Human LOX Research Articles

Design, synthesis and mechanistic study of N-4-Piperazinyl Butyryl Thiazolidinedione derivatives of ciprofloxacin with Anticancer Activity via Topoisomerase I/II inhibition

A new group of thiazolidine-2,4-dione derivatives of ciprofloxacin having butyryl linker 3a-l was synthesized via an alkylation of thiazolidine-2,4-diones with butyryl ciprofloxacin with yield range 48–77% andfully characterized by various spectroscopic and analytical tools. Anti-cancer screening outcomes indicated that 3a and 3i possess antiproliferative activities against human melanoma LOX IMVI cancer cell line with IC50 values of 26.7 ± 1.50 and 25.4 ± 1.43 µM, respectively, using doxorubicin and cisplatin as positive controls with an IC50 of 7.03 ± 0.40 and 5.07 ± 0.29 µM, respectively. Additionally, compound 3j showed promising anticancer activity against human renal cancer A498 cell line with IC50 value of 33.9 ± 1.91 µM while doxorubicin and cisplatin showed IC50 values of 3.59 ± 0.20 and 7.92 ± 0.45, respectively. On the other hand, compound 3i did not show considerable anti-bacterial activity against S. aureus, E. coli and P. aeruginosa, and only moderate activity against K. pneumoniae with only a tenth of the activity of ciprofloxacin, confirming the cytotoxicity observed. Mechanistically, compound 3i inhibited both topoisomerase I and II with IC50 of 4.77 ± 0.26 and 15 ± 0.81 µM. Furthermore, it induced cell cycle arrest at S phase in melanoma LOX IMVI cells. Moreover, 3i provoked substantial levels of early, late apoptosis and necrosis in melanoma LOX IMVI cell line comparable to that induced by doxorubicin. Furthermore, compound 3i increased the expression level of active caspase-3 by 49 folds higher in LOX IMVI cell, increased protein expression level of Bax more than the control by 3 folds and inhibited PARP-1by 33% in LOX IMVI. All results were supported by theoretical docking studies on both tested enzymes confirming potential cytotoxicity for the synthesized hybrids.

Modeling the Interaction of Human Lipoxygenase LOX 15 with Free Arachidonic Acid

Modeling the Interaction of Human Lipoxygenase LOX 15 with Free Arachidonic Acid

In Vitro Oxidative Crosslinking of Recombinant Barnacle Cyprid Cement Gland Proteins

Barnacle adhesion is a focus for fouling-control technologies as well as the development of bioinspired adhesives, although the mechanisms remain very poorly understood. The barnacle cypris larva is responsible for surface colonisation. Cyprids release cement from paired glands that contain proteins, carbohydrates and lipids, although further compositional details are scant. Several genes coding for cement gland-specific proteins were identified, but only one of these showed database homology. This was a lysyl oxidase-like protein (lcp_LOX). LOX-like enzymes have been previously identified in the proteome of adult barnacle cement secretory tissue. We attempted to produce recombinant LOX in E. coli, in order to identify its role in cyprid cement polymerisation. We also produced two other cement gland proteins (lcp3_36k_3B8 and lcp2_57k_2F5). lcp2_57k_2F5 contained 56 lysine residues and constituted a plausible substrate for LOX. While significant quantities of soluble lcp3_36k_3B8 and lcp2_57k_2F5 were produced in E. coli, production of stably soluble lcp_LOX failed. A commercially sourced human LOX catalysed the crosslinking of lcp2_57k_2F5 into putative dimers and trimers, and this reaction was inhibited by lcp3_36k_3B8. Inhibition of the lcp_LOX:lcp2_57k_2F5 reaction by lcp3_36k_3B8 appeared to be substrate specific, with no inhibitory effect on the oxidation of cadaverine by LOX. The results demonstrate a possible curing mechanism for barnacle cyprid cement and, thus, provide a basis for a more complete understanding of larval adhesion for targeted control of marine biofouling and adhesives for niche applications.

Biochemical Characterization of 13-Lipoxygenases of Arabidopsis thaliana.

13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (Vmax app) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s−1·mg protein−1), with a Km value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg2+, Ca2+, Cu2+ and Cd2+, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.

