Synthesis, characterization and biological activity of lanthanum(III) complexes containing 2-methylene-1,10-phenanthroline units bridged by aliphatic diamines

Two novel lanthanum(III) complexes containing 2-methylene-1,10-phenanthroline units bridged by aliphatic diamines were synthesized and characterized by elemental analysis, IR, NMR, thermal analysis and conductance measurements. They have been assayed for anticancer activity in vitro against HL-60 (human leukocytoma) cells, PC-3MIE8 (human prostate carcinoma) cells, BGC-823 (human stomach carcinoma) cells, MDA-MB-435 (human galactophore carcinoma) cells, Bel-7402 (human liver carcinoma) cells, and Hela (human cervix carcinoma) cells. The results show that the two complexes exhibit good cytotoxic activities against different cell lines in general, especially more effective than cisplatin against Bel-7402, BGC-823 and MDA-MB-435 cell lines. DNA-binding studies indicate that, besides the intercalation, the complexes bind to DNA by the other interaction(s), which might be responsible for the production of more compact DNA, coinciding with more A-like feature of DNA as suggested by CD spectra.     

Grading of intrinsic and acquired cisplatin-resistant human melanoma cell lines: an infrared ATR study

Attenuated total reflection (ATR) spectroscopy is used as an in vitro optical approach for the diagnosis and characterization of cell and tissue pathology. In comparison with the more conventional FTIR microspectroscopy that relies on transmission of IR radiation, ATR spectroscopy uses the evanescent wave technique, which is a step forward toward in vivo research. The aim of the present investigation was to examine the potential of ATR spectroscopy to differentiate between drug-resistant and drug-sensitive melanoma cell lines. We studied two human melanoma parental cell lines, GA and BG, and their cisplatin-resistant counterparts, GAC and BGC, respectively, which were derived by survival selection with this anticancer drug. Cisplatin cytotoxicity was measured on the four cell lines, and their relative resistance to cisplatin was established: BGC > BG > GAC > GA. Different resistance mechanisms were noticed between the two parental groups in accordance with their spectrum. ATR spectra-based cluster analysis of the selective biomarkers, such as phosphate and RNA/DNA, were found useful in differentiating sensitive from resistant cells. Normalized and absolute values of the differences between spectra were employed to compare between the two parental groups. It was possible to predict the relative cisplatin resistance between the cell lines using the discriminant classifying function. The success rates in predicting cisplatin resistance in these cells was 88 and 81% for GA versus GAC and BG versus BGC, respectively. These results support the further development of the ATR technique as a simple, in vitro, reagent-free method to identify drug resistance in cancer cells.     

In vitro cytotoxicity study on platinum (II) complexes with epoxysuccinates as leaving groups

A series of novel cisplatin-type platinum complexes were designed, characteristic of epoxysuccinates as leaving groups. The pertinent compounds were prepared and characterized by IR, (1)H NMR, and ESI-MS spectra with elementary analyses. The in vitro cytotoxic activities of compounds toward SPC-A1 human lung adenocarcinoma cell line and BGC823 human stomach adenocarcinoma cell line were determined. Biological tests have confirmed that complexes containing 4R,5R-DMID [abbreviation of (4R,5R)-4,5-bis (aminomethyl)-2-isopropyl-1,3-dioxolane] as carrier ligands have greater cytotoxicity toward tumor cells than the corresponding compounds with other carrier ligands. Most platinum complexes with trans-epoxysuccinates usually have higher cytotoxicity than those with cis-epoxysuccinates. Complex 4a shows the most effective among those tested platinum complexes in both cell lines, and its cytotoxicity approached that of cisplatin.     

Insulin receptor overexpression in 184B5 human mammary epithelial cells induces a ligand-dependent transformed phenotype

To determine the role of the insulin receptor overexpression in breast epithelial cell transformation, the 184B5 human breast epithelial cell line was transfected with human insulin receptor cDNA. In two cell lines transfected with and overexpressing human insulin receptors (IR) (223.8 and 184.5 ng IR/10⁶ cells), but not in untransfected cells, insulin binding and tyrosine kinase activity were elevated, and insulin induced a dose-dependent increase in colony formation in soft agar.     

UV mutation spectra in cell lines from patients with Cockayne's syndrome and ataxia telangiectasia, using the shuttle vector pZ189.

