BACKGROUND/OBJECTIVES: Although several recent studies have reported the anti-cancer effects of extracts or components of Citrus unshiu peel, which has been used for various purposes in traditional medicine, the molecular mechanisms for their effects remain unclear. In the present study, the anti-cancer activity of a water-soluble extract of C. unshiu peel (WECU) in MDA-MB-231 human breast carcinoma cells at the level of apoptosis induction was investigated. MATERIALS/METHODS: Cytotoxicity was evaluated using the MTT assay. Apoptosis was detected using DAPI staining and flow cytometry analyses. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, caspase activity and Western blotting were used to confirm the basis of apoptosis. RESULTS: The results indicated that WECU-induced apoptosis was related to the activation of caspase-8, and -9, representative initiator caspases of extrinsic and intrinsic apoptosis pathways, respectively, and caspase-3 accompanied by proteolytic degradation of poly(ADP-ribose) polymerase and down-regulation of the inhibitors of apoptosis protein family members. WECU also increased the pro-apoptotic BAX to anti-apoptotic BCL-2 ratio, loss of mitochondrial membrane potential and cytochrome c release from mitochondria to cytoplasm. Furthermore, WECU provoked the generation of ROS, but the reduction of cell viability and induction of apoptosis by WECU were prevented when ROS production was blocked by antioxidant N-acetyl cysteine. CONCLUSIONS: These results suggest that WECU suppressed proliferation of MDA-MB-231 cells by activating extrinsic and intrinsic apoptosis pathways in a ROS-dependent manner.
Apoptosis* ; Blotting, Western ; Breast Neoplasms* ; Breast* ; Caspase 3 ; Caspase 8 ; Caspases, Initiator ; Cell Survival ; Citrus* ; Cysteine ; Cytochromes c ; Cytoplasm ; Down-Regulation ; Flow Cytometry ; Humans* ; Medicine, Traditional ; Membrane Potential, Mitochondrial ; Mitochondria ; Oxygen* ; Poly(ADP-ribose) Polymerases ; Reactive Oxygen Species ; Water*

OBJECTIVE: To study the protective effect of isoflurane preconditioning on hepatic ischemia-reperfusion (I/R) injury mediated by the noncanonical pyroptosis pathway. METHODS: Thirty C57BL/6 mice were randomly divided into sham-operated group, isoflurane group and I/R group, and in the latter two groups, hepatic I/R injury was induced by clamping the portal vein for 30 min. In isoflurane group, the mice were pretreated with 1.4% isoflurane 30 min before the surgery. The protective effect of isoflurane preconditioning against hepatic I/R injury was evaluated by assessing the pathological score of HE staining of the liver tissue and serum ALT and AST levels. Serum IL-1β and IL-18 levels and the protein expression of GSDMS were detected by ELISA and Western blotting to evaluate the inhibitory effect of isoflurane preconditioning on pyroptosis. Western blotting and immunofluroescence were used to detect the protein expression of caspase-11 in the liver tissues to evaluate the inhibitory effect of isoflurane preconditioning on noncanonical pyroptosis pathway. RESULTS: The Suzuki's score of the liver tissue was significantly higher in I/R group than in the sham group ( < 0.05), while the score in the isoflurane group was significantly lower than that in the I/R group ( < 0.05). Serum ALT and AST levels significantly increased in the sham group ( < 0.05), and were significantly lower in isoflurane group than in I/R group ( < 0.05). The serum levels of IL-1β and IL-18 were significantly higher in I/R group than in sham group ( < 0.05), and were significantly lower in isoflurane group than in I/R group ( < 0.05). The expression of GSDMD in the I/R group was significantly higher than that in sham group, and was significantly lower in isoflurane group than in I/R group ( < 0.05). The hepatic expression of caspase-11 was significantly higher in I/R group than in sham group ( < 0.05), and was significantly lower in isoflurane group than in I/R group ( < 0.05). CONCLUSIONS Isoflurane preconditioning has protective effect against hepatic I/R injury, which is related to the inhibition of the noncanonical pyroptosis pathway.
Animals ; Caspases, Initiator ; Ischemic Preconditioning ; Isoflurane ; Liver ; Mice ; Mice, Inbred C57BL ; Pyroptosis ; Reperfusion Injury

