Lysosomes are degradation and signaling centers within the cell, and their dysfunction impairs a wide variety of cellular processes. To understand the cellular effect of lysosome damage, we screened natural small-molecule compounds that induce lysosomal abnormality using Caenorhabditis elegans (C. elegans) as a model system. A group of vobasinyl-ibogan type bisindole alkaloids (ervachinines A-D) were identified that caused lysosome enlargement in C. elegans macrophage-like cells. Intriguingly, these compounds triggered cell death in the germ line independently of the canonical apoptosis pathway. In mammalian cells, ervachinines A-D induced lysosomal enlargement and damage, leading to leakage of cathepsin proteases, inhibition of autophagosome degradation and necrotic cell death. Further analysis revealed that this ervachinine-induced lysosome damage and lysosomal cell death depended on STAT3 signaling, but not RIP1 or RIP3 signaling. These findings suggest that lysosome-damaging compounds are promising reagents for dissecting signaling mechanisms underlying lysosome homeostasis and lysosome-related human disorders.
Alkaloids ; pharmacology ; Animals ; Caenorhabditis elegans ; cytology ; drug effects ; metabolism ; Cell Death ; drug effects ; Cell Survival ; drug effects ; HeLa Cells ; Humans ; Lysosomes ; drug effects ; pathology ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; drug effects

Animals ; Antineoplastic Agents ; pharmacology ; B-Cell CLL-Lymphoma 10 Protein ; deficiency ; metabolism ; CARD Signaling Adaptor Proteins ; deficiency ; metabolism ; Cell Survival ; drug effects ; DNA Damage ; Doxorubicin ; pharmacology ; HeLa Cells ; Humans ; Mice ; Mice, Knockout ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ; deficiency ; metabolism ; NF-kappa B ; metabolism

OBJECTIVETo investigate the effect of docosahexaenoic acid (DHA) on apoptosis, migration and invasion of cervical cancer cell lines.

METHODScervical cancer cell lines Hela and Siha in logarithmic phase were treated different concentrations of DHA. The morphological changes of the cells were observed microscopically and cell apoptosis was observed using Hoechst 33258 fluorescent staining. MTT assay was used to evaluate the effect of DHA in suppressing cell growth, and flow cytometry was employed to analyze the changes of cell apoptotic rate following DHA stimulations. Wound healing assay and Transwell migration assay were used to evaluate the migration of the cell lines. The expression levels of Bax, Bcl-2 cleaved caspase3, MMP-9 and VEGF proteins were detected by Western blotting.

RESULTSDHA exposure of the cells caused obvious morphological changes and dose-dependently increased the number of apoptotic bodies in the cells. MTT assay showed that DHA inhibited the growth of the cancer cells in a time- and concentration-dependent manner. DHA also effectively suppressed migration and invasion of the cancer cells. The cells exposed to DHA showed significantly down-regulation of Bcl-2, MMP-9 and VEGF proteins and up-regulation of cleaved-caspase 3 and Bax.

CONCLUSIONDHA can promote cervical carcinoma cell apoptosis by down-regulating the anti-apoptotic proteins Bax, Bcl-2 and cleaved-caspase3 and suppress cell invasion by decreasing MMP-9 and VEGF expressions.

Apoptosis ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; drug effects ; Cell Movement ; Cell Proliferation ; Docosahexaenoic Acids ; pharmacology ; Down-Regulation ; Female ; HeLa Cells ; drug effects ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; pathology ; Vascular Endothelial Growth Factor A ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVE: To investigate the effects of 7-difluoromethy-5, 4'- dimethoxygenistein (DFMG) on inhibiting proliferation and inducing apoptosis of human cervical cancer HeLa cells and its possible molecular mechanism in vitro.
 METHODS: HeLa cells were cultured in vitro. The effect of DFMG on inhibiting proliferation was determined using MTT assay. The effects of DFMG on inducing apoptosis were assessed using flow cytometry with AV-PI staining, AO/EB staining, and agarose gel electrophoresis. Multiple molecular techniques, such as RT-PCR, Western blot, siRNA transfection, and cDNA transfection, were used to explore its possible molecular mechanism.
 RESULTS: DFMG presented with dramatically inhibiting proliferation effect of HeLa cells in a time-and dose-dependent manner ranging from 0.25 to 64 μg/mL and from 24 to 72 h in vitro, and its IC(50) was 4.62 μg/mL for 48 h. The cells treated with DFMG for 48 h showed typical morphological change of apoptosis, typical DNA ladder of agarose gel electrophoresis, and the sub-G(1) population increased in a dose-dependent manner. Simultaneously the expressions of c-myc mRNA, c-myc protein and its downstream genes, such as bax, cyto-c and caspase-9, were up-regulated, while bcl-2 protein was down-regulated. Down-regulation of c-myc by siRNA attenuated DFMG-induced cell proliferation inhibition and inducing apoptosis. Up-regulation expression of c-myc by cDNA transfection could enhance the effects of DFMG-induced cell proliferation inhibition and inducing apoptosis.
 CONCLUSION DFMG could inhibit the proliferation and induce the apoptosis of human cervical cancer HeLa cells in vitro, and its mechanism may be closely related to regulate c-myc and its down-stream gene expression.
Apoptosis ; drug effects ; Caspase 9 ; metabolism ; Cell Proliferation ; drug effects ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Genistein ; analogs & derivatives ; pharmacology ; HeLa Cells ; drug effects ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; pathology ; bcl-2-Associated X Protein ; metabolism

The protein apoptotic protease activating factor 1 (Apaf1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. We have developed a vital method that allows fluorescence-activated cell sorting of cells at different stages of the apoptotic pathway and demonstrated that upon pharmacological inhibition of Apaf1, cells recover from doxorubicin- or hypoxia-induced early apoptosis to normal healthy cell. Inhibiting Apaf1 not only prevents procaspase-9 activation but delays massive mitochondrial damage allowing cell recovery.
Adenosine Triphosphate ; metabolism ; Apoptosis ; drug effects ; Apoptotic Protease-Activating Factor 1 ; genetics ; metabolism ; Cell Hypoxia ; drug effects ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; HeLa Cells ; Humans ; Microscopy, Electron, Transmission

To explore the effect of quercetin on the proliferation and apoptosis of HeLa cells, HeLa cells were incubated with quercetin at different concentrations. Cell viability was evaluated by MTT assay, cell apoptosis was detected by Annexin-V/PI double labeled cytometry and DNA ladder assay. Cell cycle was flow cytometrically determined and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33258 staining and the apoptosis-related proteins in the HeLa cells were assessed by Western blotting. The results showed that quercetin significantly inhibited the growth of HeLa cells and induced obvious apoptosis in vitro in a time- and dose-dependent manner. Moreover, quercetin induced apoptosis of HeLa cells in cell cycle-dependent manner because quercetin could induce arrest of HeLa cells at G0/G1 phase. Quercetin treatment down-regulated the expression of the PI3K and p-Akt. In addition, quercetin could down-regulate expression of bcl-2, up-regulate Bax, but exerted no effect on the overall expression of Akt. We are led to conclude that quercetin induces apoptosis via PI3k/Akt pathways, and quercetin has potential to be used as an anti-tumor agent against human cervix cancer.
Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Flow Cytometry ; HeLa Cells ; Humans ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Quercetin ; pharmacology ; Signal Transduction ; drug effects ; Time Factors ; bcl-2-Associated X Protein ; metabolism

Vorinostat (VOR) has been reported to enhance the cytotoxic effects of doxorubicin (DOX) with fewer side effects because of the lower DOX dosage in breast cancer cells. In this study, we investigated the novel mechanism underlying the synergistic cytotoxic effects of VOR and DOX co-treatment in cervical cancer cells HeLa, CaSki and SiHa cells. Co-treatment with VOR and DOX at marginal doses led to the induction of apoptosis through caspase-3 activation, poly (ADP-ribose) polymerase cleavage and DNA micronuclei. Notably, the synergistic growth inhibition induced by the co-treatment was attributed to the upregulation of the pro-apoptotic protein Bad, as the silencing of Bad expression using small interfering RNA (siRNA) abolished the phenomenon. As siRNA against p53 did not result in an increase in acetylated p53 and the consequent upregulation of Bad, the observed Bad upregulation was mediated by acetylated p53. Moreover, a chromatin immunoprecipitation analysis showed that the co-treatment of HeLa cells with VOR and DOX increased the recruitment of acetylated p53 to the bad promoter, with consequent bad transactivation. Conversely, C33A cervical cancer cells containing mutant p53 co-treated with VOR and DOX did not exhibit Bad upregulation, acetylated p53 induction or consequent synergistic growth inhibition. Together, the synergistic growth inhibition of cervical cancer cell lines induced by co-treatment with VOR and DOX can be attributed to the upregulation of Bad, which is induced by acetylated p53. These results show for the first time that the acetylation of p53, rather than histones, is a mechanism for the synergistic growth inhibition induced by VOR and DOX co-treatments.
Acetylation ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects ; Cell Survival/drug effects ; Chromatin/metabolism ; Doxorubicin/*pharmacology ; Drug Synergism ; Female ; HeLa Cells ; Humans ; Hydroxamic Acids/*pharmacology ; Transcriptional Activation ; Tumor Suppressor Protein p53/genetics/*metabolism ; Uterine Cervical Neoplasms/metabolism ; bcl-Associated Death Protein/genetics/*metabolism

We have recently reported that Ginsenoside Rh2 (G-Rh2) induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. However, the molecular mechanism of its death-inducing function remains unclear. Here we show that G-Rh2 stimulated the activation of both caspase-8 and caspase-9 simultaneously in HeLa cells. Under G-Rh2 treatment, membrane death receptors Fas and TNFR1 are remarkably upregulated. However, the induced expression of Fas but not TNFR1 was contributed to the apoptosis process. Moreover, significant increases in Fas expression and caspase-8 activity temporally coincided with an increase in p53 expression in p53-non-mutated HeLa and SK-HEP-1 cells upon G-Rh2 treatment. In contrast, Fas expression and caspase-8 activity remained constant with G-Rh2 treatment in p53-mutated SW480 and PC-3 cells. In addition, siRNA-mediated knockdown of p53 diminished G-Rh2-induced Fas expression and caspase-8 activation. These results indicated that G-Rh2-triggered extrinsic apoptosis relies on p53-mediated Fas over-expression. In the intrinsic apoptotic pathway, G-Rh2 induced strong and immediate translocation of cytosolic BAK and BAX to the mitochondria, mitochondrial cytochrome c release, and subsequent caspase-9 activation both in HeLa and in SW480 cells. p53-mediated Fas expression and subsequent downstream caspase-8 activation as well as p53-independent caspase-9 activation all contribute to the activation of the downstream effector caspase-3/-7, leading to tumor cell death. Taken together, we suggest that G-Rh2 induces cancer cell apoptosis in a multi-path manner and is therefore a promising candidate for anti-tumor drug development.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cytochromes c ; metabolism ; Enzyme Activation ; drug effects ; Ginsenosides ; chemistry ; pharmacology ; HeLa Cells ; Humans ; Inhibitory Concentration 50 ; Mitochondria ; drug effects ; metabolism ; Protein Transport ; drug effects ; Receptors, Death Domain ; metabolism ; Receptors, Tumor Necrosis Factor, Type I ; metabolism ; Signal Transduction ; drug effects ; Tumor Suppressor Protein p53 ; metabolism ; Up-Regulation ; drug effects ; bcl-2 Homologous Antagonist-Killer Protein ; metabolism ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; metabolism

The methanolic extract obtained from the root portion of Caltha palustris var. alba was evaluated for its anthelmintic efficacy against gastrointestinal nematodes of sheep under both in vitro and in vivo conditions using worm motility inhibition (WMI) assay and fecal egg count reduction (FECR) assay, respectively. The extract was subjected to antimicrobial activity using agar-well diffusion method against different bacterial strains. In addition the extract was evaluated for cytotoxic and antioxidant activity against cultured THP-1(Leukemia), A-549 (Lung), HCT-15 (Colon), Cervix (HeLa) and PC-3(Prostrate) cell lines by SRB and DPPH radical scavenging assays. The extract used resulted in mean %WMI of 94.44%, as observed when the worms were put in lukewarm buffer for 30 min after exposure to different treatments. The mean mortality index of the sample was 0.95. The lethal concentration (LC50) was 0.11 mg·mL(-1). Cell lines were exposed to concentration of 100 μg·mL(-1) of extract for 48 h, which reduced the viability of these cell lines. The same plant extract also showed 55.58% DPPH radical scavenging activity.
Animals ; Anthelmintics ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; therapeutic use ; Antioxidants ; pharmacology ; Bacteria ; drug effects ; Biphenyl Compounds ; metabolism ; Feces ; parasitology ; Gastrointestinal Tract ; parasitology ; HeLa Cells ; Humans ; Nematoda ; drug effects ; Neoplasms ; drug therapy ; Phytotherapy ; Picrates ; metabolism ; Plant Extracts ; pharmacology ; therapeutic use ; Ranunculaceae ; Sheep ; parasitology

OBJECTIVETo investigate the regulatory effects of lanthanum chloride (LaCl3) on the activation of nuclear factor kappa B inhibitor (IκB) kinase beta (IKKβ) induced by tumor necrosis factor alpha (TNF-α).

METHODS(1) Hela cells were cultured routinely in vitro. One portion of cells were collected and divided into TNF-α group (cultured with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min), low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, LaCl3 group (cultured with serum-free RMPI 1640 medium containing 100 µmol/L LaCl3 for 30 min), and control group (cultured with serum-free RMPI 1640 medium for 30 min) according to the random number table. Cells in low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group were first cultured with serum-free RMPI 1640 medium containing 5, 25, 100 µmol/L LaCl3 for 4 h, and then stimulated with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min. There were 3 samples in each group. Cells were collected for detection of intracellular location of NF-κB/p65 protein by immunofluorescence staining. (2) Another portion of cells were collected and divided into TNF-α group, low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, and control group with the same treatment as above. There were 3 samples in each group. The protein levels of NF-κB/p65 in nuclei, and the protein levels of IκBα, phosphorylated IκBα (p-IκBα) as well as IKKβ and phosphorylated IKKβ (p-IKKβ) in cytoplasm were determined by Western blotting. The binding activity between NF-κB/p65 in the nuclear and target gene was determined by NF-κB/p65 transcription factor kit (denoted as absorption value). Data were processed with analysis of variance or LSD-t test.

RESULTS(1) High expression of NF-κB/p65 was observed in cytoplasm of control group. High expression of NF-κB/p65 was observed in nuclei of TNF-α group. The expression of NF-κB/p65 in cytoplasm of LaCl3 group was lower than that of control group. In groups treated with LaCl3 and TNF-α, NF-κB/p65 expression levels in nuclei and cytoplasm were decreased along with the increase in the concentration of LaCl3, which were all lower than those in TNF-α group. (2) There was certain amount of NF-κB/p65 protein expressed in nuclei of control group. The expression of NF-κB/p65 protein in nuclei of TNF-α group was higher than that of control group. In groups treated with LaCl3 and TNF-α, the expressions of NF-κB/p65 protein in nuclei were decreased along with an increase in the concentration of LaCl3. The level of IκBα in TNF-α group was significantly decreased but that of p-IκBα increased as compared with those in control group. Along with the increase in the concentration of LaCl3, the levels of IκBα gradually increased and the levels of p-IκBα gradually decreased in groups treated with LaCl3 and TNF-α. There were no statistical differences in expression levels of IKKβ among the 5 groups. The expression of p-IKKβ could be hardly observed in control group, but it was obviously increased in TNF-α group. The expression levels of p-IKKβ in groups treated with LaCl3 and TNF-α were gradually decreased along with the increase in the concentration of LaCl3. The absorption value in TNF-α group was 0.39 ± 0.03, which was higher than that in control group (0, t = -7.23, P<0.01). The absorption values in low-dose LaCl3 +TNF-α group, moderate-dose LaCl3 + TNF-α group, and high-dose LaCl3 +TNF-α group were respectively 0.17 ± 0.03, 0.15 ± 0.03, and 0, which were obviously lower than that in TNF-α group (with t values respectively -6.54, -5.92, -7.23, P values all below 0.01).

CONCLUSIONSLaCl3 can block the activation of NF-κB signaling pathway by blocking the phosphorylation of IKKβ of Hela cells.

Culture Media ; HeLa Cells ; Humans ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; Lanthanum ; pharmacology ; NF-KappaB Inhibitor alpha ; Signal Transduction ; drug effects ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology