chloromethyl groups at N1 of pyrazole rings, which exhibited the highest ability to form bifunctional adducts with DNA in vitro.

[Molecular mechanisms of anthracyclines action]. / Molekularne mechanizmy dzialania antracyklin.

Anthracyclines have been in use for almost 40 years for the treatment of many malignancies. During this period, hundreds of analogs of the first two anthracycline antibiotics, doxorubicin and daunorubicine, have been synthesized and evaluated. Multiple mechanisms have been proposed to explain the cytostatic and cytotoxic actions of anthracyclines. These include free radical formation, lipid peroxidation, and direct membrane effects. The best characterized, however, are interactions with the DNA-topoisomerase II complex or DNA itself via intercalation or covalent binding formation and base modification, which in turn are responsible for disturbances in DNA replication and transcription, and then the induction of DNA repair or apoptotic cell death. There is evidence that at low concentrations, anthracyclines can induce a differentiation program in proliferating cells. The search for the relationship between the structure of anthracyclines and their mode of action in vitro or their clinical effectiveness is continuing. The concentrations of the drugs is important in these studies since not all the mechanisms of action observed in vitro seem to be responsible for clinical effects and, on the other hand, the drug action associated with their clinical utilization is very complex. Because of anthracycline-induced cardiotoxicity, an important part of the research is focused on new methods of drug delivery to cancer cells. We review recent progress in understanding the molecular mechanisms of anthracycline action and the new approaches which are being undertaken to improve their therapeutic index.

Effects of anthracycline derivatives on human leukemia K562 cell growth and differentiation.

New derivatives of daunorubicin (DRB), doxorubicin (DOX), and epidoxorubicin (EDOX) with an amidine group bonded to C-3' of daunosamine moiety with either morpholine or hexamethyleneimine ring attached to the amidine group are studied in this paper. We have shown that all of these newly synthesized anthracycline derivatives inhibit human leukemia K562 cell line proliferation but only some of them induce erythroid differentiation when used at subtoxic concentrations. Morpholine derivative of DOX has the greatest potential to inhibit proliferation and to induce differentiation in vitro. The correlation between these two cellular processes was also significant for other tested compounds. In cell cycle analysis, we have demonstrated that those anthracycline derivatives that exert the greatest cytostatic potential caused G(2)/M arrest, which in turn, might contribute to the development of a differentiating phenotype. The concentrations of the compounds used in the study are pharmacologically relevant. These new potent inducers of differentiation might be exploited as anticancer drugs for treatment of leukemia by differentiation therapy.

[Induced differentiation of the K562 leukemic cell line]. / Indukowane róznicowanie komórek bialaczkowych linii K562.

Hematopoietic cells undergo three major fates: proliferation, differentiation, and apoptosis. These processes are closely intertwined. Under normal circumstances, hematopoietic cell proliferation and cell death are carefully balanced. Induction of differentiation is associated with a loss of proliferative capacity, and cell death accompanies hematopoietic cell maturation. Leukemic transformations can be related to dysregulation of each of these processes. Considerable evidence supports the notion that leukemias are likely to arise from the disruption of the differentiation process of hematopoietic progenitors, which fail to give birth to blood cells with restricted phenotypes, as well as from diminished ability to undergo apoptosis. Main results supporting such mechanisms have been obtained from studying bone marrow and analyzing the differentiation process of the human K562 and HL-60 and mouse MEL leukemic cell lines. This paper reviews the current concepts of how understanding the mechanisms of action of differentiation-inducing agents may contribute to the development of less toxic strategies to control growth and apoptosis of human cancer cells. Furthermore, the identification of new approaches to induce erythroid differentiation and reactivate fetal globin genes is crucial for the development of potential therapeutic agents in hematological disorders, including beta-thalassemia. The natural/synthetic agents inducing differentiation of human erythroleukemia K562 cells are presented.

Sequence specificity of formaldehyde-mediated covalent binding of anthracycline derivatives to DNA.

