Decrease In Plating Efficiency Research Articles

Synthesis of photoactivable Pt(IV) prodrug loaded on NaYF4 based upconversion nanoparticles functionalized with 2-deoxy-D-glucose and its evaluation for targeted cancer therapy

Nano-formulation based on Tm, Yb doped NaYF4 upconversion nanoparticles (UCNPs) functionalized with 2-deoxy-D-glucose have been synthesized to load the photoactivable Pt(IV) prodrug, cis-[PtI2(NH3)2(OCOCH2CH2COOH)2]. The Pt(IV) prodrug has been synthesized by oxidation of cis-[PtI2(NH3)2] to [PtI2(OH)2(NH3)2] and its further treatment with succinic anhydride. It is loaded through ester bond formation between the carboxyl groups of Pt(IV) prodrug with hydroxyl groups of 2-deoxy-D-glucose (2-DG) coated on UCNPs. The cytotoxicity of formulation after exposing to 385 nm UV light and in absence of light is evaluated against MCF-7 cell lines by MTT assay. The results have revealed enhanced cytotoxicity of UV exposed nano-formulation. Additionally, the clonogenic assay has exhibited the decrease in plating efficiency as inferred from decreased surviving fraction around 20% only for UV activated formulation as compared to formulation in dark, as well as merely Pt(IV) prodrug. These results are indicative that more internalization of the formulation inside the cancer cells was achieved due to the presence of 2-DG rendering more efficiency to kill cancer cells.

Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli.

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify among eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

Application of the salt stress to the protoplast cultures of the carrot (Daucus carota L.) and evaluation of the response of regenerants to soil salinity

This is the first study to generate carrot plants for enhanced salinity tolerance using a single-cell in vitro system. Protoplasts of three carrot accessions were exposed to treatment by seven different concentrations of NaCl (10–400 mM). Salt concentrations higher than 50 mM decreased plating efficiency and those of 200–400 mM of NaCl completely arrested mitotic divisions of cultured cells. The protoplast-derived plants from the control and 50–100 mM NaCl treatment were subjected to an 8-week salt stress in greenhouse conditions induced by salinized soil (EC 3 and 6 mS cm−1). 50 mM NaCl stress applied in vitro induced polyploidy among regenerated plants. The regenerants obtained from the 50 and 100 mM NaCl-treated protoplast cultures grown in saline soil had a higher survival rate compared to the regenerants from the control cultures. The salt-stressed plants accumulated anthocyanins in petioles and produced denser hairs on leaves and petioles in comparison to the control plants. Salt stress influenced pollen viability and seed setting of obtained regenerants. The results suggest that salt stress applied in vitro in protoplast cultures creates variation which allows alleviating the negative effects of salt stress on the development and reproduction of the carrot.

Radiation-induced bystander effect in non-irradiated glioblastoma spheroid cells.

Radiation-induced bystander effects (RIBEs) are detected in cells that are not irradiated but receive signals from treated cells. The present study explored these bystander effects in a U87MG multicellular tumour spheroid model. A medium transfer technique was employed to induce the bystander effect, and colony formation assay was used to evaluate the effect. Relative changes in expression of BAX, BCL2, JNK and ERK genes were analysed using RT-PCR to investigate the RIBE mechanism. A significant decrease in plating efficiency was observed for both bystander and irradiated cells. The survival fraction was calculated for bystander cells to be 69.48% and for irradiated cells to be 34.68%. There was no change in pro-apoptotic BAX relative expression, but anti-apoptotic BCL2 showed downregulation in both irradiated and bystander cells. Pro-apoptotic JNK in bystander samples and ERK in irradiated samples were upregulated. The clonogenic survival data suggests that there was a classic RIBE in U87MG spheroids exposed to 4 Gy of X-rays, using a medium transfer technique. Changes in the expression of pro- and anti-apoptotic genes indicate involvement of both intrinsic apoptotic and MAPK pathways in inducing these effects.

