Cellular accumulation and DNA platination of two new platinum(II) anticancer compounds based on anthracene derivatives as carrier ligands

The anticancer properties of two new fluorescent platinum(II) compounds, cis-[Pt(A9opy)Cl₂] and cis-[Pt(A9pyp)(dmso)Cl₂] are described. These compounds are highly active against several human tumor cell lines, including human ovarian carcinoma sensitive and cisplatin-resistant cell lines (A2780 and A2780R). To study the cellular processing of these new compounds, a series of in vitro studies have been performed, including the investigation of intracellular platinum accumulation and DNA-platination experiments in A2780 and A2780R cells. Compared to cisplatin, both compounds are accumulated highly in both sensitive and resistant cell lines, and more platinum has been found to bind to the nuclear DNA. Interestingly, cis-[Pt(A9opy)Cl₂] shows high accumulation and DNA adduct formation in the resistant cell line A2780R, as compared to the sensitive counterpart A2780 cell line. This suggests that cis-[Pt(A9opy)Cl₂] is able to overcome some of the well-known resistance mechanisms in this cell line, such as decreased cellular uptake and increased DNA repair.     

Growth,morphological and biochemical changes in oxa-spermine derivative-treated MCF-7 human breast cancer cells

The growth inhibitory properties of two oxa-spermine derivatives named compound 1 and compound 2, representatives of a novel type of polyamine derivatives, were studied. Dose-response growth inhibitory curves obtained after 48h drug exposure demonstrated the much higher cytotoxic activity of compound 1 towards MCF-7 human breast cancer cells. Further experiments with compound 1 showed that this oxa-spermine derivative exhibited considerable cytotoxicity with IC(50) values of 3.74 microM and 2.93 microM after 24h and 48h drug exposure respectively. In MCF-7 cells, after 8h drug (10 microM) exposure it caused shrinkage, chromatin condensation and nuclear fragmentation. However, no clear DNA laddering was detected in treated cells. Drug treatment provoked an increase in polyamine oxidase (PAO) activity. This enzyme is able to produce cytotoxic H(2)O(2) and 3-acetamidopropanal, catalyzing the oxidative deamination of N(1)-acetylated derivatives of spermine and spermidine to spermidine and putrescine respectively. Taken together these data demonstrate that the novel oxa-polyamine derivative compound 1 has considerable cytotoxic activity towards MCF-7 cells and indicate that an induction of PAO may be involved in its cytotoxic and apoptotic effects.     

Synthesis,characterization, cytotoxicity,and DNA binding of some new platinum(II) and platinum(IV) complexes with benzimidazole ligands

In this study, two Pt(II) and three Pt(IV) complexes with the structures of [PtL₂Cl₂] (1), [PtL₂I₂] (2), [PtL₂Cl₂(OH)₂] (3), [PtL₂Cl₂(OCOCH₃)₂] (4), and [PtL₂Cl₄] (5) (L = benzimidazole as carrier ligand) were synthesized and evaluated for their in vitro antiproliferative activities against the human MCF-7, HeLa, and HEp-2 cancer cell lines. The influence of compounds 1–5 on the tertiary structure of DNA was determined by their ability to modify the electrophoretic mobility of the form I and II bands of pBR322 plasmid DNA. The inhibition of BamH1 restriction enzyme activity of compounds 1–5 was also determined. In general, it was found that compounds 1–5 were less active than cisplatin and carboplatin against MCF-7 and HeLa cell lines (except for 1, which was found to be more active than carboplatin against the MCF-7 cell line). Compounds 1 and 3 were found to be significantly more active than cisplatin and carboplatin against the HEp-2 cell line.     

Estrogenic activities of extracts of Chinese licorice (Glycyrrhiza uralensis) root in MCF-7 breast cancer cells

Despite the wide use of Chinese licorice root (Glycyrrhiza uralensis) for the treatment of menopausal complaints, little is known on its potential estrogenic properties, and available information relative to its effects on cell proliferation is contradictory. In this study, the estrogenic properties of licorice root were evaluated in vitro by use of several assays. The effects of increasing concentrations of a DMSO extract of licorice root on the growth of MCF-7 breast cancer cells were biphasic. The extract showed an ER-dependent growth-promoting effect at low concentrations and an ER-independent anti-proliferative activity at high concentrations. In further experiments, licorice root was sequentially extracted to yield four fractions: hexane, EtOAc, methanol and H₂O. Only the EtOAc extract had effects on cell proliferation similar to the DMSO extract. The hexane extract had no effect on cell growth. In contrast, the methanol and water extracts showed an ER-independent, growth-promoting effect. Similar to its effects on cell proliferation, the EtOAc extract had a biphasic effect on S phase cell cycle distribution and the level of PCNA protein. This extract-induced transactivation of endogenous ERα in MCF-7 cells, supported by inducing down-regulation of ERα protein and mRNA levels, and up-regulation of ERα target genes pS2 and GREB1. These results suggest that the activity of licorice root and the balance between increased risk for cancer and prevention of estrogen-dependent breast cancer may depend on the amount of dietary intake.     

