Curcumae Rhizoma is a Chinese medicinal herb that is contraindicated during pregnancy. Cold-congelation and blood-stasis are corresponding syndromes to Curcumae Rhizoma. Whether syndrome-based treatment is associated with developmental neurotoxicity of Curcumae Rhizoma remains to be unclear. To verify the theory of traditional Chinese medicine of "syndrome-based treatment during pregnancy", the present study induced the mice blood stasis model by immersing mice in ice water. Pregnant C57 BL/6 wild type(WT) mice and pregnant Nrf2 knock out(KO) mice were randomly divided into control groups and Rhizoma Curcumae exposure groups. The mice were exposed to Rhizoma Curcumae during day 5 to day 18 after pregnancy. The neurodevelopment was examined to evaluate the differences of developmental neurotoxicity between normal and blood-stasis pregnant mice exposed to Rhizoma Curcumae. caspase-3 and caspase-9 activity in brain of the offspring were measured by colorimetric assays. Bcl-2 mRNA and protein expression in brain of the offspring were examined by Real-time RT-PCR and Western blot, respectively. According to the findings, C57 BL/6 mice exposed to Rhizoma Curcumae(10.0 g·kg~(-1)) had a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring, compared with the normal control group, but with no significant change in those of blood-stasis pregnant mice offspring. However, mice exposed to Rhizoma Curcumae(10.0 g·kg~(-1)) showed no change in Bcl-2 gene expression and p38 MAPK phosphorylation in brain of the offspring. Nrf2 gene knockout using CRISPR/Cas9 resulted in a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring. In conclusion, developmental neurotoxicity of the blood-stasis pregnant mice exposed to Rhizoma Curcumae was weaker than that of the normal pregnant mice. Nrf2 activation involved in the phenomenon of Rhizoma Curcumae of "syndrome-based treatment during pregnancy", but the upstream signal pathway mechanism value shall be further investigated.
Animals ; Apoptosis ; Brain ; drug effects ; Caspases ; genetics ; Curcuma ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Female ; Maternal Exposure ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NF-E2-Related Factor 2 ; genetics ; Pregnancy ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Random Allocation ; Rhizome ; chemistry ; Signal Transduction

OBJECTIVE: Reactive oxygen species (ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H₂O₂ stimuli. METHODS: Modified microfluidics and imaging techniques were used to determine O₂ ^•- levels and construct an O₂ ^•- reaction network. To elucidate the consequences of increased O₂ ^•- input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca²⁺ levels, mitochondrial Ca²⁺ uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway. RESULTS AND CONCLUSIONS Results from a modified microchip demonstrated that 1 mmol/L H₂O₂ induced a rapid increase in cellular O₂ ^•- levels (>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power (evaluated by ROS levels) and decreased reducing power (evaluated by glutathione (GSH) levels). In addition, we examined the dynamics of cytosolic Ca²⁺ and mitochondrial Ca²⁺ by confocal laser scanning microscopy and confirmed that Ca²⁺ stores in the endoplasmic reticulum were the primary source of H₂O₂-induced cytosolic Ca²⁺ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca²⁺ signals between organelles, ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.
Apoptosis ; Calcium/metabolism* ; Calcium Signaling ; Caspases/metabolism* ; Cell Line, Tumor ; Cell Lineage ; Cytosol/metabolism* ; Glutathione/metabolism* ; Humans ; Hydrogen Peroxide/chemistry* ; Mitochondria/metabolism* ; Oxidation-Reduction ; Oxidative Stress ; Reactive Oxygen Species/metabolism* ; Signal Transduction ; Superoxides/chemistry*

The underground cane of Schizocapsa plantaginea (Hance) has long been used by Chinese ethnic minority as a constituent of anti-cancer formulae. Saponins are abundant secondary metabolic products located in the underground cane of this plant. The potential therapeutic effects of total saponins isolated from Schizocapsa plantaginea (Hance) (SSPH) on human hepatocellular carcinoma (HCC) were tested in vitro in human liver cancer cell lines, SMMC-7721 and Bel-7404. Apoptosis and cell cycle arrest were determined using flow cytometry, caspase activation was determined by ELISA, and PARP, cleaved PARP, mitogen-activated protein kinase (MAPK) expression and phosphorylation were measured using Western blotting analysis. In vivo anti-HCC effects of SSPH were verified in nude mouse xenograft model. SSPH exerted markedly inhibitory effect on HCC cell proliferation in time- and concentration-dependent manner. Moreover, SSPH significantly induced apoptosis through caspase-dependent signaling and arrested cell cycle at G/M phase. These anti-proliferation effects of SSPH were associated with up-regulated phosphorylation of extracellular signal-regulated kinase-1/2 (Erk1/2) and c-jun-NH2-kinase-1/2 (JNK1/2) and reduced phosphorylation of p38MAPK. Furthermore, inhibitors of ERK, UO126, and JNK, SP600125 inhibited the anti-proliferation effects by SSPH, suggesting that Erk and JNK were the effector molecules in SSPH induced anti-proliferative action. During in vivo experiments, SSPH was found to inhibit xenograft tumor growth in nude mice, with a similar mechanism in vitro. Our study confirmed that SSPH exerted antagonistic effects on human liver cancer cells both in vitro and in vivo. Molecular mechanisms underlying SSPH action might be closely associated with MAPK signaling pathways. These results indicated that SSPH has potential therapeutic effects on HCC.
Animals ; Antineoplastic Agents ; isolation & purification ; pharmacology ; toxicity ; Apoptosis ; drug effects ; Caspases ; genetics ; metabolism ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dioscoreaceae ; chemistry ; Heterografts ; drug effects ; growth & development ; Humans ; Inhibitory Concentration 50 ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; MAP Kinase Signaling System ; drug effects ; Mice ; Mice, Nude ; Phosphorylation ; drug effects ; Plant Tubers ; chemistry ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Saponins ; isolation & purification ; pharmacology ; toxicity

OBJECTIVE: This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin (ADR) in treating proliferative vitreoretinopathy (PVR) using ARPE-19 cells. METHODS: The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B (SRB) assay and propidium iodide (PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2AX (γ-H2AX), phosphorylated checkpoint kinase 1 (p-CHK1), and phosphorylated checkpoint kinase 2 (p-CHK2) were assessed to detect DNA damage and repair. RESULTS: ADR could significantly inhibit ARPE-19 cell proliferation and induce caspase-dependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein-protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the γ-H2AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure. CONCLUSIONS The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.
Apoptosis ; Caspases/metabolism* ; Cell Proliferation ; Cell Survival/drug effects* ; Doxorubicin/pharmacology* ; Flow Cytometry ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Membrane Potential, Mitochondrial ; Oligonucleotide Array Sequence Analysis ; Oxidative Stress/drug effects* ; Phosphorylation ; Propidium/chemistry* ; RNA, Small Interfering/metabolism* ; Retinal Pigment Epithelium/metabolism* ; Rhodamines/chemistry* ; Signal Transduction/drug effects* ; Transcriptome ; Tumor Suppressor Protein p53/metabolism* ; Vitreoretinopathy, Proliferative/drug therapy*

OBJECTIVETo confirm the anticancer effect of total annonaceous acetogenins (TAAs) abstracted from Annona squamosa Linn. on human hepatocarcinoma.

METHODSThe inhibitory effect of TAAs was demonstrated in H22-bearing mice. The potency of TAAs was confirmed as its 50% inhibiting concentration (IC50) on Bel-7402 cell under Sulfur Rhodamine B staining. Both underlying mechanisms were explored as cellular apoptosis and cell cycle under flow cytometry. Mitochondrial and recipient apoptotic pathways were differentiated as mitochondrial membrane potential under flow cytometry and caspases activities under fluorescence analysis.

RESULTSThe inhibitory rate of TAAs in mice was 50.98% at 4 mg/kg dose. The IC50 of TAAs on Bel-7402 was 20.06 µg/mL (15.13-26.61µg/mL). Effective mechanisms of TAAs were confirmed as both of arresting cell cycle at G1 phase and inducing apoptosis dose- and time-dependently. Mitochondrial and recipient pathways involved in apoptotic actions of TAAs.

CONCLUSIONTAAs is effective for hepatocarcinoma, via inhibiting proliferation and inducing apoptosis.

Acetogenins ; chemistry ; pharmacology ; therapeutic use ; Animals ; Annona ; chemistry ; Antineoplastic Agents, Phytogenic ; chemistry ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; enzymology ; pathology ; Caspases ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Dose-Response Relationship, Drug ; Humans ; Liver Neoplasms ; drug therapy ; enzymology ; pathology ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Organ Specificity ; drug effects ; Spleen ; drug effects ; Thymus Gland ; drug effects ; Xenograft Model Antitumor Assays

Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.
Antineoplastic Agents ; chemistry ; pharmacology ; Apoptosis ; drug effects ; genetics ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Caspase 8 ; genetics ; metabolism ; Caspases ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cell Size ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cytochromes c ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Fucus ; chemistry ; G1 Phase Cell Cycle Checkpoints ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; MCF-7 Cells ; Microscopy, Fluorescence ; Molecular Structure ; Polysaccharides ; chemistry ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; bcl-2-Associated X Protein ; genetics ; metabolism

OBJECTIVETo investigate the protective effect of soyasaponins on acute liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in mice.

METHODThe mice were randomly divided into five groups: the normal control, the model group, the silymarin (positive control) group, and soyasaponins high and low-dose groups. They were administered with drugs once every day for 7 days. At the end of the experiment, GalN and LPS were injected intraperitoneally to all of the groups except for the normal group to establish the acute liver injury model. The pathological changes were detected with hematoxylin & eosin (HE) staining, tumor necrosis factor-alpha (TNF-alpha) was detected by ELISA method, and the alanine aminotransferase (ALT), aspartate aminotransferase (AST), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), reduced glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), and the activation of Caspase-3 and Caspase-8 were detected by the colorimetric method.

RESULTSoyasaponins could reduce the activities of serum ALT and AST, the acute hepatic injury induced by GalN/LPS, serum TNF-alpha level, hepatic NO and MDA contents, and the Caspase-3 and Caspase-8 activations of liver tissues, and increase the hepatic CAT, GPx, GST and GSH levels.

CONCLUSIONSoyasaponins shows the protective effect on acute liver injury induced by GalN and LPS in mice, which may be related to its antioxidative ability and anti-liver apoptosis.

Alanine Transaminase ; blood ; Animals ; Antioxidants ; metabolism ; Apoptosis ; drug effects ; Aspartate Aminotransferases ; blood ; Caspases ; metabolism ; Chemical and Drug Induced Liver Injury ; metabolism ; pathology ; prevention & control ; Galactosamine ; toxicity ; Lipopolysaccharides ; toxicity ; Liver ; pathology ; Male ; Mice ; Saponins ; pharmacology ; Soybeans ; chemistry

OBJECTIVETo in vitro compare the induction of extracts of Stellera chamaejasme ESC, ESC-1 and ESC-2 on NCI-H157 cell apoptotic.

METHODThe apoptosis rate was inspected by flow cytometry; caspase-3, 8, 9 activities was measured by spectrophotometry. Fas, Fas-L, TNF-alpha, Trail-R, Cyto-C, Smac/diablo protein expressions of apoptosis pathway was observed by Elisa method.

RESULTCompared with the control group, ESC, ESC-1, ESC-2 can significantly improve the apoptosis rate of NCI-H157 cell. ESC significantly improved cells caspase-3, 8 activity, ESC-2 can significantly improve the activity of caspase-3, 8, 9. ESC, ESC-1, ESC-2 significantly increased Fas expression and ESC significantly increased Fas/Fas-L ratio. ESC, ESC-1, ESC-2 significantly increased TNF-alpha protein expression. ESC-1 significantly lowered TRAIL-R expression. ESC, ESC-1, ESC-2 had no significant effect on Cyto-C. ESC-1, ESC-2 significantly reduced Smac protein expression.

CONCLUSIONThe apoptotic effect induced by ESCs may be related to the regulation of death receptor pathway proteins. Induction mechanisms of ESCs were so complicated that it may have a two-way regulatory effect. Its induction in apoptosis is a result from comprehensive regulation and control.

Apoptosis ; drug effects ; Caspases ; metabolism ; Cell Line, Tumor ; Humans ; Neoplasms ; chemistry ; drug therapy ; pathology ; Plant Extracts ; pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; analysis ; Thymelaeaceae ; chemistry ; Tumor Necrosis Factor-alpha ; analysis ; fas Receptor ; analysis

OBJECTIVETo study the total glucosides of Radix Paeoniae Rubra induced K562 tumor cell apoptosis of signaling pathways and related gene changes.

METHODBy MTT and flow cytometric, real-time quantitatie polymerase chain reaction (PCR) and Western blot methods researched the level of genes and proteins.

RESULTThe total glucosides of Radix Paeoniae Rubra could inhibit K562 cell growth by MTT method, there was the relationship of concentration-time between them, TGC prompted K562 cell translocation of phosphatidylserine, may be apoptosis through non-receptor-dependent pathways because caspase-3 mRNA, caspase-9 mRNA and cytochrome C increased, caspase-8 mRNA had no significant change. The expression of Bcl-2 protein and Bcl-X1 protein could decreased, the expression of Bax protein could increased by regulating gene expression.

CONCLUSIONTGC induced K562 cell apoptosis that might be through the mitochondria pathway, when cell apoptosis occured, Bcl-2 or Bcl-XI proteins combination of Bax protein would be displacement, then the mitochondrial membrane became permeable to release a series of material, then lead to cell death.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; genetics ; Caspases ; genetics ; Dose-Response Relationship, Drug ; Gene Expression Regulation, Neoplastic ; drug effects ; Glucosides ; pharmacology ; Humans ; K562 Cells ; Paeonia ; chemistry ; Signal Transduction ; drug effects ; Time Factors

The massive reorganization of microtubule network involves in transcriptional regulation of several genes by controlling transcriptional factor, nuclear factor-kappa B (NF-kappaB) activity. The exact molecular mechanism by which microtubule rearrangement leads to NF-kappaB activation largely remains to be identified. However microtubule disrupting agents may possibly act in synergy or antagonism against apoptotic cell death in response to conventional chemotherapy targeting DNA damage such as adriamycin or comptothecin in cancer cells. Interestingly pretreatment of microtubule disrupting agents (colchicine, vinblastine and nocodazole) was observed to lead to paradoxical suppression of DNA damage-induced NF-kappaB binding activity, even though these could enhance NF-kappaB signaling in the absence of other stimuli. Moreover this suppressed NF-kappaB binding activity subsequently resulted in synergic apoptotic response, as evident by the combination with Adr and low doses of microtubule disrupting agents was able to potentiate the cytotoxic action through caspase-dependent pathway. Taken together, these results suggested that inhibition of microtubule network chemosensitizes the cancer cells to die by apoptosis through suppressing NF-kappaB DNA binding activity. Therefore, our study provided a possible anti-cancer mechanism of microtubule disrupting agent to overcome resistance against to chemotherapy such as DNA damaging agent.
Animals ; Antibiotics, Antineoplastic/therapeutic use ; *Apoptosis ; Caspases/metabolism ; Cell Line ; Colchicine/pharmacology ; DNA/metabolism ; *DNA Damage ; Doxorubicin/therapeutic use ; Humans ; Mice ; Microtubules/chemistry/*drug effects/metabolism ; NF-kappa B/antagonists & inhibitors/*metabolism ; Neoplasms/drug therapy ; Nocodazole/pharmacology ; Protein Binding ; Signal Transduction ; Tubulin Modulators/*pharmacology ; Vinblastine/pharmacology