Autophagy is a process of cytoplasmic degradation of endogenous proteins and organelles. Although its primary role is protective, it can also contribute to cell death. Recently, autophagy was found to play a role in the activation of host defense against intracellular pathogens. The aims of our study was to investigate whether host cell autophagy influences Toxoplasma gondii proliferation and whether autophagy inhibitors modulate cell survival. HeLa cells were infected with T. gondii with and without rapamycin treatment to induce autophagy. Lactate dehydrogenase assays showed that cell death was extensive at 36-48 hr after infection in cells treated with T. gondii with or without rapamycin. The autophagic markers, LC3 II and Beclin 1, were strongly expressed at 18-24 hr after exposure as shown by Western blotting and RT-PCR. However, the subsequent T. gondii proliferation suppressed autophagy at 36 hr post-infection. Pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), down-regulated LC3 II and Beclin 1. The latter was also down-regulated by calpeptin, a calpain inhibitor. Monodansyl cadaverine (MDC) staining detected numerous autophagic vacuoles (AVs) at 18 hr post-infection. Ultrastructural observations showed T. gondii proliferation in parasitophorous vacuoles (PVs) coinciding with a decline in the numbers of AVs by 18 hr. FACS analysis failed to confirm the presence of cell apoptosis after exposure to T. gondii and rapamycin. We concluded that T. gondii proliferation may inhibit host cell autophagy and has an impact on cell survival.
Anti-Bacterial Agents/pharmacology ; Apoptosis/drug effects/physiology ; Autophagy/drug effects ; HeLa Cells ; Humans ; Sirolimus/pharmacology ; Toxoplasma/*cytology/*physiology

OBJECTIVETo evaluate feasibility of inhibiting coxsackievirus B3 (CVB3) infection at cellular, protein and gene levels by using small interfering RNA (siRNA).

METHODSAntiviral activities of siRNAs were evaluated by observing cytopathic effect (CPE), using plaque reduction Western blotting assays and RT-PCR.

RESULTSEight siRNAs were synthesized, among them, SiRNA-2, SiRNA-3, SiRNA-6 and SiRNA-7 which were targeted against sequences located in 2B, VP4, 2A and 3C section of CVB3 genome, were designed to have different effect of inhibiting CVB3 infection in vitro. SiRNA-2 showed the best protective effect, 95 percent inhibition of CVB3 cytopathic effect and plaque forming effect was observed at 0.0001 MOI, viral protein synthesis and replication were inhibited. SiRNA-2 showed 30 percent inhibition of virus at 0.1 MOI, 70 percent inhibition at 0.01 MOI, 88 percent inhibition at 0.001 MOI, and 99 percent inhibition at 0.0001 MOI 48 hours after CVB3 infection.

CONCLUSIONSiRNA could effectively inhibit CVB3 infection in vitro, siRNA-2, which is targeted against sequence in 2B section of CVB3 genome, seemed to be the best one among those synthesized in this study.

Coxsackievirus Infections ; therapy ; virology ; Cytopathogenic Effect, Viral ; drug effects ; Enterovirus ; genetics ; physiology ; HeLa Cells ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; Virus Replication ; drug effects

OBJECTIVETo seek effective drugs inhibiting herpes simplex virus (HSV-2) with the signal pathway required by virus replication as the target spot.

METHODHSV-2-induced Vero cytopathic effect was observed, and MTT method was adopted to detect call activity, in order to assess the antiviral capacity of freeze dried powder of aqueous extracts of Saururus chinensis (AESC). Western blot was used to check the effect of AESC on signal pathway induced by HSV-2 virus in HeLa cells.

RESULTAESC obviously inhibits the pathway activation of CPE induced by HSV-2 infection and NF-kappaB required for virus replication. The inhibition ratio of AESC freeze dried powder at 0.10, 0.03, 0.01 and 0.003 g x L(-1) were (70.68 +/- 3.39)%, (61.74 +/- 2.13)%, (39.31 +/- 1.10)% and (18.54 +/- 3.44)%, respectively. The IC50 was determined at (0.023 +/- 0.004) g x L(-1). The inhibition concentration of the positive control acyclovir was 0.001 g x L(-1) (5.0 x 10(-6) mol x L(-1)). The best administration time was from 2 h before infection to 6 h after infection. Western blot also showed that AESC can notably inhibit HSV-2-induced NF-kappaB nuclear transfer.

CONCLUSIONAESC can inhibit HSV-2 virus replication, which is related to the pathway activation of NF-kappaB required for virus replication.

Animals ; Antiviral Agents ; pharmacology ; toxicity ; Cercopithecus aethiops ; Drugs, Chinese Herbal ; pharmacology ; toxicity ; HeLa Cells ; Herpesvirus 2, Human ; drug effects ; physiology ; Humans ; NF-kappa B ; metabolism ; Saururaceae ; chemistry ; Signal Transduction ; drug effects ; Vero Cells ; Virus Replication ; drug effects

This study inquired into the mechanisms of co-immobilized cytokines and free cytokines-induced apoptosis on HeLa cells. With the use of photochemical fixed method, TNF-alpha/IFN-gamma were co-immobilized on a 24-well polystyrene culture plate. HeLa cells were stained with fluorescent probe JC-1 to detect the changes of mitochondrial membrane potential (deltapsim), and then were examined by flow cytometry. The results showed that co-immobilized cytokines could induce the apoptosis of HeLa cells in a dose-independent manner. When treated with low-dose of co-immobilized cytokines (20ng/ml), the mitochondrial membrane potential (deltapsim) of HeLa cells continually decreased in 6 days. These indicate that low dose co-immobilized cytokines have a long-term of apoptosis-inducing effect on HeLa cells. We assume that there is close relationship between the mitochondrial membrane potential decrease and the apoptosis of HeLa cells.
Apoptosis ; drug effects ; Dose-Response Relationship, Drug ; HeLa Cells ; Humans ; Immobilized Proteins ; pharmacology ; Interferon-gamma ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondrial Membranes ; drug effects ; physiology ; Tumor Necrosis Factor-alpha ; pharmacology

A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was used to determine that apoptosis causes HeLa cell death induced by pseudolaric acid B. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 decreased p53 protein expression during exposure to pseudolaric acid B. SP600125 decreased the phosphorylation of p53 during pseudolaric acid B exposure, indicating that JNK mediates phosphorylation of p53 during the response to pseudolaric acid B. SP600125 reversed pseudolaric acid B-induced down-regulation of phosphorylated extracellular signal-regulated protein kinase (ERK), and protein kinase C (PKC) was activated by pseudolaric acid B, whereas staurosporine, calphostin C, and H7 partly blocked this effect. These results indicate that p53 is partially regulated by JNK in pseudolaric acid B-induced HeLa cell death and that PKC participates in pseudolaric acid B-induced HeLa cell death.
Tumor Suppressor Protein p53/metabolism/*physiology ; Protein Kinase C/metabolism ; Phosphorylation ; JNK Mitogen-Activated Protein Kinases/*physiology ; Humans ; Hela Cells ; Diterpenes/*pharmacology ; DNA Fragmentation/drug effects ; Cell Death/*drug effects ; Anthracenes/pharmacology

OBJECTIVETo investigate the telomerase activity and to document biological behaviors of HeLa cells upon treatment with specific PI3K/AKT signaling pathway inhibitor, LY294002.

METHODSCCK-8 assay was used to determine IC50 of LY294002. The expressions of total AKT and phosphorylation AKT (P-AKT) were determined using Western blot. Telomerase activity of cell was measured by TRAP-ELISA assay. Cell growth curve, flow cytometry technique and Hoechst33258 stain were used to evaluate the cell growth, cell cycle and apoptosis respectively. Cell migration was determined by cell wound healing assay.

RESULTSIC50 value of LY294002 of treated HeLa cells was 1.73 mg/L. Western blot showed that LY294002 enabled to decrease P-AKT activity in the presence of same total AKT protein. The cell telomerase activity was decreased to 36.72% in contrast to 98.61% of the control. LY294002 decreased the telomerase activity in HeLa cells, and the growth capacity of the cells was significantly suppressed. The number of cells at G0/G1 phases increased to 66.88% compared with that of the control cells (47.36%). The apoptosis rate also increased from 2.4% to 14.9%. The relative migration distance decreased to 24.6% compared with that of control (62.57%).

CONCLUSIONLY294002 inhibition of PI3K/AKT signaling pathway leads to alteration of telomerase activity along with changes of the biological behaviors of the HeLa cells suggesting that regulation of telomerase activity may be closely related to PI3K/AKT signaling pathway.

Apoptosis ; drug effects ; Blotting, Western ; Cell Line ; Cell Movement ; drug effects ; physiology ; Cell Proliferation ; drug effects ; Chromones ; pharmacology ; Enzyme Inhibitors ; pharmacology ; G1 Phase ; drug effects ; HeLa Cells ; Humans ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; Signal Transduction ; drug effects ; physiology ; Telomerase

