This study aims to delve into the influences and underlying mechanisms of Banxia Xiexin Decoction(BXD) on the proliferation, apoptosis, invasion, and migration of colon cancer cells. Firstly, the components of BXD in blood were identified by UPLC-MS/MS, and subsequently the content of these components were determined by HPLC. Then, different concentrations of BXD were used to treat both the normal intestinal epithelial cells(NCM460) and the colon cancer cells(HT29 and HCT116). The cell viability and apoptosis were examined by the cell counting kit-8(CCK-8) and flow cytometry, respectively. Western blot was employed to determine the expression of the apoptosis regulators B-cell lymphoma-2(Bcl-2) and Bcl-2-associated X(Bax). The cell wound healing assay and Transwell assay were employed to measure the cell migration and invasion, respectively. Additionally, Western blot was employed to determine the expression levels of epithelial-mesenchymal transition(EMT)-associated proteins, including epithelial cadherin(E-cadherin), neural cadherin(N-cadherin), and vimentin. The protein and mRNA levels of the factors in the poly(ADP-ribose) glycohydrolase(PARG)/poly(ADP-ribose) polymerase 1(PARP1)/nuclear factor kappa-B p65(NF-κB p65) signaling pathway were determined by Western blot and RT-qPCR, respectively. The results demonstrated that following BXD intervention, the proliferation of HT29 and HCT116 cells was significantly reduced. Furthermore, BXD promoted the apoptosis, enhanced the expression of Bcl-2, and suppressed the expression of Bax in colon cancer cells. At the same time, BXD suppressed the cell migration and invasion and augmented the expression of E-cadherin while diminishing the expression of N-cadherin and vimentin. In addition, BXD down-regulated the protein and mRNA levels of PARG, PARP1, and NF-κB p65. In conclusion, BXD may inhibit the malignant phenotypes of colon cancer cells by mediating the PARG/PARP1/NF-κB signaling pathway.
Colonic Neoplasms/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Phenotype ; Signal Transduction/drug effects* ; Cell Proliferation/drug effects* ; Apoptosis ; Cell Movement/drug effects* ; Neoplasm Invasiveness ; HCT116 Cells ; Proto-Oncogene Proteins c-bcl-2/biosynthesis* ; Humans ; Poly (ADP-Ribose) Polymerase-1 ; Glycoside Hydrolases ; bcl-2-Associated X Protein ; NF-kappa B p50 Subunit

Objective: To explore the effect and mechanism of activation of peripheral blood mononuclear cell (PBMC) Toll-like receptor (TLR3) signaling pathway in recombinant HBsAg (rHBsAg) immune response. Methods: White blood cells were collected from peripheral blood of 13 healthy donors in the preparation of blood products. PBMC was isolated and treated with Poly I:C (Poly I:C group) and PBS (control group) respectively. 48 h later, some cells were collected and the expressions of TLR3 signaling pathway proteins were detected by flow cytometry. After activating (Poly I:C group)/inactivating (control group) TLR3 signaling pathway, rHBsAg was given to both groups for 72 h, and the proportions of DC, T, B cells and their subsets in PBMC were detected by flow cytometry. Paired <i>ti>-test, paired samples wilcoxon signed-rank test and canonical correlation analyses were used for statistical analysis. Results: The percentage of TLR3 protein-positive cells (19.21%) and protein expression (8 983.95), NF-κB protein expression (26 193.13), the percentage of pNF-κB protein-positive cells (13.73%) and its proportion in NF-κB (16.03%), and the percentage of pIRF3 protein-positive cells (12.64%) and its proportion in IRF3 (21.80%) in Poly I:C group were higher than those in control group (11.54%, 8 086.00, 22 340.66, 8.72%, 9.71%, 9.57%, 19.12%) (<i>Pi><0.05), and the percentage of TRIF protein-positive cells (89.75%) and protein expression (304 219.54) were higher in Poly I:C group than in the control group (89.64%, 288 149.72) (<i>Pi>>0.05). After PBMC stimulation by rHBsAg, the proportions of mDC (2.90%), pDC (1.80%), B cell (5.31%) and plasma cell (67.71%) in Poly I:C group were significantly higher than those in the control group (1.83%, 0.81%, 4.23%, 58.82%) (<i>Pi><0.05). Results of canonical correlation analysis showed that the expression of TLR3 protein was positively correlated with the proportions of plasma cells, the expression of pIRF3 protein was positively correlated with the proportions of plasma cells and mDC, and the percentage of pNF-κB protein-positive cells and the percentage of pIRF3 protein-positive cells were positively correlated with the proportion of CD4⁺T cells. Conclusions: Poly I:C can activate TLR3/TRIF/NF-κB and TLR3/TRIF/IRF3 signaling pathway, promote the function of downstream signaling molecules, and then promote the maturation of DC, induce the immune responses of CD4⁺T cell, and promote the maturation and activation of B cells and the immune response of rHBsAg.
