New Nitric Oxide Donor-¦Â-galactose Based Azo-ene Weng Diol Anti-bacterial, Anti-tumor Pathways,

NO, which is a paradoxical gaseous messenger enzymatically generated in vivo by three isoforms of nitric oxide synthase (iNOS, nNOS and eNOS), plays an important role in numerous physiological and pathophysiological processes including relaxing vascular smooth muscle, inhibiting platelet aggregation, assisting the immune system in destroying tumor cells and intracellular pathogens and participating in neuronal synaptic transmission. Unfortunately, NO has extremely short half-life and is easily converted into a variety of reactive nitrogen species (RNS), such as dinitrogen trioxide (N₂O₃), nitrogen dioxide (NO₂), and the peroxynitrite anion (ONOO^-), so it is difficult to utilize this active small molecule in therapy and research. Thus site-specific delivery of exogenous NO becomes an attractive therapeutic option in the treatment of disease. Due to the instability and inconvenient handling of aqueous NO solutions, there has been an increasing interest in using NO donors capable of generating RNS in vivo by a variety of mechanisms including decomposition,oxidation, or reduction.1. β-galactosyl-pyrrolidinyl diazeniumdiolates (β-Gal-NONOate) is a newsite-specific NO-releasing compound reported by our group, which releases RNS(NO) once activated by β-galactosidase. In Chapter 2, β-Gal-NONOate was used as a bactericidal reagent to determine its effectiveness of NO releasing. Through the evaluation of intracellular NO level and the comparison of survival of E. coli transformed with lacZ gene but treated with β-Gal-NONOate and NONOate, respectively, it's evident that β-Gal-NONOate had a higher bactericidal activity than conventional NONOate. While either β-Gal-NONOate- or NONOate-treated-E. coli without transferred lacZ gene manifested low bactericidal activity. The results revealed that β-Gal-NONOate was a high effective and promising NO donor. It was presumed that β-Gal-NONOate could be easily transported into the cells via sugar transporters. Thus, once it was hydrolyzed by β-galactosidase produced inside cells, NO was readily released from the compound. Therefore it took on a novel and efficient approach to deliver RNS (NO) into cells.2. β-Gal-NONOate, which has been reported as a site-specific NO donor with high antibacterial activity(in Chapter 2) is an intriguing agent in therapy and related research. Through this study, β-Gal-NONOate was proved to be promising in overcoming prevalent antibiotic resistance of bacteria. It was applied to get rid of ampicillin resistance of E. coli K-12 with β-galactosidase activity. Bacterial susceptibility to antibiotics was evaluated with the minimum inhibitory concentration (MIC), which could be measured with E-test After successful inducement of ampicillin-resistant strains, β-Gal-NONOate and NONOate were separately applied to restore antibiotic susceptibility of bacteria through a series of sequential subcultures. The results demonstrated that β-Gal-NONOate possessed greater potential to decrease the MIC of ampicillin-resistant strains than NONOate; while combined with ampicillin for subculture, β-Gal-NONOate was able to kill off the resistant strains within fewer passages than NONOate, that is, to greatly shorten the duration of antibiotic resistance. These results further substantiated that β-Gal-NONOate was a very prospective NO donor, which could be explored and utilized not only as antimicrobial but also as a promising agent to remove increasingly serious antibiotic resistance of bacteria in current times. Based on our previous studies, it is concluded that elimination of antibiotic resistance may be due to more site-specific NO delivery into cells, accessibility of efficaciously intracellular NO level and high efficient antimicrobial roles of β-Gal-NONOate.3. The elucidation of the molecular details of antibiotic resistance will lead to improvements in extending the efficacy of current antimicrobials. Two dimensional electrophoresis(2-DE) is a key technique in studies of proteome. In Chapter 4, proteomic methodologies were applied for the comparative analysis of proteome of E. coli K-12 responded to ampicillin(Amp) resistance and Amp re-susceptivity for understanding of universal pathways that form barriers for antimicrobial agents. For this purpose, three kinds of E. coli K-12(E coli K-12 ancestor strain, E. coli K-12 Amp resistant strain, E. coli K-12 re-susceptible strain) proteome were characterized with the use of 2-DE methods. Then, differential proteins due to Amp resistance were determined by comparing with each other using PDQuest7.2 to study the influence on bacterium in proteome by β-Gal-NONOate. Our findings will be helpful for further understanding of antibiotic-resistant / overcoming resistance mechanism related to proteome. This study also confirmed that β-Gal-NONOate certainly contributes the elimination of Amp by changing the protein expression. 