We have used the SV40-based shuttle vector pZ189 to determine ultraviolet mutation spectra in SV40-transformed cell lines from two patients with Cockayne's syndrome (CS) and ataxia telangiectasia (AT). The shuttle vector was UV-irradiated, transfected into the cells and recovered two days later, after many rounds of replication had occurred. Plasmid DNA was used to transform indicator bacteria in which plasmids containing a mutation in the supF gene resulted in white colonies. Mutant plasmids were analysed both by agarose gels and by DNA sequencing. In contrast to published spectra for xeroderma pigmentosum cells, the types of mutation induced by UV mutation in the CS and AT cell lines were similar to each other and to published spectra for normal cell lines. There were however, some differences in the sequence distribution of the mutations.     

Novel cisplatin-type platinum complexes and their cytotoxic activity

A series of novel cisplatin-type platinum complexes, formulated as [PtA2(OCOCH2OR)2] (A2 = two monoamines or one diamine, R is an alkyl group), were designed, characteristic of alkoxyacetate as carboxylato ligands. The pertinent compounds were prepared and characterized by elementary analyses, IR, 1H NMR, and ESI-MS spectra. The cytotoxic activities of compound 1a in vitro toward HL-60 human leukemia and BEL-7402 human hepatocellular carcinoma cell lines were pioneeringly studied. Then, compounds 1b-3d were evaluated for their in vitro cytotoxicity against Ramos human lymphoma, 3AO human ovarian carcinoma, and A549 human non-small cell lung cancer cell lines. Most of them showed better cytotoxic activity than carboplatin against above selected cell lines.     

Insulin/IGF-I hybrid receptors play a major role in IGF-I signaling in thyroid cancer.

The insulin-like growth factor-I (IGF-I) plays an important role in determining the biological behavior of a variety of malignancies. We measured IGF-I, its receptor and related receptors in thyroid cancer. IGF-I was present both in normal thyroid tissue and in thyroid cancer tissue and it was produced by stromal cells but not by thyrocytes. Values were significantly higher in malignant than in normal tissue. IGF-I receptors (IGF-I-Rs) and the homologous insulin receptors (IRs) were found overexpressed in both thyroid cancer cell lines (n = 4) and specimens (n = 17) as compared to normal values. In addition, high levels of hybrid IGF-I/insulin receptors (IR/IGF-I-Rs) were present in both thyroid cancer specimens and cell lines. IR/IGF-I-R hybrids were the most represented type of receptor in 14/17 specimens and exceeded the IGF-I-R content in all cases. Hybrid content correlated with the IR and IGF-I-R content, suggesting that in thyroid tissue hybrid formation occurs by random assembly of IR and IGF-I-R half receptors. Hybrid receptor autophosphorylation was stimulated by IGF-I with high affinity. In cells with a high IR/IGF-I-Rs content, blocking antibodies specific to these receptors substantially inhibited IGF-I induced cell growth. These data indicate that the IGF-I system is overactivated in thyroid cancer and that IR/IGF-I-R hybrid receptors play an important role in IGF-I mitogenic signaling in these tumors.     

New Glycerolipids from the Traditional Chinese Medicine of Syngnathus acus (Hai-Long)

Two new glycerolipids, syngaculipids A and B ( 1 and 2 ), one first naturally occurring metabolite ( 8 ), together with five known compounds ( 3 – 7 ) were isolated from the AcOEt fraction of Syngnathus acus L. (Hai-Long). Their structures were elucidated by comprehensive spectral analyses involving UV, IR, MS, 1D and 2D NMR spectra and ECD calculations. All the isolated compounds were evaluated for their cytotoxicity against A549 and HCT-116 cell lines. Compound 8 exhibited moderate cytotoxicity with IC₅₀ values of 34.5 and 38.9 μM on the A549 and HCT-116 cell lines, respectively.     

Infrared microspectroscopy of individual human cervical cancer (HeLa) cells

We report infrared (IR) spectra observed for individual, cultured human cervical cancer (HeLa) cells. Spectra were collected microscopically, in reflection/absorption modes, from cells deposited and dried on microscope slides or from cells grown directly on slides. Within the spectra of the dried cells, significant spectral heterogeneity exists that was previously attributed to different stages of the cell cycle [Boydston-White. S., et al., Biospectroscopy 1999, 5, 219-227; Holman, H. Y., et al., Biopolymers Biospectrosc 2000, 57, 329-335; Tobin, M. J., et al., in First BASIE Workshop, Karlsruhe, Germany, 2003]. The results reported here confirm earlier findings and present the possibility of determining the abundance of cells within each stage of the cycle from the IR spectra. In an accompanying paper, we show that the spectra of cells in suspension exhibit spectral intensity distributions that are different from that of the dried cells. This result has far-reaching implications for the use of infrared microspectroscopy to screen dried cell preparations for the presence of abnormal cells.     

Ataxia telangiectasia-mutated dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts

The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6, and 24 h after treatment with 1.5-Gy IR or sham treatment and were compared with those previously recognized in normal human fibroblasts. Under basal conditions, 160 genes or expressed sequence tags were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. On DNA damage, 1,091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1,811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth, and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison with normal cells. The reduced change in DNA damage response genes and the attenuated repression of cell cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells.     

Glucose-regulated glucagon secretion requires insulin receptor expression in pancreatic alpha-cells

The insulin receptor (IR) and its signaling appear to be essential for insulin secretion from pancreatic beta-cells. However, much less is known about the role of the IR in alpha-cells. To assess the role of the IR in glucagon and insulin secretion, we engineered adeno-viruses for high efficiency small interference RNA (siRNA)-IR expression in isolated mouse pancreatic islets and lentiviruses for siRNA-IR expression in pancreatic alpha- and beta-cell lines (alpha-TC6 and MIN6) with specific, long term stable IR knockdown. Western blot analysis showed that these strategies resulted in 60-80% reduction of IR protein in islets and alpha- and beta-cell lines. Cell growth was reduced by 35-50% in alpha-TC and MIN6 cells stably expressing siRNA-IR, respectively. Importantly, glucagon secretion, in response to glucose (25 to 2.8 mm), was completely abolished in islets expressing siRNA-IR, whereas secretion increased 1.7-fold in islets expressing control siRNA. In contrast, there was no difference in glucose-stimulated insulin secretion when comparing siRNA-IR and siRNA control, with both groups showing a 1.7-fold increase. Islet glucagon and insulin content were also unaffected by IR knockdown. To further explore the role of the IR, siRNA-IR was stably expressed in pancreatic cell lines, which dramatically suppressed glucose-regulated glucagon secretion in alpha-TC6 cells (3.4-fold) but did not affect GSIS in MIN6 cells. Defects in siRNA-IR-expressing alpha-cells were associated with an alteration in the activity of Akt and p70S6K where insulin-induced phosphorylation of protein kinase B/AKt was greatly reduced while p70S6K activation was enhanced, suggesting that the related pathways play important roles in alpha cell function. This study provides direct evidence that appropriate expression of the IR in alpha-cells is required for glucose-dependent glucagon secretion.     

Fluorescence spectroscopy for detection of malignancy: H-ras overexpressing fibroblasts as a model.

Autofluorescence from intracellular chromophores upon illumination of cells by monochromatic light has been studied towards the development of novel noninvasive and sensitive technology for the early detection of cancer. To investigate the relationship between biochemical and morphological changes underlying malignant disease and resulting fluorescence spectra, an in vitro model system of a paired normal and malignant murine fibroblasts cell lines, differing in cancer-associated H-ras expression was employed. A comparison of fluorescence excitation and emission spectra of proliferative cells revealed that fluorescence intensity of malignant cells was significantly less than that of normal cells upon excitation at 290 nm. Fluorescence of both cell lines decreased with decreasing cell concentration, but at each concentration, normal cells had higher fluorescence intensity than malignant cells. Similar differences between the cell lines were observed when brought to quiescence or at stationary phase. Results suggested that the chromophore contributing most significantly to these spectra is tryptophan and its moieties in proteins. This model system demonstrates the specific contribution of H-ras to subcellular chromophores, resulting in a significant difference in their autofluorescence intensity, and implies the potential use of the technique for cancer detection. This model system is potent for analysis of the contribution of other oncogenes and their combinations towards spectral detection of cancer.     

