Changes In HeLa Cells Research Articles

Effect of inhibition of intermediate-conductance-Ca2+-activated K+ channels on HeLa cell proliferation.

To explore the influence of intermediate-conductance-Ca2+-activated K+ channels. (IKCal) on HeLa cell proliferation. An IKCal blocking agent (clotrimazole (CLT)) and small hairpin ribonucleic acid interference (RNAi) was used to block IKCal in HeLa cells; subsequently, cell growth was observed. Furthermore, the messenger ribonucleic acid (mRNA) expression of IKCal was detected by reverse transcriptase polymerase chain reaction (RT-PCR) after IKCal-blocking. The obvious morphological changes in HeLa cells were observed 48 h after CLT-blocking. The PCR results indicated that CLT reduced the mRNA expression of IKCal in HeLa cells. HeLa cells were transfected with pGenesil via RNAi; the HeLa cells transfected with pGenesil-IK displayed obvious morphological changes 48 h after transfection. In addition, RT-PCR further demonstrated the reduced mRNA expression of IKCal in the pGenesil group. CLT and blocking of IKCal gene expression effectively inhibits HeLa cell proliferation; therefore, the use of a blocking agent and RNAi both effectively downregulated the mRNA expression of IKCal, which in turn mediated the proliferation of HeLa cells, producing an antitumor effect.

Novel Gold and Silver Carbene Complexes Exert Antitumor Effects Triggering the Reactive Oxygen Species Dependent Intrinsic Apoptotic Pathway.

Cisplatin and other platinum-based drugs are well-known valid anticancer drugs. However, during chemotherapy, the presence of numerous side effects and the onset of frequent phenomena of resistance has pushed many research groups to devise new metal-based compounds holding improved anticancer properties and fewer undesired effects. Amongst the variety of synthesized compounds, significant antiproliferative effects have been obtained by employing organometallic compounds, particularly those based on silver and gold. With this in mind, we synthesized four compounds, two silver complexes and two gold complexes, with good inhibitory effects on the in vitro proliferation of breast and ovarian cancer-cell models. The antitumor activity of the most active compound, that is, AuL4, was found to be ninefold higher than that of cisplatin, and this compound induced dramatic morphological changes in HeLa cells. AuL4 induced PARP-1 cleavage, caspases 3/7 and 9 activation, mitochondria disruption, cytochrome c release in cancer-cell cytoplasm, and the intracellular production of reactive oxygen species. Thus, AuL4 treatment caused cancer-cell death by the intrinsic apoptotic pathway, whereas no cytotoxic effects were recorded upon treating non-tumor cell lines. The reported outcomes may be an important contribution to the expanding knowledge of medicinal bio-organometallic chemistry and enlarge the available anticancer toolbox, offering improved features, such as higher activity and/or selectivity, and opening the way to new discoveries and applications.

Synthesis, characterization and crystal structures of two pentagonal-bipyramidal Fe(III) complexes with dihydrazone of 2,6-diacetylpyridine and Girard's T reagent. Anticancer properties of various metal complexes of the same ligand

In this work synthesis, characterization and crystal structures of two isothiocyanato Fe(III) complexes with 2,2′-[2,6-pyridinediylbis(ethylidyne-1-hydrazinyl-2-ylidene)]bis[N,N,N-trimethyl-2-oxoethanaminium] dichloride (H2LCl2) ligand, with composition [FeL(NCS)2]SCN·2H2O and [FeL(NCS)2]2[Fe(H2O)(NCS)5]·4H2O, has been reported. Both iron(III) complexes possess the same pentagonal-bipyramidal complex cation, while the nature of their anions depends on mole ratio of NH4SCN and FeCl3·6H2O used in reaction. Cytotoxic activity of new Fe(III) complexes, as well as of previously synthesized isothiocyanato Co(II), Ni(II), Mn(II), Zn(II) and Cd(II) complexes with the same ligand, was tested against five human cancer cell lines (HeLa, MDA-MB-453, K562, LS174 and A549) and normal cell line MRC-5. The best activity was observed in the case of Fe(III), Co(II) and Cd(II) complexes. The investigation of potential of these complexes to induce HeLa and K562 cell cycle perturbations was also evaluated. Mechanism of cell death mode was elucidated on the basis of morphological changes of HeLa cells as well as identification of target caspases. It was established that DNA damage could be responsible for the activity of Fe(III) and Co(II) complexes. SynopsisPentagonal-bipyramidal Fe(III) complexes with dihydrazone of 2,6-diacetylpyridine and Girard's T reagent have been synthesized and characterized. Cytotoxic activity of Fe(III) complexes and Co(II), Ni(II), Mn(II), Zn(II) and Cd(II) complexes with the same ligand was tested. The best activity was observed in the case of Fe(III), Co(II) and Cd(II) complexes.