Increased levels of tyrosinated alpha-, beta(III)-, and beta(IV)-tubulin isotypes in paclitaxel-resistant MCF-7 breast cancer cells

Paclitaxel (PTX), the diterpene alkaloid, is a potent anti-cancer drug and is routinely used for the treatment of breast and ovarian cancers. The cellular targets of PTX are microtubules, which are composed of alpha- and beta-tubulin. Development of PTX resistance in patients has been a major problem associated with cancer chemotherapy. In an effort to get insight into this phenomenon of drug resistance, a PTX-resistant cell line from MCF-7 breast cancer cells has been generated. Western analysis of the cell extracts revealed that the resistant cells contain 2-fold higher amount of tyrosinated alpha-tubulin than those of the wild-type MCF-7 cells. Similar analyses of beta-tubulin with the isotype-specific monoclonal antibodies demonstrated that the PTX-resistant cells contain 2.5-fold higher amounts of beta(III) and 1.5-fold higher amount of beta(IV)-tubulin, while no difference was observed in the level of beta(I) isotype. These results demonstrate for the first time that PTX resistance is associated with an increase in the level of tyrosinated alpha-tubulin.     

Preparation of antitumor platinum(II) complexes of 1,2-diphenylethylenediamine isomers and their interactions with DNA and its purine moieties

A series of Pt(II) complexes containing 1,2-diphenylethylenediamine (stien) isomers were synthesized and tested for their antitumor activity against leukemia L1210. Among the Pt(II) complexes examined water-soluble Pt(II) complexes with sulfate, nitrate and D-glucuronate ions as leaving groups exhibited relatively high antitumor activity. Furthermore, the interactions between calf-thymus DNA and Pt(SO4) (stein) complexes were investigated by means of circular dichroism spectrometry. Dichroism enhancements observed in the interaction between DNA and Pt(SO4) (stien) complexes were analysed to be contributable to two factors: (1) vicinal effects of DNA on the d-d transitions of Pt(II) ions and (2) conformational changes of DNA caused by the coordination of cis-configurational Pt(II) complexes.     

Probing the interaction of thionine with human serum albumin by multispectroscopic studies and its in vitro cytotoxic activity toward MCF-7 breast cancer cells

The studies on protein–dye interactions are important in biological process and it is regarded as vital step in rational drug design. The interaction of thionine (TH) with human serum albumin (HSA) was analyzed using isothermal titration calorimetry (ITC), spectroscopic, and molecular docking technique. The emission spectral titration of HSA with TH revealed the formation of HSA–TH complex via static quenching process. The results obtained from absorption, synchronous emission, circular dichroism, and three-dimensional (3D) emission spectral studies demonstrated that TH induces changes in the microenvironment and secondary structure of HSA. Results from ITC experiments suggested that the binding of TH dye was favored by negative enthalpy and a favorable entropy contribution. Site marker competitive binding experiments revealed that the binding site of TH was located in subdomain IIA (Sudlow site I) of HSA. Molecular docking study further substantiates that TH binds to the hydrophobic cavity of subdomain IIA (Sudlow site I) of HSA. Further, we have studied the cytotoxic activity of TH and TH–HSA complex on breast cancer cell lines (MCF-7) by MTT assay and LDH assay. These studies revealed that TH–HSA complex showed the higher level of cytotoxicity in cancer cells than TH dye-treated MCF-7 cells and the significant adverse effect did not found in the normal HBL-100 cells. Fluorescence microscopy analyses of nuclear fragmentation studies validate the significant reduction of viability of TH–HSA-treated human MCF-7 breast cancer cells through activation of apoptotic-mediated pathways.     

DNA crosslinking induced by 1,2-diaminocyclohexanedichloroplatinum(II) in murine leukemia L1210 cells and comparison with other platinum analogues

1,2-Diaminocyclohexanedichloroplatinum(II) (DCDP) is an analogue of the clinically efficacious cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP). DCDP is presently undergoing clinical trials at least in part because a cis-DDP-resistant murine leukemia L1210 cell line is sensitive to its action. The alkaline elution technique was used to measure DNA-protein and DNA-interstrand crosslinks induced by DCDP in sensitive and resistant L1210 cells. This was compared to the action of cis-DDP and its clinically ineffective isomer trans-DDP. The action of DCDP was similar to that for cis-DDP with maximum crosslinking occurring between 6 and 12 h after a 1 h treatment. Both cis-DDP and DCDP exhibited proportionately higher levels of interstrand crosslinking than trans-DDP. Near complete removal of both classes of DCDP-induced crosslinks was seen by 72 h. While the extent of crosslinking was different for each compound, little difference between the two cell lines was noted with respect to crosslinking by either DCDP or trans-DDP. These cell lines exhibit a 2-fold resistance to both DCDP and trans-DDP and at equitoxic doses of both drugs the resistant cells demonstrated twice the interstrand crosslinks that were seen in the sensitive cells. The extent of crosslinking related directly to the concentration of drug. When treated with equitoxic doses of DCDP, cis-DDP or trans-DDP, the resistant cells consistently exhibited more interstrand crosslinks than sensitive cells, suggesting the existence of a more critical cytotoxic lesion which was not detectable by the alkaline elution technique. These lesions could be either intrastrand crosslinks or monofunctional platination. Resistance must be due to a differential sensitivity to the lesions that form, which may be due to an altered capacity to repair the lesions.     