Adaptor Proteins, Vesicular Transport/pharmacology* ; Hepatitis B Surface Antigens ; Humans ; Immunity ; Leukocytes, Mononuclear/metabolism* ; NF-kappa B ; Poly I-C/pharmacology* ; Signal Transduction ; Toll-Like Receptor 3/metabolism* ; Toll-Like Receptors

OBJECTIVE: To observe the effect of electroacupuncture (EA) on vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor receptor-3 (VEGFR-3), proinflammatory factors and apoptosis in myocardial tissue in mice with acute myocardial ischemia (AMI), and to explore the mechanism of EA for AMI. METHODS: Fifty male C57BL/6 mice were randomly divided into a sham operation group, a model group, an EA group, an inhibitor group and an inhibitor+EA group, 10 mice in each group. Except for the sham operation group, the mice in the remaining groups were intervented with ligation at the left anterior descending (LAD) coronary artery to establish AMI model. The mice in the sham operation group were intervented without ligation after thoracotomy. The mice in the EA group were intervented with EA at "Shenmen" (HT 7) and "Tongli" (HT 5), disperse-dense wave, 2 Hz/15 Hz in frequency, 1 mA in current intensity, 30 min each time, once a day, for 3 d. The mice in the inhibitor group were treated with intraperitoneal injection of SAR 131675 (12.5 mg•kg^-1•d^-1, once a day for 3 d). The mice in the inhibitor+EA group were injected intraperitoneally with SAR 131675 30 min before EA. The ECG before modeling, 30 min after modeling and 3 d after intervention was detected, and the ST segment displacement was recorded; after the intervention, the ELISA method was applied to measure the contents of serum creatine kinase isoenzyme (CK-MB), aspartate aminotransferase (AST) as well as tumor necrosis factor-α (TNF-α) and interleukin-23 (IL-23) in myocardial tissue; the HE staining method was used to observe the morphological changes of myocardial tissue; the immunofluorescence double labeling method was applied to measure the number of co-expression positive cells of VEGF-C/VEGFR-3 in myocardial tissue; the TUNEL method was used to detect the level of cardiomyocyte apoptosis; the Western blot method was applied to measure the protein expressions of VEGF-C, VEGFR-3, b-lymphoma-2 (Bcl-2), activated caspase-3 (Cleaved Caspase-3) and activated poly adenosine diphosphate ribose polymerase-1 (Cleaved PARP-1). RESULTS: Compared with the sham operation group, in the model group the ST segment displacement was increased (<i>Pi><0.01); the contents of CK-MB, AST, TNF-α and IL-23 were increased (<i>Pi><0.01); the arrangement of myocardial fibers was disordered, and interstitial inflammatory cell infiltration was obvious; the number of co-expression positive cells of VEGF-C/VEGFR-3 was decreased (<i>Pi><0.01); the number of cardiomyocyte apoptosis was increased (<i>Pi><0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were decreased (<i>Pi><0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were increased (<i>Pi><0.01). Compared with the model group, in the EA group the ST segment displacement was decreased (<i>Pi><0.01); the contents of CK-MB, AST, TNF-α, IL-23 were decreased (<i>Pi><0.01); the severity of myocardial pathological injury was reduced; the number of co-expression positive cells of VEGF-C/VEGFR-3 was increased (<i>Pi><0.01); the number of cardiomyocyte apoptosis was reduced (<i>Pi><0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were increased (<i>Pi><0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were reduced (<i>Pi><0.01). There was no significant difference in all the