Radiosensitization by Chir-124, a selective Chk1 inhibitor: Effects of p53 and cell cycle checkpoints

Checkpoint kinase-1 (CHK1) is a key regulator of the DNA damage-elicited G2-M checkpoints. The aim of the present study was to investigate the effects of a selective CHK1 inhibitor, Chir124, on cell survival and cell cycle progression following ionizing radiation (IR). Treatment with Chir-124 resulted in reduced clonogenic survival and abrogated the IR- induced G2-M arrest in a panel of isogenic HCT116 cell lines after IR. This radiosensitizing effect was relatively similar between p53-/- and p53-sufficient wild type (WT) HCT116 cells. However, the number of mitotic cells (as measured by assessing the phosphorylation of mitotic proteins) increased dramatically in p53-/- HCT116 cells after concomitant Chir-124 exposure, compared to IR alone, while no such effect was observed in p53-sufficient WT HCT116 cells. In p53-/- cells, Chir-124 treatment induced a marked accumulation of polyploid cells that were characterized by micronucleation or multinucleation. p21-/- HCT116 cells displayed a similar pattern of response as p53-/- cells. Chir-124 was able to radiosensitize HCT116 cells that lack checkpoint kinase-2 (CHK2) or that were deficient for the spindle checkpoint protein Mad2. Finally, Chir-124 could radiosensitize tetraploid cell lines, which were relatively resistant against DNA damaging agents. Altogether these results suggest that Chir-124-mediated radiosensitization is profoundly influenced by the p53 and cell cycle checkpoint system.     

Synthesis, characterization and antitumor activity of copper(II) complex with nicotinamido-4-bis(2-chloroethyl)aminobenzaldimine.

Nicotinamido-4-bis(2-chloroethyl)aminobenzaldimine (NBAB) was synthesized and characterized by elemental analysis, IR and 1H NMR spectra. The complex of Cu(NBAB)2(NO3)2 was prepared in ethanol and characterized by elemental analysis, conductivity, cyclic voltammetry, IR, UV-Vis, fluorescence, CD and EPR spectra. The characteristic data suggest that the complex has an elongated octahedral structure, and NBAB behaves as bidentate in the keto form. The antitumor activities of NBAB and the complex against L1210 murine leukemia and K562 were investigated with both the MTT method and the colony formation test. The results in vitro indicate that antitumor activities of NBAB are superior to 2,2'-chlorodiethylamine hydrochloride (nitrogen mustard) for L1210, and inferior to nitrogen mustard for K562, but the antitumor activities of the complex for both cell lines are superior to nitrogen mustard.     

CHK1 Affecting Cell Radiosensitivity is Independent of Non-Homologous End Joining

CHK1 is one of the most important checkpoint proteins in mammalian cells for responding toDNA damage. Cells defective in CHK1 are sensitive to ionizing radiation (IR). The mechanismby which CHK1 protects cells from IR-induced killing remains unclear. DNA double strandbreaks (DSBs) induced by IR are critical lesions for cell survival. Two major complementaryDNA DSBs repair pathways exist in mammalian cells, homologous recombination repair (HRR)and non-homologous end joining (NHEJ). By using CHK1 kinase dead human cell linesestablished in our laboratory, we show here that although these human cell lines have differentCHK1 activities with different sensitivities to IR-induced killing and G2 accumulation, all thesecell lines show similar inductions and rejoining rates of DNA DSBs. These results indicate thatthe different radiosensitivities and G2 checkpoint responses in these cell lines are independent ofNHEJ, suggesting that CHK1-regulated checkpoint facilitates HRR and therefore protects cellsfrom IR-induced killing.     

5-Azacytidine Enhances the Radiosensitivity of CNE2 and SUNE1 Cells In Vitro and In Vivo Possibly by Altering DNA Methylation

The radioresistance of tumor cells remains a major cause of treatment failure in nasopharyngeal carcinoma (NPC). Recently, several reports have highlighted the importance of epigenetic changes in radiation-induced responses. Here, we investigated whether the demethylating agent 5-azacytidine (5-azaC) enhances the radiosensitivity of NPC cells. The NPC cell lines CNE2 and SUNE1 were treated with 1 μmol/L 5-azaC for 24 h before irradiation (IR); clonogenic survival was then assessed. Tumor growth was investigated in a mouse xenograft model in vivo. The apoptosis, cell cycle progression and DNA damage repair were examined using flow cytometry, immunofluorescent staining and western blotting. Promoter methylation and the expression of four genes epigenetically silenced during the development of NPC were evaluated by pyrosequencing and real-time PCR. We found that pretreatment with 5-azaC significantly decreased clonogenic survival after IR compared to IR alone; the sensitivity-enhancement ratio of 5-azaC was 1.4 and 1.2 for CNE2 and SUNE1 cells, respectively. The combined administration of 5-azaC and IR significantly inhibited tumor growth in the mouse xenograft model, and enhanced radiation-induced apoptosis in vitro compared to 5-azaC alone or IR alone. 5-AzaC also decreased promoter methylation and upregulated the expression of genes which are epigenetically silenced both in vitro and in vivo in NPC. Thus, 5-azaC enhance the radiosensitivity of both the CNE2 and SUNE1 cell lines, possibly by altering DNA methylation levels and increasing the ability of irradiated cells to undergo apoptosis. The use of 5-azaC combined with IR maybe represent an attractive strategy for the treatment of NPC.     

