Thomas R. Tritton

Inhibition of epidermal growth factor receptor-associated tyrosine kinase blocks glioblastoma invasion of the brain.

OBJECTIVE Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival despite aggressive treatment, in part because of its propensity to aggressively infiltrate into brain tissue. Glioblastoma multiforme is also unique because it is the only nonepithelial human tumor for which excessive activation of epidermal growth factor receptor (EGFR) has been consistently linked to tumor growth and patient survival, and EGFR activation promotes glioblastoma multiforme infiltration in vitro. METHODS Cocultures of human glioblastoma spheroids (derived from three separate patients) and fetal rat brain aggregates were examined for infiltration using confocal microscopy, in the presence of 0 to 100 mumol/L genistein, a tyrosine kinase (TK) inhibitor, and 3 mumol/L tyrphostin A25, a specific EGFR-TK inhibitor. RESULTS Infiltration (not attachment) was completely inhibited by genistein at 10 mumol/L, the IC20 for monolayer growth inhibition in two cell lines. Tyrphostin A25 at 3 mumol/L (the IC20 for monolayers) reduced invasion in a third cell line from 38.8 +/- 6.1% invasion-hour per hour (n = 5) to 2.9 +/- 1.2% invasion-hour per hour (n = 6) (P = 0.0002, two-tailed t test, 93% inhibition), and from 0.54 +/- 0.065% per hour (slope) to 0.028 +/- 0.018% per hour (P = 0.00001, 95% inhibition). Maximal percent invasion was reduced from 100 +/- 0 to 7.4 +/- 5.6% of the fetal rat brain aggregate. No change was detected in EGFR-associated tyrosine phosphorylation at those doses in monolayers by 32P immunolabeling, consistent with the known effects of low concentrations of TK inhibitors. An increase in expression of wild-type and truncated EGFR was demonstrated by Western blotting. Invasion was equally well inhibited by a monoclonal antibody to the high-affinity ligand binding domain of EGFR and not by antibody to an inactive domain. CONCLUSION Our observations support the role of EGFR activation as a determinant by which glioblastoma invades normal brain tissue, and we show that invasion can be effectively inhibited at much lower concentrations of TK inhibitors than are necessary for growth suppression.

Cell surface actions of adriamycin

Adriamycin has a vast range of reported actions on the structural and functional properties of cells. This review summarizes the literature on the ability of the drug to modulate the cell surface membrane and attempts to address the question of how such actions could be linked to cytotoxicity. In addition, we consider the use of polymer immobilization of adriamycin to separate intracellular from plasma membrane effects of the drug, and show how this approach has been helpful in interpreting the pharmacology of adriamycin. Finally, a range of biophysical and spectroscopic approaches to defining the molecular details of adriamycin-bilayer interactions is surveyed, and the results used to discuss a model for how this antineoplastic agent binds to membranes.

Tamoxifen induces TGF‐β1 activity and apoptosis of human MCF‐7 breast cancer cells in vitro

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF‐7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF‐7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM‐2 and TGF‐β1 mRNAs in MCF‐7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen‐induced pS2 gene was strongly suppressed. The biological activity of TGF‐β was increased at least fourfold in the media from MCF‐7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF‐7 cells and it may be mediated by the secretion of active TGF‐β.

Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis

We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c-fos/c-jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR (P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c-fos but not of c-jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c-fos and the development of apoptosis. The ability to block asbestos-induced elevations in c-fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.

Ultrasound-enhanced hydroxyl radical production from two clinically employed anti-cancer drugs, adriamycin and mitomycin C

Abstract Continuous-wave 1 MHz ultrasound at the therapeutic intensity of 1 W cm −2 was found to enhance significantly the hydroxyl radical production from two clinically employed redox cycling drugs, viz. adriamycin (doxorubicin) and mitomycin C, with respect to the control drug-free insonicated phosphate buffer suspension. Benzoic acid (Bz) was employed as a sensitive chemical probe to detect hydroxyl radicals (HO ■ ). Bz is initially non-fluorescent and upon aromatic hydroxylation becomes permanently fluorescent. A series of time course studies up to 30 min were performed on drug suspensions to characterize the HO ■ generation in the presence and absence of ultrasound at 37°C. Identical ultrasound treatments on non-redox cycling clinical drugs, 5-fluorouracil and methotrexate, did not yield any significant enhancement in the production of HO ■ in comparison to the drug-free insonicated phosphate buffer suspension. Ultrasound exposures of 30 min did not yield measurable changes in the chemical constitution of the four drugs as assessed through high-performance liquid chromatography. Identical ultrasound treatments at 3 MHz did not produce any HO ■ in the presence or absence of these four anti-cancer drugs. Free radical scavengers such as mannitol, superoxide dismutase, catalase and a transition metal chelating agent were employed independently to elucidate the chemical species and pathways involved in the production of the HO ■ . The findings strongly implicate an active role of acoustically induced cavitation in potentiating redox cycling drugs via chemical reduction and, thereafter, production of the OH ■ via Fentons pathway.

