Cytotoxic effects of two organotin compounds and their mode of inflicting cell death on four mammalian cancer cells

In this report, we have tested the cytotoxicity of two organotin (OT) compounds by flow cytometry on a panel of immortalized cancer cell lines of human and murine origin. Although the OT compounds exhibited varying levels of cytotoxicity, diphenylmethyltin chloride was more toxic than 1,4-bis (diphenylchlorostannyl)p-xylene on all cell lines tested. The OT compounds were found to be highly cytotoxic to lymphoma cell lines with lower toxicity toward the HeLa cervical cancer cell line. In order to discern the mechanism by which cell death was induced, additional experiments were conducted to monitor characteristic changes consistent with apoptosis and/or necrosis. Cell lines treated with the experimental compounds indicated that there was no consistent mode of cell death induction. However, both compounds induced apoptosis in the pro-B lymphocyte cell line, NFS-70. The work presented here also demonstrates that the two OT compounds possess selective cytotoxicity against distinct transformed cell lines.     

Synthesis of (1,3,4-thiadiazol-2-yl)-acrylamide derivatives as potential antitumor agents against acute leukemia cells

A lead compound with the (1,3,4-thiadiazol-2-yl)-acrylamide scaffold was discovered to have significant cytotoxicity on several tumor cell lines in an in-house cell-based screening. A total of 60 derivative compounds were then synthesized and tested in a CCK-8 cell viability assay. Some of them exhibited improved cytotoxic activities. The most potent compounds had IC₅₀ values of 1–5 μM on two acute leukemia tumor cell lines, i.e. RS4;11 and HL-60. Flow cytometry analysis of several active compounds and detection of caspase activation indicated that they induced caspase-dependent apoptosis. It was also encouraging to observe that these compounds did not have obvious cytotoxicity on normal cells, i.e. IC₅₀ > 50 μM on HEK-293T cells. Although the molecular targets of this class of compound are yet to be revealed, our current results suggest that this class of compound represents a new possibility for developing drug candidates against acute leukemia.     

Analysis of the cytotoxic effects of ruthenium–ketoconazole and ruthenium–clotrimazole complexes on cancer cells

Ruthenium-based compounds have intriguing anti-cancer properties, and some of these novel compounds are currently in clinical trials. To continue the development of new metal-based drug combinations, we coupled ruthenium (Ru) with the azole compounds ketoconazole (KTZ) and clotrimazole (CTZ), which are well-known antifungal agents that also display anticancer properties. We report the activity of a series of 12 Ru–KTZ and Ru–CTZ compounds against three prostate tumor cell lines with different androgen sensitivity, as well as cervical cancer and lymphoblastic lymphoma cell lines. In addition, human cell lines were used to evaluate the toxicity against non-transformed cells and to establish selectivity indexes. Our results indicate that the combination of ruthenium and KTZ/CTZ in a single molecule results in complexes that are more cytotoxic than the individual components alone, displaying in some cases low micromolar CC₅₀ values and high selectivity indexes. Additionally, all compounds are more cytotoxic against prostate cell lines with lower cytotoxicity against non-transformed epidermal cell lines. Some of the compounds were found to primarily induce cell death via apoptosis yet weakly interact with DNA. Our studies also demonstrate that the cytotoxicity induced by our Ru-based compounds is not directly related to their ability to interact with DNA.     

Synthesis and biological evaluation of cytotoxic activity of novel anthracene l-rhamnopyranosides

A series of anthracene l-rhamnopyranosides were designed and synthesized in a practical way and their cytotoxic activity was examined in vitro. Most compounds exhibited both potent cytotoxicity against several tumor cell lines and high DNA binding capacity. The preliminary results showed that subtle modifications of rhamnosyl moiety in anthracene rhamnosides with acetyl group had a selective toxicity for different tumor cells and the displacement of C-10 carbonyl group in emodin by acetylmethylene group was helpful to improve the inhibitory activity. Lipophilicity of the anthracene glycosides was not a crucial factor for cytotoxicity and most molecules with good cytotoxicity could inhibit the catalytic activity of Top2α.     

The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity

Since the major approach in searching for potential anticancer agents over the last 50 years has been based on selective cytotoxic effects on mammalian cancer cell lines, cell-based methods for cytotoxicity are described and compared. The sulphorhodamine B (SRB) assay is described in detail as the preferred method and also a novel approach has been developed which is based on the hypothesis that, in some circumstances, the naturally occurring compounds act as prodrugs rather than active compounds in their own right. Consequently, extracts or compounds are pre-incubated with systems modelling metabolic processes in the body before being tested. The methods have been validated using known compounds and Iris tectorum extracts have been shown to be more cytotoxic after treatment with beta-glucosidase. In addition bioassays based on mammalian cells involving antioxidant and upregulation of some cellular self-defence mechanisms are discussed which are related to prevention as well as treatment of cancer. Extracts of Alpinia officinarum induced glutathione-S-transferase (GST) activity in cultured hepatocytes and this was traced to the phenylpropanoids present, especially 1'-acetoxychavicol acetate.     

The role of heme and the mitochondrion in the chemical and molecular mechanisms of mammalian cell death induced by the artemisinin antimalarials

The artemisinin compounds are the frontline drugs for the treatment of drug-resistant malaria. They are selectively cytotoxic to mammalian cancer cell lines and have been implicated as neurotoxic and embryotoxic in animal studies. The endoperoxide functional group is both the pharmacophore and toxicophore, but the proposed chemical mechanisms and targets of cytotoxicity remain unclear. In this study we have used cell models and quantitative drug metabolite analysis to define the role of the mitochondrion and cellular heme in the chemical and molecular mechanisms of cell death induced by artemisinin compounds. HeLa ρ(0) cells, which are devoid of a functioning electron transport chain, were used to demonstrate that actively respiring mitochondria play an essential role in endoperoxide-induced cytotoxicity (artesunate IC(50) values, 48 h: HeLa cells, 6 ± 3 μM; and HeLa ρ(0) cells, 34 ± 5 μM) via the generation of reactive oxygen species and the induction of mitochondrial dysfunction and apoptosis but do not have any role in the reductive activation of the endoperoxide to cytotoxic carbon-centered radicals. However, using chemical modulators of heme synthesis (succinylacetone and protoporphyrin IX) and cellular iron content (holotransferrin), we have demonstrated definitively that free or protein-bound heme is responsible for intracellular activation of the endoperoxide group and that this is the chemical basis of cytotoxicity (IC(50) value and biomarker of bioactivation levels, respectively: 10β-(p-fluorophenoxy)dihydroartemisinin alone, 0.36 ± 0.20 μM and 11 ± 5%; and with succinylacetone, >100 μM and 2 ± 5%).     

Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro-in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro-in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.     

Progression of conventional hepatic cell culture models to bioengineered HepG2 cells for evaluation of herbal bioactivities

Cancer cell lines of human tissue origin have been extensively used to investigate antiproliferative activity and toxicity of herbal extracts, isolated compounds, and anticancer drugs. These cell lines are genetically and/or epigenetically well characterized to determine the altered expression of proteins within given cellular pathways and critical genes in cancer. Human derived hepatoma (HepG2) cell line has been extensively exploited to examine cytoprotective, antioxidative, hepatoprotective, anti-hepatoma, hypocholesterolemic, anti-steatosis, bioenergetic homeostatic and anti-insulin resistant properties. Moreover, mechanism of action of various botanicals and bioactive constituents has been reported using these cells. HepG2 cells have significant differences as compared to primary hepatocytes with respect to expression of cytochrome P450 enzymes and xenobiotic receptors in conventional in vitro culture conditions. Therefore, strategies have been employed to overcome limitations of two dimensional (2D) in vitro HepG2 cell culture in order to recognize functional biomarkers more accurately and to boost its predictive value in clinical research. In consequence, three dimensional (3D) human hepatoma cell culture models are being developed as a resource to achieve these goals of simulating the in vivo tumor microenvironment. It is assumed that bioengineered 3D hepatoma cell culture models can provide significant assistance in scrutinizing the molecular response of herbal natural products to recognize novel prognostic targets and crucial biomarkers in treatment strategies for cancer patients in near future.     

Novel inhibitors of human histone deacetylases: Design,synthesis and bioactivity of 3-alkenoylcoumarines

Histone deacetylases (HDACs) are well-established, promising targets for anticancer therapy due to their critical role in cancer development. Accordingly, an increasing number of HDAC inhibitors displaying cytotoxic effects against cancer cells have been reported. Among them, a large panel of chemical structures was described including coumarin-containing molecules. In this study, we described synthesis and biological activity of new coumarin-based derivatives as HDAC inhibitors. Among eight derivatives, three compounds showed HDAC inhibitory activities and antitumor activities against leukemia cell lines without affecting the viability of peripheral blood mononuclear cells from healthy donors.     

Biological evaluation of novel estrogen-platinum(II) hybrid molecules on uterine and ovarian cancers-molecular modeling studies

We have recently reported the synthesis of a series of original 17beta-estradiol-linked platinum(II) hybrid molecules. The biological activity of these compounds was evaluated in vitro on estrogen dependent and independent (ER(+) and ER(-)) human uterine and ovarian cancers. The hybrid molecules present higher affinity than that of 17beta-estradiol for the estrogen receptor alpha (ERalpha). The cytotoxicity and the affinity of the hybrid molecules are explained using molecular modeling analysis. This study further confirms that the derivatives made of a 2-(2'-aminoethyl)pyridine ligand displayed superior activity against the cell lines particularly when the connecting arm is 8-10 carbon atoms long. Molecular modeling shows that a long side chain can facilitate the access of the platinum(II) moiety to DNA. The novel compounds also prove to be moderately cytotoxic against platinum resistant endometrial and ovarian cancer cell lines.     

Expression of adhesion molecules and ligands for activating and costimulatory receptors involved in cell-mediated cytotoxicity in a large panel of human melanoma cell lines

Knowledge of the interactions between MHC-unrestricted cytotoxic effector cells and solid tumour cells is essential for introducing more effective NK cell-based immunotherapy protocols into clinical practise. Here, to begin to obtain an overview of the possible universe of molecules that could be involved in the interactions between immune effector cells and melanoma, we analyse the surface expression of adhesion and costimulatory molecules and of ligands for NK-activating receptors on a large panel of cell lines from the “European Searchable Tumour Cell Line and Data Bank” (ESTDAB, http://www.ebi.ac.uk/ipd/estdab/) and discuss their potential role in the immune response against this tumour. We show that most melanoma cell lines express not only adhesion molecules that are likely to favour their interaction with cells of the immune system, but also their interaction with endothelial cells potentially increasing their invasiveness and metastatic capacity. A high percentage of melanoma cell lines also express ligands for the NK-activating receptor NKG2D; whereas, the majority express MICA/B molecules, ULBP expression, however, was rarely found. In addition to these molecules, we also found that CD155 (poliovirus receptor, PVR) is expressed by the majority of melanoma cell lines, whereas CD112 (Nectin-2) expression was rare. These molecules are DNAM-1 ligands, a costimulatory molecule involved in NK cell-mediated cytotoxicity and cytokine production that also mediates costimulatory signals for triggering naïve T cell differentiation. The phenotypical characterisation of adhesion molecules and ligands for receptors involved in cell cytotoxicity on a large series of melanoma cell lines will contribute to the identification of markers useful for the development of new immunotherapy strategies.     

Target fishing and docking studies of the novel derivatives of aryl-aminopyridines with potential anticancer activity

A set of 16 previously synthesized aryl-aminopyridine and aryl-aminoquinoline derivatives have been evaluated for cytotoxic activity against three cancer cell lines (human cervical cancer-HeLa; human chronic myeloid leukemia-K562; human melanoma-Fem-x) and two types of normal peripheral blood mononuclear cells, with and without phytohemaglutinin (PBMC-PHA; PBMC+PHA). Twelve of the studied compounds showed moderate cytotoxicity, with selectivity against K562 but not the remaining two cancer cell lines. Four compounds were not active in cytotoxicity assays, presumably due to high predicted lipophilicity and low solubility. To rationalize the observed cytotoxic effects, structure-based virtual screening was carried out against a pool of potential targets constructed using the inverse docking program Tarfisdock and bibliographical references. The putative targets were identified on the basis of the best correlation between docking scores and in vitro cytotoxicity. It is proposed that the mechanism of action of the studied aminopyridines involves the disruption of signaling pathways and cancer cell cycle through the inhibition of cyclin-dependent kinases and several tyrosine kinases, namely Bcr-Abl kinase and KIT receptor kinase. The obtained results can guide further structural modifications of the studied compounds aimed at developing selective agents targeting proteins involved in cancer cell survival and proliferation.     

A survey of antiprion compounds reveals the prevalence of non-PrP molecular targets

Prion diseases are fatal neurodegenerative diseases caused by the accumulation of the misfolded isoform (PrP(Sc)) of the prion protein (PrP(C)). Cell-based screens have identified several compounds that induce a reduction in PrP(Sc) levels in infected cultured cells. However, the molecular targets of most antiprion compounds remain unknown. We undertook a large-scale, unbiased, cell-based screen for antiprion compounds and then investigated whether a representative subset of the active molecules had measurable affinity for PrP, increased the susceptibility of PrP(Sc) to proteolysis, or altered the cellular localization or expression level of PrP(C). None of the antiprion compounds showed in vitro affinity for PrP or had the ability to disaggregate PrP(Sc) in infected brain homogenates. These observations suggest that most antiprion compounds identified in cell-based screens deploy their activity via non-PrP targets in the cell. Our findings indicate that in comparison to PrP conformers themselves, proteins that play auxiliary roles in prion propagation may be more effective targets for future drug discovery efforts.     

Effective treatment of advanced solid tumors by the combination of arsenic trioxide and L-buthionine-sulfoximine

Clinical application of anticancer agents has been often hampered by toxicity against normal cells, so the achievement of their cancer-specific action is still one of the major challenges to be addressed. Previously, we reported that arsenic trioxide (As2O3) could be a promising new drug against not only leukemia but also solid tumors. The cytotoxicity of As2O3 occurred through the generation of reactive oxygen species (ROS), thus inhibiting radical scavenging systems would enhance the therapeutic efficacy of As2O3 provided that normal cells were relatively resistant to such a measure. Here, we report that the combination therapy of As2O3 with L-buthionine-sulfoximine (BSO), which inhibits a critical step in glutathione synthesis, effectively enhanced in vitro growth inhibition effect of As2O3 on all 11 investigated cell lines arising from prostate, breast, lung, colon, cervix, bladder, and kidney cancers, compared with As2O3 treatment alone. Furthermore, this combination enhanced cytotoxicity to cell lines from prostate cancer with less toxicity to those from normal prostate. In vitro cytotoxic assay using ROS-related compounds demonstrated that hydrogen peroxide (H2O2) is a major cytotoxic mediator among ROS molecules. Biochemical analysis showed that combined use of As2O3 and BSO blocked H2O2-scavenging systems including glutathione, catalase, and glutathione peroxidase, and that the degree of this blockade was well correlated with intracellular ROS levels and sensitivity to this treatment. Finally, the effectiveness of the combination therapy of As2O3 with BSO was demonstrated with an orthotopic model of prostate cancer metastasis. We propose that the combination therapy of As2O3 with BSO is a valid means of blockade of H2O2-scavenging system, and that the combination of a ROS-generating agent with an inhibitor of major scavenging systems is effective in terms of both efficacy and selectivity. Furthermore, because the effective doses of both compounds are within clinically achievable range, this report will lead to immediate benefit for the development of a new cancer therapy.     

Characterization of a Novel Anti-Cancer Compound for Astrocytomas

The standard chemotherapy for brain tumors is temozolomide (TMZ), however, as many as 50% of brain tumors are reportedly TMZ resistant leaving patients without a chemotherapeutic option. We performed serial screening of TMZ resistant astrocytoma cell lines, and identified compounds that are cytotoxic to these cells. The most cytotoxic compound was an analog of thiobarbituric acid that we refer to as CC-I. There is a dose-dependent cytotoxic effect of CC-I in TMZ resistant astrocytoma cells. Cell death appears to occur via apoptosis. Following CC-I exposure, there was an increase in astrocytoma cells in the S and G2/M phases. In in vivo athymic (nu/nu) nude mice subcutaneous and intracranial tumor models, CC-I completely inhibited tumor growth without liver or kidney toxicity. Molecular modeling and enzyme activity assays indicate that CC-I selectively inhibits topoisomerase IIα similar to other drugs in its class, but its cytotoxic effects on astrocytoma cells are stronger than these compounds. The cytotoxic effect of CC-I is stronger in cells expressing unmethylated O⁶-methylguanine methyltransferase (MGMT) but is still toxic to cells with methylated MGMT. CC-I can also enhance the toxic effect of TMZ on astrocytoma when the two compounds are combined. In conclusion, we have identified a compound that is effective against astrocytomas including TMZ resistant astrocytomas in both cell culture and in vivo brain tumor models. The enhanced cytotoxicity of CC-I and the safety profile of this family of drugs could provide an interesting tool for broader evaluation against brain tumors.     

Synthesis,cytotoxic activity,DNA topoisomerase-II inhibition,molecular modeling and structure–activity relationship of 9-anilinothiazolo[5,4-b]quinoline derivatives

Some novel 9-anilinothiazolo[5,4-b]quinoline derivatives were synthesized and their cytotoxic activities were examined. The inhibition of some of the most active compounds over human topoisomerase II (Topo II) activity was assessed with the kDNA decatenation assay. The novel compounds differ in the substituents attached to the anilino ring, a dialkylamino alkylamino group, a saturated heterocyclic moiety, a methylthio group at position 2 and a fluorine atom present or absent at 7-position. According to the data, compounds with a diethylaminopropylamino group and a chlorine atom at 4′-position of the anilino ring were the most cytotoxic. The molecular models of all compounds indicated a correlation between hydrophobicity and cytotoxic activity although the direction and magnitude of the dipole moment also had a significant influence on its cytotoxicity. The 2-dialkylaminoalkylamino substituent is flexible and is known to facilitate the crossing of cell membranes; thus, this last barrier may be a limiting step in the mechanisms mediating the cytotoxicity. On the other hand, the activity of 2-methylthio derivatives seems to rely more on the electronic effects brought about by the substitution of the aniline ring. The synthesis, cytotoxicity against cancer cell lines, in vitro inhibition of human topoisomerase II, molecular modeling and the preliminary analysis of structure–activity relationships are presented.     

Sequential cytotoxicity: a theory examined using a series of 3,5-bis(benzylidene)-1-diethylphosphono-4-oxopiperidines and related phosphonic acids

The concept of sequential cytotoxicity, which states that successive chemical attacks on cellular constituents can be more deleterious to neoplasms than normal cells, was evaluated using a series of 3,5-bis(benzylidene)-1-diethylphosphono-4-oxopiperidines 1 and related phosphonic acids 2, which were screened against a panel of malignant and normal cell lines. The compounds proved to be not only potent cytotoxins (71% of the CC(50) figures are submicromolar) but to display greater cytotoxicity to the neoplastic cells. QSAR revealed that both cytotoxic potencies and selective toxicity were increased by a rise in the electron-withdrawing properties and a decrease in the hydrophobicity of the aryl substituents. Utilisation of the PL10 concept and evaluation of druglike properties revealed 1c as the lead tumour-specific cytotoxin. This molecule activated caspase-3 in HL-60 cells but not in the HSC-2 cell line. While 1c caused internucleosomal DNA fragmentation in HL-60 cells, it did not elicit this effect in either HSC-2 and HSC-4 cells. Clearly 1c exerts its cytotoxic potencies by different mechanisms and such pleiotropy is likely the principal reason for the remarkable display of preferential toxicity towards malignant cells of the compounds in series 1 and 2.