Pretreatment Of HeLa Cells Research Articles

Characteristics of quercetin interactions with liposomal and vacuolar membranes

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is claimed to exert many beneficial health effects. With application of 1H NMR (nuclear magnetic resonance) and FTIR (Fourier-transform infrared) techniques, quercetin interaction with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was analyzed. Patch-clamp technique was employed to study quercetin effects at single channel level of vacuolar membranes in the liverwort Conocephalum conicum. Light and electron microscopy were applied to study quercetin effects on human negroid cervix carcinoma cells (HeLa). Enzymatic measurements along with DPPH (1,1-diphenyl-2-picrylhydrazyl) bioassay were performed to investigate the influence of quercetin on antioxidant enzymes and reactive oxygen species (ROS) production. The inclusion of quercetin to the membrane exerted pronounced ordering effect on the motional freedom of lipids in the head group region as manifested by broadening of the 1H NMR spectral line representing the choline groups. FTIR analysis revealed quercetin incorporation into DPPC liposomes via hydrogen bonding between its own hydroxyl groups and lipid polar head groups in the C–O–P–O–C segment. Both, FTIR and NMR techniques indicated also quercetin spectral effects in the region corresponding to alkyl chains. Patch-clamp experiments showed that quercetin stabilizes tonoplast and promotes a close state of SV channels. Microscopic observations of HeLa cells revealed characteristic changes in ultrastructure and morphology of the examined cells in comparison to control cells. Pretreatment of HeLa cells with quercetin alleviated H2O2-induced cell injury by improving redox balance as indicated by the increase in glutathione content and SOD (superoxide dismutase) levels as well as by the decrease in ROS level. \\In conclusion, the incorporation, distribution and the changes of biophysical properties of the membranes are very important for the effectiveness of phenolic compounds as antioxidant and anticancer factors.

Essential role of autophagy in fucoxanthin-induced cytotoxicity to human epithelial cervical cancer HeLa cells

To investigate the effects and the molecular mechanisms of fucoxanthin, a major carotenoid found in edible seaweed, on HeLa cells. The cytotoxicity of fucoxanthin was evaluated using MTT assay. Cell cycle and apoptosis were evaluated using flow cytometric analysis. Autophagy was detected with acridine orange staining and transient transfection of the GFP-LC3 plasmid into the cells. Protein expression was detected with Western blotting. Treatment of HeLa cells with fucoxanthin (10-80 μmol/L) for 48 h caused dose-dependent cytotoxicity with an IC50 value of 55.1±7.6 μmol/L. Fucoxanthin (10, 20, and 40 μmol/L) dose-dependently induced G0/G1 arrest, but did not change the apoptosis of HeLa cells. The same concentrations of fucoxanthin dose-dependently increased the protein expression of LC3 II (the autophagosome marker) and Beclin 1 (the initiation factor for autophagosome formation) in HeLa cells. Moreover, fucoxanthin dose-dependently decreased the levels of phosphorylated Akt and its downstream proteins p53, p70S6K, and mTOR, and increases the expression of PTEN in HeLa cells. Pretreatment of HeLa cells with 3-methyladenine (5 mmol/L) blocked the cytotoxic effect of fucoxanthin as well as fucoxanthin-induced autophagy. Fucoxanthin exerts autophagy-dependent cytotoxic effect in HeLa cells via inhibition of Akt/mTOR signaling pathway.

Both PI3K- and mTOR-Signaling Pathways Take Part in CVB3-Induced Apoptosis of HeLa Cells