Time course of global gene expression alterations in Candida albicans during infection of HeLa cells.

Candida albicans (C. albicans) is an opportunistic fungus that quickly adapts to various microniches. It causes candidiasis, a common fungal infection for which the pathogenic mechanism has not been elucidated yet. To explore the pathogenic mechanism of candidiasis we used several methods, including microscopic observation of morphological changes of HeLa cells and fungus, analysis of differentially expressed genes using gene chips, and a series of biological and bioinformatic analyses to explore genes that are possibly involved in the pathogenesis of C. albicans. During the C. albicans infection, significant morphological changes of the fungus were observed, and the HeLa cells were gradually destroyed. The gene chip experiments showed upregulated expression of 120 genes and downregulated expression of 178 genes. Further analysis showed that some genes may play an important role in the pathogenesis of C. albicans. Overall, morphological variation and adaptive gene expression within a particular microniche may exert important effects during C. albicans infections.

Synthesis and in vitro photodynamic therapy of chlorin derivative 131-ortho-trifluoromethyl-phenylhydrazone modified pyropheophorbide-a.

Photodynamic therapy (PDT) is entering the mainstream of the cancer treatments recently. Pyropheophorbide-a (Pa), as a degradation product of chlorophyll-a, has been shown to be a potent photosensitizer in photodynamic therapy. In this paper, we investigated the in vitro photodynamic therapy of 131-ortho-trifluoromethyl-phenylhydrazone modified pyropheophorbide-a (PHPa) against human HeLa cervical cancer cell line, together with ultraviolet-visible spectra, fluorescence emission spectra, stability in various solvents, and single oxygen quantum yield. The results indicated that PHPa not only showed a greater molar extinction coefficient reached 4.55×104 Lmol-1cm-1, the long absorption wavelength (681nm) as we expected that makes it potential in deep tumor treatment, but also showed better stability in near neutral phosphate buffers (pH 7.4) and culture medium, as well as higher single oxygen quantum yield (ФD=40.5%) in DMF solutions. Moreover, cell experiments suggested that PHPa could be uptaken by HeLa cells successfully, and has low dark toxicity without irradiation, but remarkable photo-cytotoxicity (IC50, 1.92±0.59μM) that the inhibition rate of HeLa cells could increase up 91.4% at 30μM of PHPa after irradiation. In addition, morphological changes of HeLa cells further demonstrated that PHPa can induce damage and apoptotic cell death. Furthermore, the mechanism of photochemical processes was investigated by using specific quenching agent sodium azide (SA) and D-mannitol (DM), respectively, which showed the formation of singlet oxygen (Type II reaction mechanism) may play a predominant role, Type I and Type II photodynamic reactions could occur simultaneously in this PHPa mediated PDT process.