Biochemical and Cellular Characterization and Inhibitor Discovery of Pseudomonas aeruginosa 15-Lipoxygenase.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunocompromised patients. P. aeruginosa secretes a lipoxygenase, LoxA, but the biological role of this enzyme is currently unknown. LoxA is poorly similar in sequence to both soybean LOX-1 (s15-LOX-1) and human 15-LOX-1 (37 and 39%, respectively) yet has kinetics comparably fast versus those of s15-LOX-1 (at pH 6.5, Kcat = 181 ± 6 s(-1) and Kcat/KM = 16 ± 2 μM(-1) s(-1)). LoxA is capable of efficiently catalyzing the peroxidation of a broad range of free fatty acid (FA) substrates (e.g., AA and LA) with high positional specificity, indicating a 15-LOX. Its mechanism includes hydrogen atom abstraction [a kinetic isotope effect (KIE) of >30], yet LoxA is a poor catalyst against phosphoester FAs, suggesting that LoxA is not involved in membrane decomposition. LoxA also does not react with 5- or 15-HETEs, indicating poor involvement in lipoxin production. A LOX high-throughput screen of the LOPAC library yielded a variety of low-micromolar inhibitors; however, none selectively targeted LoxA over the human LOX isozymes. With respect to cellular activity, the level of LoxA expression is increased when P. aeruginosa undergoes the transition to a biofilm mode of growth, but LoxA is not required for biofilm growth on abiotic surfaces. However, LoxA does appear to be required for biofilm growth in association with the host airway epithelium, suggesting a role for LoxA in mediating bacterium-host interactions during colonization.

133 : In vitro eradication of serous ovarian cancer with IFN-α2α and IFN-γ in combination with monocytes

Interferons (IFNs) have been utilized for their antiproliferative and antitumor effects against human cancers, including hairy cell leukemia, chronic myelogenous leukemia, and malignant melanoma. We have previously shown that in combination with human elutriation-purified monocytes, IFN- α 2a and IFN- γ have nearly eradicated certain cancer cell types in vitro and in vivo in a nude mouse model. More specifically, previous in vitro studies have shown the eradication of human osteosarcoma, LOX melanoma, and A549 lung tumor cells, while no such effect seen on the human diploid cell lines WI-38 and MRC-5. Further in vivo analysis of OVCAR-3 and LOX melanoma inoculated intratumorally in female BALB/c nude mice showed that combination therapy significantly reduced tumor growth more profoundly than IFNs or monocytes alone. Such finding may have clinical implications. We have adapted this IFN and monocyte combination treatment model towards human serous ovarian cancer, a subtype accounting for approximately 60–80% of ovarian cancer diagnoses. We further validated IFN-primed monocytes as a novel antitumor treatment against ovarian tumor cells in vitro . Combination treatment has antitumor and antiproliferative effects towards the ovarian cancer cell lines SKOV-3, CaOV3, OVCAR-4, OVCAR-5, and OVCAR-8. These serous tumor cell lines show varying, though prevalent, anti-tumor and anti-proliferative effects following IFN-primed monocyte treatment. SKOV-3, OVCAR-4, and OVCAR-8 cells are moderately sensitive to treatment; however, CaOV3 and OVCAR-5 cells are highly sensitive, with near complete eradication. Results demonstrate that such treatment is a novel candidate for ovarian cancer. Future studies will include in vivo IFN-primed monocyte treatment of intraperitoneal xenografts of serous ovarian cancer cell lines in female BALB/c nude mice, treatment of tumor cells with IFN-primed monocytes in combination with cancer therapeutics, and treatment of ovarian cancer cells with IFN-primed monocytes extracted from patients diagnosed with ovarian cancer.

Combinatorial treatment with carboxyamidotriazole- orotate and temozolomide in sc-implanted human LOX IMVI melanoma xenografts

Background: Carboxyamidotriazole orotate (CTO) is the orotic acid salt of 5-amino-1(4-(4-chlorobenzoyl)-3, 5-dichlorobenzyl)-1, 2, 3-triazole-4-carboxamide (CAI). CTO possesses increased solubility as compared to CAI as the free base. The antiproliferative and antimetastatic effects of CTO are related to inhibition of receptor-operated, calcium-channel-mediated calcium influx. CTO can inhibit calcium-sensitive signal transduction in the VEGF and the PI3K pathways, inhibition of FGF-2-induced tyrosine kinase, VEGF-mediated activation of phospholipase Cy, generation of IP3 and nitric oxide synthase activation, and induction of apoptosis in imatinib mesylate resistant CML cells by downregulating bcr-abl. Methods: Different combinations of CTO and temozolomide were first tested in female athymic NCr- nu/nu mice to evaluate tolerance of the combination. The tolerated combinations were then tested to evaluate the antitumor activity against subcutaneously implanted human LOX IMVI melanoma xenografts. Results: Oral CTO at doses of 513 or 342 mg/kg/dose Q1D × 14 resulted in inhibition of tumor growth ( p <0.001 and p =0.004). Oral TEM at doses of 90 and 60 mg/kg/dose Q4D × 3 resulted in dose-dependent inhibition of tumor growth ( p <0.001 and p <0.001). Oral CTO at 513 or 342 mg/kg/dose in combination with temozolomide 90 mg/kg/dose resulted in comparable tumor inhibition to temozolomide alone. However, oral CTO at 513 mg/kg/dose in combination with temozolomide 60mg/kg/dose resulted in additive antitumor activity compared to each drug alone. Also, CTO at 342 mg/kg/dose in combination with temozolomide 60mg/kg/dose had more than additive antitumor activity and was statistically different from the group treated with temozolomide 60 mg/kg/dose alone ( p =0.001). Conclusions: These results suggest that this combination of previously configured therapeutic doses of temozolomide with CTO enhances the sensitivity of temozolomide and may permit use of lower temozolomide doses to obtain an optimum antitumor effect in combination therapy. This dose-dilution combinatorial strategy fulfills the need for increased tumor sensitivity and efficacy. Additionally, this strategy reduces drug resistance and toxicity associated with dose dense strategy of temozolomide treatment in this melanoma model.

Combinatorial treatment with CTO and temozolomide in sc-implanted human LOX 1MVI melanoma.

e19006 Background: Carboxyamidotriazole orotate (CTO) is the orotic acid salt of 5-amino-1.(4-(4-chlorobenzoyl)-3,5-dichlorobenzyl)-1, 2, 3-triazole-4-carboxamide. CTO possesses increased solubility. The antiproliferative and antimetastatic effects of CTO are related to inhibition of receptor –opertated calcium channel- mediated calcium influx. CTO can inhibit calcium sensitive signal transduction in the VEGF and the PI3K pathways, inhibition of FGF-2-induced tyrosine kinase, VEGF-mediated activation of phospholipase Cγ, generation of IP3 and nitric oxide synthase activation, and induction of apoptosis in imatinib mesylate resistant CML cells by downregulating bcr-abl. Methods: Different combinations of CTO and temozolomide (TEM) were first tested in female athymic NCr-nu/nu mice to evaluate tolerance of the combination. The tolerated combinations were then tested to evaluate the antitumor activity against subcutaneously –implanted human LOX 1MVI melanoma xenografts. Results: Oral CTO at doses of 513 or 342 mg/kg/dose Q1Dx14 resulted in inhibition of tumor growth (p&lt;0.001 and p=0.004). Oral TEM at doses of 90 and 60mg/kg/dose Q4Dx3 resulted in dose-dependent inhibition of tumor growth (p&lt;0.001 and p&lt;0.001). Oral CTO at 513 or 342 mg/kg/dose in combination with TEM 90mg/kg/dose resulted in comparable tumor inhibition to TEM alone. However, oral CTO at 513mg/kg/dose in combination with TEM 60mg/kg/dose resulted in additive antitumor activity compared to each drug alone. Also, CTO at 342mg/kg/dose in combination with TEM 60mg/kg/dose had more than additive antitumor activity and was statistically different from the group treated with TEM 60mg/kg/dose alone (p=0.001). Conclusions: These results suggest that CTO enhances the sensitivity of TEM and may permit use of lower doses of TEM to obtain an optimum antitumor effect in combination therapy thus reducing toxicity of high TEM doses in this melanoma model.