BACKGROUND: Although pulmonary resection is the standard approach for the management of pulmonary metastases from soft tissue sarcoma, most of them are unresectable and chemotherapy remains the only option. The effectiveness of the cytotoxic drugs may be limited by the toxicities that occur before the therapeutic dose is reached. The regional administration of doxorubicin using pulmonary arterial perfusion in a rodent model can produce 10 to 25 times higher concentrations in the lung than systemic administration with minimal systemic toxicities. However, it is unclear whether a high concentration of doxorubicin has beneficial effects for killing cancer cells. MATERIAL AND METHOD: We studied this to evaluate the dose-dependent cytotoxic and apoptotic effects of doxorubicin on methylcholanthrene-induced rat fibrosarcoma(MCA) cells. This study examined the cytotoxicity and apoptosis-related gene expressions(Fas, FasL, Bax, caspase 1, caspase 2, caspase 8, Bcl-2, Bcl-xL, Bcl-xS) in MCA cells after 24 hours exposure to various concentrations of doxorubicin such as 1, 5, 10, 50, and 100 micrometer. RESULT: Dose-dependent cytotoxicity was observed after 24 hours exposure to doxorubicin. However, peak apoptosis after 24 hours exposure was observed at 5 micrometer of doxorubicin. Above 5 micrometer, apoptotic activity was decreased with dose-increment. All mRNA levels of apoptosis-related genes after 24 hours exposure were up-regulated above the control level at 1 micrometer of doxorubicin and then decreased by doxorubicin dose-increment except caspase 8, which showed higher levels than the control level at 5 micrometer. Apoptosis-related protein levels were highest at 1 micrometer of doxorubicin and then decreased by doxorubicin dose-increment. However, Bax and Bcl-xL proteins steadily showed higher levels than the control throughout the different concentrations of doxorubicin. CONCLUSION: These results suggest that apoptosis is the main cytotoxic mechanism in low concentrations of doxorubicin in MCA cells and apoptosis-related genes, such as Bax, caspase 8, a can kill MCA cells, even when apoptosis is inhibited, and have its propriety for achieving much cytotoxicity against MCA cells.
Animals ; Apoptosis* ; bcl-X Protein ; Caspase 1 ; Caspase 2 ; Caspase 8 ; Doxorubicin* ; Drug Therapy ; Fibrosarcoma ; Homicide ; Lung ; Neoplasm Metastasis ; Perfusion ; Rats* ; RNA, Messenger ; Rodentia ; Sarcoma

The aim of this study was to determine the raising anticancer effects of resveratrol (Res) on paclitaxel (PA) in non-small cell lung cancer (NSCLC) cell line A549. The 10 µg/ml of Res had no effect on human fetal lung fibroblast MRC-5 cells or on A549 cancer cells and the 5 or 10 µg/ml of PA also had no effect on MRC-5 normal cells. PA-L (5 µg/ml) and PA-H (10 µg/ml) had the growth inhibitory effects in NSCLC cell line A549, and Res increased these growth inhibitory effects. By flow cytometry experiment, after Res (5 µg/ml)+PA-H (10 µg/ml) treatment, the A549 cells showed the most apoptosic cells compared to other group treatments, and after additional treatment with Res, the apoptosic cells of both two PA concentrations were raised. Res+PA could reduce the mRNA and protein expressions of COX-2, and Res+PA could reduce the COX-2 related genes of VEGF, MMP-1, MMP-2, MMP-9, NF-κB, Bcl-2, Bcl-xL, procollagen I, collagen I, collagen III and CTGF, TNF-α, IL-1β, iNOS and raise the TIMP-1, TIMP-2, TIMP-3, IκB-α, p53, p21, caspase-3, caspase-8, caspase-9, Bax genes compared to the control cells and the PA treated cells. From these results, it can be suggested that Res could raise the anticancer effects of PA in A549 cells, thus Res might be used as a good sensitizing agent for PA.
Carcinoma, Non-Small-Cell Lung ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cell Line* ; Collagen ; Fibroblasts ; Flow Cytometry ; Humans ; In Vitro Techniques* ; Lung ; Paclitaxel* ; Procollagen ; RNA, Messenger ; Tissue Inhibitor of Metalloproteinase-1 ; Tissue Inhibitor of Metalloproteinase-2 ; Tissue Inhibitor of Metalloproteinase-3 ; Vascular Endothelial Growth Factor A

BACKGROUNDAngiopoietin-1 (Ang-1) is an endothelial-specific growth factor that can promote angiogenesis. Studies demonstrated that Ang-1 can inhibit apoptosis of umbilical endothelial cells, but so far little is known about its effects on apoptosis of microvascular endothelial cells. With the apoptotic model of murine-cerebral-derived microvascular endothelial cells (bEnd.3) induced by serum-free culture, we attempted to clarify the molecular mechanism of bEnd.3 apoptosis, particularly its relation to cytochrome C (Cyt C).

METHODSThe cultured microvascular endothelial cell strain, bEnd.3 cell, was employed. An apoptotic model of bEnd.3 was established by serum-free culture. Flow cytometry after Annexin labeling and PI staining were used to assess the apoptotic effects of Ang-1 on bEnd.3, and the expression of Bax/Bcl-2, caspase 8, caspase 3, and Cyt C were detected with Western blotting and ELISA.

RESULTSThe apoptotic rate of bEnd.3 cells after stimulation with Ang-1 (100 ng/L) in serum-free medium was significantly higher than that in control group. Ang-1 inhibited early-stage apoptosis more than late-stage apoptosis provided by propidium iodide (PI) and AnnexinV double staining. The inhibition of Ang-1 on bEnd.3 cell apoptosis was strengthened with the increase in concentration (0 - 400 ng/ml). Ang-1 could decrease the expression of Bax, caspase3 and 8, and increase that of Bcl-2. The results of ELISA indicated that Ang-1 significantly decreased CytC content in cytoplasm and increase that in mitochondria.

CONCLUSIONSAng-1 could inhibit bEnd.3 apoptosis induced by serum-free medium culture. The apoptosis was associated with decreased Bax expression, increased Bcl-2 expression, which result in Cyt C transferring from mitochondria to cytoplasm, and then caspases activation are reduced and cell apoptosis is suppressed.

Angiopoietin-1 ; pharmacology ; Animals ; Annexin A5 ; analysis ; Apoptosis ; drug effects ; Caspase 3 ; Caspase 8 ; Caspases ; analysis ; Cells, Cultured ; Cytochromes c ; physiology ; Endothelial Cells ; cytology ; drug effects ; Mice ; Microcirculation ; drug effects ; bcl-2-Associated X Protein ; analysis

OBJECTIVETo explore the role of Bid protein in the mitochondria and endoplasmic reticulum (ER) associated apoptotic pathway.

METHODSApoptosis of MUTZ-1 cells induced by homoharringtonine (HHT) was measured by FACS. Mitochondria and ER associated apoptotic pathway was detected by RT-PCR and Western blotting. And the translocation of Bid protein was measured by laser scanning confocal microscope (LSCM).

RESULTSAfter exposure of MUTZ-1 to HHT at 0.05 microg/ml for 24 h, typical ER-stress phenomenon induced apoptotic cells and release of Ca2+ from the cytosolic Ca2+ storage and the loss of mitochondrial membrane potential were observed. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor were markedly increased at 4 h after 0.05 microg/ml HHT treatment and peaked at 12 h, then decreased steady. Activation of caspase protein was also observed at 8 h. The translocation of Bid protein from ER to mitochondria was observed at 12 h after HHT treatment.

CONCLUSIONHHT can induce MUTZ-1 cells apoptosis. The cell death may be likely mediated by the ER stress pathway as well as mitochondrial pathway and Bid protein may be the cross talk of the two apoptotic pathways.

Apoptosis ; drug effects ; physiology ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; physiology ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Caspases, Initiator ; metabolism ; Cell Line ; DNA-Binding Proteins ; metabolism ; Endoplasmic Reticulum ; metabolism ; physiology ; Harringtonines ; pharmacology ; Humans ; Mitochondria ; metabolism ; physiology ; Regulatory Factor X Transcription Factors ; Transcription Factors ; metabolism

The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits. Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation, although their precise roles remain to be established. In this study, we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells. Flow cytometric analysis revealed that the sub G(1) fraction was increased in CNOT1-depleted cells. Virtually, the same level of the sub G1 fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits, suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity. Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits. Importantly, the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells. The formation of P-bodies, where mRNA decay is reported to take place, was largely suppressed in CNOT1-depleted cells. Therefore, CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex, and thus is critical in control of mRNA deadenylation and mRNA decay. We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4, which is associated with endoplasmic reticulum ER stress-induced apoptosis. Taken together, CNOT1 depletion structurally and functionally deteriorates the CCR4-NOTcomplex and induces stabilization of mRNAs, which results in the increment of translation causing ER stress-mediated apoptosis. We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.
Apoptosis ; Caspases, Initiator ; genetics ; metabolism ; Cell Survival ; Endoplasmic Reticulum ; enzymology ; Enzyme Activation ; Flow Cytometry ; HEK293 Cells ; HeLa Cells ; Humans ; Protein Subunits ; genetics ; metabolism ; RNA Stability ; RNA, Messenger ; analysis ; RNA, Small Interfering ; genetics ; metabolism ; Ribonucleases ; metabolism ; Stress, Physiological ; Transcription Factor CHOP ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transfection

OBJECTIVETo observe the effect of sodium valproate (VPA) on the proliferation and apoptosis of human lung carcinoma SPC-A1 cells and the underlying mechanism.

METHODSThe effect of VPA on the proliferation of SPC-A1 cells was evaluated by MTT assay and clone formation assay. Flow cytometry was used to analyze the apoptosis of the cells exposed to VPA. The changes in the expressions of Bcl-xl, Bcl-2, Mcl-1, caspase-9, and caspase-3 in the exposed cells were detected by Western blotting.

RESULTSIncubation with VPA for 48 h resulted in a significant inhibition of SPC-A1 cell proliferation, with a IC(50) of 1.8 mmol/L. VPA treatment also inhibited cell colony formation and induced obvious cell apoptosis. Exposure to 8 mmol/L VPA for 48 h caused a percentage of early apoptotic cells of 60.44%. VPA treatment at different concentrations for 48 h obviously lowered the protein levels of Bcl-xl, Bcl-2, and Mcl-1 and induced caspase-9 and caspase-3 activation in SPC-A1 cells.

CONCLUSIONVPA can inhibit the proliferation of SPC-A1 cells by triggering mitochondrion-dependent apoptosis.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Myeloid Cell Leukemia Sequence 1 Protein ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Valproic Acid ; pharmacology ; bcl-X Protein ; metabolism

This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.
Humans ; Female ; Breast Neoplasms/metabolism* ; MCF-7 Cells ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Beclin-1/pharmacology* ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Cell Proliferation

This study was purposed to explore the effects of hyperbaric oxygen (HBO) combined with adriamycin (ADM) on inducing apoptosis of multidrug resistant cells line K562/A02. The cell apoptosis and expression of caspase-3 activity were analyzed by flow cytometry and transmission electron microscopy; the expression levels of HIF-1α, BCL-2 and BAX mRNA were detected by quantitative real time PCR; the caspase 8 activity was determined by using caspase 8 kit; the expression level of P-gp was detected by Western blot. The results showed that the apoptosis rate of K562/A02 cells in combination group (0.2 MPa HBO + ADM) was higher than that in ADM group [(47.36 ± 3.87) % vs (28.51 ± 1.09) %], the difference was statistical significant (p < 0.05); the expression levels of HIF-1α mRNA, P-gp and BCL-2 in combination group were lower than those in ADM group, there were significant differences (p < 0.05); the activities of BAX, caspase 3 and caspase 8 proteins in combination group were higher than those in ADM group, the difference was statistical significant (p < 0.05). It is concluded that 0.2 MPa HBO combined with ADM can reverse the drug-resistance of K562/A02 cells to ADM, enhance the apoptosis rate of cells. The molecular mechanism may be related with down-regulation of P-gp and BCL-2 expression, and up-regulation of caspase-3 and caspase-8 activities by HIF-1α.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Gene Expression Regulation, Leukemic ; Humans ; Hyperbaric Oxygenation ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; K562 Cells ; Proto-Oncogene Proteins c-bcl-2 ; metabolism