RESUMO
In this work, simultaneous targeted metabolic profiling by isotope dilution and non-targeted fingerprinting is proposed for cancer cell studies. The novel streamlined metabolomics workflow was established using anion-exchange chromatography (IC) coupled to high-resolution mass spectrometry (MS). The separation time of strong anion-exchange (2 mm column, flow rate 380 µL min-1, injection volume 5 µL) could be decreased to 25 min for a target list comprising organic acids, sugars, sugar phosphates, and nucleotides. Internal standardization by fully 13C labeled Pichia pastoris extracts enabled absolute quantification of the primary metabolites in adherent cancer cell models. Limits of detection (LODs) in the low nanomolar range and excellent intermediate precisions of the isotopologue ratios (on average <5%, N = 5, over 40 h) were observed. As a result of internal standardization, linear dynamic ranges over 4 orders of magnitude (5 nM-50 µM, R2 > 0.99) were obtained. Experiments on drug-sensitive versus resistant SW480 cancer cells showed the feasibility of merging analytical tasks into one analytical run. Comparing fingerprinting with and without internal standard proved that the presence of the 13C labeled yeast extract required for absolute quantification was not detrimental to non-targeted data evaluation. Several interesting metabolites were discovered by accurate mass and comparing MS2 spectra (acquired in ddMS2 mode) with spectral libraries. Significant differences revealed distinct metabolic phenotypes of drug-sensitive and resistant SW480 cells.
Assuntos
Metabolômica , Isótopos de Carbono , Cromatografia por Troca Iônica , Humanos , Espectrometria de Massas , Pichia/metabolismo , Células Tumorais CultivadasRESUMO
A new series of N-aryl-N'-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)ureas bearing an alkoxycarbonylamino group at the 6-position were synthesized and examined as putative anticancer agents targeting sirtuins in glioma cells. On the basis of computational docking combined to in vitro sirtuin 1/2 inhibition assays, we selected compound 18 [R/S-N-3-cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea] which displays a potent antiproliferative activity on various glioma cell types, assessed by quantitative videomicroscopy, eventually triggering senescence. The impact on normal glial cells was lower with a selectivity index of >10. Furthermore, human U373 and Hs683 glioblastoma cell lines served to demonstrate the inhibitory activity of 18 against histone deacetylase (HDAC) class III sirtuins 1 and 2 (SIRT1/2) by quantifying acetylation levels of histone and non-histone proteins. The translational potential of 18 was validated by an NCI-60 cell line screen and validation of growth inhibition of drug resistant cancer cell models. Eventually, the anticancer potential of 18 was validated in 3D glioblastoma spheroids and in vivo by zebrafish xenografts. In summary, compound 18 is the first representative of a new class of SIRT inhibitors opening new perspectives in the medicinal chemistry of HDAC inhibitors.
Assuntos
Benzopiranos/química , Benzopiranos/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , HumanosRESUMO
Triapine, an anticancer thiosemicarbazone, is currently under clinical investigation. Whereas promising results were obtained in hematological diseases, trials in solid tumors widely failed. To understand mechanisms causing triapine insensitivity, we have analysed genomic alterations in a triapine-resistant SW480 subline (SW480/tria). Only one distinct genomic loss was observed specifically in SW480/tria cells affecting the phosphodiesterase 4D (PDE4D) gene locus. Accordingly, pharmacological inhibition of PDE4D resulted in significant triapine resistance in SW480 cells. Hence, we concluded that enhanced cyclic AMP levels might confer protection against triapine. Indeed, hyperactivation of both major downstream pathways, namely the protein kinase A (PKA)-cAMP response element-binding protein (Creb) and the exchange protein activated by cAMP (Epac)-Ras-related protein 1 (Rap1) signaling axes, was observed in SW480/tria cells. Unexpectedly, inhibition of PKA did not re-sensitize SW480/tria cells against triapine. In contrast, Epac activation resulted in distinct triapine resistance in SW480 cells. Conversely, knock-down of Epac expression and pharmacological inhibition of Rap1 re-sensitized SW480/tria cells against triapine. Rap1 is a well-known regulator of integrins. Accordingly, SW480/tria cells displayed enhanced plasma membrane expression of several integrin subunits, enhanced adhesion especially to RGD-containing matrix components, and bolstered activation/expression of the integrin downstream effectors Src and RhoA/Rac. Accordingly, integrin and Src inhibition resulted in potent triapine re-sensitization especially of SW480/tria cells. In summary, we describe for the first time integrin activation based on cAMP-Epac-Rap1 signaling as acquired drug resistance mechanism. combinations of triapine with inhibitors of several steps in this resistance cascade might be feasible strategies to overcome triapine insensitivity of solid tumors.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Integrinas/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Piridinas/farmacologia , Tiossemicarbazonas/farmacologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HCT116 , Humanos , Integrinas/genética , Masculino , Camundongos SCID , Interferência de RNA , Rolipram/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
One of the most promising classes of iron chelators are α-N-heterocyclic thiosemicarbazones with Triapine as the most prominent representative. In several clinical trials Triapine showed anticancer activity against hematological diseases, however, studies on solid tumors failed due to widely unknown reasons. Some years ago, it was recognized that "terminal dimethylation" of thiosemicarbazones can lead to a more than 100-fold increased activity, probably due to interactions with cellular copper depots. To better understand the structural requirements for the switch to nanomolar cytotoxicity, we systematically synthesized all eight possible N-methylated derivatives of Triapine and investigated their potential against Triapine-sensitive as well as -resistant cell lines. While only the "completely" methylated compound exerted nanomolar activity, the data revealed that all compounds with at least one N-dimethylation were not affected by acquired Triapine resistance. In addition, these compounds were highly synergistic with copper treatment accompanied by induction of reactive oxygen species and massive necrotic cell death.