This work illustrated the mechanism contributing to the process of Phosphatidylinostiol 3-kinase (PI3K)/protein kinase B (PKB)/mammalian target of rapamycin (mTOR) signaling pathway, which has been demonstrated to play an important role in virus-induced apoptosis, which contributes to the Viral Myocarditis (VMC) pathogeneses. We examined the expression of Bax, Bim, caspase-3, caspase-9, and viral replication after Coxsackievirus B3 (CVB3) infection using the mTOR inhibitor and PI3K inhibitor pretreated HeLa cells, respectively. Apoptosis in different groups was determined by flow cytometry. Bax, Bim, caspase-9, and caspase-3 were examined by semiquantitative polymerase chain reaction (PCR) and Western blot analysis. The expression of CVB3 mRNA and viral capsid protein VP1 were analyzed by semiquantitative PCR and Western blot analysis distinctively. We found that rapamycin and LY294002 promote CVB3-induced cytopathic effect (CPE) and apoptosis. CVB3 replication in host cells is mediated in mRNA and protein expression by rapamycin and LY294002. Moreover, comparing with controls, at 12 and 24 h of postinfection (p.i.), Bim and Bax expression increased in cells after treated with rapamycin or LY294002, which also stimulates the activation of procaspase-9, and the CVB3-induced caspase-3 self-cleavage. However, in the meantime, the mRNA expression of caspase-9 and caspase-3 did not have an obvious change. In summary, our results demonstrated that the mTOR-signaling pathway plays an important role in CVB3-induced CPE and apoptosis, which is indispensable in VMC, via regulating Bim, Bax, caspase-9, caspase-3, and viral replication. Our findings may provide a new perspective and a deeper understanding of the mechanism of CVB3-induced apoptosis which, in turn, may help with the development of new therapy for the CVB3 infection.

Disulfide crosslinked PEGylated starch micelles as efficient intracellular drug delivery platforms

Novel reduction-responsive disulfide core-crosslinked micelles based on amphiphilic starch-graft-poly(ethylene glycol) (starch-g-PEG) were prepared and used for efficient intracellular drug delivery. The starch-g-PEG copolymers can be conveniently prepared by grafting starch with carboxyl group terminated PEG, and subsequently conjugated with lipoic acid for disulfide crosslinking. The self-assembled starch-g-PEG micelles and the corresponding disulfide core-crosslinked micelles were then characterized by transmission electron microscopy, dynamic laser scattering and fluorescence techniques. It is interesting to observe that the hydrodynamic radii of disulfide core-crosslinked micelles would increase gradually in phosphate buffered saline (PBS) due to the cleavage of the disulfide bond in the micellar core, caused by the presence of reductive glutathione (GSH). The glutathione-responsive behaviors of the disulfide core-crosslinked micelles should be attractive for intracellular drug delivery. Thus, a model anticancer drug doxorubicin (DOX) was loaded into micelles and the in vitro drug release in response to GSH was also studied. The results showed that only a small amount of loaded DOX was released from the core-crosslinked starch-g-PEG micelles in PBS solution without GSH, while quick release occurred in the presence of 10.0 mM GSH. Confocal laser scanning microscopy and flow cytometry analyses further demonstrate that the disulfide crosslinked micelles exhibited a faster drug release behavior in glutathione monoester (GSH-OEt) pretreated HeLa cells than that in the nonpretreated and buthionine sulfoximine (BSO) pretreated cells. In addition, the DOX-loaded crosslinked micelles show higher cellular proliferation inhibition against GSH-OEt pretreated HeLa and HepG2 than against the nonpretreated and BSO pretreated ones. These results suggest that such disulfide crosslinked starch-g-PEG micelles, which can efficiently release the loading drug in response to intracellular GSH concentration, may provide favorable platforms for cancer therapy.

Chlamydial infection in vitamin D receptor knockout mice is more intense and prolonged than in wild-type mice

Vitamin D hormone (1,25-dihydroxyvitamin D) is involved in innate immunity and induces host defense peptides in epithelial cells, suggesting its involvement in mucosal defense against infections. Chlamydia trachomatis is a major cause of bacterial sexually transmitted disease worldwide. We tested the hypothesis that the vitamin D endocrine system would attenuate chlamydial infection. Vitamin D receptor knock-out mice (VDR−/−) and wild-type mice (VDR+/+) were infected with 103 inclusion forming units of Chlamydia muridarum and cervical epithelial cells (HeLa cells) were infected with C. muridarum at multiplicity of infection 5:1 in the presence and absence of 1,25-dihydroxyvitamin D3. VDR−/− mice exhibited significantly higher bacterial loading than wild-type VDR+/+ mice (P<0.01) and cleared the chlamydial infection in 39 days, compared with 18 days for VDR+/+ mice. Monocytes and neutrophils were more numerous in the uterus and oviduct of VDR−/− mice than in VDR+/+ mice (P<0.05) at d 45 after infection. Pre-treatment of HeLa cells with 10nM or 100nM 1,25-dihydroxyvitamin D3 decreased the infectivity of C. muridarum (P<0.001). Several differentially expressed protein spots were detected by proteomic analysis of chlamydial-infected HeLa cells pre-treated with 1,25-dihydroxyvitamin D3. Leukocyte elastase inhibitor (LEI), an anti-inflammatory protein, was up-regulated. Expression of LEI in the ovary and oviduct of infected VDR+/+ mice was greater than that of infected VDR−/− mice. We conclude that the vitamin D endocrine system reduces the risk for prolonged chlamydial infections through regulation of several proteins and that LEI is involved in its anti-inflammatory activity.

