Betsy T. Kren

A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation.

The hydrophilic bile salt ursodeoxycholic acid (UDCA) protects against the membrane-damaging effects associated with hydrophobic bile acids. This study was undertaken to (a) determine if UDCA inhibits apoptosis from deoxycholic acid (DCA), as well as from ethanol, TGF-beta1, Fas ligand, and okadaic acid; and to (b) determine whether mitochondrial membrane perturbation is modulated by UDCA. DCA induced significant hepatocyte apoptosis in vivo and in isolated hepatocytes determined by terminal transferase-mediated dUTP-digoxigenin nick end-labeling assay and nuclear staining, respectively (P < 0.001). Apoptosis in isolated rat hepatocytes increased 12-fold after incubation with 0.5% ethanol (P < 0.001). HuH-7 cells exhibited increased apoptosis with 1 nM TGF-beta1 (P < 0. 001) or DCA at >/= 100 microM (P < 0.001), as did Hep G2 cells after incubation with anti-Fas antibody (P < 0.001). Finally, incubation with okadaic acid induced significant apoptosis in HuH-7, Saos-2, Cos-7, and HeLa cells. Coadministration of UDCA with each of the apoptosis-inducing agents was associated with a 50-100% inhibition of apoptotic changes (P < 0.001) in all the cell types. Also, UDCA reduced the mitochondrial membrane permeability transition (MPT) in isolated mitochondria associated with both DCA and phenylarsine oxide by > 40 and 50%, respectively (P < 0.001). FACS(R) analysis revealed that the apoptosis-inducing agents decreased the mitochondrial transmembrane potential and increased reactive oxygen species production (P < 0.05). Coadministration of UDCA was associated with significant prevention of mitochondrial membrane alterations in all cell types. The results suggest that UDCA plays a central role in modulating the apoptotic threshold in both hepatocytes and nonliver cells, and inhibition of MPT is at least one pathway by which UDCA protects against apoptosis.

Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation

The hydrophilic bile salt ursodeoxycholic acid (UDCA) is a potent inhibitor of apoptosis. In this paper, we further characterize the mechanism by which UDCA inhibits apoptosis induced by deoxycholic acid, okadaic acid and transforming growth factor β1 in primary rat hepatocytes. Our data indicate that coincubation of cells with UDCA and each of the apoptosis-inducing agents was associated with an approximately 80% inhibition of nuclear fragmentation (P<0.001). Moreover, UDCA prevented mitochondrial release of cytochrome c into the cytoplasm by 70–75% (P<0.001), thereby, inhibiting subsequent activation of DEVD-specific caspases and cleavage of poly(ADP-ribose) polymerase. Each of the apoptosis-inducing agents decreased mitochondrial transmembrane potential and increased mitochondrial-associated Bax protein levels. Coincubation with UDCA was associated with significant inhibition of these mitochondrial membrane alterations. The results suggest that the mechanism by which UDCA inhibits apoptosis involves an interplay of events in which both depolarization and channel-forming activity of the mitochondrial membrane are inhibited.

MicroRNAs identified in highly purified liver-derived mitochondria may play a role in apoptosis.

MicroRNAs (miRNAs) are a class of small ~22 nt noncoding (nc) RNAs that regulate gene expression post-transcriptionally by direct binding to target sites on mRNAs. They comprise more than 1,000 novel species in mammalian cells and exert their function by modulating gene expression through several different mechanisms, including translational inhibition, and/or degradation of target mRNAs. Mitochondria maintain and express their own genome, which is distinct from the nuclear transcriptional and translational apparatus. Thus, they provide a potential site for miRNA mediated post-transcriptional regulation. To determine whether they maintain a unique miRNA population, we examined the miRNA profile from highly purified and RNase treated mitochondria from adult rat liver. Fifteen miRNAs were identified by microarray analysis of which, five were confirmed by TaqMan® 5’nuclease assays using rat specific probes. Functional analysis of the miRNAs indicated that they were not targeted to the mitochondrial genome nor were they complementary to nuclear RNAs encoding mitochondrial proteins. Rather, the mitochondria-associated miRNAs appear to be involved in the expression of genes associated with apoptosis, cell proliferation, and differentiation. Given the central role that mitochondria play in apoptosis, the results suggest that they may serve as reservoirs of select miRNAs that may modulate these processes in a coordinate fashion.

MicroRNA-143 reduces viability and increases sensitivity to 5-fluorouracil in HCT116 human colorectal cancer cells

MicroRNAs are aberrantly expressed in cancer; microRNA‐143 (miR‐143) is down‐regulated in colon cancer. HCT116 human colorectal cancer cells were used to investigate the biological role of miR‐143. Transient miR‐143 overexpression resulted in an approximate 60% reduction in cell viability. In addition, stable miR‐143 overexpressing cells were selected with G418 and exposed to 5‐fluorouracil. Increased stable expression of miR‐143 was associated with decreased viability and increased cell death after exposure to 5‐fluorouracil. These changes were associated with increased nuclear fragmentation and caspase ‐3, ‐8 and ‐9 activities. In addition, extracellular‐regulated protein kinase 5, nuclear factor‐κB and Bcl‐2 protein expression was down‐regulated by miR‐143, and further reduced by exposure to 5‐fluorouracil. In conclusion, miR‐143 modulates the expression of key proteins involved in the regulation of cell proliferation, death and chemotherapy response. In addition, miR‐143 increases the sensitivity of colon cancer cells to 5‐fluorouracil, probably acting through extracellular‐regulated protein kinase 5/nuclear factor‐κB regulated pathways. Collectively, the data obtained in the present study suggest anti‐proliferative, chemosensitizer and putative pro‐apoptotic roles for miR‐143 in colon cancer.

Nucleotide Exchange in Genomic DNA of Rat Hepatocytes Using RNA/DNA Oligonucleotides TARGETED DELIVERY OF LIPOSOMES AND POLYETHYLENEIMINE TO THE ASIALOGLYCOPROTEIN RECEPTOR

Chimeric RNA/DNA oligonucleotides have been shown to promote single nucleotide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser365 position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and negatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stability and efficiency in targeting hepatocytes via the asialoglycoprotein receptor. Physical characterization and electron microscopy revealed that the oligonucleotides were efficiently encapsulated within the liposomes, with the positive and negative formulations remaining stable for at least 1 month. Transfection efficiencies in isolated rat hepatocytes approached 100% with each of the formulations. However, the negative liposomes and 25-kDa lactosylated polyethyleneimine provided the most intense nuclear fluorescence with the fluorescein-labeled oligonucleotides. The lactosylated polyethyleneimine and the three different liposomal formulations resulted in A to C conversion efficiencies of 19–24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatocytes. The results suggest that both the liposomal and polyethyleneimine formulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the introduction or repair of genetic mutations by the chimeric RNA/DNA oligonucleotides.

Emergence of protein kinase CK2 as a key target in cancer therapy

Protein kinase CK2, a protein serine/threonine kinase, plays a global role in activities related to cell growth, cell death, and cell survival. CK2 has a large number of potential substrates localized in diverse locations in the cell including, for example, NF‐κB as an important downstream target of the kinase. In addition to its involvement in cell growth and proliferation it is also a potent suppressor of apoptosis, raising its key importance in cancer cell phenotype. CK2 interacts with diverse pathways which illustrates the breadth of its impact on the cellular machinery of both cell growth and cell death giving it the status of a “master regulator” in the cell. With respect to cancer, CK2 has been found to be dysregulated in all cancers examined demonstrating increased protein expression levels and nuclear localization in cancer cells compared with their normal counterparts. We originally proposed CK2 as a potentially important target for cancer therapy. Given the ubiquitous and essential for cell survival nature of the kinase, an important consideration would be to target it specifically in cancer cells while sparing normal cells. Towards that end, our design of a tenascin based sub‐50 nm (i.e., less than 50 nm size) nanocapsule in which an anti‐CK2 therapeutic agent can be packaged is highly promising because this formulation can specifically deliver the cargo intracellularly to the cancer cells in vivo. Thus, appropriate strategies to target CK2 especially by molecular approaches may lead to a highly feasible and effective approach to eradication of a given cancer.

Developmental regulation of CRD-BP, an RNA-binding protein that stabilizes c-myc mRNA in vitro.