Focus on Molecules: Lysyl oxidase

1. StructureLysyl Oxidase (LOX, also called protein-lysine 6-oxidase)(Genbank accession numbers: Nucleotide NM_002317.5, ProteinNP_002308.2, EC 1.4.3.13) is one of the five LOX family members(LOX and LOX Like1-4). The human LOX gene is located onchromosome 5q23.2. LOX is synthesized as a 50 kDa pre-proteincontaining 3 domains; the N-terminal signal peptide sequence(aa1-21), the N-terminal pro-peptide domain (aa 22-162), and theC-terminal catalytic domain (aa 162-417). The signal peptide isremoved by cleavage at the Cys21-Ala22 bond and the pro-peptidedomain is N-glycosylated in the endoplasmic reticulum and Golgiapparatus yielding a pro-enzyme, which is then secreted fromcells as a catalytically inactive protein. The 32 kDa active enzyme(C-terminal domain) is released by proteolytic cleavage of thepro-peptide at Gly162eAsp163 by procollagen C-proteinase (bonemorphogenetic protein 1; BMP-1) (Kagan and Li, 2003)(Fig. 1).2. FunctionLOX belongs to a family of amine oxidases. LOX was initiallyreported to be expressed andsecreted by fibrogenic cells but is nowknown to be expressed in several other cell types. LOX oxidizespeptidyl lysine and hydroxylysine residues in collagen and lysineresidues in elastin to produce peptidyl alpha-aminoadipic-delta-semialdehydes. These aldehyde modifications can spontaneouslycombine with vicinal peptidyl aldehydes or with epsilon-aminogroups of peptidyl lysine to form covalent cross-links that stabilizeand cause collagen and elastin fibers to be insoluble in the extra-cellular matrix (ECM). The significance of LOX was demonstrated inthe LOX knockout mouse, which could not survive at birth, due torupture of the aorta and diaphragm from incomplete elastin cross-linking. LOX is also essential for development of the distal andproximal airways, and the formation of alveoli in the lungs. LOX isalso critical for notochord formation and muscle development. LOXaffects differentiation of osteoblasts by forming cross-links in thesurrounding collagen matrix. These results underline the impor-tance of LOX in the morphogenesis and repair of connective tissuesof the cardiovascular, respiratory, skeletal, and other organ systems(Fongetal.,2009).LOXcanalsoinducecross-linkingbetweenlysinesin histones in the nucleus (Kagan and Li, 2003). LOX has also beenreported as a potent chemotactic agent for monocytes and vascularsmooth muscle cells.3. Disease involvementDuetotherangeofLOXbiologicalfunctions,abnormalitiesofLOXexpression underlie the development of a number of pathologicalprocesses related with an imbalance in ECM synthesis/degradation.LOX is expressed in several ocular tissues, and the followingdiseases of the eye have been linked with LOX:A. Keratoconus: Keratoconus is a corneal degeneration in whichthe extracellular matrix of the cornea loses its integrity, slowlychanging from the normal round shape to a cone shape. Onestudy reported that corneal LOX mRNA expression was signif-icantly lower in keratoconus patients when compared to theirage-matched controls. Another study showed that reducingcopper levels in tears results in keratoconus in an animalmodel. Copper is an essential cofactor for LOX activity.B. Diabetic retinopathy (DR): There are a number of reports oflower expression of LOX co-factors in DR retina samples andlower LOX cross-linking activity in vitreous samples of patientswith DR. However, theeffectofhigh glucose onLOXexpressionand enzymatic activity in cultured human retinal endothelialcells is less clear, with conflicting evidence suggesting eitherincreased or decreased LOX expression. Although discrepant,these studies indicate a potential involvement of LOX in theobserved DR phenotype.

GLO1 overexpression in human malignant melanoma

Glyoxalase I [lactoylglutathione lyase (EC 4.4.1.5) encoded by GLO1] is a ubiquitous cellular defense enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis. Accumulative evidence suggests an important role of GLO1 expression in protection against methylglyoxal-dependent protein adduction and cellular damage associated with diabetes, cancer, and chronological aging. On the basis of the hypothesis that GLO1 upregulation may play a functional role in glycolytic adaptations of cancer cells, we examined GLO1 expression status in human melanoma tissue. Quantitative reverse transcription polymerase chain reaction analysis of a cDNA tissue array containing 40 human melanoma tissues (stages III and IV) and 13 healthy controls revealed pronounced upregulation of GLO1 expression at the mRNA level. Immunohistochemical analysis of a melanoma tissue microarray confirmed upregulation of glyoxalase I protein levels in malignant melanoma tissue versus healthy human skin. Consistent with an essential role of GLO1 in melanoma cell defense against methylglyoxal cytotoxicity, siRNA interference targeting GLO1-expression (siGLO1) sensitized A375 and G361 human metastatic melanoma cells towards the antiproliferative, apoptogenic, and oxidative stress-inducing activity of exogenous methylglyoxal. Protein adduction by methylglyoxal was increased in siGLO1-transfected cells as revealed by immunodetection using a monoclonal antibody directed against the major methylglyoxal-derived epitope argpyrimidine that detected a single band of methylglyoxal-adducted protein in human LOX, G361, and A375 total cell lysates. Using two-dimensional proteomics followed by mass spectrometry the methylglyoxal-adducted protein was identified as heat shock protein 27 (Hsp27; HSPB1). Taken together, our data suggest a function of GLO1 in the regulation of detoxification and target adduction by the glycolytic byproduct methylglyoxal in malignant melanoma.

