Mucosal TLR5 activation controls healthspan and longevity.

Addressing age-related immunological defects through therapeutic interventions is essential for healthy aging, as the immune system plays a crucial role in controlling infections, malignancies, and in supporting tissue homeostasis and repair. In our study, we show that stimulating toll-like receptor 5 (TLR5) via mucosal delivery of a flagellin-containing fusion protein effectively extends the lifespan and enhances the healthspan of mice of both sexes. This enhancement in healthspan is evidenced by diminished hair loss and ocular lens opacity, increased bone mineral density, improved stem cell activity, delayed thymic involution, heightened cognitive capacity, and the prevention of pulmonary lung fibrosis. Additionally, this fusion protein boosts intestinal mucosal integrity by augmenting the surface expression of TLR5 in a certain subset of dendritic cells and increasing interleukin-22 (IL-22) secretion. In this work, we present observations that underscore the benefits of TLR5-dependent stimulation in the mucosal compartment, suggesting a viable strategy for enhancing longevity and healthspan.

Prometheus, an omics portal for interkingdom comparative genomic analyses.

Functional analyses of genes are crucial for unveiling biological responses, genetic engineering, and developing new medicines. However, functional analyses have largely been restricted to model organisms, representing a major hurdle for functional studies and industrial applications. To resolve this, comparative genome analyses can be used to provide clues to gene functions as well as their evolutionary history. To this end, we present Prometheus, a web-based omics portal that contains more than 17,215 sequences from prokaryotic and eukaryotic genomes. This portal supports interkingdom comparative analyses via a domain architecture-based gene identification system and Gene Search, and users can easily and rapidly identify single or entire gene sets in specific pathways. Bioinformatics tools for further analyses are provided in Prometheus or through Bio-Express, a cloud-based bioinformatics analysis platform. Prometheus is a new paradigm for comparative analyses of large amounts of genomic information.

Comprehensive analysis of Translationally Controlled Tumor Protein (TCTP) provides insights for lineage-specific evolution and functional divergence.

BACKGROUND: Translationally controlled tumor protein (TCTP) is a conserved, multifunctional protein involved in numerous cellular processes in eukaryotes. Although the functions of TCTP have been investigated sporadically in animals, invertebrates, and plants, few lineage-specific activities of this molecule, have been reported. An exception is in Arabidopsis thaliana, in which TCTP (AtTCTP1) functions in stomatal closuer by regulating microtubule stability. Further, although the development of next-generation sequencing technologies has facilitated the analysis of many eukaryotic genomes in public databases, inter-kingdom comparative analyses using available genome information are comparatively scarce. METHODOLOGY: To carry out inter-kingdom comparative analysis of TCTP, TCTP genes were identified from 377 species. Then phylogenetic analysis, prediction of protein structure, molecular docking simulation and molecular dynamics analysis were performed to investigate the evolution of TCTP genes and their binding proteins. RESULTS: A total of 533 TCTP genes were identified from 377 eukaryotic species, including protozoa, fungi, invertebrates, vertebrates, and plants. Phylogenetic and secondary structure analyses reveal lineage-specific evolution of TCTP, and inter-kingdom comparisons highlight the lineage-specific emergence of, or changes in, secondary structure elements in TCTP proteins from different kingdoms. Furthermore, secondary structure comparisons between TCTP proteins within each kingdom, combined with measurements of the degree of sequence conservation, suggest that TCTP genes have evolved to conserve protein secondary structures in a lineage-specific manner. Additional tertiary structure analysis of TCTP-binding proteins and their interacting partners and docking simulations between these proteins further imply that TCTP gene variation may influence the tertiary structures of TCTP-binding proteins in a lineage-specific manner. CONCLUSIONS: Our analysis suggests that TCTP has undergone lineage-specific evolution and that structural changes in TCTP proteins may correlate with the tertiary structure of TCTP-binding proteins and their binding partners in a lineage-specific manner.

Targeting Heat Shock Protein 27 in Cancer: A Druggable Target for Cancer Treatment?

Heat shock protein 27 (HSP27), induced by heat shock, environmental, and pathophysiological stressors, is a multi-functional protein that acts as a protein chaperone and an antioxidant. HSP27 plays a significant role in the inhibition of apoptosis and actin cytoskeletal remodeling. HSP27 is upregulated in many cancers and is associated with a poor prognosis, as well as treatment resistance, whereby cells are protected from therapeutic agents that normally induce apoptosis. This review highlights the most recent findings and role of HSP27 in cancer, as well as the strategies for using HSP27 inhibitors for therapeutic purposes.

Parkin in the regulation of fat uptake and mitochondrial biology: emerging links in the pathophysiology of Parkinson's disease.

PURPOSE OF REVIEW: Perturbations in fatty acid levels and in regulatory proteins linked to fat and mitochondrial homeostasis are associated with modifying the risk of Parkinson's disease . Findings, that are not surprising, based on the high fat content of the brain, the myriad of neurological functions dependent on polyunsaturated fatty acids and the role of mitochondria in energy supply and stress amelioration. Nevertheless, dissecting out the molecular links between lipid biology, mitochondrial regulation and Parkinson's disease is complicated by the divergent causes underpinning Parkinson's disease pathophysiology. Here, we summarize aspects of fatty acid biology relevant to Parkinson's disease; the known links between the modulation of fat and Parkinson's disease and introduce mechanisms whereby the E3-ubiquitin ligase, Parkin known to be mutated as a genetic predisposing factor in Parkinson's disease, modulates fat uptake and mitochondrial control. RECENT FINDINGS: Prior evidence supports that Parkin, under mitochondrial stress conditions, plays a pivotal role in the mitophagy mitochondrial housekeeping program. Recent evidence now demonstrates a broader role of Parkin in controlling fat uptake and mitochondrial regulatory programs. SUMMARY: The identification that Parkin has a multifunctional role in modulating cellular fatty acid uptake and mitochondrial biology further strengthens the pathophysiologic link between fat metabolism, mitochondria and Parkinson's disease.

Parkin is a lipid-responsive regulator of fat uptake in mice and mutant human cells.

It has long been hypothesized that abnormalities in lipid biology contribute to degenerative brain diseases. Consistent with this, emerging epidemiologic evidence links lipid alterations with Parkinson disease (PD), and disruption of lipid metabolism has been found to predispose to α-synuclein toxicity. We therefore investigated whether Parkin, an E3 ubiquitin ligase found to be defective in patients with early onset PD, regulates systemic lipid metabolism. We perturbed lipid levels by exposing Parkin+/+ and Parkin-/- mice to a high-fat and -cholesterol diet (HFD). Parkin-/- mice resisted weight gain, steatohepatitis, and insulin resistance. In wild-type mice, the HFD markedly increased hepatic Parkin levels in parallel with lipid transport proteins, including CD36, Sr-B1, and FABP. These lipid transport proteins were not induced in Parkin-/- mice. The role of Parkin in fat uptake was confirmed by increased oleate accumulation in hepatocytes overexpressing Parkin and decreased uptake in Parkin-/- mouse embryonic fibroblasts and patient cells harboring complex heterozygous mutations in the Parkin-encoding gene PARK2. Parkin conferred this effect, in part, via ubiquitin-mediated stabilization of the lipid transporter CD36. Reconstitution of Parkin restored hepatic fat uptake and CD36 levels in Parkin-/- mice, and Parkin augmented fat accumulation during adipocyte differentiation. These results demonstrate that Parkin is regulated in a lipid-dependent manner and modulates systemic fat uptake via ubiquitin ligase-dependent effects. Whether this metabolic regulation contributes to premature Parkinsonism warrants investigation.

Organoplatinum chromophores for application in high-performance nonlinear absorption materials.

