OBJECTIVETo investigate the effects and underlying mechanism of notoginsenoside R1 on amyloid-β (1-42) (Aβ(1-42)) induced mitochondrial apoptotic death in SH-SY5Y cells.

METHODCell viability was assayed by MTT, apoptotic rates were analyzed with PI/Annexin V flow cytometry, Bax and Bcl-2 expression were detected with Western blotting, enzymatic activity of caspase-3, caspase-8 and caspase-9 were measured by ELISA assay.

RESULTThe 6.25-100 nmol x L(-1) of notoginsenoside R1 attenuate Aβ(1-42) induced apoptotic death of SH-SY5Y in dose dependent manner. The ratio of Bcl-2/Bax was elevated in SH-SY5Y with notoginsenoside R1 treatment. Caspase-3 and caspase-9 were activated with notoginsenoside R1 treatment while caspase-8 was not affected.

CONCLUSIONNotoginsenoside R1 could protect SH-SY5Y cells from Aβ(1-42) induced apoptosis via mitochondria related apoptotic pathway.

Amyloid beta-Peptides ; antagonists & inhibitors ; Apoptosis ; drug effects ; Caspases ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cytoprotection ; Ginsenosides ; pharmacology ; Humans ; Mitochondria ; drug effects ; Peptide Fragments ; antagonists & inhibitors

The mitochondria-mediated caspase activation pathway is a major apoptotic pathway characterized by mitochondrial outer membrane permeabilization (MOMP) and subsequent release of cytochrome c into the cytoplasm to activate caspases. MOMP is regulated by the Bcl-2 family of proteins. This pathway plays important roles not only in normal development, maintenance of tissue homeostasis and the regulation of immune system, but also in human diseases such as immune disorders, neurodegeneration and cancer. In the past decades the molecular basis of this pathway and the regulatory mechanism have been comprehensively studied, yet a great deal of new evidence indicates that cytochrome c release from mitochondria does not always lead to irreversible cell death, and that caspase activation can also have non-death functions. Thus, many unsolved questions and new challenges are still remaining. Furthermore, the dysfunction of this pathway involved in cancer development is obvious, and targeting the pathway as a therapeutic strategy has been extensively explored, but the efficacy of the targeted therapies is still under development. In this review we will discuss the mitochondria-mediated apoptosis pathway and its physiological roles and therapeutic implications.
Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Caspases ; metabolism ; Cytochromes c ; metabolism ; Humans ; Inhibitor of Apoptosis Proteins ; antagonists & inhibitors ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Neoplasms ; drug therapy ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

OBJECTIVETo investigate the effect of aurora kinase inhibitor ENMD-2076 on human acute myelogenous leukemia (AML) cell lines.

METHODSAML THP-1 and Kasumi-1 cells were treated with ENMD-2076 for 24 h and 48 h,respectively. Cell growth was measured by MTT assay. Apoptosis was determined using Hoechst staining apoptosis detection kit. Activation of Caspase pathway and expression of apoptosis regulator proteins were detected by Western blot.

RESULTSENMD-2076 significantly induced growth arrest and apoptosis in THP-1 and Kasumi-1 cells. Enhanced apoptosis was observed in ENMD-2076 group evidenced by strong activation of Caspase-9,Caspase-3 and PARP. Furthermore,the ENMD-2076 treatment resulted in down-regulation of anti-apoptotic protein Mcl-1 expression. Also,up-regulated expression of pro-apoptotic protein Bak,Bad and Bax was detected after ENMD-2076 treatment.

CONCLUSIONENMD-2076 can kill effectively AML cells by inhibiting cell growth and inducing apoptosis,which is associated with activation of Caspase pathway and regulation of pro-apoptotic and anti-apoptotic proteins.

Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Aurora Kinases ; Caspases ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Protein-Serine-Threonine Kinases ; antagonists & inhibitors ; Pyrazoles ; pharmacology ; Pyrimidines ; pharmacology

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

BACKGROUND/AIMS: We examined the effects of cilostazol on mitogen-activated protein kinase (MAPK) activity and its relationship with cilostazol-mediated protection against apoptosis in lipopolysaccharide (LPS)-treated endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to LPS and cilostazol with and without specific inhibitors of MAPKs; changes in MAPK activity in association with cell viability and apoptotic signaling were investigated. RESULTS: Cilostazol protected HUVECs against LPS-induced apoptosis by suppressing the mitochondrial permeability transition, cytosolic release of cytochrome c, and subsequent activation of caspases, stimulating extracellullar signal-regulated kinase (ERK1/2) and p38 MAPK signaling, and increasing phosphorylated cAMPresponsive element-binding protein (CREB) and Bcl-2 expression, while suppressing Bax expression. These cilostazol-mediated cellular events were effectively blocked by MAPK/ERK kinase (MEK1/2) and p38 MAPK inhibitors. CONCLUSIONS: Cilostazol protects HUVECs against LPS-induced apoptosis by suppressing mitochondriadependent apoptotic signaling. Activation of ERK1/2 and p38 MAPKs, and subsequent stimulation of CREB phosphorylation and Bcl-2 expression, may be responsible for the cellular signaling mechanism of cilostazolmediated protection.
Apoptosis/*drug effects ; Caspases/metabolism ; Cell Line ; Cell Survival/drug effects ; Cyclic AMP Response Element-Binding Protein/metabolism ; Cytochromes c/metabolism ; Dose-Response Relationship, Drug ; Endothelial Cells/*drug effects/enzymology/pathology ; Humans ; Lipopolysaccharides/*toxicity ; Mitochondrial Membrane Transport Proteins/drug effects/metabolism ; Mitogen-Activated Protein Kinase 1/antagonists & inhibitors/*metabolism ; Mitogen-Activated Protein Kinase 3/antagonists & inhibitors/*metabolism ; Phosphodiesterase Inhibitors/*pharmacology ; Phosphorylation ; Protein Kinase Inhibitors/pharmacology ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Signal Transduction/*drug effects ; Tetrazoles/*pharmacology ; Time Factors ; bcl-2-Associated X Protein/metabolism ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism

AIMTo determine how SDF-1 alpha/CXCR4 activates nuclear factor-kappa B (NF-kappaB) and promotes oral squamous cell carcinoma (OSCC) invasion.

METHODOLOGYA lentivirus-based knockdown approach was utilized to deplete gene expression. NF-kappaB activation was evaluated by Western blot analysis and electrophoretic mobility shift (EMSA).

RESULTSWe show that the activation of NF-kappaB by CXCR4 occurs through the Carma3/Bcl10/Malt1 (CBM) complex in OSCC. We found that loss of components of the CBM complex in HNSCC can inhibit SDF-1 alpha induced phosphorylation and degradation of IkappaBalpha, while TNF alpha induced IKK activation remains unchanged. Further, we identified a role for novel and atypical, but not classical, PKCs in activating IKK through CXCR4. Importantly, inhibition of the CBM complex leads to a significant decrease in SDF-1 alpha mediated invasion of OSCC.

CONCLUSIONThe CBM complex plays a critical role in CXCR4-induced NF-kappaB activation in OSCC. Targeting molecular components of the NF-kappaB signaling pathway may provide an important therapeutic opportunity in controlling the progression and metastasis of OSCC mediated by SDF-1 alpha.

Adaptor Proteins, Signal Transducing ; antagonists & inhibitors ; physiology ; B-Cell CLL-Lymphoma 10 Protein ; CARD Signaling Adaptor Proteins ; antagonists & inhibitors ; physiology ; Carcinoma, Squamous Cell ; pathology ; Caspase Inhibitors ; Caspases ; physiology ; Cell Line, Tumor ; Chemokine CXCL12 ; antagonists & inhibitors ; physiology ; Enzyme Activation ; drug effects ; Gene Silencing ; Genetic Vectors ; genetics ; Humans ; I-kappa B Kinase ; drug effects ; I-kappa B Proteins ; metabolism ; Isoenzymes ; antagonists & inhibitors ; Lentivirus ; genetics ; Membrane Proteins ; antagonists & inhibitors ; physiology ; Mouth Neoplasms ; pathology ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; physiology ; Neoplasm Invasiveness ; Neoplasm Proteins ; antagonists & inhibitors ; physiology ; Phosphorylation ; Plasmids ; genetics ; Protein Kinase C ; antagonists & inhibitors ; Receptors, CXCR4 ; physiology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate the mechanism of U87 cell apoptosis induced by inhibiting miR-21 expression.

METHODSAntisense oligonucleotides of miR-21 were chemically synthesized and transfected into U87 cells. The apoptosis, proliferation, and invasion of the cells were evaluated. The relationship between miR-21 and PTEN or caspase was identified by bioinformatics and Western blot.

RESULTSInhibiting miR-21 expression led to U87 cell growth suppression, apoptosis induction, invasion reduction, caspase-3 activity elevation and caspase-9 activation, but did not affect PTEN and caspase-8 expression.

CONCLUSIONmiR-21 may function as an antiapoptotic miRNA in U87 cells. Inhibiting miR-21 expression could induce U87 cell apoptosis via caspase-9 and 3 activation, but not PTEN activation.

Animals ; Apoptosis ; drug effects ; genetics ; Caspases ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Enzyme Activation ; genetics ; Gene Expression Regulation, Neoplastic ; Glioma ; genetics ; pathology ; Humans ; MicroRNAs ; antagonists & inhibitors ; genetics ; Oligoribonucleotides, Antisense ; genetics ; pharmacology ; PTEN Phosphohydrolase ; metabolism ; Transfection

The mechanism of apoptosis induced by SIRT1 deacetylase inhibitors in both human breast cancer MCF-7 and MCF-7 doxorubicin-resistant cells was studied. MTT assay was used to detect growth-inhibitory effect on the cells. Protein expression was detected by Western blotting. Chromatin condensation was detected by a fluorescent microscope after Hoechst 33342 staining. Cell cycle distribution was analyzed with flow cytometry. Apoptotic cells were detected with Annexin V staining. Nicotinamide (NAM) and Sirtinol, two SIRT1 deacetylase inhibitors, exhibited the similar growth-inhibitory effects on MCF-7/DOX cells and MCF-7 cells, but no potentiation of DOX activities. The arrest at G2/M phase was detected by flow cytometry in both MCF-7 and MCF-7/DOX cells after NAM treatment. Activation of caspase pathway in MCF-7 cells, such as the cleavages of PARP, caspase-6, -7, -9, were observed after exposure to NAM 50 mmol x L(-1), accompanied by the occurrence of chromatin condensation and Annexin V positive cells. However, the cleavages of PARP, caspase-6 and -7 in MCF-7/DOX cells delayed after exposure to NAM for 24 h and obviously increased at 48 h with appearance of chromatin condensation and Annexin V positive cells. SIRT1 deacetylase inhibitors show no cross resistance to MCF-7 drug-resistant cells, and the similar growth-inhibitory actions of them to MCF-7 sensitive and drug-resistant cells by which it is mediated by activation of apoptotic caspase pathway.
Apoptosis ; drug effects ; Benzamides ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Caspases ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Enzyme Inhibitors ; pharmacology ; Female ; Humans ; Naphthols ; pharmacology ; Niacinamide ; pharmacology ; Sirtuin 1 ; antagonists & inhibitors

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS: To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS: hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS: These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.
Amino Acid Chloromethyl Ketones/pharmacology ; Antineoplastic Agents/*pharmacology ; Caspases/antagonists & inhibitors/metabolism ; Cell Line, Tumor ; Cisplatin/*pharmacology ; Cysteine Proteinase Inhibitors/pharmacology ; Cytochrome c Group/metabolism ; Drug Resistance, Neoplasm/genetics ; Humans ; Neoplasms/therapy ; RNA, Small Interfering ; Telomerase/*antagonists & inhibitors/genetics/metabolism ; bcl-2-Associated X Protein/metabolism

Suppression of apoptosis is one of the hallmarks of carcinogenesis. Tumor cells endure apoptotic pressure by overexpressing several antiapoptotic proteins, and FLICE inhibitory protein (FLIP) is one of the important antiapoptotic proteins that have been shown to be overexpressed in various primary tumor cells. FLIP has two death-effector domains in tandem, mimicking the prodomain of procaspase-8. It is recruited to Fadd in death-inducing signaling complex, thereby preventing the activation of procaspase-8. To date, three isoforms of human cytosolic FLIP (c-FLIP) and six viral homologs (v-FLIP) have been identified. Recently, the crystal structure of v-FLIP MC159 was determined for the first time as an atomic-detail FLIP structure, which revealed that two death effector domains are packed tightly against each other mainly through conserved hydrophobic interactions. The overexpression of c-FLIP in tumor cells has been shown to be the determinant of the tumor's resistance to death ligands such as FasL and TRAIL. It has also been shown that the down-regulation of c-FLIP results in sensitizing resistant tumor cells. Therefore, the agents directly targeting c-FLIP at mRNA and protein levels are expected to be developed in near future and tested for the potential as a new class of anti-cancer drugs.
Apoptosis ; CASP8 and FADD-Like Apoptosis Regulating Protein/antagonists & inhibitors/chemistry/*metabolism ; Caspases/antagonists & inhibitors/metabolism ; Humans ; Neoplasms/*metabolism/pathology/*therapy ; Signal Transduction