Abstract A228: Noninvasive magnetic resonance spectroscopic PD markers of a minor-groove interstrand cross-linking agent (BN2629) in human colon carcinoma and melanoma xenografts

Abstract BN2629 (also known as SJG-136) is a novel pyrrolobenzodiazepine dimer analogue that binds covalently in the minor groove of DNA, inducing little or no distortion of the double helix. Hence, it evades the p53-mediated detection and up-regulation of nucleotide excision repair mechanisms but induces apoptosis. It has just completed Phase 1 clinical trials in the USA (NCI) and UK (CR-UK), and has been approved by the NCI for Phase II evaluation. The aim of this work was to develop a robust and non-invasive surrogate marker for tumor response following BN2629 treatment. We carried out an in vivo 31phosphorus magnetic resonance spectroscopy (31P MRS) study of BN2629 (80 g/kg via ip for 3 days) and vehicle in HT29 human colon carcinoma and LOX IMVI melanoma xenograft models. In vitro 31P MRS were performed on tumor extracts. Significant growth delays (p&amp;lt;0.0001) were observed in both BN2629-treated HT29 and LOX IMVI xenografts when compared with controls. In vivo31P MRS of the HT29 xenografts showed an increase in the phosphomonoester/total phosphorus signals (PME/TotP) (p=0.04) and PME/ - NTP ratios (p&amp;lt;0.02) after 3 days of BN2629 treatment. No significant changes were observed in the control group. In vitro 31P MRS of extracts from BN2629-treated HT29 tumors showed significant increases in phosphoethanolamine (PE; p&amp;lt;0.01), glycerophosphocholine (GPC; p&amp;lt;0.01) and glycerophosphoethanolamine (GPE; p&amp;lt;0.03) when compared with controls. In vivo 31P MRS of the LOX IMVI xenografts showed an increase in - NTP/TotP ratio ( -NTP/TotP) (p&amp;lt;0.01) after 3 days of BN2629 treatment. No significant changes were observed in the control group. In vitro 31P MRS of extracts from BN2629-treated LOX IMVI tumors showed significant increases in GPC (p&amp;lt;0.001), GPE (p&amp;lt;0.01) and ATP (p=0.04) when compared with controls. Treatment with BN2629 resulted in tumor growth delay and altered bioenergetic and phospholipid metabolism in vivo, which are consistent with metabolic changes observed following treatment with agents such as 5-FU [1] and cyclophosphamide [2]. The phospholipid changes may be associated with alteration in membrane turnover following treatment, and these changes may have potential as surrogate non-invasive markers for determining tumor response following treatment with BN2629. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A228.

Assembly of Mutant-Template Telomerase RNA into Catalytically Active Telomerase Ribonucleoprotein That Can Act on Telomeres Is Required for Apoptosis and Cell Cycle Arrest in Human Cancer Cells

The telomerase ribonucleoprotein is a promising target for cancer therapy, as it is highly active in many human malignancies. A novel telomerase targeting approach combines short interfering RNA (siRNA) knockdown of endogenous human telomerase RNA (hTer) with expression of a mutant-template hTer (MT-hTer). Such combination MT-hTer/siRNA constructs induce a rapid DNA damage response, telomere uncapping, and inhibition of cell proliferation in a variety of human cancer cell lines. We tested which functional aspects of the protein catalytic component of telomerase [human telomerase reverse transcriptase (hTERT)] are required for these effects using human LOX melanoma cells overexpressing various hTERTs of known properties. Within 3 days of MT-hTer/siRNA introduction, both growth inhibition and DNA damage responses were significantly higher in the setting of wild-type hTERT versus catalytically dead hTERT or mutant hTERT that is catalytically competent but unable to act on telomeres. These effects were not attenuated by siRNA-induced knockdown of the telomeric protein human Rap1 and were additive with knockdown of the telomere-binding protein TRF2. Hence, the effects of MT-hTer/siRNA require a telomerase that is both catalytically competent to polymerize DNA and able to act on telomeres in cells.

Lo, and the Niche Is Knit: Lysyl Oxidase Activity and Maintenance of Lung, Aorta, and Skin Integrity

Lo, and the Niche Is Knit: Lysyl Oxidase Activity and Maintenance of Lung, Aorta, and Skin Integrity

Synthesis and evaluation of novel rhodacyanine dyes that exhibit antitumor activity.

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) = 50 nM, IC50(CV-1) = 17.3 microM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agents candidates.

Crosslinked gelatin beads: A culture method for the study of cell adhesion and invasion

A method is described for culturing invasive cell lines on crosslinked gelatin beads and preparing them for immunocytochemical and morphological observation. Very invasive cells such as Rous sarcoma virus-transformed chicken embryo fibroblasts, and human melanoma LOX and RPMI7951 and breast carcinoma MDA-MB-231 cells will actively degrade this matrix, extending cellular protrusions, called invadopodia, into the sites of degradation. Normal chicken embryo fibroblasts and other non-invasive cell lines do not disrupt the surface of these beads and do not form invadopodia. Invadopodia extending into the bead can be visualized by electron microscopy. Cellular removal of fluorescent fibronectin that has been covalently coupled to the bead surface can be monitored using fluorescence microscopy of frozen-thin-sections. In double label experiments, immunocytochemistry is used to localize antigens in invadopodia at sites of membrane invasion. The materials for bead preparation are inexpensive, and this method has the advantage that many cell types will attach and spread readily on the beads, while only highly invasive cells will invade into the bead.

Nucleolar and nuclear aberrations in human lox tumor cells following treatment with p120 antisense oligonucleotide ISIS-3466

Previous reports from this laboratory have shown marked cytocidal effects of the ISIS-3466 antisense phosphorothioate oligodeoxynucleotide to the human nucleolar protein p120 on human cancer cell lines in vitro and inhibition of tumor growth in vivo in an i.p./i.p. LOX cell model (L. Perlaky et al. Anti-Cancer Drug Design 8:3-14, 1993). In this study, light and fluorescence microscopy showed that the number of LOX cells in mitosis decreased by 50% after incubation for 4 h in 0.2–0.4 μM antisense oligonucleotide; a 70% reduction in cell number was found from 8–72 h post-treatment. In addition, marked unravelling of nucleolar structures and chromatin fragmentation was found after a 4-h incubation. The nucleolar unravelling occurred in varying degrees ranging from partial unfolding to almost complete separation of the strands of nucleolar residues. Twenty four hours post-treatment, immunofluorescence staining with the anti-p120 monoclonal antibody showed reduced nucleolar protein p120 and translocation of the p120 protein from the nucleoli to the nucleoplasm. Analysis of the mechanisms of the nucleolar unravelling and inhibition of mitosis will provide further understanding of the cytocidal effects of the ISIS-3466 antisense oligonucleotide.

Location of P-II and AlPCS 4 in human tumor LOX in vitro and in vivo by means of computer-enhanced video fluorescence microscopy

The patterns of in vitro intracellular and in vivo intratumoral localization of Photofrin II (P-II) and aluminum phthalocyanine tetrasulfonate (AlPCS 4) in human melanoma LOX were studied by means of computer-enhanced video fluorescence microscopy (CEVFM). The hydrophobic drug P-II localized diffusely in the perinuclear fraction of the cytoplasm of the LOX cells cultivated in vitro. Light exposure did not result in any observable change in the localization pattern. The hydrophilic dye AlPCS 4 was distributed as granular and grain patterns in the cytoplasm before light exposure, in exactly the same pattern as that of acridine orange incubated in the same cells, which is known to emit red fluorescence from lysosomes, thus indicating that AlPCS 4 was also primarily localized in the lysosomes of the LOX cells. After light exposure the distribution of the intracellular AlPCS 4 fluorescence was altered and the intensity increased. In vivo, P-II had a combined cellular localization pattern (i.e. a strongly cytoplasmic membrane-localizing pattern and a weakly intracellular distribution pattern) and an extracellular distribution pattern in the tumor tissue, while the AlPCS 4 fluorescence was seen mainly in the stroma of the tumor. The total fluorescence intensity of P-II and AlPCS 4 in the LOX tumor tissue at different times after injection was quantitatively determined by means of CEVFM.