Chromophores and materials that exhibit nonlinear absorption over a broad spectrum and with high temporal dynamic range are of interest for application in materials engineering and biology. Recent work by a number of research groups has led to the development of a new family of organometallic chromophores and materials featuring interesting and useful nonlinear absorption properties. These systems contain the platinum acetylide moiety as a fundamental molecular unit, combined with delocalized, π-conjugated electron systems. These organometallic chromophores provide a unique combination of properties, such as negligible ground state absorption in the visible region, large spin-orbit coupling giving rise to high triplet excited state yield, triplet lifetime in the microsecond domain, high two-photon cross-section in the visible and near-infrared regions, and high triplet-triplet absorption cross-section in the visible and near-infrared region. This Spotlight on Application highlights recent developments in this area, combining background and review on nonlinear absorption in platinum acetylide chromophores and describing significant recent results from our own laboratory.

Mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in TNFR1-associated periodic syndrome (TRAPS).

Reactive oxygen species (ROS) have an established role in inflammation and host defense, as they kill intracellular bacteria and have been shown to activate the NLRP3 inflammasome. Here, we find that ROS generated by mitochondrial respiration are important for normal lipopolysaccharide (LPS)-driven production of several proinflammatory cytokines and for the enhanced responsiveness to LPS seen in cells from patients with tumor necrosis factor receptor-associated periodic syndrome (TRAPS), an autoinflammatory disorder caused by missense mutations in the type 1 TNF receptor (TNFR1). We find elevated baseline ROS in both mouse embryonic fibroblasts and human immune cells harboring TRAPS-associated TNFR1 mutations. A variety of antioxidants dampen LPS-induced MAPK phosphorylation and inflammatory cytokine production. However, gp91(phox) and p22(phox) reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits are dispensable for inflammatory cytokine production, indicating that NADPH oxidases are not the source of proinflammatory ROS. TNFR1 mutant cells exhibit altered mitochondrial function with enhanced oxidative capacity and mitochondrial ROS generation, and pharmacological blockade of mitochondrial ROS efficiently reduces inflammatory cytokine production after LPS stimulation in cells from TRAPS patients and healthy controls. These findings suggest that mitochondrial ROS may be a novel therapeutic target for TRAPS and other inflammatory diseases.

Synthesis and properties of a solution-processable truxene derivative for OLED devices.

A soluble truxene derivative (TR1) attached with triphenylamine at the peripheral position was designed and synthesized. The structure and purity of TR1 were carefully characterized by 1H NMR, UV/vis and photoluminescent spectroscopy, mass spectroscopy, and thermal analyses. It exhibited good solubility in common organic solvents and good film forming properties. The maximum absorption and emission peaks in THF solution were shown at 358 nm and 415 nm, respectively. Bright blue emission was observed in both solution and solid states under UV excitation. The fluorescent quantum efficiency was 0.46. The best luminous efficiency was found to be 3.65 cd/A with CIE coordinates of (0.163, 0.260) in electroluminescence devices.

Optimizing simultaneous two-photon absorption and transient triplet-triplet absorption in platinum acetylide chromophores.

A series of platinum-containing organometallic dimer complexes has been synthesized and the photophysical properties have been investigated under one- and two-photon (2PA) absorption conditions. The complexes have the general structure [DPAF-C[triple bond]C-Pt(PBu(3))(2)-C[triple bond]C-Ar-C[triple bond]C-Pt(PBu(3))(2)-C[triple bond]C-DPAF], where Ar is a pi-conjugated unit, Bu = n-butyl, and DPAF = diphenylamino-2,7-fluorenylene. The core Ar units include 1,4-phenylene, 2,5-thienylene, 5,5'-(2,2'-bithienylene), 2,5-(3,4-ethylenedioxythiophene, 2,1,3-benzothiadiazole, and 4,7-dithien-2-yl-2,1,3-benzothiadiazole. Absorption and photoluminescence spectroscopy indicates that the complexes feature low-lying excited states based on both the core [-Pt(PBu(3))(2)-C[triple bond]C-Ar-C[triple bond]C-Pt(PBu(3))(2)-] chromophore as well as the DPAF units. Photoexcitation of the complexes produces a singlet state excited state, which rapidly undergoes intersystem crossing to afford a triplet state that has a lifetime in the microsecond time domain. In most cases, the lowest energy triplet state is localized on the core chromophore. Femtosecond 2PA spectra are measured along with triplet-triplet absorption spectra and nanosecond intensity-dependent transmission for solutions of the complexes. Each of the complexes features a 2PA absorption band in the near-infrared region (lambda approximately 700-750 nm) with a cross section 50-200 GM that is ascribed to the DPAF chromophore. The complexes also feature broad triplet-triplet absorption throughout the visible and near-infrared regions (lambda approximately 500-800 nm, (TT) approximately 5-10 x 10(4) M(-1) cm(-1)). Each of the complexes exhibits efficient nonlinear absorption of nanosecond pulses in the near-infrared region (600-800 nm), and we demonstrate that effect is most efficient in the chromophores where the 2PA cross section maxima coincides spectrally with the excited triplet state absorption.

An alternatively spliced isoform of non-muscle myosin II-C is not regulated by myosin light chain phosphorylation.

We report a novel isoform of non-muscle myosin II-C (NM II-C), NM II-C2, that is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans). The 41 amino acids are inserted into loop 2 of the NM II-C heavy chain within the actin binding region. Unlike most vertebrate non-muscle and smooth muscle myosin IIs, baculovirus-expressed mouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatory myosin light chain (MLC(20)) phosphorylation for maximum actin-activated MgATPase activity or maximum in vitro motility as measured by the sliding actin filament assay. In contrast, noninserted HMM II-C0 and another alternatively spliced isoform HMM II-C1, which contains 8 amino acids inserted into loop 1, are dependent on MLC(20) phosphorylation for both actin-activated MgATPase activity and in vitro motility ( Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol. Chem. 280, 22769-22775 ). HMM II-C1C2, which contains both the C1 and C2 inserts, does not require MLC(20) phosphorylation for full activity similar to HMM II-C2. These constitutively active C2-inserted isoforms of NM II-C are expressed only in neuronal tissue. This is in contrast to NM II-C1 and NM II-C0, both of which are ubiquitously expressed. Full-length NM II-C2-GFP expressed in COS-7 cells localizes to filaments in interphase cells and to the cytokinetic ring in dividing cells.

The B2 alternatively spliced isoform of nonmuscle myosin II-B lacks actin-activated MgATPase activity and in vitro motility.

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the nonspliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells [X. Ma, S. Kawamoto, J. Uribe, R.S. Adelstein, Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development, Mol. Biol. Cell 15 (2006) 2138-2149]. In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acid II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific.

Galanin is up-regulated in colon adenocarcinoma.

The early diagnosis of colorectal cancer and the early detection of recurrence are central to effective treatment, as prognosis is directly related to the stage of the disease. When colorectal cancer is diagnosed at an early, localized stage, 5-year survival is 90%. There is substantial interest in the identification of circulating human tumor-derived proteins in serum for the purposes of early cancer diagnosis. We have implemented an approach based on the analysis of microarray data for the identification of tumor proteins that may have utility as biomarkers in colon cancer. Expression analysis of microarray data obtained from a variety of 290 tumors and normal tissues revealed that galanin was maximally expressed in colon cancer. These findings were corroborated by real-time quantitative PCR, in which the colon cancer cell lines LOVO, HCT15, SW480, and SW620 cell showed significantly higher levels of galanin expression than did noncolon cancer cell lines. To evaluate galanin as a potential biomarker of colon cancer, a preliminary "training" set of serum from 40 healthy donors and 55 colon cancer patients was analyzed by ELISA. The data pattern was confirmed by an independent set of 90 masked serum samples: 24 from healthy donors and 66 from colon cancer patients. This result yielded a sensitivity of 69.7% [95% confidence interval (95% CI), 57.1-80.4], specificity of 75.0% (95% CI, 53.3-90.2), and positive predictive value of 88.5% (95% CI, 76.6-95.7). The galanin expression level was significantly increased with tumor size and tumor stage. These findings justify a prospective assessment of serum galanin protein as a screening tool for colon cancer.

Platinum acetylide two-photon chromophores.

To explore the photophysics of platinum acetylide chromophores with strong two-photon absorption cross-sections, we have investigated the synthesis and spectroscopic characterization of a series of platinum acetylide complexes that feature highly pi-conjugated ligands substituted with pi-donor or -acceptor moieties. The molecules (numbered 1-4) considered in the present work are analogs of bis(phenylethynyl)bis(tributylphosphine)platinum(II) complexes. Molecule 1 carries two alkynyl-benzothiazolylfluorene ligands, and molecule 2 has two alkynyl-diphenylaminofluorene ligands bound to the central platinum atom. Compounds 3 and 4 possess two dihexylaminophenyl substituents at their ends and differ by the number of platinum atoms in the oligomer "core" (one vs two in 3 and 4, respectively). The ligands have strong effective two-photon absorption cross-sections, while the heavy metal platinum centers give rise to efficient intersystem crossing to long-lived triplet states. Ultrafast transient absorption and emission spectra demonstrate that one-photon excitation of the chromophores produces an S1 state delocalized across the two conjugated ligands, with weak (excitonic) coupling through the platinum centers. Intersystem crossing occurs rapidly (Kisc approximately 1011 s-1) to produce the T1 state, which is possibly localized on a single conjugated fluorenyl ligand. The triplet state is strongly absorbing (epsilonTT > 5 x 104 M-1 cm-1), and it is very long-lived (tau > 100 micro s). Femtosecond pulses were used to characterize the two-photon absorption properties of the complexes, and all of the chromophores are relatively efficient two-photon absorbers in the visible and near-infrared region of the spectrum (600-800 nm). The complexes exhibit maximum two-photon absorption at a shorter wavelength than 2lambda for the one-photon band, consistent with the dominant two-photon transition arising from a two-photon-allowed gerade-gerade transition. Nanosecond transient absorption experiments carried out on several of the complexes with excitation at 803 nm confirm that the long-lived triplet state can be produced efficiently via a sequence involving two-photon excitation to produce S1, followed by intersystem crossing to produce T1.

An iridium(III) complex that exhibits dual mechanism nonlinear absorption.

The photophysical properties of the complex (L)Ir(ppy)(2)(+), where ppy = 2-phenylpyridine and L = 4,4'-(2,2'-bipyridine-5,5'-diylbis(ethyne-2,1-diyl))bis(N,N-dihexylaniline), have been investigated under one- and two-photon excitation conditions. In THF solution, the complex exhibits broad ground-state absorption with lambda(max) approximately 500 nm and weak photoluminescence with lambda(max) approximately 730 nm. Excitation of (L)Ir(ppy)(2)(+) at 355 nm produces a long-lived excited state (tau approximately 1 mus) that features a strong excited-state absorption in the near-infrared (lambda(max) approximately 875 nm, Deltaepsilon approximately 6.1 x 10(4) M(-1) cm(-1)). Photoluminescence and transient absorption studies of (L)Ir(ppy)(2)(+) carried out using 5 ns, 1064 nm pulsed excitation demonstrate that the same long-lived and strongly absorbing excited state can be efficiently produced by two-photon absorption. Solutions of the complex in THF display nonlinear absorption of 5 ns, 1064 nm pulses in a process that is believed to involve a combination of two-photon absorption and reverse saturable absorption.

Photophysics of platinum-acetylide substituted hexa-peri-hexabenzocoronenes.