Fatty acid metabolism in human breast cancer cells (MCF7) transfected with heart-type fatty acid binding protein

The human breast cancer cell line MCF7 does not express heart-type fatty acid binding protein (H-FABP), a marker protein for differentiated mammary gland. MCF7 cells transfected with the bovine H-FABP cDNA expressed the corresponding protein and were characterized by growth inhibition and lower tumorgenicity in nude mice [22]. By enzyme linked immunoassay we now determined the amount of bovine H-FABP in these cells as 638 ± 80 ng/mg protein and used the transfected cells to study the role of H-FABP in fatty acid metabolism. Compared to control cells the uptake of radioactively labelled palmitic acid and oleic acid into MCF7 cells after 30 or 60 min was increased by 67% in H-FABP expressing transfectants, demonstrating a stimulatory role for this FABP-type in fatty acid metabolism. However, preferential targeting of [¹⁴C]oleic acid into neutral or phospholipid classes was not observed by the criterion of high performance thin layer chromatography followed by autoradiography. A reason for the modest increase of fatty acid uptake in H-FABP transfected MCF7 cells may be the basal expression of epidermal-type FABP, which was detected for the first time in these cells. It appears that the small amount of E-FABP expressed in MCF7 cells fulfils the need of the cells for a cytosolic fatty acid carrier under culture conditions and that even high concentrations of another FABP do only slightly increase the uptake due to limitations of fatty acid transport through the plasma membrane or of metabolism.     

Synthesis,structural characterization and cell death-inducing effect of novel palladium(II) and platinum(II) saccharinate complexes with 2-(hydroxymethyl)pyridine and 2-(2-hydroxyethyl)pyridine on cancer cells in vitro

Four palladium(II) and platinum(II) saccharinate (sac) complexes with 2-(hydroxymethyl)pyridine (2-hmpy) and 2-(2-hydroxyethyl)pyridine (2-hepy), namely trans-[Pd(2-hmpy)₂(sac)₂]·H₂O (1), trans-[Pt(2-hmpy)₂(sac)₂]·3H₂O (2), trans-[Pd(2-hepy)₂(sac)₂] (3) and trans-[Pt(2-hepy)₂(sac)₂] (4), have been synthesized and characterized by elemental analysis, UV–vis, IR and NMR. Single crystal X-ray analysis reveals that the metal(II) ions in each complex are coordinated by two sac and two 2-hmpy or 2-hepy ligands with a trans arrangement. Anticancer effects of 1–4 were tested against four different cancer cell lines (A549 and PC3 for lung cancer, C6 for glioblastoma, and Hep3B for liver cancer). Cytotoxicity was first screened by the MTT assay and the results were further confirmed by the ATP assay. The mode of cell death was determined by both histological and biochemical methods. Among the metal complexes, complex 2 resulted in relatively stronger anti-growth effect in a dose-dependent manner (3.13–200 μM), compared to the others, by inducing apoptosis.     

A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcinoma breast cancer cells

In vitro assays involving primary cells are used routinely to evaluate organ-specific toxic effects, for instance, the use of primary hepatocytes to evaluate hepatotoxicity. A major drawback of an in vitro system is the lack of multiple organ interactions as observed in a whole organism. A novel cell culture system, the integrated discrete multiorgan cell culture system (IdMOC), is described here. The IdMOC is based on the "wells within a well" concept, consisting of a cell culture plate with larger, containing wells, within each of which are multiple smaller wells. Cells from multiple organs can be cultured initially in the small wells (one organ per well, each in its specialized medium). On the day of toxicity testing, a volume of drug-containing medium is added to the containing well to flood all inner wells, thereby interconnecting all the small wells. After testing, the overlying medium is removed and each cell type is evaluated for toxicity using appropriate endpoints. We report here the application of IdMOC in the evaluation of the cytotoxicity of tamoxifen, an anticancer agent with known human toxicity, on primary cells from multiple human organs: liver (hepatocytes), kidney (kidney cortical cells), lung (small airway epithelial cells), central nervous system (astrocytes), blood vessels (aortic endothelial cells) as well as the MCF-7 human breast adenocarcinoma cells. IdMOC produced results that can be used for the quantitative evaluation of its anticancer effects (i.e., cytotoxicity towards MCF-7 cells) versus its toxicity toward normal organs (i.e., liver, kidney, lung, CNS, blood vessels).     

DNA repair and replication in human fibroblasts treated with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 μM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4–6 h after treatment with 0.7 μM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 μM) were found to inhibit replicon initiation by up to 50% within 30–60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1·10⁷ Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 μM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 μM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.