Aloe-Emodin Influence on the Lysosomal Compartment of Hela Cells

Background:Aloe-emodin belongs to the group of anthraquinones having extremely high biological activity. The aim of this study was to evaluate the range of morphological and biochemical changes in HeLa cells treated with aloe-emodin, especially with regard to the lysosomal compartment.Methods:Marking of lysosomes was performed with neutral red staining for conventional light microscopy and acridine orange staining for confocal microscopy. To evaluate ctivity of lysosomal enzymes and permeability of the lysosomal membrane, spectrophotometric techniques were employed.Results:Aloe-emodin caused increased permeability of lysosomal membranes in HeLa cells, expressed inter alia by extinction of the orange color of acridine orange (lysosomal marker) and in reduction of neutral red uptake by lysosomes. These changes are accompanied by release of cathepsins from the interior of the lysosomes with a simultaneous highly significant increase in their activity in the cytoplasm.Conclusion:The results indicate that aloe-emodin can activate lysosomal pathway-dependent apoptosis in HeLa cells.

Preferential cytotoxicity of ZnO nanoparticle towards cervical cancer cells induced by ROS-mediated apoptosis and cell cycle arrest for cancer therapy

The present study aimed to synthesize multifunctional ZnO-NP samples, namely ZnO-20, ZnO-40, and ZnO-80 nm, using different approaches, to be used as efficient anticancer agents. Systematic characterizations revealed their particle sizes and demonstrated nanostructures of nanorods (ZnO-80 nm) and nanogranules (ZnO-20 and ZnO-40 nm). They exhibited significant (p < 0.05) toxicity to HeLa cancer cells. HeLa cell viabilities at 1 mM dose reduced to 37, 32, 15 %, by ZnO-80, ZnO-40, and ZnO-20 nm, respectively, at 48 h. However, the same dose exerted different effects of 79.6, 76, and 75 % on L929 normal cells at 48 h. Measurement of reactive oxygen species (ROS) showed a considerable ROS yields on HeLa cells by all samples with a pronounced percentage (50 %) displayed by ZnO-20 nm. Moreover, ROS-mediated apoptosis induction and blocked cell cycle progression in the S, G2/M, and G0/G1 phases significantly (p < 0.05). Apoptosis induction was further confirmed by DNA fragmentation and Hoechst–PI costained images viewed under fluorescence microscope. Additionally, morphological changes of HeLa cells visualized under light microscope showed assortment of cell death involved shrinkage, vacuolization and apoptotic bodies’ formation. Most importantly, results exposed the impact of size and morphology of ZnO samples on their toxicity to Hela cells mediated mainly by ROS production. ZnO-20 nm in disk form with its nanogranule shape and smallest particle size was the most toxic sample, followed by ZnO-40 nm and then ZnO-80 nm. An additional proposed mechanism contributed in the cell death herein was ZnO decomposition producing zinc ions (Zn2+) into the acidic cancer microenvironment due to the smaller sizes of ZnO-NPs. This mechanism has been adopted in the literatures as a size-dependent phenomenon. The emerged findings were suggested to provide new platforms in the development of therapeutics as selective agents to the fatal cervical cancer, and to benefit from the synergistic influence of size and nanostructure when designing anticancer agents. Mechanism of cell death by ZnO-NPs, displaying ROS formation and Zn2+ release that induced apoptosis and arrested cell cycle progression. Apoptosis involved DNA fragmentation, chromatin condensation, membrane shrinkage, and formation of apoptotic bodies. The synergistic effect of ZnO-NPs size (20, 40, 80 nm) and nanostructure (nanorods, nanogranules) impacted the inhibition of HeLa cells growth and eventually death.

Volume measurements and fluorescent staining indicate an increase in permeability for organic cation transporter substrates during apoptosis

Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. Blebbing caused by actinomycin D or camptothecin, but not by anti-Fas antibody, is accompanied by an almost 10% volume increase as measured by transmission-through-dye microscopy. When the experiment is carried out in DMEM medium, the swelling appears to result from activation of amiloride-sensitive channels. Low-sodium choline-, but not N-methyl−D-glucamine-based, medium, also supports swelling during the blebbing phase of apoptosis; this indicates that the membrane becomes permeable to choline as well. Because choline can enter the cells through organic cation transporters (OCT), we tested three fluorescent dyes (2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide, rhodamine 123 and ethidium bromide) that have been reported to utilize OCT for cell entry. Intact HeLa cells are poorly permeable for these fluorophores, and initially they accumulate on the plasma membranes. Blebbing results in an enhanced penetration of these dyes into the cell interior, as was demonstrated both by direct observation and by FRET. The increased membrane permeability is specific for OCT substrates; the other tested cationic dyes apparently cross the membrane by other routes and exhibit a markedly different behavior. Our results reveal a previously unknown feature of apoptosis and the utility of cationic dyes for studying membrane transport.