Genetic interactions among homologous recombination mutants in Candida albicans

rad52-ΔΔ and, to a lesser extent, rad51-ΔΔ deletants of Candidaalbicans displayed slow growth and aberrant filamentous morphology whereas rad59-ΔΔ mutants, both by growth rate and morphology resembled wild type. In this study, we have constructed pair-wise double deletants to analyze genetic interactions among these homologous recombination (HR) proteins that affect growth and morphology traits. When grown in liquid YPD medium, double mutant rad51-ΔΔ rad59-ΔΔ exhibited growth rates, cell and colony morphologies, and plating efficiencies that were not significantly different from those observed for rad51-ΔΔ. The same was true for rad52-ΔΔ rad59-ΔΔ compared to rad52-ΔΔ. Slow growth and decreased plating efficiency were caused, at least in part, by a decreased viability, as deduced from FUN1 staining. Flow cytometry and microscopic studies of filamentous mutant populations revealed major changes in cell ploidy, size and morphology, whereas DAPI staining identified complex nuclear rearrangements in yeast and filamentous cells. These phenotypes were not observed in the rad59-ΔΔ mutant populations. Our results show that abolishing Rad51 functions induces the appearance of a subpopulation of aberrant yeast and filamentous forms with increased cell size and ploidy. The size of this complex subpopulation was exacerbated in rad52-ΔΔ mutants. The combination of filamentous cell morphology and viability phenotypes was reflected on the colony morphology of the respective mutants. We conclude that the rad52 mutation is epistatic to rad51 for all the morphological traits analyzed. We discuss these results in the light of the several functions of these recombination genes.

Mixed effects of long-term frozen storage on cord tissue stem cells

Background aimsCord tissue (CT) storage is promoted as an opportunity to preserve a source of mesenchymal stromal cells (MSCs) for future use. We analyzed maximal MSC yields from fresh and frozen CT including functional capacity after long-term cryopreservation as a means of assessing potential utility. MethodsCT was evaluated immediately upon harvest or frozen and banked for 5 years before analysis. Upon thawing, cell viability and yield were determined, as were growth characteristics and the ability to differentiate into various tissues. After thawing, enzymatic digestion of CT to release MSCs resulted in poor cell recoveries and few viable cells, requiring explant cultures to recover sufficient cell numbers for analysis. Upon expansion of surviving cells, fluorescence-activated cell sorter analysis showed the cells to be MSCs based on phenotype (CD34–, 45–, 44+, 90+, 105+) and function (ability to form adipocytes and osteoblasts). Frozen CT, however, exhibited decreased plating efficiency, increased doubling times but near equivalent maximum cell expansion, compared with fresh CT. ConclusionsPoor cell yields and recoveries, along with slower growth characteristics, make frozen CT a less-than-optimal choice for MSC banking, despite good functional recovery. In addition, because the amount of fresh CT available at birth is limited and total MSC yields are low, even fresh CT-MSC requires extensive in vitro expansion before clinical use, which limits it application.

Effect of β-lactam antibiotics on plant regeneration in carrot protoplast cultures.

Protoplasts of three carrot cultivars were isolated from in vitro-grown plantlets by overnight incubation in an enzyme mixture composed of 1% (w/v) cellulase Onozuka R-10 and 0.1% (w/v) pectolyase Y-23. After cell immobilization in modified thin alginate layers, three types of β-lactam antibiotics (cefotaxime, carbenicillin, or timentin) at five different concentrations (100, 200, 300, 400, or 500 mg L−1) were added to the culture medium. In 20-d-old cultures, a different number of cell colonies had formed and varied on average from 27 to 56% in carbenicillin- and cefotaxime-containing media, respectively. Supplementation of the culture media with antibiotics at concentrations higher than 100 mg L−1 resulted in a decrease in plating efficiency in comparison with the controls. However, from all antibiotic treatments, except carbenicillin at concentrations of 400–500 mg L−1, efficient plant regeneration occurred. For this reason, we believe that cefotaxime and timentin in the concentrations analyzed here may be used in complex in vitro procedures or valuable carrot cultures as a prophylactic agent for prevention against occasional contaminations.