Transverse location of anthracyclines in lipid bilayers paramagnetic quenching studies

Quenching of anthracycline fluorescence by a series of spin-labeled fatty acids was used to probe the transverse location of the drug in phosphatidylcholine bilayers in the form of small unilamellar vesicles. Stern-Volmer plots of the quenching data indicate that the fluorophore moiety of the anthracycline is intercalated into the hydrocarbon region of the bilayer, with deeper penetration observed in fluid-phase than in solid-phase vesicles. 31P-NMR parameters (T1 and nuclear Overhauser enhancement (NOE] are unaffected by the presence of drug, consistent with a binding site removed from the interfacial region. Comparison of intensity (F0/F) plots with lifetime (tau 0/tau) data shows that the predominant mechanism of anthracycline quenching by membrane-bound nitroxides is static. Since the membrane-bound drug is also accessible to quenching by I-, the binding site in the membrane must create a channel which is accessible to solvent. Two other fluorescent probes, 12-(9-anthroyloxy)stearate (12-AS) and diphenylhexatriene (DPH), were employed to confirm the results obtained with the anthracyclines, giving quenching data representative of their location in the bilayer.

Selectivity of the anthracyclines for negatively charged model membranes: role of the amino group.

SummaryThe equilibrium-binding affinities of six adriamycin analogues and four daunomycin derivatives for negatively charged dimyristoyl phosphatidylcholine/dimyristoyl phosphatidic acid (DMPC/DMPA) small unilamellar vesicles are compared with values for electroneutral DMPC liposomes. Binding of the daunomycin series to negatively charged dimyristoyl phosphatidyl glycerol (DMPG) vesicles was also examined. Under physiological conditions of pH and ionic strength, substitution of the amino group of adriamycin or daunomycin resulted in a reduced affinity for negatively charged bilayers, even if the substituent enhanced the degree of ionization of the amine. Decreasing the ionic strength increases the binding affinity for acidic membranes but decreases the drug affinity for neutral membranes. We propose that the electrostatic bond of the phosphate-amino group that has been shown to exist between anthracyclines and phosphatidic acid is sterically destabilized by substitution of the amino group. The results are consistent with a mode of anthracycline binding to negatively charged membranes which is driven by hydrophobic and electrostatic considerations but is destabilized by steric bulk at the amino group. THe data also provide insight into the design of new anthracyclines with high membrane affinities and reduced uptake; such directed interaction with plasma membranes may enhance antineoplastic potential while reducing cardiac toxicity.

Activity of two novel anthracene-9,10-diones against human leukemia cells containing intercalator-sensitive or -resistant forms of topoisomerase II

We have examined the activities of two novel aza-anthracene-9,10-diones (aza), 1-aza and 2-aza, in HL-60 human leukemia cell lines containing type II topoisomerases with different sensitivities to inhibition by other intercalating agents. The sensitive line, HL-60, was sensitive to 2-aza but not to 1-aza, whereas the resistant HL-60/AMSA was sensitive to neither agent. Measurements of 1- and 2-aza-induced, topoisomerase II-mediated DNA cross-linking in the cells revealed patterns of resistance and sensitivity that paralleled the results in the cytotoxicity assays. However, measurements of drug-induced topoisomerase II-mediated DNA cross-linking using purified HL-60 and HL-60/AMSA topoisomerase II indicated that both agents could stabilize a covalent complex between DNA and the HL-60 enzyme. HL-60/AMSA topoisomerase II resisted stabilization by either agent. This suggests that the resistance of HL-60 cells to 1-aza is not due to the inability of this drug to inhibit topoisomerase II but rather to another, undefined mechanism.

Interaction of Adriamycin with Single and Multibilayer Dipalmitoylphosphatidylcholine Vesicles: Spin-Labeling and Calorimetric Study

AbstractThe effects of adriamycin on the organization of dipalmitoylphosphatidylcholine (DPPC) membranes alone or in the presence of 1 mol% cardiolipin (CL) in the form of single and multibilayer vesicles has been studied by spin-labeling, and by high sensitivity differential scanning calorimetry. With sonicated small unilamellar vesicles (SUVs), adriamycin increased the order parameter of 5-doxylstearate spin-labeled vesicles, an effect primarily observed in the gel phase of DPPC. thermal transition profiles, obtained by high-sensitivity differential scanning calorimetry and by 2,2,6,6-tetramethylpiperidinyl-l-oxy (TEMPO) partitioning studies, indicated that the drug induced aggregation and fusion of SUVs, especially at high drug-lipid molar ratios, and this phenomenon was further verified by negative-staining electron microscopy. the presence of J mol% CL in the lecithin bilayer markedly enhanced the effect of adriamycin on membrain order and fusion, especially under conditions of low ionic strength. th...

Correlation of altered tyrosine phosphorylation with methotrexate resistance in a cisplatin-resistant subline of L1210 cells

Collateral resistance to cisplatin and methotrexate has been reported in several cell lines. A murine leukemia cell line (L1210/DDP) selected for cisplatin resistance also has been shown to be highly resistant to methotrexate. Of the mechanisms proposed for methotrexate resistance, only changes in methotrexate transport into the cells were found in an earlier report. Methotrexate enters mammalian cells via an active transport system. In the present study, we demonstrated that the transport into the cell may be impaired in the resistant cells due to altered tyrosine phosphorylation of a membrane protein with a molecular mass of 66 kDa. This alteration was manifested by altered tyrosine phosphorylation of the 66 kDa protein and may be an underlying modification that renders the cells resistant to methotrexate. These results suggest involvement of tyrosine phosphorylation in folate transport and methotrexate resistant in L1210/DDP cells.