A gold nanocluster-based fluorescent probe for simultaneous pH and temperature sensing and its application to cellular imaging and logic gates.

Metal nanocluster-based nanomaterials for the simultaneous determination of temperature and pH variations in micro-environments are still a challenge. In this study, we develop a dual-emission fluorescent probe consisting of bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) and fluorescein-5-isothiocyanate (FITC) as temperature- and pH-responsive fluorescence signals. Under single wavelength excitation the FITC/BSA-AuNCs exhibited well-separated dual emission bands at 525 and 670 nm. When FITC was used as a reference fluorophore, FITC/BSA-AuNCs showed a good linear response over the temperature range 1-71 °C and offered temperature-independent spectral shifts, temperature accuracy, activation energy, and reusability. The possible mechanism for high temperature-induced fluorescence quenching of FITC/BSA-AuNCs could be attributed to a weakening of the Au-S bond, thereby lowering the charge transfer from BSA to AuNCs. Additionally, the pH- and temperature-responsive properties of FITC/BSA-AuNCs allow simultaneous temperature sensing from 21 to 41 °C (at intervals of 5 °C) and pH from 6.0 to 8.0 (at intervals of 0.5 pH unit), facilitating the construction of two-input AND logic gates. Three-input AND logic gates were also designed using temperature, pH, and trypsin as inputs. The practicality of using FITC/BSA-AuNCs to determine the temperature and pH changes in HeLa cells is also validated.

A molecular fluorescent probe for targeted visualization of temperature at the endoplasmic reticulum.

The dynamics of cellular heat production and propagation remains elusive at a subcellular level. Here we report the first small molecule fluorescent thermometer selectively targeting the endoplasmic reticulum (ER thermo yellow), with the highest sensitivity reported so far (3.9%/°C). Unlike nanoparticle thermometers, ER thermo yellow stains the target organelle evenly without the commonly encountered problem of aggregation, and successfully demonstrates the ability to monitor intracellular temperature gradients generated by external heat sources in various cell types. We further confirm the ability of ER thermo yellow to monitor heat production by intracellular Ca2+ changes in HeLa cells. Our thermometer anchored at nearly-zero distance from the ER, i.e. the heat source, allowed the detection of the heat as it readily dissipated, and revealed the dynamics of heat production in real time at a subcellular level.

Gold nanocluster-based fluorescence biosensor for targeted imaging in cancer cells and ratiometric determination of intracellular pH

The dysregulated pH is working as a mark of cancer. It is a challenge for developing a biosensor for targeted imaging in cancer cells and monitoring of intracellular pH. Here, a ratiometric fluorescence biosensor for pH determination was developed with targeted imaging into folate acceptor (FR)-rich cancer cells at the same time. AuNCs protected by bovine serum albumin (BSA) worked as reference fluorophore and fluorescein-isothiocyanate (FITC) acted as the response signal for pH. For targeted imaging of cancer cells, the AuNCs were simultaneously conjugated with folic acid (FA). The developed ratiometric biosensor can monitor pH with a wide linear range from 6.0–7.8 with a pKa at 6.84. Under every different pH condition, the probe showed high selectivity over various metal ions and amino acids with its fluorescence ratio stayed almost constant (<5%). It also showed good cyclic accuracy when pH switched between 6.0 and 8.0, as well as low cytotoxicity. The AuNC-based inorganic–organic nanohybrid biosensor showed good cell-permeability, low cytotoxicity, and long-term photostability. Accordingly, the pH biosensor was employed to gain targeted imaging in FR+ve Hela cells with FR−ve lung carcinoma cells A549 as comparison, and achieved to monitor the pH changes in Hela cells.

The extracts of Holotrichia diomphalia larvae inhibit proliferation and induce apoptosis of cancer cells in vitro and in vivo

Grub extract comes from Holotrichia diomphalia larvae. Numerous studies have demonstrated that grub extracts inhibit the growth of several types of cancer cells in vitro. Here, the anti-tumor activities of grub extracts were observed in experiments performed in vitro and in vivo. Human cervical carcinoma HeLa cells were treated with a range of grub extract doses. Grub extract treatment also resulted in characteristic apoptotic morphological changes in HeLa cells. In vivo acute oral toxicity tests showed that the median lethal dose (LD50) at 5 d was 48.73 g/kg (with 95% confident limits of 46.43–51.14 g/kg) in Kunming mice. Moreover, when the mice were treated with grub extracts at doses of 1.95, 3.9, and 7.8 g/(kg·d) for 10 consecutive days, the solid tumor growth of mice bearing Sl80 was 18.64%, 39.47%, and 53.24%, respectively, relative to the control (P<0.05 or 0.01). These data indicate that grub extracts may inhibit tumor growth and induce apoptotic cell death, and may be a potential and promising natural product to treat tumors.

Proteomic investigation into betulinic acid-induced apoptosis of human cervical cancer HeLa cells.

Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.

Proliferation Inhibition and Apoptosis Induction of Juglone on Human Cervical Cancer HeLa Cells

Abstract: To investigate whether juglone could inhibits the proliferation on human cervical cancer cells (HeLa) in vitro. Cells were divided into control group, different concentration (10μM, 20μM, 50μM, 100μM and200μM) juglone groups for different durations. The viability of HeLa cells was detected by methyl thiazolyl tetrazolium (MTT) assay. The morphology changes of HeLa cells were observed by inverted microscope .The results showed that the viability of HeLa cells was decreased and the cell morphology was changed in a dose-dependent manner after treatment different concentration juglone for 24h when compared with control group. The results suggest that Juglone may be effective for the treatment of HeLa cells.

Morphological Change of Cell Membrane by Interaction with Domain‐Swapped Cytochrome c Oligomers

Monomeric cyt c has been reported to bind to the mitochondrial membrane by electrostatic and hydrophobic interactions with anionic phospholipids. We have previously shown that domain-swapped oligomeric cyt c retains the secondary structure of the monomer, and its surface possesses a larger area and more charges compared to the monomer. However, the effect of oligomerization of cyt c on cells has yet to be revealed. Herein, we investigated the interaction of oligomeric cyt c with anionic phospholipid-containing vesicles and the outer membrane of HeLa cells. Oligomeric cyt c interacted more strongly than monomeric cyt c with anionic phospholipid-containing vesicles and the outer membrane of HeLa cells. Oligomeric cyt c induced lateral phase separation of lipids in LUVs and GUVs, thereby leading to membrane disruption, whereas monomeric cyt c did not. Morphological changes in HeLa cells resulted from interaction with oligomeric cyt c, but little from interaction with the monomer. These results show that domain-swapped oligomeric proteins might exhibit properties different to those of monomer in cell systems.

High throughput cell cycle analysis using microfluidic image cytometry (μFIC)

Microfluidic image cytometry (μFIC) is a novel approach for the cytotoxicity assessment of the cells cultured and treated within microfluidic channels under a precisely controlled chemical environment. Here, following our previous morphology-, and MTT absorbance-based μFIC, we are presenting our recent effort to develop, evaluate, and apply a high throughput cell cycle analysis method using fluorescence-based μFIC. A microfluidic device with a concentrantion gradient generator (CGG) and eight straight cell culture channels was fabricated, optimized, and applied for the assessment of paclitaxel-induced cell cycle changes of HeLa cells. Throughout this study, we have shown that the cell cycle analysis using fluorescence-based μFIC was able to provide comparable experimental data with those of flow cytometry. Moreover, cell cycle analysis using μFIC can also provide further advantages over flow cytometry, such as higher throughput, lower assay cost, less generation of toxic waste, and etc., which should have significant implications in pharmaceutical and biological applications as a future high throughput cell cycle analysis platform.