Monoglucosyl-rutin as a potential radioprotector in mammalian cells.

In the present study, the role of monoglucosyl-rutin as a potential radioprotector was investigated using mammalian cell culture models. Cell survival and DNA damage were assessed using colony formation, sister chromatid exchange and γH2AX assays. It was demonstrated that monoglucosyl-rutin was able to increase cell survival when exposed to ionizing radiation, possibly by decreasing the amount of base damage experienced by the cell. However, the present study also demonstrated that, despite monoglucosyl-rutin exhibiting radioprotective effects at low doses, high doses of monoglucosyl-rutin led to a decrease in plating efficiency and an increased doubling time. This effect may be due to double-strand breaks caused by high concentrations of monoglucosyl-rutin.

Decreased plating efficiency, proliferation and osteogenic differentiation of synovial fluid mesenchymal progenitors as a marker of severity of juvenile idiopathic arthritis

Decreased plating efficiency, proliferation and osteogenic differentiation of synovial fluid mesenchymal progenitors as a marker of severity of juvenile idiopathic arthritis

Expression and role in glycolysis of human ADP-dependent glucokinase

A novel murine enzyme, ADP-dependent glucokinase (ADPGK), has been shown to catalyse glucose phosphorylation using ADP as phosphoryl donor. The ancestral ADPGK gene appears to have been laterally transferred from Archaea early in metazoan evolution, but its biological role has not been established. Here, we undertake an initial investigation of the functional properties of human ADPGK in human tumour cell lines and specifically test the hypothesis that ADPGK might prime glycolysis using ADP under stress conditions such as hypoxia. Recombinant human ADPGK was confirmed to catalyse ADP-dependent glucose phosphorylation in vitro, with an apparent K (M) for glucose of 0.29 mM. Expression databases and western blotting of surgical samples demonstrated high expression in many human tissues, including tumours. Unlike hexokinase-2 (HK2), RNAi studies with exon arrays showed that ADPGK is not a transcriptional target of hypoxia inducible factor-1. Consistent with this, ADPGK protein was not upregulated by hypoxia or anoxia. Surprisingly, stable fivefold overexpression of ADPGK in H460 or HCT116 cells had no apparent effect on proliferation or glycolysis, and did not rescue clonogenicity or glycolysis when HK2 was suppressed by siRNA. Furthermore, suppression of ADPGK by siRNA did not cause detectable inhibition of glycolysis or cell killing by anoxia, although it did induce a statistically significant decrease in plating efficiency of H460 cells under aerobic conditions. Thus, human ADPGK catalyses ADP-dependent phosphorylation of glucose in vitro, but despite its high expression in human tumour cell lines it appears not to make a quantifiable contribution to glycolysis under the conditions evaluated.

Inhibition of NF-κB and DNA double-strand break repair by DMAPT sensitizes non-small-cell lung cancers to X-rays

We investigated the efficacy and mechanism of dimethylaminoparthenolide (DMAPT), an NF-κB inhibitor, to sensitize human lung cancer cells to X-ray killing in vitro and in vivo. We tested whether DMAPT increased the effectiveness of single and fractionated X-ray treatment through inhibition of NF-κB and/or DNA double-strand break (DSB) repair. Treatment with DMAPT decreased plating efficiency, inhibited constitutive and radiation-induced NF-κB binding activity, and enhanced radiation-induced cell killing by dose modification factors of 1.8 and 1.4 in vitro. X-ray fractionation demonstrated that DMAPT inhibited split-dose recovery/repair, and neutral DNA comet assays confirmed that DMAPT altered the fast and slow components of X-ray-induced DNA DSB repair. Knockdown of the NF-κB family member p65 by siRNA increased radiation sensitivity and completely inhibited split-dose recovery in a manner very similar to DMAPT treatment. The data suggest a link between inhibition of NF-κB and inhibition of DSB repair by DMAPT that leads to enhancement of X-ray-induced cell killing in vitro in non-small-cell lung cancer cells. Studies of A549 tumor xenografts in nude mice demonstrated that DMAPT enhanced X-ray-induced tumor growth delay in vivo.

CXXC Finger Protein 1 Contains Redundant Functional Domains That Support Embryonic Stem Cell Cytosine Methylation, Histone Methylation, and Differentiation

CXXC finger protein 1 (Cfp1) is a regulator of both cytosine methylation and histone methylation. Murine embryonic stem (ES) cells lacking Cfp1 exhibit a decreased plating efficiency, decreased cytosine methylation, elevated global levels of histone H3-Lys4 trimethylation, and a failure to differentiate in vitro. Remarkably, transfection studies reveal that expression of either the amino half of Cfp1 (amino acids 1 to 367 [Cfp1(1-367)]) or the carboxyl half of Cfp1 (Cfp1(361-656)) is sufficient to correct all of the defects observed with ES cells that lack Cfp1. However, a point mutation (C169A) that abolishes DNA-binding activity of Cfp1 ablates the rescue activity of the Cfp1(1-367) fragment, and a point mutation (C375A) that abolishes the interaction of Cfp1 with the Setd1 histone H3-Lys4 methyltransferase complexes ablates the rescue activity of the Cfp1(361-656) fragment. Introduction of both the C169A and C375A point mutations ablates the rescue activity of the full-length Cfp1 protein. These results indicate that retention of either the Cfp1 DNA-binding domain or Setd1 interaction domain is required for Cfp1 rescue activity, and they illustrate the functional complexity of this critical epigenetic regulator. A model is presented for how epigenetic cross talk may explain the finding of redundant functional domains within Cfp1.

Mitochondrial DNA Depletion Induces Radioresistance by Suppressing G2Checkpoint Activation in Human Pancreatic Cancer Cells

We hypothesized that mitochondrial function regulates cell cycle checkpoint activation and radiosensitivity. Human pancreatic tumor cells (MiaPaCa-2, rho(+)) were depleted of mitochondrial DNA (rho degrees ) by culturing cells in the presence of ethidium bromide. Depletion of mitochondrial DNA was verified by PCR amplification of total DNA using primer pairs specific for mitochondrial DNA. Loss of mitochondrial DNA decreased plating efficiency and the percentage of cells in S phase. Exponential cultures were irradiated with 2, 4 and 6 Gy (dose rate: 0.83 Gy/min) of ionizing radiation and harvested for determination of cell viability, growth and cell cycle phase distributions. Rho degrees cells were radioresistant compared to rho(+) cells, with a dose-modifying factor (DMF) of 1.6. Although cell growth was significantly inhibited in irradiated rho(+) cells compared to unirradiated control cells, the inhibition in Rho degrees cells was minimal. In addition, mitochondrial DNA depletion suppressed radiation-induced G(2) checkpoint activation, which was accompanied by increases in both cyclin B1 and CDK1. These results suggest that mitochondrial function may regulate cell cycle checkpoint activation and radiosensitivity in pancreatic cancer cells.