Assuntos
Antineoplásicos/farmacologia , Piridinas/farmacologia , Tiossemicarbazonas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Metilação , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Relação Estrutura-Atividade , Tiossemicarbazonas/síntese química , Tiossemicarbazonas/química , Células Tumorais CultivadasRESUMO
Triapine has been investigated as anticancer drug in multiple clinical phase I/II trials. Although promising anti-leukemic activity was observed, Triapine was ineffective against solid tumors. The reasons are currently widely unknown. The biological activity of Triapine is strongly connected to its iron complex (Fe-Triapine) which is pharmacologically not investigated. Here, novel analytical tools for Triapine and Fe-Triapine were developed and applied for cell extracts and body fluids of treated mice. Triapine and its iron complex showed a completely different behavior: for Triapine, low protein binding was observed in contrast to fast protein adduct formation of Fe-Triapine. Notably, both drugs were rapidly cleared from the body (serum half-life time <1h). Remarkably, in contrast to Triapine, where (in accordance to clinical data) basically no renal excretion was found, the iron complex was effectively excreted via urine. Moreover, no Fe-Triapine was detected in serum or cytosolic extracts after Triapine treatment. Taken together, our study will help to further understand the biological behavior of Triapine and its Fe-complex and allow the development of novel thiosemicarbazones with pronounced activity against solid tumor types.
Assuntos
Antineoplásicos/farmacocinética , Neoplasias do Colo/tratamento farmacológico , Complexos de Coordenação/farmacocinética , Ferro/farmacocinética , Piridinas/farmacocinética , Tiossemicarbazonas/farmacocinética , Animais , Antineoplásicos/sangue , Antineoplásicos/urina , Proteínas Sanguíneas/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaios Clínicos como Assunto , Neoplasias do Colo/patologia , Complexos de Coordenação/sangue , Complexos de Coordenação/urina , Feminino , Meia-Vida , Ferro/sangue , Ferro/urina , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Piridinas/sangue , Piridinas/urina , Tiossemicarbazonas/sangue , Tiossemicarbazonas/urina , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ecdysteroids, analogs of the insect molting hormone, are known for their various mild, nonhormonal bioactivities in mammals. Previously, we reported that less-polar ecdysteroids can modulate the doxorubicin resistance of a multidrug resistant (MDR) mouse lymphoma cell line expressing the human ABCB1 transporter. Here, we describe the ability of 20-hydroxyecdysone (1) and its mono- (2) and diacetonide (3) derivatives to sensitize various MDR and non-MDR cancer cell lines towards doxorubicin, paclitaxel, vincristine, or cisplatin. Drug IC50 values with or without ecdysteroid were determined by MTT assay. Compound 3 significantly sensitized all cell lines to each chemotherapeutic except for cisplatin, whose activity was decreased. In order to overcome solubility and stability issues for the future in vivo administration of compound 3, liposomal formulations were developed. By means of their combination index values obtained via checkerboard microplate method, a formulation showed superior activity to that of compound 3 alone. Because ecdysteroids act also on non-ABCB1 expressing (sensitive) cell lines, our results demonstrate that they do not or not exclusively exert their adjuvant anticancer activity as ABCB1 inhibitors, but other mechanisms must be involved, and they opened the way towards their in vivo bioactivity testing against various cancer xenografts.
Assuntos
Cisplatino/farmacologia , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ecdisteroides/farmacologia , Neoplasias/tratamento farmacológico , Paclitaxel/farmacologia , Animais , Humanos , Células MCF-7 , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite the recent advances in the treatment of tumors with intrinsic chemotherapy resistance, such as melanoma and renal cancers, their prognosis remains poor and new chemical agents with promising activity against these cancers are urgently needed. Sphaeropsidin A, a fungal metabolite whose anticancer potential had previously received little attention, was isolated from Diplodia cupressi and found to display specific anticancer activity in vitro against melanoma and kidney cancer subpanels in the National Cancer Institute (NCI) 60-cell line screen. The NCI data revealed a mean LC50 of ca. 10 µM and a cellular sensitivity profile that did not match that of any other agent in the 765,000 compound database. Subsequent mechanistic studies in melanoma and other multidrug-resistant in vitro cancer models showed that sphaeropsidin A can overcome apoptosis as well as multidrug resistance by inducing a marked and rapid cellular shrinkage related to the loss of intracellular Cl(-) and the decreased HCO3 (-) concentration in the culture supernatant. These changes in ion homeostasis and the absence of effects on the plasma membrane potential were attributed to the sphaeropsidin A-induced impairment of regulatory volume increase (RVI). Preliminary results also indicate that depending on the type of cancer, the sphaeropsidin A effects on RVI could be related to Na-K-2Cl electroneutral cotransporter or Cl(-)/HCO3 (-) anion exchanger(s) targeting. This study underscores the modulation of ion-transporter activity as a promising therapeutic strategy to combat drug-resistant cancers and identifies the fungal metabolite, sphaeropsidin A, as a lead to develop anticancer agents targeting RVI in cancer cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Diterpenos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular Tumoral , Diterpenos/química , Células HEK293 , Humanos , Camundongos , Microscopia de Vídeo , Estrutura Molecular , Propídio , Azul TripanoRESUMO
Treatment options for hepatocellular carcinoma using chemotherapeutics at intermediate and advanced stages of disease are limited as patients most rapidly escape from therapy and succumb to disease progression. Mechanisms of the hepatic xenobiotic metabolism are mostly involved in providing chemoresistance to therapeutic compounds. Given the fact that the aberrant activation of cyclin-dependent kinases (CDK) is frequently observed in hepatocellular carcinomas, we focused on the efficacy of the novel compounds BA-12 and BP-14 that antagonize CDK1/2/5/7 and CDK9. Inhibition of those CDKs in human hepatocellular carcinoma cell lines reduced the clonogenicity by arresting cells in S-G2 and G2-M phase of the cell cycle and inducing apoptosis. In contrast, primary human hepatocytes failed to show cytotoxicity and apoptosis. No loss of chemosensitivity was observed in hepatocellular carcinoma cells after long-term exposure to inhibitors. In vivo, treatment of xenografted human hepatocellular carcinomas with BA-12 or BP-14 effectively repressed tumor formation. Moreover, BA-12 or BP-14 significantly diminished diethylnitrosamine (DEN)-induced hepatoma development in mice. These data show that BA-12 or BP-14 exhibit strong antitumorigenic effects in the absence of chemoresistance, resulting in a superior efficacy compared with currently used chemotherapeutics in hepatocellular carcinomas.
Assuntos
2-Aminopurina/análogos & derivados , Carcinoma Hepatocelular/tratamento farmacológico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , 2-Aminopurina/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dietilnitrosamina/toxicidade , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Camundongos , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1ß1 NaK. METHODS: The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1ß1, α2ß1 and α3ß1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. RESULTS: Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2ß1 and α3ß1 than for the α1ß1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1ß1 than for the α2ß1 and α3ß1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. CONCLUSIONS: Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2ß1 and α3ß1 than for the α1ß1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.