Histone deacetylase inhibitor BML-210 induces growth inhibition and apoptosis and regulates HDAC and DAPC complex expression levels in cervical cancer cells

Histone deacetylase inhibitors (HDACIs) represent a new class of targeted anti-cancer agents and different other diseases, like muscular disorders. A number of studies have shown that extracellular signal-activated kinases can target chromatin-modifying complexes directly and regulate their function. The molecular connection between the dystrophin-associated protein complex (DAPC) and chromatin has been described, by showing that NO signaling regulates histone deacetylase (HDAC) activity and influences gene expression in different cell types. In present study, we investigated HDACs changes in HeLa cells undergoing growth inhibition and apoptosis, caused by HDACI BML-210 and retinoic acid (ATRA). Cell cycle analysis indicated that HeLa cell treatment with 20 and 30 μM concentration of BML-210 increased the proportion of cells in G0/G1 phase, and caused accumulation in subG1, indicating that the cells are undergoing apoptosis. We determined down-regulation of HDAC 1-5 and 7 after treatment with BML-210. Also, we demonstrated expression of different isoforms of alpha-dystrobrevin (α-DB) and other components of DAPC such as syntrophin, dystrophin, beta-dystrobrevin (β-DB) and NOS in HeLa cells after treatments. We determined changes in protein expression level of dystrophin, NOS1, α- and β-DB and in subcellular localization of α-DB after treatments with BML-210 and ATRA. In conclusion, these results suggest that HDACI BML-210 can inhibit cell growth and induce apoptosis in cervical cancer cells, what correlates with down-regulation of HDAC class I and II and changes in the DAPC expression levels. This can be important for identifying target proteins in DAPC signaling to HDACs, as a target of pharmacological intervention for treatment of muscular dystrophies and other diseases.

&lt;i&gt;In Vitro&lt;/i&gt; Evaluation of Folate-Targeted Chitosan Nanoparticles Loaded with Hydroxycamptothecin

We prepared FA-targeted and 10-hydroxycamptothecin loaded chitosan nanoparticles (FA-HCPT-NPs) with a combination of emulsion-solvent evaporation and chemical crosslinking method. In vitro cytotoxicity test to the Human Cervix Carcinoma cells (HeLa) was evaluated by cell morphology and internalization observation. The specicity of the FA-HCPT-NPs targeting cancerous cells was demonstrated by comparative intracellular uptake of HCPT-NPs and commercial availible HCPT injection. Laser confocal scanning imaging proved that FA-HCPT-NPs could greatly enhance up-take by HeLa cells. The morphological changes of HeLa cells showed the FA-HCPT-NPs could inhibit HeLa cells more effectively than HCPT-NPs and HCPT. The results indicated that the novel FA-HCPT-NPs could be a potential drug delivery system for tumor cell-selective targeting therapy.

Metabolism of HeLa cells revealed through autofluorescence lifetime upon infection with enterohemorrhagic &lt;italic&gt;Escherichia coli&lt;/italic&gt;

Fluorescence lifetime imaging microscopy (FLIM) is a sensitive technique in monitoring functional and conformational states of nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD),main compounds participating in oxidative phosphorylation in cells. In this study, we have applied FLIM to characterize the metabolic changes in HeLa cells upon bacterial infection and made comparison with the results from the cells treated with staurosporine (STS), a well-known apoptosis inducer. The evolving of NADH's average autofluorescence lifetime during the 3 h after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components' relative contributions of NADH increases with time, a fact indicating cellular metabolic activity, such as a decrease of oxidative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. Being associated with mitochondria, FAD lifetimes and redox ratio could indicate heterogeneous mitochondrial function, microenvironment with bacterial infection, and further pathway to cell death. The redox ratios for both EHEC-infected and STS-treated HeLa cells have been observed and these observations also indicate possible apoptosis induced by bacterial infection.

Characterization of Cholix Toxin-induced Apoptosis in HeLa Cells

Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways.