Among the second-generation photosensitizers (PS) developed for the treatment of neoplastic diseases by photodynamic therapy (PDT) (Kessel, 1996;Ochsner, 1997), metallo-phthalocyanines (MePc) have been proposed (Spikes, 1986;van Lier, 1990;Rosenthal, 1991) as an alternative to Photofrin (PII), the only PS currently approved for clinical use. Their stronger absorption in the red part of the spectrum (molar extinction coefficient, 2.5 × 10 5 M -1 cm -1 at 675 nm), where the depth of light penetration in tissues is twice that obtained at 630 nm with PII (Svaasand, 1984), together with their chemical homogeneity and their lower potential to induce cutaneous photosensitivity (Roberts et al, 1989;Tralau et al, 1989) are major advantages over PII.Unsubstituted MePc are not soluble in physiological solvents and their in vivo administration relies upon their incorporation into carriers, such as the liposomal formulation of ZnPc (Isele et al, 1995), or their chemical conversion into water-soluble dyes by the attachment of selected substituents onto the benzene rings of the macrocycle. An inverse relationship was related between the degree of substitution by sulphonato groups of MePc and their hydrophobicity and photodynamic activities, both at the cellular level and in tumour-bearing mice (Brasseur et al, 1987(Brasseur et al, , 1988Paquette et al, 1988;Berg et al, 1989;Chan et al, 1990Chan et al, , 1991Margaron et al, 1996a). The potential of the water-soluble aluminium sulphophthalocyanine (AlPcS) to generate activated oxygen species (Sharman et al, 1999) and to induce a photodynamic response in vivo has been widely evaluated, and a mixture of AlPcS bearing 2-4 sulphonato groups per Pc (Photosense) is used extensively and successfully in clinical PDT in Russia (Zharkova et al, 1994). Furthermore, the di-sulphonated derivative has been shown to induce tumour regression mainly via direct tumour cell kill rather than damage to the tumour vasculature, as observed for PII (Chan et al, 1996;Margaron et al, 1996b). The unsubstituted AlClPc has, however, attracted less attention even though it has been shown that this compound formulated as a Cremophor emulsion was preferentially retained by a gliosarcoma and was able to induce tumour necrosis in this model (Dereski et al, 1994). More recently, we demonstrated that the Cremophorformulated AlClPc was more effective in inducing tumour regression in EMT-6 tumour-bearing mice than the mono-through tetrasulphonated derivatives, while exerting relatively minor effects against normal tissues (Chan et al, 1997). The low dosage required together with the absence of systemic toxicity, even at much higher doses, renders the AlClPc Cremophor emulsion one of the most potent photosensitizer preparations currently available in terms of therapeutic window. However, Cremophor oil is known to induce unwanted side-effects in patients (Dye and Watkins, Summary The potential use of unsubstituted aluminium phthalocyanine (AlClPc) as a sensitizer for photodynamic therapy (PDT) of cancer has not been f...
To identify optimal features of metalated sulfophthalocyanine dyes for their use as photosensitizers in the photodynamic therapy of cancer, we synthesized a series of alkynyl-substituted trisulfonated phthalocyanines and compared their amphiphilic properties to a number of parameters related to their photodynamic potency. Varying the length of the substituted alkynyl side-chain modulates the hydrophobic/hydrophilic properties of the dyes providing a linear relationship between their n-octanol/water partition coefficients and retention times on reversed-phase HPLC. Aggregate formation of the dyes in aqueous solution increased with increasing hydrophobicity while monomer formation was favored by the addition of serum proteins or organic solvent. Trisulfonated zinc phthalocyanines bearing hexynyl and nonynyl substituents exhibited high cellular uptake with strong localization at the mitochondrial membranes, which coincided with effective photocytotoxicity toward EMT-6 murine mammary tumor cells. Further increase in the length of the alkynyl chains (dodecynyl, hexadecynyl) did not improve their phototoxicity, likely resulting from extensive aggregation of the dyes in aqueous medium and reduced cell uptake. Aggregation was evident from shifts in the electronic spectra and reduced capacity to generate singlet oxygen. When monomerized through the addition of Cremophor EL all sulfonated zinc phthalocyanines gave similar singlet oxygen yields. Accordingly, differences in the tendency of the dyes to aggregate do not appear to be a determining factor in their photodynamic potency. Our results confirm that the latter in particular relates to their amphiphilic properties, which facilitate cell uptake and intracellular localization at photosensitive sites such as the mitochondria. Combined, these factors play a significant role in the overall photodynamic potency of the dyes.
