In vitro demonstration of the heavy-atom effect for photodynamic therapy

Photodynamic therapy (PDT) is an emerging treatment modality for a range of disease classes, both cancerous and noncancerous. This has brought about an active pursuit of new PDT agents that can be optimized for the unique set of photophysical characteristics that are required for a successful clinical agent. We now describe a totally new class of PDT agent, the BF2-chelated 3,5-diaryl-1H-pyrrol-2-yl-3,5-diarylpyrrol-2-ylideneamines (tetraarylazadipyrromethenes). Optimized synthetic procedures have been developed to facilitate the generation of an array of specifically substituted derivatives to demonstrate how control of key therapeutic parameters such as wavelength of maximum absorbance and singlet-oxygen generation can be achieved. Photosensitizer absorption maxima can be varied within the body's therapeutic window between 650 and 700 nm, with high extinction coefficients ranging from 75,000 to 85,000 M(-1) cm(-1). Photosensitizer singlet-oxygen generation level was modulated by the exploitation of the heavy-atom effect. An array of photosensitizers with and without bromine atom substituents gave rise to a series of compounds with varying singlet-oxygen generation profiles. X-ray structural evidence indicates that the substitution of the bromine atoms has not caused a planarity distortion of the photosensitizer. Comparative singlet-oxygen production levels of each photosensitizer versus two standards demonstrated a modulating effect on singlet-oxygen generation depending upon substituent patterns about the photosensitizer. Confocal laser scanning microscopy imaging of 18a in HeLa cervical carcinoma cells proved that the photosensitizer was exclusively localized to the cellular cytoplasm. In vitro light-induced toxicity assays in HeLa cervical carcinoma and MRC5-SV40 transformed fibroblast cancer cell lines confirmed that the heavy-atom effect is viable in a live cellular system and that it can be exploited to modulate assay efficacy. Direct comparison of the efficacy of the photosensitizers 18b and 19b, which only differ in molecular structure by the presence of two bromine atoms, illustrated an increase in efficacy of more than a 1000-fold in both cell lines. All photosensitizers have very low to nondeterminable dark toxicity in our assay system.

Oxovanadium(IV) complexes of phenanthroline bases: the dipyridophenazine complex as a near-IR photocytotoxic agent

Oxovanadium(IV) complexes [VOCl(B)(2)]Cl (1-3) of phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d: 2', 3'-f] quinoxaline (dpq in 2) and dipyrido[3,2-a: 2', 3'-c] phenazine (dppz in 3), have been prepared, characterized and their DNA and protein binding, photo-induced DNA and protein cleavage activity andm photocytotoxicity have been studied. Complex 2, structurally characterized by X-ray crystallography, shows the presence of a vanadyl group in VOClN4 coordination geometry. The dpq ligand displays a chelating mode of binding with a N-donor site trans to the oxo-group. The chloride ligand is cis to the oxo-group. The one-electron paramagnetic complexes show a d-d band near 715 nm in 15% DMF-Tris-HCl buffer. The complexes are redox active exhibiting a V(IV)/V(III) redox couple within -0.5 to -0.7 V vs. SCE in 20% DMF-Tris-HCl/0.1 M KCl. The complexes bind to calf thymus (CT) DNA in the order: 3 (dppz) > 2 (dpq) > 1 (phen). The binding data reveal the groove and/or partial intercalative DNA binding nature of the complexes. The complexes show chemical nuclease'' activity in the dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide via a hydroxyl radical pathway. The dpq and dppz complexes are efficient photocleavers of DNA in UV-A light of 365 nm forming reactive singlet oxygen (O-1(2)) and hydroxyl radical ((OH)-O-center dot) species. Complexes 2 and 3 also show DNA cleavage activity in red light (> 750 nm) by an exclusive (OH)-O-center dot pathway. The complexes display a binding propensity to bovine serum albumin (BSA) protein giving K-BSA values in the range of 7.1 x 10(4)-1.8 x 10(5) M-1. The dppz complex 3 shows BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via (OH)-O-center dot pathway. The dppz complex 3 exhibits significant PDT effect in human cervical cancer HeLa cells giving IC50 values of 1.0 mu M and 12.0 mu M in UV-A and visible light, respectively (IC50 = > 100 mu M in the dark).

Identification of carbohydrate structures as receptors for localised adherent enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli strains of diffused adherent (DA) and localised adherent (LA) phenotypes were tested for their ability to bind to glycolipids. DA strains did not bind to the glycolipids tested, while LA strains bound to asialo GM1, asialo GM2, globoside and lacto-N-neotetraose in decreasing order of avidity. The minimum common sequence among the four glycolipids could be delineated as GalNac β 1–4 Gal as the binding epitope with GalNac β 1–3 Gal and GlcNac β 1–3 Gal serving as relatively weaker binders. The binding was not inhibited by a variety of free oligosaccharides or by the neoglycoproteins tested. Adhesion-negative mutants of an enteropathogenic LA strain showed a markedly reduced binding to asialo GM1 indicating that the recognition of GalNac β 1–4 Gal was correlated with the ability to adhere to HeLa cells. Thus recognition and binding to glycolipids could play an important role in colonisation through adherence to intestinal surfaces.

Photocytotoxic Lanthanum(III) and Gadolinium(III) Complexes of Phenanthroline Bases Showing Light-Induced DNA Cleavage Activity

Lanthanide complexes of formulation [La(B)(2)(NO3)(3)] (1-3) and [Gd(B)(2)(NO3)(3)] (4-6), where B is a N,N-donor phenanthroline base, namely, 1,10-phenanthroline (phen in 1, 4),dipyrido[3,2-d2',3'-f]quinoxaline (dpq in 2,5) and dipyrido[3,2-a2',3'-c]phenazine (dppz in 3, 6), have been prepared, characterized from physicochemical data, and their photoinduced DNA and protein cleavage activity studied The photocytotoxicity of the dppz complexes 3 and 6 has been studied using HeLa cancer cells. The complexes exhibitligand centered bands in the UV region The dppz complexes show thelowest energy band at 380 nm in N,N-dimethylformamide (DMF) The La(III)complexes are diamagnetic. The Gd(III) complexes (4-6) have magneticmoments that correspond to seven unpaired electrons The complexes are1(.)1 electrolytic in aqueous DMF The dpq and dppz complexes in DMFshow ligand-based reductions. The complexes display moderate binding propensity to calf thymus DNA giving binding constant values in the range of 5.7 x 10(4)-5.8 x 10(5) M-1 with a relative order. 3, 6 (dppz)> 2, 5 (dpq) > 1, 4 (phen) The binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes do not show any hydrolytic cleavage of plasmid supercoiled pUC19 DNA. The dpq and dppz complexes efficiently cleave SC DNA to its nicked circular form onexposure to UV-A light of 365 nm at nanomolar complex concentration. Mechanistic studies reveal the involvement of singlet oxygen (O-1(2)) and hydroxyl radical (HO center dot) as the cleavage active species.The complexes show binding propensity to bovine serum albumin (BSA)protein giving K-BSA values of similar to 10(5) M-1. The dppz complexes 3 and 6 show BSA protein cleavage activity in UV-A light of 365 nm The dppz complexes 3 and 6 exhibit significant photocytotoxicity in HeLa cells giving respective IC50 values of 341 nM and 573 nM in UV-A light of 365 nm for an exposure time of 15 min (IC50 > 100 mu M in dark for both the complexes) Control experiments show significant dark and phototoxicity of the dppz base alone (IC50 = 413 nM in light with 4 h incubation in dark and 116 mu M in dark with 24 h incubation). A significant decrease in the dark toxicity of the dppz base is observedon binding to the lanthanide ions while retaining similar phototoxicity.