This manuscript reports the synthesis and photophysical investigation of two hexa-peri-hexabenzocoronenes (HBCs) that are functionalized with platinum(II) acetylide units of the type trans-(Ar-CC-)2Pt(PBu3)2. In one complex, the platinum is directly linked to the HBC chromophore by an ethynyl spacer, whereas in the second, the platinum is separated from the HBC via a 1,4-phenylene ethynylene spacer. The Pt-acetylide units introduce strong spin-orbit coupling into the HBC chromophore, giving rise to high yields of the triplet excited state along with moderately intense phosphorescence at ambient temperature. On the basis of emission spectroscopy, the triplet state of the HBC chromophore is located at 2.14 eV and the S-T splitting is 0.6 eV. The triplet-triplet absorption and radical cation absorption spectra of the Pt-HBCs are determined by laser flash photolysis. Aggregation of the Pt-HBCs in a poor solvent such as hexane leads to quenching of the triplet state, but spectroscopy provides no evidence for the formation of a triplet excimer, even under conditions where the molecules are strongly aggregated.

Expression of a thioredoxin-related protein-1 is induced by prostaglandin E(2).

Prostaglandin E(2) (PGE(2)) plays an important role in protection of the gastric mucosa against various damaging agents and growth-inhibitory activity on tumor cells. However, the precise regulation mechanism of PGE(2) in gastric cancer cells is still unclear. In this study, we isolated a gene, which is regulated by PGE(2) in SNU-1, human gastric adenocarcinoma cells, using differential display RT-PCR (DD RT-PCR) and characterized the function of the gene induced by PGE(2). The full-length cDNA of the gene was cloned by the rapid amplification of cDNA ends method. The 1659 base pair cDNA consists of a 30-nt 5'-noncoding region, an 891-nt open reading frame and a 738-nt 3'noncoding region that includes a poly (A) signal. As a result of protein motif search, we found that it has a conserved thioredoxin-active site, Cys-Gly-Pro-Cys and a Myb-DNA binding domain repeat signature. Thus, we designated this gene product as thioredoxin-related protein-1, TRP-1. TRP-1 was expressed in a lower extent in renal, gastric and colon cancer tissues and is translated into 33 kDa protein in nuclear and cytoplasmic fractions. TRP-1 has a thioredoxin activity, which was detected using the insulin disulfide reduction assay. Another potential role of TRP-1 is repression of B-Myb activity through direct binding to B-Myb, a transcriptional factor induced at G1-S transition. Finally, TRP-1 overexpression inhibits mammalian cell proliferation and specifically predispose to G0/G1 phase arrest. In conclusion, these results imply that TRP-1 is a mammalian thioredoxin and plays as a transcriptional repressor through direct binding to the transcription factor B-Myb.

Disease-associated mutations and alternative splicing alter the enzymatic and motile activity of nonmuscle myosins II-B and II-C.

Human families with single amino acid mutations in nonmuscle myosin heavy chain (NMHC) II-A (MYH9) and II-C (MYH14) have been described as have mice generated with a point mutation in NMHC II-B (MYH10). These mutations (R702C and N93K in human NMHC II-A, R709C in murine NMHC II-B, and R726S in human NMHC II-C) result in phenotypes affecting kidneys, platelets, and leukocytes (II-A), heart and brain (II-B), and the inner ear (II-C). To better understand the mechanisms underlying these defects, we characterized the in vitro activity of mutated and wild-type baculovirus-expressed heavy meromyosin (HMM) II-B and II-C. We also expressed two alternatively spliced isoforms of NMHC II-C which differ by inclusion/exclusion of eight amino acids in loop 1, with and without mutations. Comparison of the actin-activated MgATPase activity and in vitro motility shows that mutation of residues Asn-97 and Arg-709 in HMM II-B and the homologous residue Arg-722 (Arg-730 in the alternatively spliced isoform) in HMM II-C decreases both parameters but affects in vitro motility more severely. Analysis of the transient kinetics of the HMM II-B R709C mutant shows an extremely tight affinity of HMM for ADP and a very slow release of ADP from acto-HMM. Although mutations generally decreased HMM activity, the R730S mutation in HMM II-C, unlike the R730C mutation, had no effect on actin-activated MgATPase activity but decreased the rate of in vitro motility by 75% compared with wild type. Insertion of eight amino acids into the HMM II-C heavy chain increases both actin-activated MgATPase activity and in vitro motility.

Membrane-bound alkaline phosphatase gene induces antitumor effect by G2/M arrest in etoposide phosphate-treated cancer cells.

Gene therapy is used to induce immune responses, regulate tumor growth, or sensitize tumor cells to specific treatment. For sensitizing tumor cells to specific drug, we considered a prodrug-converting system using membrane-bound intestinal alkaline phosphatase (IAP) as the prodrug-activating genes. The IAP is capable of converting a relatively non-cytotoxic prodrug, etoposide phosphate (EP), into etoposide with a significant antitumor activity. We used the retroviral vector for transducing IAP gene into SNU638 gastric cancer cells and EP was prepared by phosphorylation of etoposide. To determine the chromosomal incorporation of membrane-bound IAP gene and AP activity in IAP gene-transduced cells (SNU638/IAP), we performed genomic PCR and AP activity analysis. In genomic DNA of SNU638/IAP cells, full cDNA fragment of a 2.5 kb IAP was detected, and AP activity was shown at most 15 approximately 18-fold increase compared with control cells. According to the in vitro cytotoxicity study, SNU638/IAP cells greatly enhanced the cytotoxic effect in proportion to the concentration of EP, while control cells didn't cause any cytotoxic effects after EPtreatment. Especially, the cell population of G2/M phase was increased in EP-treated SNU638/ IAP cells because P4 DNA unknotting activity of topoisomerase II was decreased by EP treatment such as the action mechanism of etoposide. Finally, a strong antitumor response was observed in SNU638/IAP cancer cells-bearing nude mice that were treated with EP. These results suggest that the prodrug-converting system by membrane-bound IAP gene and EP prodrug is useful as the strong strategy of gene therapy for cancer treatment.

Loss of endogenous TGF-beta effect induces mouse hepatoma malignancy by correlation with cyclooxygenase-2 and VEGF.

The relation between transforming growth factor-beta (TGF-beta) and cyclooxygenase (COX) in hepatoma malignancy is not understood yet. To investigate regulation mechanism of endogenous TGF-beta on hepatoma, we established MH129F mouse hepatoma cell overexpressing the cytoplasmic domain of type II TGF-beta receptor (TRII). MH129F cell apoptosis was elevated almost 20% after 5 ng/ml TGF-beta1 treatment. However, soluble TRII-overexpressing cells (MH129F/TRIIs) did not show any change of growth pattern after TGF-beta1 treatment because MH129F/TRIIs cells blocked the growth inhibitory effect of TGF-beta1. In MH129F/TRIIs cells, expression of cycooxygenase-2 (COX-2) and bcl-2 was remarkably elevated, and then enhancement of COX-2 mediated induction of prostaglandin E(2) (PGE(2)) production up to 7-fold. Especially, vascular endothelial growth factor (VEGF) expression was regulated by COX-2 in MH129F/TRIIs cells, which were inhibited endogenous TGF-beta response. Implantation of 5x10(6) MH129F/TRIIs cells into nude mice showed the significantly enhanced tumor formation, and intensity of COX-2 expression was slightly higher in MH129F/TRIIs tumor section than control. Moreover, a strong antitumor response was observed in MH129F/TRIIs-bearing mice that were treated with a specific COX-2 inhibitor, celecoxib. Therefore, we suggest that COX-2 mediate the tumorigenicity of hepatoma cells blocking endogenous TGF-beta effect via VEGF regulation.