DNA interstrand cross-links (ICL) can be induced both by natural products (e.g., psoralens with UVA) and by chemical agents, some of which are used in chemotherapy (e.g., Carboplatin and mitomycin C). Here, we report the formation of ICL by UV radiation in brominated DNA, but only for very specific conformations. The quantum yields for strand break and cross-link formation depend on the wavelength with a maximum near 280 nm. It is known that the photosensitization of DNA by bromodeoxyuridine (BrdUrd) results mainly from the electron affinity of bromine, leading to the irreversible formation of 2'-deoxyuridin-5-yl radicals (dUrd*) upon the addition of an electron from an adjacent adenosine. It is well documented that the photolytic loss of the bromine atom is greatly suppressed in single-stranded DNA versus that in double-stranded DNA. To study this behavior, we have used two models of BrdUrd-mediated sensitization: one consists of a DNA duplex containing a bulge, formed by five mismatched bases, including the BrdUrd, and the other consists of completely duplex DNA. UV irradiation induces much higher levels of single-strand breaks (ssb) in the completely duplex DNA at the BrdUrd site compared to the DNA with a bulge. However, in completely duplex DNA, ssb appear only in the brominated strand, whereas in the bulged duplex DNA, ssb occur on both strands. Most importantly, we also observe formation of interstrand cross-links in bulged duplex DNA in the BrdUrd region. Thus, we propose that UV irradiation of cells containing BrdUrd incorporated randomly into duplex DNA will create many ssb, whereas BrdUrd present in DNA bulges or open regions in double-stranded DNA (transcription bubbles, replication forks) will lead to potentially lethal damage in both strands in the form of ICL. These findings may help explain the potent clinical antiviral activity of IdUrd and BrdUrd (e.g., IdUrd is used to treat eye infections caused by the herpes virus) and suggest that ICL formation may be a very specific probe for identifying single-stranded regions in the DNA of living cells. In addition, this model system provides an excellent means of introducing ICL for studies on their repair and biological consequences.
Although ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ domain-containing protein known to bind to various channels, receptors, cytoskeletal elements, and cytoplasmic proteins, there is still very little evidence for a role of EBP50 in the regulation of receptor signal transduction. In this report, we show that EBP50 inhibits the phospholipase C (PLC)--mediated inositol phosphate production of a G␣ q -coupled receptor as well as PLC- activation by the constitutively active G␣ q -R183C mutant. Coimmunoprecipitation experiments revealed that EBP50 interacts with G␣ q and to a greater extent with G␣ q -R183C. Agonist stimulation of the thromboxane A 2 receptor (TP receptor) resulted in an increased interaction between EBP50 and G␣ q , suggesting that EBP50 preferentially interacts with activated G␣ q . We also demonstrate that EBP50 inhibits G␣ q signaling by preventing the interaction between G␣ q and the TP receptor and between activated G␣ q and PLC-1. Investigation of the EBP50 regions involved in G␣ q binding indicated that its two PDZ domains are responsible for this interaction. This study constitutes the first demonstration of an interaction between a G protein ␣ subunit and another protein through a PDZ domain, with broad implications in the regulation of diverse physiological systems. EBP501 (also known as NHERF1), a 55-kDa phosphoprotein, was first identified as a cofactor essential for protein kinase A-mediated inhibition of Na ϩ /H ϩ exchanger isoform 3 (NHE3) (1). EBP50 contains two PDZ domains (PDZ1 and PDZ2) implicated in multiple protein-protein interactions, and an ERM domain, which binds to the actin-associated ERM proteins (ezrin, radixin, moesin, and merlin) (2, 3). EBP50 was also found to interact with a small number of transmembrane proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) (4), the P2Y1 purinergic receptor (5), the platelet-derived growth factor receptor (6), the  2 -adrenergic receptor (5), the B1 subunit of the H ϩ -ATPase (7), and the type IIa sodium phosphate cotransporter (8). EBP50 also associates with the phospholipases C (PLC)-1/2, and with the TRP4 and TRP5 calcium channels to form a PLC-1/2-TRP4/5-EBP50 protein complex (9). The physiological role of this interaction on the regulation of PLC-1/2 remains undefined. The EBP50 protein can also bind through its PDZ domains to various intracellular proteins, including GRK6A (10), EPI64 (11), and Yes-associated protein 65 (12). A close relative of EBP50 has been identified and is known as E3KARP (13), SIP-1 (14), and NHERF2 (15). EBP50 and NHERF2 share 52% amino acid identity and a conserved domain architecture (13). It has been shown recently that the PDZ domains of EBP50 can homooligomerize and also hetero-oligomerize with the PDZ domains of NHERF2 (16). Despite the growing evidence suggesting the role of EBP50 as a scaffolding protein involved in the formation of signaling complexes, there is still little evidence for a role of EBP50 in the regulation of transmembrane recep...
Targeted delivery of aluminum tetrasulfophthalocyanine (AlPcS4) to the scavenger receptor of macrophages, via coupling to maleylated bovine serum albumin (mal-BSA), was explored as a means to improve photodynamic efficacy. The AlPcS4 was covalently coupled to BSA (9:1 molar ratio) via one or two sulfonamide-hexanoic-amide spacer chains, followed by treatment with maleic anhydride to yield the mal-BSA-phthalocyanine conjugates. The latter were tested for singlet oxygen production, receptor-mediated cell uptake and phototoxicity toward J774 cells of macrophage origin and nonphagocytic EMT-6 cells. Cell uptake of 125I-mal-BSA showed specific binding for J774 cells but not for EMT-6 cells. Competition studies of the conjugates with 125I-mal-BSA showed that coupling of AlPcS4 to BSA resulted in recognition of the conjugate by the scavenger receptor, whereas coupling to mal-BSA further enhanced its binding affinity. This suggests that affinity for the scavenger receptor is related to the overall negative charge of the protein. Phototoxicity of the conjugates toward J774 cells paralleled their relative affinity, with mal-BSA-AlPcS4 coupled via two spacer chains showing the highest activity. The conjugates were less phototoxic toward the EMT-6 cell line. The activities in both cell lines of all conjugated AlPcS4 preparations were, however, lower than that of the free disulfonated AlPcS2. Possible implications for the in vivo use of protein-photosensitizer conjugates to target selectively various macrophage-associated disorders is discussed.
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