Functions of Alphavirus Macrodomain-containing protein nsP3

Alphaviruses are positive strand RNA viruses that replicate in association with cellular membranes. The viral RNA replication complex consists of four non-structural proteins nsP1-nsP4 which are essential for viral replication. The functions of nsP1, nsP2 and nsP4 are well established, but the roles of nsP3 are mainly unknown. In this work I have clarified some of the functions of nsP3 in order to better understand the importance of this protein in virus replication. Semliki Forest virus (SFV) has been mostly used as a model alphavirus during this work, but some experiments have also been conducted with Sindbis and Chikungunya viruses. NsP3 is composed of three different protein domains. The N-terminus of nsP3 contains an evolutionarily conserved macrodomain, the central part of nsP3 contains a domain that is only found in alphaviruses, and the C-terminus of the protein is hypervariable and predicted to be unstructured. In this work I have analyzed the functions of nsP3 macrodomain, and shown that viral macrodomains bind poly(ADP-ribose) and that they do not resemble cellular macrodomains in their properties. Furthermore, I have shown that some macrodomains, including viral macrodomains of SFV and hepatitis E virus, also bind poly(A). Mutations in the ligand binding pocket of SFV macrodomain hamper virus replication but do not confer lethality, indicating that macrodomain function is beneficial but not mandatory for virus replication. The hypervariable C-terminus of nsP3 is heavily phosphorylated and is enriched in proline residues. In this work it is shown that this region harbors an SH3 domain binding motif (Sh3BM) PxRxPR through which cellular amphiphysin is recruited to viral replication sites and to nsP3 containing cytoplasmic aggregate structures. The function of Sh3BM was destroyed by a single point mutation, which led to impaired viral RNA replication in HeLa cells, pointing out the functional importance of amphiphysin recruitment by the Sh3BM. In addition, evidence is provided tho show that the endosomal localization of alphavirus replication is mediated by nsP3 and that the phosphorylation of hypervariable region might be important for the endosomal targeting. Together these findings demonstrate that nsP3 contains multiple important host interaction motifs and domains, which facilitate successful viral propagation in host cells.

Ferrocene-Promoted Photoactivated DNA Cleavage and Anticancer Activity of Terpyridyl Copper(II) Phenanthroline Complexes

Ferrocene-appended copper(II) complexes [Cu( Fc-tpy)(B)](ClO4)(2) (1-3) and [Cu(Ph-tpy)(dppz)](ClO4)(2) (4) as control, where Fc-tpy is 4'-ferroceny1-2,2':6',2 ''-terpyridine, Ph-tpy is 4'-pheny1-2,2':6',2 ''-terpyridine, and B is a phenanthroline base, viz., 1,10-phenanthroline (phen, 1), dipyridoquinoxaline (dpq, 2), and dipyridophenazine (dppz, 3), were prepared and structurally characterized, and their DNA binding, photoactivated DNA cleavage activity, and cytotoxic properties were studied [Fe = (eta(5)-C5H4)Fe-11(eta(5)-C5H5)]. Complexes 1 and 3 as hexafluorophosphate salts were structurally characterized by X-ray crystallography. Molecular structures of [Cu(Fc-tpy)(phen)](PF6)(2) (1a) and [Cu(Fc-tpy)(dppz)](PF6)(2)center dot MeCN (3a center dot MeCN) show a distorted square-pyramidal geometry at copper(II), with the Fc-tpy ligand and the phenanthroline base showing respective tridentate and bidentate binding modes. The phenanthroline base exhibits axial-equatorial bonding, while the Fc-tpy ligand binds at the basal plane. The complexes showed quasi-reversible cyclic voltammetric responses near 0.45 and -0.3 V vs SCE in aqueous DMF-0.1 M KCl assignable to the Fc(+)-Fc and Cu(II) Cu(1) redox couples, respectively. The complexes bind to DNA, giving K-b values of 1.4 x 10(4) to 5.6 x 10(5) M-1 in the order 4 similar to 3 > 2 > 1. Thermal denaturation and viscometric titration data suggest groove and/or partial intercalative mode of DNA binding of the complexes. The complexes showed chemical nuclease activity in the presence of 3-mercaptopropionic acid (0.5 mM) or H2O2 (0.25 mM). Complexes 2-4 showed plasmid DNA cleavage activity in visible light, forming (OH)-O-center dot radicals. The Fc-tpy complex 3 showed better DNA photocleavage activity than its Ph-tpy analogue. The ferrocene moiety in the dppz complex 3 makes it more photocytotoxic than the Ph-tpy analogue 4 in HeLa cells.

Identification of TGFβ signaling, p53, and actin stress fibers as targets of LKB1 tumor suppressor activity