We previously isolated and characterized a coding region determinant-binding protein (CRD-BP) that might regulate c-myc mRNA post-transcriptionally. CRD-BP binds specifically to the coding region of c-myc mRNA and might stabilize c-myc mRNA in vitro by protecting it from endonucleolytic cleavage. Since c-myc abundance is regulated during embryonic development and cell replication, we investigated whether CRD-BP is also regulated in animal tissues. We focused on CRD-BP expression during rat liver development and liver regeneration, because c-myc mRNA is regulated post-transcriptionally in both cases. CRD-BP expression parallels c-myc expression during liver development; the protein is present in fetal and neonatal liver but is absent or in low abundance in adult liver. In contrast, the up-regulation of c-myc mRNA following partial hepatectomy is not accompanied by up-regulation of CRD-BP. To our knowledge, CRD-BP is the first example of a putative mammalian mRNA-binding protein that is abundant in a fetal tissue but either absent from or scarce in adult tissues. Its expression in fetal liver and in transformed cell lines suggests CRD-BP is an oncofetal protein.

Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice

Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type-specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain-deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.

A Bile Acid Protects against Motor and Cognitive Deficits and Reduces Striatal Degeneration in the 3-Nitropropionic Acid Model of Huntington's Disease

There is currently no effective treatment for Huntingtons disease (HD), a progressive, fatal, neurodegenerative disorder characterized by motor and cognitive deterioration. It is well established that HD is associated with perturbation of mitochondrial energy metabolism. Tauroursodeoxycholic acid (TUDCA), a naturally occurring bile acid, can stabilize the mitochondrial membrane, inhibit the mitochondrial permeability transition, decrease free radical formation, and derail apoptotic pathways. Here we report that TUDCA significantly reduced 3-nitropropionic acid (3-NP)-mediated striatal neuronal cell death in cell culture. In addition, rats treated with TUDCA exhibited an 80% reduction in apoptosis and in lesion volumes associated with 3-NP administration. Moreover, rats which received a combination of TUDCA + 3-NP exhibited sensorimotor and cognitive task performance that was indistinguishable from that of controls, and this effect persisted at least 6 months. Bile acids have traditionally been used as therapeutic agents for certain liver diseases. This is the first demonstration, however, that a bile acid can be delivered to the brain and function as a neuroprotectant and thus may offer potential therapeutic benefit in the treatment of certain neurodegenerative diseases.

Tauroursodeoxycholic acid partially prevents apoptosis induced by 3-nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability transition.

Abstract: Ursodeoxycholic acid (UDCA) has been shown to be a strongmodulator of the apoptotic threshold in both hepatic and nonhepatic cells.3‐Nitropropionic acid (3‐NP), an irreversible inhibitor of succinatedehydrogenase, appears to cause apoptotic neuronal cell death in the striatum,reminiscent of the neurochemical and anatomical changes associated withHuntingtons disease (HD). This study was undertaken (a) to characterizefurther the mechanism by which 3‐NP induces apoptosis in rat neuronal RN33Bcells and (b) to determine if and how the taurine‐conjugated UDCA,tauroursodeoxycholic acid (TUDCA), inhibits apoptosis induced by 3‐NP. Ourresults indicate that coincubation of cells with TUDCA and 3‐NP was associatedwith an ∼80% reduction in apoptosis (p < 0.001), whereasneither taurine nor cyclosporin A, a potent inhibitor of the mitochondrialpermeability transition (MPT), inhibited cell death. Moreover, TUDCA, as wellas UDCA and its glycine‐conjugated form, glycoursodeoxycholic acid, preventedmitochondrial release of cytochrome c (p < 0.001), whichprobably accounts for the observed inhibition of DEVD‐specific caspaseactivity and poly(ADP‐ribose) polymerase cleavage. 3‐NP decreasedmitochondrial transmembrane potential (p < 0.001) and increasedmitochondrial‐associated Bax protein levels (p < 0.001).Coincubation with TUDCA was associated with significant inhibition of thesemitochondrial membrane alterations (p < 0.01). The results suggestthat TUDCA inhibits 3‐NP‐induced apoptosis via direct inhibition ofmitochondrial depolarization and outer membrane disruption, together withmodulation of Bax translocation from cytosol to mitochondria. In addition,cell death by 3‐NP apparently occurs through pathways that are independent ofthe MPT.