4. So far, nitric oxide (NO) donors have been applied to various aspects of antitumor therapy. To selectively sensitize tumor cells and avoid unwanted side effects, β-Gal-NONOate was firstly used in the cancer research. In this study, we first verified its superiority over its parent NONOate in terms of targeted intracellular NO-releasing and antitumor activity with 9L/LacZ cells in vitro. β-Gal-NONOate only released NO when hydrolyzed by β-galactosidase in 9L/LacZ cells, which led to its more powerful cytotoxicity than that of NONOate. The results showed that β-Gal-NONOate produced higher NO levels than NONOate in 9L/LacZ cells at equal concentration, and hence induced optimal NO levels for antitumor activity. However, in 9L cells, β-Gal-NONOate showed less toxicity than NONOate. Therefore, it is demonstrated that β-Gal-NONOate is a site-specific prodrug for targeting NO intracellularly as a β-galactosidase-sensitive NO donor, and it is also expected to be a promising probe in numerous experimental settings and a potential therapeutic drug for antitumor treatment.5. In order to evaluate the antitumor effects of β-Gal-NONOate in details and to clarify its mechanism to action as an antitumor prodrug, four cell lines were used in Chapter 6. Through cationic liposome-mediated transfection, the eukaryotic expression vector pcDNA3-LacZ was transferred into the HeLa cells; through G418 screening, the HeLa/LacZ cell line steadily expressing β-galactosidase was obtained. With C6/LacZ, C6, HeLa/LacZ and HeLa cells as in vitro model, β-galactosidase activities of tumor cells were determined through X-gal staining. NO levels in these four cell lines released from β-Gal-NONOate and NONOate were evaluated with Griess assay. Antitumor effects of NO donors on the four cell lines were estimated by cell counting, cell colony forming rate and MTT (Methylthiazoletetrazolium) assay, respectively. We found that NO levels released from β-Gal-NONOate in C6/LacZ and HeLa/LacZ cells were dependent on its concentration and evidently higher than that from NONOate. And similarly, its antitumor activity to C6/LacZ and HeLa/LacZ cells were obviously more powerful than NONOate(P < 0.05), but it did not exhibit conspicuous cytotoxicity to C6 cells. However, the NO released from NONOate inside the four cell lines and the effects on all kinds of cells didn't have great difference. We could draw a conclusion that β-Gal-NONOate was more stable and possessed higher antitumor activity than NONOate. In addition, its action greatly depended on β-galactosidase. It was an effective intracellularly NO release compound, which would be very promising in tumor therapy and relevant research.6. The apoptosis effects of 9L/LacZ, C6/LacZ and HeLa/LacZ cells induced by β-Gal-NONOate were investigated in Chapter 7. Typical apoptotic morphological features included cell shrinkage and condensation and margination of nuclear chromatin were showed by light microscopy. Condensed nuclear chromatin and the morphology of nuclei were demonstrated by fluorescent microscopy with AO and Hoechst 33342 staining. β-Gal-NONOate induced apoptotic DNA breaks were confirmed by a typical "DNA ladder" on agarose gel electrophoresis as well as evidenced by TUNEL assay. β-Gal-NONOate induced apoptosis in a concentration-dependent manner. The apoptotic rates of three cells were measured by flow cytometry with Annexin V-FITC and PI staining. The changes of intracellular [Ca²⁺]_i and mitochondrial transmembrane potential (ΔΨm) were detected using fluorescence indicator Fluo-3/AM and Rhodamine 123 with laster scanning confocal microscopy. β-Gal-NONOate resulted in a rapid increase in [Ca²⁺]_i and a great decrease in ΔΨm. The results of this study confirmed the ability of β-Gal-NONOate to suppress the proliferation of three kinds of cells with typical apoptosis feature in vitro. Disturbance of homeostasis in calcium signaling system might play pivotal roles in apoptosis of three cells. Effects of β-Gal-NONOate were assessed both alone and in paired combination with cisplatin. Q values were used to characterize the interactions as synergistic, additive, or antagonistic. Significant synergistic effects in growth inhinition of β-Gal-NONOate (0.25-10 mM) with cisplatin (2.5-10 μM) on three cell lines were observed (q>1). We conclude that β-Gal-NONOate may be worth of further studies assessing its value in cancer therapy in combination with the other chemotherapeutic agents.