delta12-Prostaglandin (PG) J2 is known to elicit an anti-neoplastic effects via apoptosis induction. Previous study showed delta12-PGJ2-induced apoptosis utilized caspase cascade through cytochrome c-dependent pathways in HeLa cells. In this study, the cellular mechanism of delta12-PGJ2- induced apoptosis in HeLa cells, specifically, the role of two mitochondrial factors; bcl-2 and apoptosis-inducing factor (AIF) was investigated. Bcl-2 attenuated delta12-PGJ2-induced caspase activation, loss of mitochondrial transmembrane potential (delta psi m), nuclear fragmentation, DNA laddering, and growth curve inhibition for approximately 24 h, but not for longer time. AIF was not released from mitochondria, even if the delta psi m was dissipated. One of the earliest events observed in delta12-PGJ2-induced apoptotic events was dissipation of delta psi m, the process known to be inhibited by bcl-2. Pre-treatment of z-VAD- fmk, the pan-caspase inhibitor, resulted in the attenuation of delta psi m depolarization in delta12-PGJ2- induced apoptosis. Up-regulation of Sox-4 protein by delta12-PGJ2 was observed in HeLa and bcl-2 overexpressing HeLa B4 cell lines. Bcl-2 overexpression did not attenuate the expression of Sox-4 and its expression coincided with other apoptotic events. These results suggest that delta12-PGJ2 induced Sox-4 expression may activate another upstream caspases excluding the caspase 9-caspase 3 cascade of mitochondrial pathway. These and previous findings together suggest that delta12-PGJ2-induced apoptosis in HeLa cells is caspase-dependent, AIF-independent events which may be affected by Sox-4 protein expression up-regulated by delta12-PGJ2.
Amino Acid Chloromethyl Ketones/pharmacology ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects/*physiology ; Caspases/physiology ; Cytochromes c/physiology ; Female ; Flavoproteins/metabolism/*physiology ; Hela Cells ; High Mobility Group Proteins/*physiology ; Humans ; Membrane Proteins/metabolism/*physiology ; Mitochondria/metabolism/physiology ; Prostaglandin D2/*pharmacology ; Protein Transport/physiology ; Proto-Oncogene Proteins c-bcl-2/biosynthesis/*physiology ; Research Support, Non-U.S. Gov't ; Trans-Activation (Genetics) ; Trans-Activators/*physiology

BACKGROUNDThe role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin.

METHODSThe inhibitory effect of shikonin on the growth of HeLa cells was measured by MTT assay. Fluorescent microscopic analysis of apoptotic cells stained with 4',6'-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis.

RESULTSShikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK), inhibitor PD 98059.

CONCLUSIONShikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.

Apoptosis ; drug effects ; Caspases ; physiology ; Cell Proliferation ; drug effects ; DNA Damage ; Flavonoids ; pharmacology ; HeLa Cells ; Humans ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Naphthoquinones ; pharmacology ; Phosphorylation ; Tumor Suppressor Protein p53 ; analysis ; Up-Regulation

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (IICR) and the IICR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP(3)R(1) (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.
Adenosine Triphosphate ; pharmacology ; Amino Acid Substitution ; Biological Transport ; drug effects ; physiology ; Blotting, Western ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; physiology ; HeLa Cells ; Humans ; Immunoprecipitation ; Inositol 1,4,5-Trisphosphate ; metabolism ; Inositol 1,4,5-Trisphosphate Receptors ; physiology ; Membrane Potentials ; drug effects ; physiology ; Membrane Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Molecular Chaperones ; genetics ; metabolism ; Mutation ; Plasmids ; Transfection

BACKGROUNDThe second mitochondria-derived activator of caspases (Smac) is a novel proapoptotic gene, which plays an important role in the apoptosis-inducing effects of irradiation on tumor cells. The purpose of this study was to investigate the effects of extrinsic Smac gene transfer and its over-expression in radiotherapeutic sensitivities of cervical cancer cells.

METHODSAfter the Smac gene was transferred into the cervical cancer cell line HeLa, subcloned cells were obtained by persistent G418 selection. Cellular Smac gene expression was detected by RT-PCR and Western blot, while in vitro cell viabilities were detected by trypan blue staining assay. After treatment with X-ray irradiation, cellular radiotherapeutic sensitivities were investigated by tetrazolium bromide colorimetry. Cellular apoptosis and its rate were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. The expression and activities of cellular caspase-3 were assayed by Western blot and colorimetry.

RESULTSSmac mRNA and protein levels in HeLa/Smac cells and the selected subclone cell line of cervical cancer were significantly higher than those of HeLa (P < 0.01). There was no significant difference in cellular viabilities between them (P > 0.05). However, after irradiation with 8 Gy X-ray, growth activities of HeLa/Smac were reduced by 22.42% (P < 0.01). When compared with those of HeLa, partial HeLa/Smac cells presented characteristic morphological changes of apoptosis under electronic microscope, with higher apoptosis rates (16.4% vs. 6.2%, P < 0.01); the caspase-3 expression levels in HeLa/Smac cells were improved significantly (P < 0.01), while its activities were increased by 3.42 times (P < 0.01).

CONCLUSIONSStable transfer of the extrinsic Smac gene and its over-expression in cervical cancer cell line could significantly enhance the expression and activities of cellular caspase-3 and ameliorate apoptosis-inducing effects of irradiation on cancer cells, which was a novel strategy to improve radiotherapeutic effects on cervical cancer.

Apoptosis ; radiation effects ; Carrier Proteins ; genetics ; physiology ; Caspase 3 ; Caspases ; metabolism ; Female ; Gene Transfer, Horizontal ; HeLa Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; Mitochondrial Proteins ; genetics ; physiology ; Radiation Tolerance ; Uterine Cervical Neoplasms ; drug therapy ; enzymology ; pathology