Heterodimerization of glycosylated insulin-like growth factor-1 receptors and insulin receptors in cancer cells sensitive to anti-IGF1R antibody

Background

Identification of predictive biomarkers is essential for the successful development of targeted therapy. Insulin-like growth factor 1 receptor (IGF1R) has been examined as a potential therapeutic target for various cancers. However, recent clinical trials showed that anti-IGF1R antibody and chemotherapy are not effective for treating lung cancer.

Methodology/Principal Findings

In order to define biomarkers for predicting successful IGF1R targeted therapy, we evaluated the anti-proliferation effect of figitumumab (CP-751,871), a humanized anti-IGF1R antibody, against nine gastric and eight hepatocellular cancer cell lines. Out of 17 cancer cell lines, figitumumab effectively inhibited the growth of three cell lines (SNU719, HepG2, and SNU368), decreased p-AKT and p-STAT3 levels, and induced G 1 arrest in a dose-dependent manner. Interestingly, these cells showed co-overexpression and altered mobility of the IGF1R and insulin receptor (IR). Immunoprecipitaion (IP) assays and ELISA confirmed the presence of IGF1R/IR heterodimeric receptors in figitumumab-sensitive cells. Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model. We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells. In particular, mass spectrometry showed that IGF1R had N-linked glycans at N913 in three figitumumab-sensitive cell lines. We observed that an absence of N-linked glycosylation at N913 led to a lack of membranous localization of IGF1R and figitumumab insensitivity.

Conclusion and Significance

The data suggest that the level of N-linked glycosylated IGF1R/IR heterodimeric receptor is highly associated with sensitivity to anti-IGF1R antibody in cancer cells.     

Distinguishing cell types or populations based on the computational analysis of their infrared spectra

Infrared (IR) spectroscopy of intact cells results in a fingerprint of their biochemistry in the form of an IR spectrum; this has given rise to the new field of biospectroscopy. This protocol describes sample preparation (a tissue section or cytology specimen), the application of IR spectroscopy tools, and computational analysis. Experimental considerations include optimization of specimen preparation, objective acquisition of a sufficient number of spectra, linking of the derived spectra with tissue architecture or cell type, and computational analysis. The preparation of multiple specimens (up to 50) takes 8 h; the interrogation of a tissue section can take up to 6 h (～100 spectra); and cytology analysis (n = 50, 10 spectra per specimen) takes 14 h. IR spectroscopy generates complex data sets and analyses are best when initially based on a multivariate approach (principal component analysis with or without linear discriminant analysis). This results in the identification of class clustering as well as class-specific chemical entities.     

Influence of double-strand-break repair pathways on radiosensitivity throughout the cell cycle in CHO cells

Unrepaired DNA double-strand breaks (DSBs) produced by ionizing radiation (IR) are a major determinant of cell killing. To determine the contribution of DNA repair pathways to the well-established cell cycle variation in IR sensitivity, we compared the radiosensitivity of wild-type CHO cells to mutant lines defective in nonhomologous end joining (NHEJ), homologous recombination repair (HRR), and the Fanconi anemia pathway. Cells were irradiated with IR doses that killed approximately 90% of each asynchronous population, separated into synchronous fractions by centrifugal elutriation, and assayed for survival (colony formation). Wild-type cells had lowest resistance in early G1 and highest resistance in S phase, followed by declining resistance as cells move into G2/M. In contrast, HR-defective cells (xrcc3 mutation) were most resistant in early G1 and became progressively less resistant in S and G2/M, indicating that the S-phase resistance in wild-type cells requires HRR. Cells defective in NHEJ (dna-pk(cs) mutation) were exquisitely sensitive in early G1, most resistant in S phase, and then somewhat less resistant in G2/M. Fancg mutant cells had almost normal IR sensitivity and normal cell cycle dependence, suggesting that Fancg contributes modestly to survival and in a manner that is independent of cell cycle position.