Tumorigenesis is a consequence of inactivating mutations of tumor suppressor genes and activating mutations of proto-oncogenes. Most of the mutations compromise cell autonomous and non-autonomous restrains on cell proliferation by modulating kinase signal transduction pathways. LKB1 is a tumor suppressor kinase whose sporadic mutations are frequently found in non-small cell lung cancer and cervical cancer. Germ-line mutations in the LKB1 gene lead to Peutz-Jeghers syndrome with an increased risk of cancer and development of benign gastrointestinal hamartomatous polyps consisting of hyperproliferative epithelia and prominent stromal stalk composed of smooth muscle cell lineage cells. The tumor suppressive function of LKB1 is possibly mediated by 14 identified LKB1 substrate kinases, whose activation is dependent on the LKB1 kinase complex. The aim of my thesis was to identify cell signaling pathways crucial for tumor suppression by LKB1. Re-introduction of LKB1 expression in the melanoma cell line G361 induces cell cycle arrest. Here we demonstrated that restoring the cytoplasmic LKB1 was sufficient to induce the cell cycle arrest in a tumor suppressor p53 dependent manner. To address the role of LKB1 in gastrointestinal tumor suppression, Lkb1 was deleted specifically in SMC lineage in vivo, which was sufficient to cause Peutz-Jeghers syndrome type polyposis. Studies on primary myofibroblasts lacking Lkb1 suggest that the regulation of TGFβ signaling, actin stress fibers and smooth muscle cell lineage differentiation are candidate mechanisms for tumor suppression by LKB1 in the gastrointestinal stroma. Further studies with LKB1 substrate kinase NUAK2 in HeLa cells indicate that NUAK2 is part of a positive feedback loop by which NUAK2 expression promotes actin stress fiber formation and, reciprocally the induction of actin stress fibers promote NUAK2 expression. Findings in this thesis suggest that p53 and TGFβ signaling pathways are potential mediators of tumor suppression by LKB1. An indication of NUAK2 in the promotion of actin stress fibers suggests that NUAK2 is one possible mediator of LKB1 dependent TGFβ signaling and smooth muscle cell lineage differentiation.

The function of NR3B and NR4A orphan nuclear receptors in osteoblasts

Nuclear receptors (NRs) comprise a large family of proteins that mediate the effects of small lipophilic molecules such as steroid hormones. In addition, there are a group of NRs which lack identified natural ligands and are referred as orphan NRs. In this thesis, the function of two such orphan NR families, the NR3B (ERRα, ERRβ and ERRγ) and the NR4A family (NGFI-B, Nurr1 and Nor1), was studied. NR3B and NR4A receptors regulate many biological processes such as energy metabolism and carcinogenesis. In addition, NR3B and NR4A receptors are expressed in bone. Therefore, the signaling and function of NR3B and NR4A orphan nuclear receptors was studied specifically in osteoblasts. NR4A receptors were found to be regulated by NR3B receptors and the Wnt/β-catenin signaling pathway as ERRα, ERRγ and β-catenin repressed the transcriptional activity of NR4A receptors in U2-OS cells. NGFI-B was found to repress the transcriptional activity of ERRγ in HeLa cells. The phytoestrogen equol was identified as a new agonist for ERRγ and ERRβ in PC-3, U2-OS, and SaOS-2 cells. Equol increased the transcriptional activity of ERRγ by increasing ERRγ co-activator binding and by inducing a conformational change in the ligand binding pocket of ERRγ. The growth inhibitory effect of equol on PC-3 prostate cancer cells was decreased by blocking ERRγ expression by siRNA. Therefore, ERRγ could mediate some of the beneficial health effects of equol. The Wnt/β-catenin signaling pathway is important for the differentiation and function of osteoblasts. NR3B and NR4A receptors were found to repress the transcriptional activity mediated by β-catenin in U2-OS cells. The mesenchymal stem cells (MSCs) isolated from ERRα knockout (KO) mice showed diminished proliferation and osteoblastic differentiation compared to the wild-type cells. The overexpression of ERRα in osteoblastic MC3T3-E1 cell line increased their mineralization. Bone sialoprotein (BSP) was shown to be a direct target gene for ERRα and ERRγ as the BSP promoter was activated by ERRα or ERRγ and PGC-1α in HeLa cells. The adipogenic differentiation of ERRα KO MSCs was also decreased and they expressed less adipogenic marker genes. In conclusion, the studies described in this thesis demonstrated that the transcriptional activity of NR3B and NR4A receptors can be regulated by other orphan NRs and signaling pathways in osteoblasts. NR3B receptors can also be regulated by ligands and a new agonist, equol, was identified for ERRβ and ERRγ. New roles for NR3B and NR4A were also identified as they were shown to converge with the Wnt signaling pathway in osteoblasts, ERRγ was shown to mediate the growth inhibitory effect of equol in prostate cancer cells, and ERRα was shown to regulate positively MSC proliferation, osteoblastic differentiation and adipogenesis.

Photocytotoxicity and near-IR light DNA cleavage activity of oxovanadium(IV) Schiff base complexes having phenanthroline bases

Oxovanadium(IV) complexes VO(L)(B)] (1-3), where H2L is a Schiff base ligand 2-(2-hydroxybenzylideneamino) phenol and B is 1,10-phenanthroline (phen for 1), dipyrido3,2-d:2',3'-f]quinoxaline (dpq for 2) or dipyrido3,2-a:2',3'-c]phenazine (dppz for 3), have been prepared, characterized and their DNA binding property and photo-induced DNA cleavage activity studied. Complex 3 which is structurally characterized by X-ray crystallography shows the presence of an oxovanadium(IV) moiety in a six coordinate VO3N3 coordination geometry. The complexes show a d-d band within 800-850 nm in DMF. The complexes display an oxidative response near 0.7 V versus SCE for V(V)-V(IV) and a reductive response within -1.1 to -1.3 V due to V(IV)-V(III) couple in DMF-0.1 M TBAP. The complexes are avid binders to calf thymus DNA giving binding constant values of 4.2 x 10(4) to 1.2 x 10(5) M (1). The complexes do not show any ``chemical nuclease'' activity in dark. The dpq and dppz complexes are photocleavers of plasmid DNA in UV-A light of 365 nm via O-1(2) pathway and in near-IR light (752.5 to 799.3 nm IR optics) by HO* pathway. Complex 3 exhibits significant photocytotoxicity in visible light in HeLa cells giving IC50 value of 13 mu M, while it is less toxic in dark (IC50 = 97 mu M). (C) 2010 Elsevier B.V. All rights reserved.

Antimicrobial activity of highly stable silver nano particles embedded in agar-agar matrix as a thin film

Highly stable silver nanoparticles (Ag NPs) in agar-agar (Ag/agar) as inorganic-organic hybrid were obtained as free-standing film by in situ reduction of silver nitrate by ethanol. The antimicrobial activity of Ag/agar film on Escherichia coli (E. coil), Staphylococcus aureus (S. aureus), and Candida albicans (C albicans) was evaluated in a nutrient broth and also in saline solution. In particular, films were repeatedly tested for antimicrobial activity after recycling. UV-vis absorption and TEM studies were carried out on films at different stages and morphological studies on microbes were carried out by SEM. Results showed spherical Ag NPs of size 15-25 nm, having sharp surface plasmon resonance (SPR) band. The antimicrobial activity of Ag/agar film was found to be in the order, C. albicans > E. coil > S. aureus, and antimicrobial activity against C. albicans was almost maintained even after the third cycle. Whereas, in case of E. coil and S. aureus there was a sharp decline in antimicrobial activity after the second cycle. Agglomeration of Ag NPs in Ag/agar film on exposure to microbes was observed by TEM studies. Cytotoxic experiments carried out on HeLa cells showed a threshold Ag NPs concentration of 60 mu g/mL, much higher than the minimum inhibition concentration of Ag NPs (25.8 mu g/mL) for E. coli. The mechanical strength of the film determined by nanoindentation technique showed almost retention of the strength even after repeated cycle. (C) 2010 Elsevier Ltd. All rights reserved.

Cobalt(II) complexes of terpyridine bases as photochemotherapeutic agents showing cellular uptake and photocytotoxicity in visible light