Localization of potent photosensitizers in human tumor LOX by means of laser scanning microscopy

By means of laser scanning fluorescence microscopy the intratumoral localization patterns of several photosensitizers in LOX tumors in nude mice were studied. Lipophilic dyes such as TPPS 1 (tetraphenylporphine monosulfonate), TPPS 2a (tetraphenylporphine disulfonates with the sulfonate groups on adjacent rings), AlPCS 1 (aluminium phthalocyanine monosulfonate) and AlPCS 2 (aluminium phthalocyanine disulfonates) localized mainly in tumor cells. The fluorescence intensity of these dyes increased from 4 h to 48 h postinjection and the fluorescence was still observable 120 h postinjection. The more hydrophilic dyes such as TPPS 3 (tetraphenylporphine trisulfonates), TPPS 4 (tetraphenylporphine tetrasulfonates), and AlPCS 4 (aluminium phthalocyanine tetrasulfonates) localized mainly extracellularly in the tumorous stroma. The fluorescence intensity of these dyes decreased from 4 h to 48 h postinjection. 120 h postinjection no significant fluorescence of these dyes could be seen in the tumors. P-II (Photofrin II), 3-THPP [tetra(3-hydroxyphenyl)porphine], TPPS 20 (tetraphenylporphine disulfonates with the sulfonate groups on opposite rings) and AlPCS 3 (aluminum phthalocyanine trisulfonates) had a combined localization pattern, i.e. a strongly cytoplasmic membrane-localizing pattern and a weakly intracellular distribution pattern, although some fluorescence could be seen in the tumorous stroma. The data are discussed in relation to what is known about the in vivo photosensitizing efficiency of some of the dyes.

The effect of glucose administration on the uptake of Photofrin II in a human tumor xenograft

Athymic BALB/c nude mice, bearing a human melanoma LOX, were given the photosensitizing drug Photofrin II (10 mg/kg body wt.) intraperitoneally. The mice were also given one of the following chemicals intraperitoneally: glucose, galactose and glucose plus nordihydroguaiaretic acid (NDGA) which is an inhibitor of glycolysis. Multiple injections of glucose (3 g/kg body wt. given at −1, 0, +1 and +3 h relative to the injection of 10 mg/kg of Photofrin II at time 0) resulted in a significant increase in the uptake of Photofrin II in the tumor 4 h after a Photofrin II injection, while the uptake of Photofrin II in the other tissues remained unchanged. Administration of galactose had no significant effect on the uptake of Photofrin II in the tissues studied (tumor, muscle, skin and liver). NDGA seemed to abolish the effect of glucose injection.

Schedule dependence, activity against natural metastases, and cross-resistance of pyrazine diazohydroxide (sodium salt, NSC 361456) in preclinical models in vivo

Pyrazine diazohydroxide (sodium salt, NSC 361456; PZDH) is a new antitumor drug with relatively broad activity in initial evaluations against murine leukemias, solid tumors, and two human tumor xenografts in vivo. The present studies were designed to address questions about PZDH activity on different treatment schedules, its activity against metastases, and the extent of its cross-resistance with established drugs. Human LOX amelanotic melanoma xenografts in athymic mice were used to explore schedule dependence and activity against natural metastases, and a series of drug-resistant murine leukemias provided an in vivo cross-resistance profile. Single-dose treatment and prolonged treatment provided equivalent therapeutic responses to PZDH by both the i.p. and i.v. routes in the i.p. LOX model. A s.c. LOX model resulting in spontaneous pulmonary metastases was adapted for bioassay and quantitation of the numbers of LOX cells killed by PZDH among both primary and metastatic cell populations. It was demonstrated that PZDH afforded about 2-log10 orders of magnitude greater cell kill among pulmonary metastases than against primary s.c. LOX tumors in the same mouse. Murine leukemias resistant to doxorubicin (ADR), vincristine (VCR), cisplatin (DDPt), methotrexate (MTX), N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), and cyclophosphamide (CPA) were not cross-resistant to PZDH. However, both P388 and L1210 leukemia sublines resistant to melphalan (L-PAM) were cross-resistant to PZDH, suggesting that patients previously treated with L-PAM might have less likelihood of response to PZDH than those who had had no opportunity to develop L-PAM resistance. Although these observations should not be applied to clinical studies without due caution, they support clinical evaluation of PZDH as well as continued investigation of its molecular pharmacology.