Steven M. D'Ambrosio

Celecoxib derivatives induce apoptosis via the disruption of mitochondrial membrane potential and activation of caspase 9

Celecoxib is a potent nonsteroid antiinflammatory drug (NSAID) that has shown great promise in cancer chemoprevention and treatment. The tumor suppression activity of celecoxib and other NSAIDs have been related to the induction of apoptosis in many cancer cell lines and animal models. While celecoxib is a specific inhibitor of cyclooxygenase (COX)‐2, recent data indicate that its apoptotic properties may also be mediated through COX‐independent pathways. In our study, we evaluated second generation celecoxib derivatives, lacking COX‐2 inhibitory activity, in a premalignant and malignant human oral cell culture model to determine their potential anticancer effect and mechanisms responsible for the COX‐independent apoptotic activity. Celecoxib and its derivatives delayed the progression of cells through the G2/M phase and induced apoptosis. The derivatives with apolar substituents at the terminal phenyl moiety of celecoxib greatly enhanced apoptosis and cell cycle delay. Apoptosis and cell cycle arrest appeared to be independent of derivative induced inhibition of PDK1 and phosphorylation of Akt and Erk1/2. Derivatives induced apoptosis was mediated by the cleavage and activation of caspase‐9 and caspase‐3, but not caspase 8, implicating the mitochondrial pathway for apoptosis induction. Inhibitors of caspase‐3 and caspase‐9 and cyclosporin A, a mitochondrial membrane potential stabilizer, attenuated derivative induced apoptosis. Inhibition of caspase‐3 prevented the activation of caspase 8, while the inhibition of caspase‐9 inhibitor blocked activation of both caspase 3 and 8 by the derivatives. Apoptosis was independent of Bcl‐2. These results indicate that the second generation celecoxib derivatives induce apoptosis in human oral cancer lines by the disruption of mitochondrial membrane potential activating caspase 9 and downstream caspase 3 and 8. This suggests that the modification of the celecoxib structure can lead to highly effective COX‐independent growth inhibitory and apoptotic agents in chemoprevention and therapy.

Effects of stress on methyltransferase synthesis: an important DNA repair enzyme.

The enhancement of tumor development following acute stress has been demonstrated in some animal studies. This study was designed to explore mechanisms that would account in part for the relationship between stress and tumor development at the level of DNA repair, using a rat model. Forty-four rats were given the carcinogen dimethylnitrosamine in their drinking water, and half were randomly assigned to a rotational stress condition. The levels of methyltransferase, a DNA repair enzyme induced in response to carcinogen damage, were significantly lower in spleens from the stressed animals. These data suggest that stress may impair DNA repair.

PHOTOREPAIR OF PYRIMIDINE DIMERS IN HUMAN SKIN IN VIVO

Abstract— The exposure of human skin in vivo to UV radiation emitted from a sunlamp induces the formation of pyrimidine dimers. The number of dimers, as detected by UV‐endonuclease, decreases following exposure of the UV–irradiated skin to visible wavelengths of light. These results suggest that humans possess a mechanism by which pyrimidine dimers are photorepaired upon illumination of human skin in vivo with visible light.

Inhibition of the growth of premalignant and malignant human oral cell lines by extracts and components of black raspberries.

Abstract: Black raspberries are a rich natural source of chemopreventive phytochemicals. Recent studies have shown that freeze-dried black raspberries inhibit the development of oral, esophageal, and colon cancer in rodents, and extracts of black raspberries inhibit benzo(a)pyrene-induced cell transformation of hamster embryo fibroblasts. However, the molecular mechanisms and the active components responsible for black raspberry chemoprevention are unclear. In this study, we found that 2 major chemopreventive components of black raspberries, ferulic acid and β-sitosterol, and a fraction eluted with ethanol (RO-ET) during silica column chromatography of the organic extract of freeze-dried black raspberries inhibit the growth of premalignant and malignant but not normal human oral epithelial cell lines. Another fraction eluted with CH2Cl2/ethanol (DM:ET) and ellagic acid inhibited the growth of normal as well as premalignant and malignant human oral cell lines. We investigated the molecular mechanisms by which ferulic acid and β-sitosterol and the RO-ET fraction selectively inhibited the growth of premalignant and malignant oral cells using flow cytometry and Western blotting of cell cycle regulatory proteins. There was no discernable change in the cell cycle distribution following treatment of cells with the RO-ET fraction. Premalignant and malignant cells redistributed to the G2/M phase of the cell cycle following incubation with ferulic acid. β-sitosterol treated premalignant and malignant cells accumulated in the G0/G1 and G2/M phases, respectively. The RO-ET fraction reduced the levels of cyclin A and cell division cycle gene 2 (cdc2) in premalignant cells and cyclin B1, cyclin D1, and cdc2 in the malignant cell lines. This fraction also elevated the levels of p21waf1/cip1 in the malignant cell line. Ferulic acid treatment led to increased levels of cyclin B1 and cdc2 in both cell lines, and p21waf1/cip1 was induced in the malignant cell line. β-sitosterol reduced the levels of cyclin B1 and cdc2 while increasing p21waf1/cip1 in both the premalignant and malignant cell lines. These results show for the first time that the growth inhibitory effects of black raspberries on premalignant and malignant human oral cells may reside in specific components that target aberrant signaling pathways regulating cell cycle progression.