Enhancing effect of Thevetia peruviana flower extract on TNF-α induced apoptosis in human cervical cancer cells

Tumor necrosis factor (TNF-α) is a pleiotropic cytokine triggers several intracellular signaling pathways including mitogen-activated protein kinases, nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase, which involve in survival and apoptosis of cancers. This study was conducted to investigate the effect of ethanolic extract from the flowers of Thevetia peruviana on TNF-α induced apoptosis in human cervical cancer (HeLa) cells by determinations of cell viability and apoptosis using WST-1 cell proliferating assay and immunoblot analysis, respectively. Pretreatment of HeLa cells with various concentrations (3–300µg/mL) of flower ethanolic extracts of T. peruviana enhanced TNF-α induced antiproliferation in HeLa cells. Extracts at concentration of 30, 100 and 300µg/mL together with TNF-α significantly decreased viability of HeLa cells compared with that treated with only TNF-α. These extracts strongly promoted TNF-α induced cleavage of caspase-3 and PARP-1. Compared with HeLa cells treated with T. peruviana extract alone, only extract at concentration of 30µg/mL together with TNF-α strongly decreased cancer cell viability. The results suggested that ethanolic extract from the flowers of Thevetia peruviana showed enhancing effect on TNF-α induced apoptosis in HeLa cells by activation of caspase cascade.

Intracellular microenvironment responsive PEGylated polypeptide nanogels with ionizable cores for efficient doxorubicin loading and triggered release

Two kinds of reduction and pH responsive disulfide-cross-linked poly(ethylene glycol)-polypeptide copolymers were prepared through one-step ring-opening polymerization of γ-benzyl-L-glutamate N-carboxyanhydride (BLG NCA) or e-benzyloxycarbonyl-L-lysine N-carboxyanhydride (ZLL NCA) and L-cystine N-carboxyanhydride (LC NCA) with amino group terminated monomethoxy poly(ethylene glycol) (mPEG-NH2) as macroinitiator. Then, the copolymers were deprotected and dispersed in phosphate buffered saline, yielding PEG-polypeptide nanogels. Doxorubicin (DOX), a model anticancer drug, was effectively loaded into nanogels via electrostatic and hydrophobic interactions. The DOX release from all DOX-loaded nanogels was accelerated in intracellular reductive and acidic conditions, which controlled by Fickian diffusion and nanogels swelling. The enhanced intracellular DOX release was observed in glutathione monoester (GSH-OEt) pretreated HeLa cells. DOX-loaded nanogels showed higher cellular proliferation inhibition towards GSH-OEt pretreated HeLa and HepG2 cells than to unpretreated or buthionine sulfoximine (BSO) pretreated cells. Hemolysis tests indicated that nanogels were hemocompatible, and the presence of nanogels could reduce the hemolysis ratio (HR) of DOX significantly. These features suggest that the nanogels can efficiently load and deliver DOX into tumor cells and enhance the inhibition of cellular proliferation in vitro, providing a favorable platform to construct an efficient drug delivery system for cancer therapy.

Anti-Rhinovirus-specific Activity of the Alpha-sympathomimetic Oxymetazoline

Oxymetazoline (CAS 2315-02-8, OMZ, Nasivin) known as the active ingredient in nose drops and sprays demonstrates excellent efficacy in the treatment of rhinitis symptoms that are mainly caused by Rhinovirus infections. To elucidate possible modes of action, the antiviral activity of OMZ was studied in vitro on human pathogenic viruses. No in vitro effects were detected against enveloped RNA viruses, Parainfluenza Virus and Respiratory Syncytial Virus and against Adenovirus, a non-enveloped DNA-virus. In contrast, OMZ showed a specific inhibition of Human Rhinovirus (HRV). Analysis of production of HRV-14 and HRV-39 after treatment of infected HeLa cells using plaque-reduction assay and virus titration showed a strong dose-dependent antiviral activity of OMZ. Additional data demonstrated that OMZ did also directly affect HRV-14 infectivity in a dose-dependent manner. Analysis of a cell-protective effect of OMZ showed that pre-treatment of HeLa cells decreased virus adsorption as well as virus replication. Furthermore, OMZ induced a down-regulation of ICAM-1 expression on Tumor Necrosis Factor-alpha (TNF-alpha)-stimulated HeLa cells and human umbilical vein endothelial cells. Taken together, these results show that OMZ besides its vasoconstrictive action also possesses potent antiviral and anti-inflammatory activities. Therefore, OMZ does not only reduce rhinitis symptoms but additionally offers a causal therapeutic approach.

Melatonin protects against rotenone‐induced cell injury via inhibition of Omi and Bax‐mediated autophagy in Hela cells

Parkinson's disease is the second most common neurodegenerative disease, and environmental toxins such as rotenone play an important role in causing degeneration of dopaminergic neurons. Melatonin, a major secretory product of pineal, is recently reported to protect against rotenone-induced cell death in animal models. Yet, the mechanism involved in this protection needs to be elucidated. Here, we report that rotenone treatment (0-100 μM) decreased cell survival of Hela cells in a dose-dependent manner. At concentrations ranging from 0.1 to 100 μM, rotenone induced a dose-dependent increase in the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, a protein associated with the autophagosomal membrane. Knockdown of Bax or Omi using shRNA inhibited 1 μM rotenone-induced autophagy. To determine whether melatonin would protect cells against rotenone-induced cell death and autophagy, we pretreated Hela cells with 250 μM melatonin for 24 hr in the presence of rotenone. Melatonin inhibited Bax expression and the release of the omi/HtrA2 into the cytoplasm induced by 1 μM rotenone. Melatonin 250 μM treatment also suppressed cell death induced by 0.1-100 μM rotenone and protected against the formation of LC3-II in cells exposed to 1 μM rotenone. This work demonstrates a novel role for melatonin as a neuroprotective agent against rotenone.

Investigation of antioxidant activity of alcoholic extract of Globularia alypum L.

Hydroethanolic extract from leaves of Globularia alypum L. (HEGA), Globulariaceae, was screened for its content on total polyphenols, flavonoids and anthocyanins. Its antioxidant activity was also assessed in chemical and biological systems. Analysis of the antioxidant compounds reveals that the HEGA contains high amounts of polyphenols, flavonoids and anthocyanins. Besides, it shows a significant antioxidant activity. When tested in HeLa cells, the extract exerts an antiproliferative effect. The concentration leading to a 50% inhibition was estimated at 1.53 mg/ml. The pretreatment of HeLa cells with non cytotoxic concentration of the leaf extract protects them from Fe2+ and hydrogen peroxide induced oxidative stress. A good protection against lipid peroxidation was obtained and evidenced by a decrease in the level of two lipid peroxidation (LPO) products, Malodialdehyde (MDA) and conjugated dienes (CD). Besides, a decrease in catalase and superoxide dismutase (SOD) activities was obtained. This suggests a recovery of the oxidative balance in the treated cells. Key words: Globularia alypum, oxidative stress, cell culture, malondialdehyde, superoxide dismutase, catalase.

Redox-Responsive Polyphosphate Nanosized Assemblies: A Smart Drug Delivery Platform for Cancer Therapy

Novel redox-responsive polyphosphate nanosized assemblies based on amphiphilic hyperbranched multiarm copolyphosphates (HPHSEP-star-PEP(x)) with backbone redox-responsive, good biocompatibility, and biodegradability simultaneously have been designed and prepared successfully. The hydrophobic core and hydrophilic multiarm of HPHSEP-star-PEP(x) are composed of hyperbranched and linear polyphosphates, respectively. Benefiting from the amphiphilicity, HPHSEP-star-PEP(x) can self-assemble into spherical micellar nanoparticles in aqueous media with tunable size from about 70 to 100 nm via adjusting the molecular weight of PEP multiarm. Moreover, HPHSEP-star-PEP(x) micellar structure can be destructed under reductive environment and result in a triggered drug release behavior. The glutathione-mediated intracellular drug delivery was investigated against a HeLa human cervical carcinoma cell line, and the results indicate that doxorubicin-loaded (DOX-loaded) HPHSEP-star-PEP(x) micelles show higher cellular proliferation inhibition against glutathione monoester pretreated HeLa cells than that of the nonpretreated ones. In contrast, the DOX-loaded micelles exhibit lower inhibition against buthionine sulfoximine pretreated HeLa cells. These results suggest that such redox-responsive polyphosphate micelles can rapidly deliver anticancer drugs into the nuclei of tumor cells enhancing the inhibition of cell proliferation and provide a favorable platform to construct excellent drug delivery systems for cancer therapy.

CD4-Independent Human Immunodeficiency Virus Infection Involves Participation of Endocytosis and Cathepsin B

During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1) strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 µM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1.

Bioreducible Micelles Self-Assembled from Amphiphilic Hyperbranched Multiarm Copolymer for Glutathione-Mediated Intracellular Drug Delivery

A new type of biodegradable micelles for glutathione-mediated intracellular drug delivery was developed on the basis of an amphiphilic hyperbranched multiarm copolymer (H40-star-PLA-SS-PEP) with disulfide linkages between the hydrophobic polyester core and hydrophilic polyphosphate arms. The resulting copolymers were characterized by nuclear magnetic resonance (NMR), Fourier transformed infrared (FTIR), gel permeation chromatography (GPC), and differential scanning calorimeter (DSC) techniques. Benefiting from amphiphilic structure, H40-star-PLA-SS-PEP was able to self-assemble into micelles in aqueous solution with an average diameter of 70 nm. Moreover, the hydrophilic polyphosphate shell of these micelles could be detached under reduction-stimulus by in vitro evaluation, which resulted in a rapid drug release due to the destruction of micelle structure. The glutathione-mediated intracellular drug delivery was investigated against a Hela human cervical carcinoma cell line. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements demonstrated that H40-star-PLA-SS-PEP micelles exhibited a faster drug release in glutathione monoester (GSH-OEt) pretreated Hela cells than that in the nonpretreated cells. Cytotoxicity assay of the doxorubicin-loaded (DOX-loaded) micelles indicated the higher cellular proliferation inhibition against 10 mM of GSH-OEt pretreated Hela cells than that of the nonpretreated ones. As expected, the DOX-loaded micelles showed lower inhibition against 0.1 mM of buthionine sulfoximine (BSO) pretreated Hela cells. These reduction-responsive and biodegradable micelles show a potential to improve the antitumor efficacy of hydrophobic chemotherapeutic drugs.

One-step preparation of reduction-responsive poly(ethylene glycol)-poly(amino acid)s nanogels as efficient intracellular drug delivery platforms

A series of disulfide-core-cross-linked poly(ethylene glycol)-poly(amino acid)s star copolymers were synthesized through one-step ring-opening polymerization of L-phenylalanine N-carboxyanhydride (L-Phe NCA) and L-cystine N-carboxyanhydride (L-Cys NCA) with amino group terminated poly(ethylene glycol) monomethyl ether (mPEG-NH2) as macroinitiator. The reduction-responsive PEG-poly(amino acid)s nanogels (NGs) were prepared by directly dispersing the resultant PEG-poly(amino acid)s in phosphate buffer solution at pH 7.4. Dynamic light scattering (DLS) measurements showed that the reducible NG swelled in response to 10 mM glutathione (GSH). Doxorubicin (DOX), an anthracycline anticancer drug, was loaded into the NGs. The in vitro release results revealed that the release behaviors could be adjusted by GSH concentration, and poly(amino acid)s content and composition. The intracellular DOX release results showed that enhanced intracellular DOX release occurred in GSH pretreated Henrietta Lacks (HeLa) cells. In vitro methyl thiazolyl tetrazolium (MTT) assays indicated that the NGs were biocompatible, and DOX-loaded NG showed higher cellular proliferation inhibition towards GSH pretreated HeLa cells than that of non-pretreated cells. Therefore, the NGs can efficiently deliver anticancer drugs into tumor cells and inhibit cell proliferation, rendering highly promising for targeted intracellular delivery of operative chemotherapeutic drugs in cancer therapy.

Double-Stranded RNA-Dependent Protein Kinase-Dependent Apoptosis Induction by a Novel Small Compound

The interferon-induced, double-stranded RNA-dependent protein kinase (PKR) can play critical roles in inhibiting virus replication and inducing apoptosis. To develop new agents that may inhibit viral replication or induce apoptosis in cancer cells via the PKR signaling pathway, we screened a chemical library for compounds that have differential cytotoxic effects on wild-type [mouse embryonic fibroblast (MEF)/PKR(+/+)] and PKR-knockout [MEF/PKR(-/-)] mouse embryonic fibroblast cells. We identified a synthetic compound, BEPP [1H-benzimidazole1-ethanol,2,3-dihydro-2-imino-a-(phenoxymethyl)-3-(phenylmethyl)-,monohydrochloride], that induces a cytotoxic effect more effectively in MEF/PKR(+/+) cells than in MEF/PKR(-/-) cells. BEPP also relatively effectively inhibited the growth of a human lung cancer cell line overexpressing PKR, compared with other cancer cell lines. In sensitive cells, BEPP induced apoptosis with activation of caspase-3. Treatment with BEPP led to increased phosphorylation of PKR and eIF2alpha, increased expression of BAX, and decreased expression of Bcl-2. BEPP-induced apoptosis was PKR dependent and was blocked by the adenovector expressing the dominant-negative PKR. Furthermore, pretreatment of HeLa cells at a noncytotoxic dose of BEPP effectively inhibited Vaccinia virus replication. Together, our results suggest that BEPP and its analogs may induce PKR-dependent apoptosis and inhibition of viral replication and that they can be a potential anticancer or anti-virus agent.

IKK antagonizes CD95 ligation-mediated apoptosis by regulating NF-κB activity

The CD95 (Apo1/Fas)/CD95 ligand system plays pivotal roles in various aspects of immune regulation and function by triggering apoptosis. Besides the apoptosis signaling pathway, CD95 ligation also induces the activation of NF-κB. Previous studies suggest that IκB kinase (IKK) may be a key player in cell survival by mediating NF-κB activation. However, the roles of IKK in CD95 ligation-mediated apoptosis and NF-κB activation are still not clear. In this report, we show that expression of the caspase-resistant uncleavable IKKβ (UCIKKβ) mutant suppressed CD95 ligation-mediated cell death in HeLa cells. Furthermore, CD95 ligation induced much more cell death in IKKβ−/− murine embryonic fibroblasts (MEFs) than in wild type MEFs, despite that IKK was only marginally activated upon CD95 ligation. Pretreatment of HeLa cells with a specific IKK inhibitor NEMO-binding domain (NBD) peptide blocked CD95 ligation-induced NF-κB transcriptional activity. And UCIKKβ enhanced the basal NF-κB activity, and consequently led to higher NF-κB activity upon CD95 ligation in HeLa cells. Therefore, IKK antagonizes CD95 ligation-mediated apoptosis by regulating NF-κB activity.

A Role for PtdIns(4,5)P2 and PIP5Kα in Regulating Stress-Induced Apoptosis

The phosphoinositide phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P(2)) is essential for many cellular processes and is linked to the etiology of numerous human diseases . PtdIns(4,5)P(2) has been indirectly implicated as a negative regulator of apoptosis ; however, it is unclear if apoptotic stimuli negatively regulate PtdIns(4,5)P(2) levels in vivo. Here, we show that two apoptotic-stress stimuli, hydrogen peroxide (H(2)O(2)) and UV irradiation, cause PtdIns(4,5)P(2) depletion during programmed cell death independently of and prior to caspase activation. Depletion of PtdIns(4,5)P(2) is essential for apoptosis because maintenance of PtdIns(4,5)P(2) levels by overexpression of PIP5Kalpha rescues cells from H(2)O(2)-induced apoptosis. PIP5Kalpha expression promotes both basal and sustained ERK1/2 activation after H(2)O(2) treatment, and importantly, pharmacological inhibition of ERK1/2 signaling blocks PIP5Kalpha-mediated cell survival. H(2)O(2) induces tyrosine phosphorylation and translocation of PIP5Kalpha away from its substrate at the plasma membrane, and both are dependent upon the activity of c-src family kinases. Furthermore, constitutively active c-src enhances tyrosine phosphorylation of PIP5Kalpha in vivo and is sufficient for the translocation of PIP5Kalpha away from the plasma membrane. These observations demonstrate that certain apoptotic stimuli initiate an essential signaling pathway during cell death, and this pathway leads to caspase-independent downregulation of PIP5Kalpha and its product PtdIns(4,5)P(2).

Dependence on p38 MAPK signalling in the up‐regulation ofTLR2,TLR4andTLR9gene expression inTrichomonas vaginalis‐treated HeLa cells

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that recognize conserved pathogen-associated molecular patterns (PAMPs) synthesized by micro-organisms. Despite the essential requirement for TLRs in prokaryotic infection, the pattern and regulation of TLR gene expression by Trichomonas vaginalis in the mucocutaneous barrier are still unknown. Our hypothesis is that T. vaginalis-infected epithelial cells are major effector cells in the skin barrier. These cells function as a central regulator of TLR gene expression, thus accelerating the process of barrier dysfunction via increased release of chemokines and proinflammatory cytokines. To test this hypothesis, RT-PCR was performed on TLRs, interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha. Stimulation of HeLa cells by T. vaginalis was observed to up-regulate TLR2, 4 and 9 mRNA expression as well as that of IL-8 and TNF-alpha. To further clarify the molecular mechanism of barrier devastation triggered by these up-regulatory stimuli, we examined the profiles of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB activation in HeLa cells using specific inhibitors. Interestingly, pretreatment of HeLa cells with the p38 MAPK inhibitor SB203580 demonstrated inhibition of T. vaginalis-induced up-regulation of TLR2, 4, and 9 mRNA expression. By contrast, inhibition of ERK or NF-kappaB activation failed to block T. vaginalis-induced up-regulation of TLR9 mRNA expression or TLR2 and TLR4 mRNA expression, respectively. In addition, pretreatment with SB203580 reduced epithelium-derived IL-8 and TNF-alpha release evoked by T. vaginalis. Our results show that T. vaginalis infection of the mucocutaneous barrier could up-regulate TLR2, 4 and 9 gene expression via the p38 MAPK signalling pathway in epithelial cells; this process then leads to modulation of p38 MAPK-dependent IL-8 and TNF-alpha release from the epithelium.

Nuclear factor-kappaB mediates up-regulation of cathepsin B by doxorubicin in tumor cells.

Anthracyclines such as doxorubicin remain among the most effective agents for the treatment of solid tumors and hematological malignancies. To overcome dose-limiting side effects like cardiotoxicity, an intensive effort has been undertaken to develop promising doxorubicin prodrugs that are specifically activated at the tumor site. One approach is the application of peptide prodrugs of doxorubicin. The enzyme cathepsin B catalyzes the activation of these prodrugs, and hence, the regulation of cathepsin B by antitumor agents could influence the efficacy of peptide prodrugs using this protease. In the present investigation, the effects of doxorubicin on cathepsin B expression in the human cervix carcinoma cell line HeLa were examined. Exposure to doxorubicin induced a time- and dose-dependent up-regulation of cathepsin B expression on mRNA, protein, and activity levels. In the cathepsin B gene promoter region, a potential nuclear factor kappaB (NF-kappaB) binding site could be identified. Pretreatment of HeLa cells with specific NF-kappaB inhibitors abrogated the induction of cathepsin B expression. Doxorubicin-induced degradation of the inhibitory protein IkappaB could be prevented by pretreatment with a specific proteasome inhibitor, resulting in a significant reduction of the doxorubicin-induced cathepsin B expression. Finally, binding of NF-kappaB subunits p50 and p65 to the NF-kappaB binding site in the cathepsin B gene promoter region could be demonstrated by electrophoretic mobility shift assay. In summary, our data clearly indicate that doxorubicin induces cathepsin B expression and activity via NF-kappaB. These findings contribute to a better understanding of tumor targeting with peptide prodrugs and help to define a possible mechanism of doxorubicin toxicity in tumor cells.

Involvement of Reactive Oxygen Species in Bacterial Killing within Epithelial Cells

Several non-phagocytic cells can actively generate the superoxide anion by NAD(P)H oxidases resembling the enzymatic complex typical of phagocytes. Overexpression of periplasmic Cu,ZnSOD rescues invasive E. coli strains from killing within epithelial cells, suggesting that superoxide generation by such cells can oxidatively damage invading bacteria. Pre-treatment of HeLa cells with diphenyl iodonium or 4'-hydroxy-3'-methoxyacetophenone, two inhibitors of NAD(P)H oxidase, significantly enhances intracellular survival of wild type invasive E. coli cells. On the contrary, these inhibitors have no effect on the intracellular survival of an invasive E. coli strain engineered to overexpress Cu,ZnSOD. These results support the hypothesis that superoxide generation by a NAD(P)H oxidase-like complex can limit bacterial survival within epithelial cells and suggest that the role of periplasmic Cu,ZnSOD in bacterial infections is not simply that of conferring protection against the phagocytic oxidative burst.