Cobalt(II) complexes of terpyridine bases Co(L)(2)](ClO4)(2) (1-3), where L is 4'-phenyl-2,2':6',2''-terpyridine (ph-tpy in 1), 4'-(9-anthracenyl)-2,2':6',2''-terpyridine (an-tpy in 2) and 4'-(1-pyrenyl)-2,2':6',2''-terpyridine (py-tpy in 3), are prepared and their photo-induced DNA and protein cleavage activity and photocytotoxic property in HeLa cells studied. The 1 : 2 electrolytic and three-electron paramagnetic complexes show a visible band near 550 nm in DMF-Tris-HCl buffer. The complexes 1-3 show emission spectral bands at 355, 421 and 454 nm, respectively, when excited at 287, 368 and 335 nm. The quantum yield values for 1-3 in DMF-H2O (2 : 1 v/v) are 0.025, 0.060 and 0.28, respectively. The complexes are redox active in DMF-0.1 M TBAP. The Co(III)-Co(II) and Co(II)-Co(I) couples appear as quasi-reversible cyclic voltammetric responses near 0.2 and -0.7 V vs. SCE, respectively. Complexes 2 and 3 are avid binders to calf thymus DNA giving K-b value of similar to 10(6) M-1. The complexes show chemical nuclease activity. Complexes 2 and 3 exhibit oxidative cleavage of pUC19 DNA in UV-A and visible light. The DNA photocleavage reaction of 3 at 365 nm shows formation of singlet oxygen and hydroxyl radical species, while only hydroxyl radical formation is evidenced in visible light. Complexes 2 and 3 show non-specific photo-induced bovine serum albumin protein cleavage activity at 365 nm. The an-tpy and py-tpy complexes exhibit significant photocytotoxicity in HeLa cervical cancer cells on exposure to visible light giving IC50 values of 24.2 and 7.6 mu M, respectively. Live cell imaging study shows accumulation of the complexes in the cytosol of HeLa cancer cells.

DNA Photocleavage and Cytotoxic Properties of Ferrocene Conjugates

Ferrocenyl conjugates 2-ferrocenylimidazophenanthroline (1) and 2-ferrocenylimidazophenanthrene (2) were prepared, characterized, and their photoinduced DNA cleavage and photocytotoxic activity were studied. 2-Phenylimidazophenanthroline (3) was used as a control species. Compound 2 was characterized by X-ray crystallography. The interaction of the compounds with double-stranded calf thymus DNA (CT DNA) was studied. The compounds show good binding affinity to CT DNA with K-b values of approximately 10(5) M-1. Thermal denaturation data suggest the groove binding nature of the compounds. The redox-active compounds show poor chemical nuclease activity in the presence of hydrogen peroxide and glutathione (GSH). Compound 1 exhibits significant DNA photocleavage activity in visible light of 476 and 532 nm. Compound 3 shows only moderate DNA cleavage activity. The positive effect of the ferrocenyl moiety is demonstrated by the DNA photocleavage data. Mechanistic investigations reveal the formation of superoxide as well as hydroxyl radicals as the active species. The photocytotoxicity of the compounds in HeLa cells was studied upon irradiation with visible light (400-700 nm). Compound 1 shows efficient photocytotoxic activity with an IC50 value of 13 mu M, while compounds 2 and 3 are less active with IC50 values of > 50 and 22 mu M, respectively.

Structure-Activity Relationship of Photocytotoxic Iron(III) Complexes of Modified Dipyridophenazine Ligands

Iron(III) complexes FeL(B)] (1-5) of a tetradentate trianionic phenolate-based ligand (L) and modified dipyridophenazine bases (B), namely, dipyrido-6,7,8,9-tetrahydrophenazine (dpqC in 1), dipyrido3,2-a:2',3'-c]phenazine-2-carboxylic acid (dppzc in 2), dipyrido3,2-a:2',3'-c]phenazine-11-sulfonic acid (dppzs in 3), 7-aminodipyrido3,2-a:2',3'-c]phenazine (dppza in 4) and benzoi]dipyridro3,2-a:2',3'-c]phenazine (dppn in 5), have been synthesized, and their photocytotoxic properties studied along with their dipyridophenazine analogue (6). The complexes have a five. electron paramagnetic iron(III) center, and the Fe(III)/Fe(II) redox couple appears at about 0.69 V versus SCE in DMF-0.1 M TBAP. The physicochemical data also suggest that the complexes possess similar structural features as that of its parent complex FeL(dppz)] with FeO3N3 coordination in a distorted octahedral geometry. The DNA-complex and protein-complex interaction studies have revealed that the complexes interact favorably with the biomolecules, the degree of which depends on the nature of the substituents present on the dipyridophenazine ring. Photocleavage Of pUC19 DNA by the complexes has been studied using visible light of 476, 530, and 647 nm wavelengths. Mechanistic investigations with inhibitors show formation of HO center dot radicals via a photoredox pathway. Photocytotoxicity study of the complexes in HeLa cells has shown that the dppn complex (5) is highly active in causing cell death in visible light with sub micromolar IC50 value. The effect of substitutions and the planarity of the phenazine moiety on the cellular uptake are quantified by determining the total Cellular iron content using the inductively coupled plasma-optical emission spectrometry (ICP-OES) technique. The cellular uptake increases marginally with an increase in the hydrophobicity of the dipyridophenazine ligands whereas complex 3 with dppzs shows very high uptake. Insights into the cell death mechanism by the dppn complex 5, obtained through DAFT nuclear staining in HeLa cells, reveal a rapid programmed cell death mechanism following photoactivation of complex 5 with visible light. The effect of substituent on the DNA photocleavage activity of the complexes has been rationalized from the theoretical studies.