Longevity, stability and DNA repair

The functional capacity of a cell, tissue, organ, or organism is dependent upon its ability to maintain the stability of its unit components. The higher the differentiated state of the system, the greater the amount of stability required to maintain that state as a function of time. Stability can be achieved via either redundancy or repair. Redundancy while easily achievable in biological systems is both costly and limited by thermodynamic considerations. Repair, in its general sense, has no such limitations. Repair at the cellular and macromolecular level is multiple in its forms and varies as a function of species, tissue, and stage of the cell cycle. The repair of DNA damage is a dynamic process with many components and subcomponents, each interacting with one another in order to achieve a balance between individual stability and evolutionary diversity. Thus, between internal and external factors which damage DNA and the subsequent expression of alterations in the functional stability of DNA lie the multi-functional pathways which attempt to maintain DNA fidelity. A strong correlation between ulta-violet light induced excision or pre-replication repair, as measured by autoradiogrphy and maximum species lifespan has been reported within different strains of the same species, between related species (e.g. Mus musculus and Peromyscus leucopus), between five orders of mammals, and most recently within members of the primate family. As has been demonstrated by the authors and others, differences in excision repair between species and tissues may relate to the turning off of portions of the repair processes during embryogenesis. Regardless of why such correlations exist or the nature of their mechanisms, it is naive to either assert or deny a causal role for DNA repair in longevity assurance systems. For example, while species-related differences in DNA repair may reflect the turning off of such repair processes during fetal development this does not mean that rates of accumulation of DNA damage are not altered by such changes. Indeed, such a phenomena might well explain the rapid evolution of lifespan within the primates without a concurrent input of new genes.

QUANTITATION OF PYRIMIDINE DIMERS BY IMMUNOSLOT BLOT FOLLOWING SUBLETHAL UV‐IRRADIATION OF HUMAN CELLS

An immunoslot blot assay was developed to detect pyrimidine dimers induced in DNA by sublethal doses of UV (254 nm) radiation. Using this assay, one dimer could be detected in 10 megabase DNA using 200 ng or 0.5 megabase DNA using 20 ng irradiated DNA. The level of detection, as measured by dimer specific antibody binding, was proportional to the dose of UV and amount of irradiated DNA used. The repair of pyrimidine dimers was measured in human skin fibroblastic cells in culture following exposure to 0.5 to 5 J m‐2 of 254 nm UV radiation. The half‐life of repair was approximately 24, 7 and 6 h in cells exposed to 0.5, 2 and 5 J m‐2 UV radiation, respectively. This immunological approach utilizing irradiated DNA immobilized to nitrocellulose should allow the direct quantitation of dimers following very low levels of irradiation in small biological samples and isolated gene fragments.

Antibodies to UV irradiated DNA: the monitoring of DNA damage by Elisa and indirect immunofluorescence

Abstract The enzyme‐linked immunosorbant assay (ELISA) was modified to (1) characterize antibodies raised in rabbits against UV‐irradiated single‐stranded DNA (UVssDNA) complexed with methylated BSA and (2) directly detect pyrimidine dimers in irradiated DNA. The antisera specifically bound to UVssDNA, UVpoly(dT) and to a limited extent to UVdsDNA and UVpoly(dC) immobilized on protamine sulfate coated microliter wells. Fifty percent of the maximum antibody binding was observed at a 1‐5000 dilution against UVssDNA. Binding to ssDNA and poly(dT) was observed only at much higher concentrations of antibody (1:500 dilution), whereas no binding to double stranded DNA (dsDNA) was observed. The extent of binding of the antibody was dependent on the dose of UV radiation to DNA, as well as, to the concentration of antigen immobilized on the plate. Specific binding to DNA irradiated with 5.0 J/m2 was detected with as little as 10 ng of DNA. The sensitivity was further extended to less than 1 J/m2 by using higher concentrations (100 ng) of UVssDNA. The ability of various irradiated molecules, DNA, homopolymers and linkers to act as inhibitors of antibody binding establish that the antigenic determinants are mainly thymine homodimers with lower affinity for cytosine dimers. Potential usefulness of the antibodies to directly quantitate pyrimidine dimers in cells exposed to UV radiation was determined by indirect immunofluorescence. Flow cytometric analysis of immunostained human lymphocytes irradiated with 254 nm radiation indicated that greater than 50% of the population had significantly higher fluorescent intensity than unirradiated control cells.