Direct Confirmation of Quiescent Property of AML Stem Cells by Showing Markedly Decreased Plating Efficiency in Single Leukemic Stem Cell Culture

Cancer stem cell, recently discovered to exist in colon cancer and brain tumor, is resistant to anti-cancer drugs and radiotherapy, demanding the development of new drugs and treatment strategies targeting tumor stem cells. Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). Various anti-cancer medicines, particularly anti-proliferative agents, have been ineffective in treating LSC due to its slower division process and longer interphase compared to normal stem cell and hematopoietic cell. This study comparatively examined growth and proliferation capacity (plating efficiency) of clonogenic hematopoietic progenitors and LSC from healthy donors and AML patients using single cell sorting and culture system (BD FACS Aria cell sorter; BD Biosciences, San Jose, CA). A total of 384 normal hematopoietic stem cells (CD34+CD38+/CD38−) were obtained from peripheral bloods and cord bloods donated by four donors using single cell sorter, and individual single cells were cultured in 96-well plates with each well containing 100ul of serum media, 100ng/ml of stem cell factor, 100ng/ml of Flt-3, 100ng/ml of thrombopoietin and 50ng/ml of G-CSF for five days. 768 single LSC (CD34+CD38−) and 384 single CD34+CD38+ cells were obtained from three AML patients. Growth and proliferation capacities of normal hematopoietic stem cell and LSC were determined in terms of plating efficiency (number of the wells in which more than two cells grew/total number of cells in 96-well plate culture × 100). Plating efficiency of individual normal single hematopoietic stem cells varied between samples. Eighty eight out of 192 single stem cells originated from cord blood grew into more than two cells, yielding plating efficiency of 45.8% and cells from the peripheral blood of two healthy donors 30.2% (58/192). In contrast, single LSC originated from the AML patients showed significantly lower plating efficiency with 14.6% (42/288), 3.6% (7/192) and 8.0% (23/288). These results directly confirmed quiescent and slowly dividing properties of LSC. In addition, plating efficiency of normal hematopoietic stem cells was shown to vary between their originating locations in healthy donors.

A Role for Mitochondrial Dysfunction in Perpetuating Radiation-Induced Genomic Instability

Radiation-induced genomic instability (RIGI) manifests as a heritable increased rate of genetic alterations in the progeny of irradiated cells generations after the initial insult. The progeny can show an increased frequency of chromosomal translocations, deletions, mutations, micronuclei, and decreased plating efficiency. What perpetuates RIGI is unclear; however, persistently increased levels of reactive oxygen species (ROS) are frequently associated with genomically unstable clones. Furthermore, addition of free radical scavengers (e.g., DMSO, glycerol, and cationic thiol cysteamine) reduces the incidence of instability after irradiation, implicating a ROS-mediated role in RIGI induction. Because mitochondria are a major natural cellular source of ROS, we tested the hypothesis that mitochondrial dysfunction has a role in maintaining the elevated ROS levels in our irradiated, genetically unstable GM10115 Chinese hamster ovary cells. Amplex Red fluorometry measurements indicate that the relative contribution of uncoupler-sensitive mitochondrial hydrogen peroxide production to total cellular hydrogen peroxide generation is greater in unstable cells. Measurements of mitochondrial DNA levels and cell cytometric fluorescent measurements of Mitotracker Green FM indicate that differences in mitochondrial ROS production are not due to varying mitochondrial levels. However, mitochondrial respiration measured in digitonin-permeabilized cells is impaired in unstable clones. In addition, manganese superoxide dismutase, a major mitochondrial antioxidant enzyme, exhibits increased immunoreactivity but decreased enzyme activity in unstable clones, which along with decreased respiration rates may explain the increased levels of cellular ROS. These studies show that mitochondria from unstable cells are abnormal and likely contribute to the persistent oxidative stress in the unstable clones.

Enzymatic Activity Is Necessary for the Tumor-Suppressive Effects of MnSOD

The antioxidant protein manganese-containing superoxide dismutase (MnSOD) has been found to be a new type of tumor-suppressor protein. Overexpression of the cDNA for this gene in various types of cancer via plasmid transfection or adenovirus transduction leads to growth suppression both in vitro and in vivo. The growth-suppressive effect of MnSOD overexpression has been presumed to be due to the enzymatic activity of the MnSOD protein, but could be due to a number of other mechanisms, including a regulatory effect of the RNA or protein produced. To examine this question, we used site-directed mutagenesis to produce a mutant form of human MnSOD that has a leucine at amino acid 26 in the active site rather than the usual histidine. We demonstrate that plasmid transfection or adenoviral transduction of this mutant MnSOD cDNA leads to a large increase in immunoreactive MnSOD protein, but little or no increase in enzymatic activity. In contrast, overexpression of wild-type MnSOD leads to cells with both increased MnSOD protein and activity. Overexpression of wild-type, but not mutant, MnSOD leads to decreased plating efficiency and growth. These results clearly demonstrate that the tumor-suppressive effect of MnSOD protein is largely due to its enzymatic activity.

Suppression of the Malignant Phenotype in Pancreatic Cancer by Overexpression of Phospholipid Hydroperoxide Glutathione Peroxidase

Phospholipid glutathione peroxidase (PhGPx) reduces lipid hydroperoxides generated in biomembranes and also uses a wide range of reducing cofactors in addition to glutathione. PhGPx is synthesized as a mitochondrial PhGPx form (L-form) and as a nonmitochondrial PhGPx form (S-form). Our aims were to determine whether overexpression of PhGPx altered pancreatic tumor cell behavior. Pancreatic cancer cell lines were found by Western blotting to have diminished levels of PhGPx-immunoreactive protein compared with normal human pancreas. To normalize the levels of this protein, PhGPx was overexpressed in MIA PaCa-2 and AsPC-1 human pancreatic cancer cells by infection with an adenovirus-PhGPx L-form construct (AdPhGPx- L-form) (0-200 MOI) or with an adenovirus-PhGPx S-form construct (AdPhGPx-S-form) (0-200 MOI), and cell growth, plating efficiency, and growth in soft agar were determined. Pancreatic cancer cells were also injected subcutaneously into nude mice and tumor volume was calculated. Single direct injections of the adenoviral- PhGPx constructs were made into preestablished tumors. In vitro, AdPhGPx-S-form demonstrated 80% tumor growth inhibition, whereas AdPhGPx-L-form demonstrated 95% tumor growth inhibition. Ad- PhGPx-L-form or AdPhGPx-S-form also decreased plating efficiency and growth in soft agar. AdPhGPx-Lform decreased in vivo tumor growth to a greater extent than did AdPhGPx-S-form. Because of the growthinhibitory effects of PhGPx, lipid hydroperoxides may play an important role in the growth of pancreatic cancer.

Human Aldehyde Dehydrogenase 3A1 Inhibits Proliferation and Promotes Survival of Human Corneal Epithelial Cells

Aldehyde dehydrogenase 3A1 (ALDH3A1) is a NAD(P)+-dependent enzyme that is highly expressed in mammalian corneal epithelial cells and has been shown to protect against UV- and 4-hydroxynonenal-induced cellular damage, mainly by metabolizing toxic lipid peroxidation aldehydes. Here we report a novel function of ALDH3A1 as a negative cell cycle regulator. We noticed a reduction in ALDH3A1 gene expression in actively proliferating primary human corneal epithelium explant cultures, indicating that ALDH3A1 expression is inversely correlated with replication. To examine this further, a human corneal epithelial cell line (HCE) lacking endogenous ALDH3A1 was stably transfected to express ALDH3A1 at levels similar to those found in vivo. ALDH3A1-transfected cells exhibited an elongated cell cycle, decreased plating efficiency, and reduced DNA synthesis compared with the mock-transfected cells. These effects were associated with reduced cyclin A- and cyclin B-dependent kinase activities and reduced phosphorylation of the retinoblastoma protein (pRb) as well as decreased protein levels of cyclins A, B, and E, the transcription factor E2F1, and the cyclin-dependent kinase inhibitor p21. These observations were further expanded and confirmed on human keratinocyte cells (NCTC-2544) overexpressing ALDH3A1. Consistent with a protective role of an elongated cell cycle, ALDH3A1-transfected cells exhibited increased resistance to the cytotoxic effects of the DNA-damaging agents mitomycin C and Vp-16. Immunohistochemistry and biochemical fractionation revealed that ALDH3A1 is localized both in the nucleus and cytosol of ALDH3A1-transfected cells, implying a possible association between the nuclear localization of the enzyme and its proliferation-suppressive functions. In conclusion, these results suggest that ALDH3A1 may protect corneal epithelial cells against oxidative damage not only through its metabolic function but also by prolonging the cell cycle.

Synergistic effect of histone deacetylase inhibitors FK228 and m-carboxycinnamic acid bis-hydroxamide with proteasome inhibitors PSI and PS-341 against gastrointestinal adenocarcinoma cells.

We investigated whether the histone deacetylase inhibitors m-carboxycinnamic acid bis-hydroxamide (CBHA) and a bicyclic depsipeptide, FK228, can enhance the anticancer effect of the proteasome inhibitors PSI and PS-341 in gastrointestinal carcinoma cells. The anticancer effect of CBHA or FK228 and PSI or PS-341 was evaluated by cell death, caspase-3 activity, externalization of phosphatidylserine and DNA fragmentation, and colony formation assay. Expression of apoptosis-related molecules and cell cycle regulatory molecules, as well as phosphorylation of p38 were investigated by immunoblots. Generation of reactive oxygen species (ROS) was detected by intracellular oxidation of 5- (and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate. CBHA or FK228 plus PSI or PS-341 synergistically induced apoptosis in human colonic DLD-1 and gastric MKN45 carcinoma cell lines. CBHA or FK228, but not 5-fluorouracil, plus PS-341 strongly decreased plating efficiency of DLD-1 cells. FK228 elicited ROS generation, and the free radical scavenger l-N-acetylcysteine inhibited the synergistic anticancer effect of combined therapy. In addition, l-N-acetylcysteine inhibited the combined therapy-mediated elevation of a proapoptotic BH3-only protein Bim expression, phosphorylation of H2AX, and accumulation of 8-hydroxydeoxyguanosine. FK228 or CBHA and PSI or PS-341 synergistically induce apoptosis in DLD-1 and MKN45 cells depending on ROS-mediated signals. Our data suggest that a combination of FK228 or CBHA with PSI or PS-341 may be a valuable therapy against gastrointestinal adenocarcinoma cells.

Redox regulation of pancreatic cancer cell growth: role of glutathione peroxidase in the suppression of the malignant phenotype.

Pancreatic cancer has low levels of antioxidant enzymes including manganese superoxide dismutase (MnSOD), which converts superoxide radical (O(2)(*-)) into hydrogen peroxide (H(2)O(2)), and glutathione peroxidase (GPx), which converts H(2)O(2) into water. Recent studies have demonstrated that overexpression of MnSOD has a tumor-suppressive effect in pancreatic cancer. However, GPx overexpression has been shown to reverse the tumor cell growth inhibition caused by MnSOD overexpression in other types of cancer. Our aims were to determine if overexpression of GPx alters in vitro pancreatic cancer cell behavior and if delivering the GPx gene directly to tumor xenografts alters growth and survival. In vitro, AdGPx slowed tumor growth by 39% and AdMnSOD slowed tumor growth by 35%. AdGPx also decreased plating efficiency and growth in soft agar. The combination of AdGPx and AdMnSOD had the greatest effect on tumor cell growth suppression with a 71% reduction in cell growth compared to controls. In vivo, either AdGPx or AdMnSOD alone slowed tumor growth by 51% and 54%, respectively, while the combination of AdGPx and AdMnSOD potentiated tumor growth suppression by 81% of controls and increased animal survival. GPx may be a tumor suppressor gene in pancreatic cancer. Delivery of the GPx gene alone or in combination with the MnSOD gene may prove beneficial for treatment of pancreatic cancer.