In Vitro and In Vivo Demonstration of Human-Ovarian-Cancer Necrosis through a Water-Soluble and Near-Infrared-Absorbing Chlorin.

With the objective of developing efficient sensitizers for therapeutic applications, we synthesized a water-soluble 5,10,15,20-tetrakis(3,4-dihydroxyphenyl)chlorin (TDC) and investigated its in vitro and in vivo biological efficacy, comparing it with the commercially available sensitizers. TDC showed high water solubility (6-fold) when compared with that of Foscan and exhibited excellent triplet-excited-state (84%) and singlet-oxygen (80%) yields. In vitro photobiological investigations in human-ovarian-cancer cell lines SKOV-3 showed high photocytotoxicity, negligible dark toxicity, rapid cellular uptake, and specific localization of TDC in neoplastic cells as assessed by flow-cytometric cell-cycle and propidium iodide staining analysis. The photodynamic effects of TDC include confirmed reactive-oxygen-species-induced mitochondrial damage leading to necrosis in SKOV-3 cell lines. The in vivo photodynamic activity in nude-mouse models demonstrated abrogation of tumor growth without any detectable pathology in the skin, liver, spleen, or kidney, thereby demonstrating TDC application as an efficient and safe photosensitizer.

In vitro demonstration of apoptosis mediated photodynamic activity and NIR nucleus imaging through a novel porphyrin.

We synthesized a novel water-soluble porphyrin THPP and its metalated derivative Zn-THPP having excellent triplet excited state quantum yields and singlet oxygen generation efficiency. When compared to U.S. Food and Drug Administration approved and clinically used sensitizer Photofrin, THPP showed ca. 2-3-fold higher in vitro photodynamic activity in different cell lines under identical conditions. The mechanism of the biological activity of these porphyrin systems has been evaluated through a variety of techniques: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, comet assay, poly(ADP-ribose)polymerase (PARP) cleavage, CM-H(2)DCFDA assay, DNA fragmentation, flow cytometric analysis, fluorescence, and confocal microscopy, which confirm the apoptotic cell death through predominantly reactive oxygen species (ROS). Moreover, THPP showed rapid cellular uptake and are localized in the nucleus of the cells as compared to Hoechst dye and Photofrin, thereby demonstrating its use as an efficient sensitizer in photodynamic therapy and live cell NIR nucleus imaging applications.

Exoribonuclease R in Pseudomonas syringae is essential for growth at low temperature and plays a novel role in the 3' end processing of 16 and 5 S ribosomal RNA.

The (3'-->5') exoribonuclease RNase R interacts with the endoribonuclease RNase E in the degradosome of the cold-adapted bacterium Pseudomonas syringae Lz4W. We now present evidence that the RNase R is essential for growth of the organism at low temperature (4 degrees C). Mutants of P. syringae with inactivated rnr gene (encoding RNase R) are cold-sensitive and die upon incubation at 4 degrees C, a phenotype that can be complemented by expressing RNase R in trans. Overexpressing polyribonucleotide phosphorylase in the rnr mutant does not rescue the cold sensitivity. This is different from the situation in Escherichia coli, where rnr mutants show normal growth, but pnp (encoding polyribonucleotide phosphorylase) and rnr double mutants are nonviable. Interestingly, RNase R is not cold-inducible in P. syringae. Remarkably, however, rnr mutants of P. syringae at low temperature (4 degrees C) accumulate 16 and 5 S ribosomal RNA (rRNA) that contain untrimmed extra ribonucleotide residues at the 3' ends. This suggests a novel role for RNase R in the rRNA 3' end processing. Unprocessed 16 S rRNA accumulates in the polysome population, which correlates with the inefficient protein synthesis ability of mutant. An additional role of RNase R in the turnover of transfer-messenger RNA was identified from our observation that the rnr mutant accumulates transfer-messenger RNA fragments in the bacterium at 4 degrees C. Taken together our results establish that the processive RNase R is crucial for RNA metabolism at low temperature in the cold-adapted Antarctic P. syringae.