Selective induction of apoptosis of human oral cancer cell lines by avocado extracts via a ROS-mediated mechanism.

Avocados have a high content of phytochemicals with potential chemopreventive activity. Previously we reported that phytochemicals extracted from avocado meat into a chloroform partition (D003) selectively induced apoptosis in cancer but not normal, human oral epithelial cell lines. In the present study, we observed that treatment of human oral cancer cell lines containing high levels of reactive oxygen (ROS) with D003 increased ROS levels twofold to threefold and induced apoptosis. In contrast, ROS levels increased only 1.3-fold, and apoptosis was not induced in the normal cell lines containing much lower levels of basal ROS. When cellular ROS levels in the malignant cell lines were reduced by N-acetyl-l-cysteine (NAC), cells were resistant to D003 induced apoptosis. NAC also delayed the induction of apoptosis in dominant negative FADD-expressing malignant cell lines. D003 increased ROS levels via mitochondrial complex I in the electron transport chain to induce apoptosis. Normal human oral epithelial cell lines transformed with HPV16 E6 or E7 expressed higher basal levels of ROS and became sensitive to D003. These data suggest that perturbing the ROS levels in human oral cancer cell lines may be a key factor in selective apoptosis and molecular targeting for chemoprevention by phytochemicals.

Enhanced expression of the 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase gene in human breast tumor cells

The expression of the 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase) gene in human breast tumors was evaluated at the level of the single cell to better understand how breast tumor cells regulate expression in response to oxidative stress. Compared to normal breast ductal cells, the level of 8-oxo-dGTPase expression in the breast tumor cells increased from non-detectable levels in normal breast to expression in 30-85% of the tumor cells in individual tumors. There was no significant association between 8-oxo-dGTPase expression and tumor grade and metastatic malignancy. The upregulation of 8-oxo-dGTPase was not directly linked to the expression of cyclins D1 and D3, estrogen receptor, p53, Ki-67 and c-erbB-2, which are genes involved in cell cycle regulation and tumor growth. The elevated expression of 8-oxo-dGTPase in human breast ductal carcinoma cells appears to be a general characteristic of breast tumors and may provide the tumor cell with a cellular defense mechanism to prevent the incorporation of 8-hydroxy-deoxyguanosine during DNA replication.

Mechanisms of nitric oxide–induced cytotoxicity in normal human hepatocytes

Chronic exposure of hepatocytes to reactive nitrogen species (RNS) following liver injury and inflammation leads not only to functional and morphological alterations in the liver but also to degenerative liver diseases and hepatocellular carcinoma. Previously, we showed that S‐nitroso‐N‐acetylpenicillamine‐amine (SNAP), which generates nitric oxide, and 3‐morpholinosydnonimine (Sin‐1), which generates equal molar concentrations of superoxide and nitric oxide resulting in peroxynitrite production, exhibited different levels of cytotoxicity to normal human hepatocytes in culture. The aim of the present study was to elucidate some of the molecular and cellular pathways leading to hepatocyte cell death induced by RNS. Following treatment of the hepatocytes with SNAP or Sin‐1, gene‐specific DNA damage was measured in mtDNA and a hprt gene fragment using a quantitative Southern blot analysis. Both agents induced dose‐dependent increases in DNA damage that was alkaline labile, but not sensitive to both formamidopyrimidine‐DNA glycosylase (fpg) and endonuclease III, which recognize 8‐oxoguanine, thymine glycol, and other oxidized pyrimidines. DNA damage was two‐ to fivefold greater in mtDNA than in the hprt gene fragment. There was a persistent and marked increase in DNA damage posttreatment that appeared to arise from the disruption of electron transport in the mitochondria, generating reactive species that saturated the repair system. DNA damage induced by Sin‐1 and SNAP led to cell‐cycle arrest in the S‐phase, growth inhibition, and apoptosis. The data support the hypothesis that the functional and morphological changes observed in liver following chronic exposure to RNS are, in part, the result of persistent mitochondrial and nuclear DNA damage. Environ. Mol. Mutagen. 37:46–54, 2001