Assuntos
Bufanolídeos/farmacologia , Neoplasias/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Bufanolídeos/química , Bufanolídeos/metabolismo , Bufanolídeos/toxicidade , Glicosídeos Cardíacos/química , Glicosídeos Cardíacos/metabolismo , Glicosídeos Cardíacos/farmacologia , Glicosídeos Cardíacos/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Glicosilação , Células HT29 , Humanos , Concentração Inibidora 50 , Isoenzimas , Ácido Láctico/metabolismo , Metaboloma , Oxirredução/efeitos dos fármacos , Consumo de Oxigênio , Ligação Proteica , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Extracellular acidity is a frequent pathophysiological condition of solid tumors offering possibilities for improving the tumor selectivity of molecular therapy. This might be accomplished by prodrugs with low systemic toxicity, attaining their full antitumor potency only under acidic conditions, such as bis(2-aminoalcoholato-κ(2)N,O)platinum(II) complexes that are activated by protonation of alcoholato oxygen, resulting in cleavage of platinum-oxygen bonds. In this work, we examined whether the pH dependency of such compounds is reflected in differential biological activity in vitro. In particular, the pH dependence of cytotoxicity, cellular accumulation, DNA platination, GMP binding, effects on DNA secondary structure, cell cycle alterations, and induction of apoptosis was investigated. Enhanced cytotoxicity of five of these complexes in non-small-cell lung cancer (A549) and colon carcinoma (HT-29) cells at pH 6.0 in comparison with pH 7.4 was confirmed: 50 % growth inhibition concentrations ranged from 42 to 214 µM in A549 cells and from 35 to 87 µM in HT-29 cells at pH 7.4 and decreased at pH 6.0 to 11-50 and 7.3-25 µM, respectively. The effects induced by all five pH-sensitive compounds involve increased 5'-GMP binding, cellular accumulation, and DNA platination as well as stronger effects on DNA secondary structure at pH 6.0 than at pH 7.4. As exemplified by treatment of A549 cells with a 2-amino-4-methyl-1-pentanolato complex, induction of apoptosis is enhanced at pH 6.5. These results confirm the increased reactivity and in vitro activity of these compounds under slightly acidic conditions, encouraging further evaluation of ring-closed aminoalcoholatoplatinum(II) derivatives in solid tumors in vivo.
Assuntos
Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/análogos & derivados , Cisplatino/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Ciclinas/metabolismo , DNA Circular/química , DNA Super-Helicoidal/química , Estabilidade de Medicamentos , Guanosina Monofosfato/química , Células HT29 , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Plasmídeos/química , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Triapine is an α-N-heterocyclic thiosemicarbazone with promising anticancer activity against hematologic malignancies but widely ineffective against solid tumor types in clinical trials. The anticancer activity of thiosemicarbazones can be dramatically increased by terminal dimethylation. KP1089 is a gallium compound containing two terminal dimethylated thiosemicarbazone ligands. To gain insights on the vulnerability of this highly active terminal dimethylated thiosemicarbazone to drug resistance mechanisms, a new cell model with acquired resistance against the lead compound KP1089 was established. Subsequent genomic analyses (arrayCGH and FISH) revealed amplification of the ABCC1 gene on double minute chromosomal DNA in KP1089-resistant cells as well as overexpression of ABCC1 and ABCG2 on the protein level. KP1089 was further confirmed as a substrate of ABCC1 and ABCG2 but not of ABCB1 using a panel of ABC transporter-overexpressing cell models as well as ABC transporter inhibitors. Moreover, glutathione depletion strongly enhanced KP1089 activity, although no glutathione conjugate formation by glutathione-S-transferase was observed. Thus, a co-transport of KP1089 together with glutathione is suggested. Finally, a panel of thiosemicarbazone derivatives was tested on the new KP1089-resistant cell line. Notably, KP1089-resistant cells were not cross-resistant against thiosemicarbazones lacking terminal dimethylation (e.g. Triapine) which are less active than KP1089. This suggests that terminal dimethylation of thiosemicarbazones - linked with distinctly enhanced anticancer activity - leads to altered resistance profiles compared to classical thiosemicarbazones making this compound class of interest for further (pre)clinical evaluation.
Assuntos
Antineoplásicos/química , Resistencia a Medicamentos Antineoplásicos , Compostos Heterocíclicos/química , Tiossemicarbazonas/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Glutationa/metabolismo , Compostos Heterocíclicos/farmacologia , Humanos , Hibridização in Situ Fluorescente , Metilação , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Neoplasias/genética , Tiossemicarbazonas/farmacologiaRESUMO
Intracellular generation of reactive oxygen species (ROS) via thiol-mediated reduction of copper(II) to copper(I) has been assumed as the major mechanism underlying the anticancer activity of copper(II) complexes. The aim of this study was to compare the anticancer potential of copper(II) complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; currently in phase II clinical trials) and its terminally dimethylated derivative with that of 2-formylpyridine thiosemicarbazone and that of 2,2'-bipyridyl-6-carbothioamide. Experiments on generation of oxidative stress and the influence of biologically relevant reductants (glutathione, ascorbic acid) on the anticancer activity of the copper complexes revealed that reductant-dependent redox cycling occurred mainly outside the cells, leading to generation and dismutation of superoxide radicals resulting in cytotoxic amounts of H(2)O(2). However, without extracellular reductants only weak intracellular ROS generation was observed at IC(50) levels, suggesting that cellular thiols are not involved in copper-complex-induced oxidative stress. Taken together, thiol-induced intracellular ROS generation might contribute to the anticancer activity of copper thiosemicarbazone complexes but is not the determining factor.