Schiff base oxovanadium(IV) complexes of phenanthroline bases showing DNA photocleavage activity at near-IR light and photocytotoxicity

Oxovanadium(IV) complexes VO(L)(B)](ClO4) (1-3) of N-2-pyridylmethylidine-2-hydroxyphenylamine (HL) Schiff base and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido3,2-d: 2',3'-f] quinoxaline (dpq in 2) or dipyrido3,2-a: 2',3'-c] phenazine (dppz in 3), were prepared, characterized and their DNA binding property, photo-induced DNA cleavage activity and photocytotoxicity in HeLa cells studied. The crystal structure of 1 shows the presence of a VO2+ moiety in VO2N4 coordination geometry. The complexes show a d-d band at similar to 830 nm in DMF. The complexes display an oxidative V(V)-V(IV) response near 0.5 V versus SCE and a reductive V(IV)/V(III) response near -0.65 V in DMF -0.1 M TBAP. The complexes that are avid binders to CT DNA giving K-b values within 7.1 x 10(4) to 3.2 x 10(5) M-1, do not show any significant chemical nuclease activity in presence of 3-mercaptopropionic acid or glutathione. The dpq and dppz complexes are photocleavers of pUC19 DNA in UV-A light of 365 nm forming both O-1(2) and (OH)-O-center dot radicals and in near-IR light of 785 nm forming (OH)-O-center dot radicals. The dppz complex exhibits photocytotoxicity in visible light in HeLa cells (IC50 = 6.8 mu M). Flow-cytometric study on this complex shows a high sub-G1 phase in light compared to dark indicating PDT effect. (C) 2011 Elsevier B. V. All rights reserved.

Ferrocene-Conjugated L-Tryptophan Copper(II) Complexes of Phenanthroline Bases Showing DNA Photocleavage Activity and Cytotoxicity

Ferrocene-conjugated L-tryptophan (L-Trp) reduced Schiff base (Fc-TrpH) copper(II) complexes [Cu(Fc-Trp)(L)](ClO(4)) of phenanthroline bases (L), viz. 2,2'-bipyridine (bpy in 1), 1,10-phenanthroline (phen in 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in 3), and dipyrido[3,2-a:2',3'-c]phenazine (dppz in 4), were prepared and characterized and their photocytotoxicity studied. Cationic reduced Schiff base (Ph-TrpH) complexes [Cu(Ph-Trp)(L)(H(2)O)] (ClO(4)) (L = phen in 5; dppz in 6) having the ferrocenyl moiety replaced by a phenyl group and the Zn(II) analogue (7) of complex 4 were prepared and used as control species. The crystal structures of 1 and 5 with respective square-planar CuN(3)O and square-pyramidal CuN(3)O(2) coordination geometry show significantly different core structures. Complexes 1-4 exhibit a Cu(II)-Cu(I) redox couple near -0.1 V and the Fc(+)-Fc couple at similar to 0.5 V vs SCE in DMF-0.1 M [Bu(4)(n)N] (ClO(4)) (Fc = ferrocenyl moiety). The complexes display a copper(II)-based d-d band near 600 nm and a Fc-centered band at similar to 450 nm in DMF-Tris-HCl buffer. The complexes are efficient binders to calf thymus DNA. They are synthetic chemical nucleases in the presence of thiol or H(2)O(2), forming hydroxyl radicals. The photoactive complexes are cleavers of pUC19 DNA in visible light, forming hydroxyl radicals. Complexes 2-6 show photocytotoxicity in HeLa cancer cells, giving IC(50) values of 4.7, 10.2, 1.3, 4.8, and 4.3 mu M, respectively, in visible light with the appearance of apoptotic bodies. The complexes also show photocytotoxicity in MCF-7 cancer cells. Nuclear chromatin cleavage has been observed with acridine orange/ethidium bromide (AO/EB) dual staining with complex 4 in visible light. The complexes induce caspase-independent apoptosis in the HeLa cells.