Daunorubicin (DRB) and doxorubicin (DOX) in the presence of formaldehyde (CH2O) form covalent adducts with DNA. A G-specific adduct is formed by producing an aminal bridge between the C-3' of daunosamine and the C-2 of guanine. New derivatives of DRB, DOX and epidoxorubicin (EDOX) with an amidine group bonded to the C-3' of the daunosamine moiety, with either a morpholine or hexamethyleneimine ring attached to the amidine group, were studied in this paper. DNase I footprinting and analyses with restriction endonucleases were applied to compare the specificity of adduct formed by the amidine derivatives and their parent compounds. These approaches provide consistent results, proving that a GC pair is required for covalent binding of anthracycline derivatives to DNA and that different flanking sequences are able to modify the sequence preference of the drugs. The 5'-GC-3', 5'-CG-3' and 5'-TC-3' sequences were protected most efficiently by the parent compounds and their morpholine derivatives and some increased protection of 5'-TC-3' sequence was observed for morpholine analogues. Hexamethyleneimine derivatives bind to DNA with much lower efficiency. Finally, the sequence specificity of anthracycline derivatives was correlated with their ability to inhibit binding of transcription factors Sp1 and AP-1 to their DNA recognition sequences. The anthracycline derivatives were more potent in inhibiting Sp1 binding to its cognate GC box than in preventing AP-1 from binding to its mixed A.T and G.C site. Overall, the results indicate that the amidine derivatives of anthracyclines show similar, but not identical sequence specificity as parent compounds, though they exert their effect at a higher concentration.

Biological evaluation of novel Pt(II) and Pd(II) complexes with pyrazole-containing ligands.

The new platinum (II) and palladium (II) complexes (2-4) with ligands 5-(2-hydroxyphenyl)-1,3-dimethyl-4-(dimethoxy)phosphonyl-1H]-pyrazole (1a) and 5-(2-hydroxyphenyl)-1,3-dimethyl-4-methoxycarbonyl-1H]-2-pyrazole (1b) were screened in a search for novel anticancer agents. Thus, alkylating activity, cytotoxicity, ability for induction of apoptosis and binding to DNA were tested. The cis-[Pt(1b)2Cl2] complex (3b) was the most potent alkylating agent in a Preussmann test, in comparison with the other test compounds and cis-platin. The highest cytotoxicity against the HL-60 and NALM-6 leukemia cell lines was observed for complexes 3b and 4b (trans-[Pd(1b)2Cl2]), although the extent of the effect was lower relative to cis-platin. Moreover, both complexes were remarkably less toxic to human umbilical vein endothelial cells (HUVECs) with IC50 values of 3b 14 and 20 times higher than that ones for HL-60 and NALM-6 cells, respectively. Complexes 3b and 4b induced caspase-3 activity. Apoptosis occurred in a strictly dose-dependent manner and required only low concentrations of 4b. However, compounds 3b and 4b showed lower binding affinity to double-stranded DNA than cis-platin.

In vitro antileukemic, antioxidant and prooxidant activities of Antoksyd S (C/E/XXI): a comparison with baicalin and baicalein.

There is increasing interest concerning the use of natural antioxidants as low toxic antileukemic compounds. Antoksyd S (C/E/XXI), is a novel herbal drug derived in Poland from the powdered roots of Scutellaria baicalensis, and the biological activities of its major components (baicalin and baicalein) were compared on the human leukemia cell line HL-60. On MTT assay, Antoksyd S (C/E/XXI) showed an obvious cytotoxic effect on HL-60 cells, which was compared with those caused by cisplatin and doxorubicin under the same experimental conditions. A comparative assay of the antioxidative and prooxidative capacity of Antoksyd S (C/E/XXI) was also undertaken using two different reactive species: superoxide (O2-) and a transition metal (Cu2+). Antoksyd S (C/E/XXI) has low toxicity, acting as a modifier of HL-60 cells proliferation and as an antioxidant, which could act prooxidatively in the presence of transition metal ions. Taken together, it seems reasonable to suggest that Antoksyd S (C/E/XXI) as compared to baicalin and baicalein, or to the cytostatics cisplatin and doxorubicin, might be an especially good candidate for the future development of new therapeutic techniques, alone or in "combination treatment regimens", to enhance leukemia cell killing.