indexes between the model group and the inhibitor group (<i>Pi>>0.05). Compared with the model group, the protein expression of VEGF-C was increased in the inhibitor+EA group (<i>Pi><0.01). Compared with the inhibitor group, in the EA group the ST segment displacement was decreased (<i>Pi><0.01); the contents of CK-MB, AST, TNF-α, IL-23 were decreased (<i>Pi><0.01); the severity of myocardial pathological injury was reduced; the number of co-expression positive cells of VEGF-C/VEGFR-3 was increased (<i>Pi><0.05); the number of cardiomyocyte apoptosis was reduced (<i>Pi><0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were increased (<i>Pi><0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were reduced (<i>Pi><0.01). Compared with the inhibitor+EA group, all the indexes in the EA group were improved except the protein expression of VEGF-C (<i>Pi><0.01). CONCLUSION EA could relieve the inflammatory reaction and apoptosis in AMI mice, and its mechanism may be related to activating VEGF-C/VEGFR-3 pathway and promoting lymphangion genesis.
Mice ; Male ; Animals ; Electroacupuncture ; Vascular Endothelial Growth Factor Receptor-3 ; Caspase 3 ; Vascular Endothelial Growth Factor C ; Tumor Necrosis Factor-alpha/genetics* ; Vascular Endothelial Growth Factor A/genetics* ; Poly(ADP-ribose) Polymerase Inhibitors ; Mice, Inbred C57BL ; Myocardial Ischemia/metabolism* ; Apoptosis ; Interleukin-23 ; Proto-Oncogene Proteins c-bcl-2

Lnc-HUR1 is an HBV-related long non-coding RNA, which can promote the proliferation of hepatoma cells and the occurrence and development of liver cancer. In this study we explored the effect of lnc-HUR1 on the apoptosis of hepatocellular carcinoma cells by taking the approach of immunoblotting, quantitative real time PCR, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and flow cytometry. We found that overexpression of lnc-HUR1 significantly reduced the activity of caspase3/7 and the cleavage of PARP-1, while knocking down of lnc-HUR1 significantly increased the activity of caspase3/7 and promoted the cleavage of PARP-1 in HepG2 cells treated with TGF-β, pentafluorouracil or staurosporine. Consistently, the data from Annexin-V/PI staining showed that overexpression of lnc-HUR1 inhibited apoptosis, while knockdown of lnc-HUR1 promoted apoptosis. Moreover, overexpression of lnc-HUR1 up-regulated the apoptosis inhibitor Bcl-2 and down-regulated the pro-apoptotic factor BAX at both RNA and protein levels. In the CCL4-induced acute liver injury mice model, the expression of Bcl-2 in the liver tissue of lnc-HUR1 transgenic mice was higher than that of the control mice. The data from ChIP assay indicated that lnc-HUR1 reduced the enrichment of p53 on Bcl-2 and BAX promoters. All these results indicated that lnc-HUR1 inhibited the apoptosis by promoting the expression of apoptosis inhibitor Bcl-2 and inhibiting the expression of apoptosis promoting factor BAX. Further studies showed that lnc-HUR1 regulated the transcription of Bcl-2 and BAX in HCT116 cells, but had no effect on the expression of Bcl-2 and BAX in HCT116 p53^-/- cells, indicating that lnc-HUR1 regulates the transcription of Bcl-2 and BAX dependent upon the activity of p53. In conclusion, HBV upregulated lnc-HUR1 can inhibit the apoptosis of hepatoma cells. Lnc-HUR1 inhibits apoptosis by inhibiting the transcriptional activity of p53. These results suggest that lnc-HUR1 plays an important role in the occurrence and development of HBV-related hepatocellular carcinoma.
Animals ; Annexins/pharmacology* ; Apoptosis ; Carcinoma, Hepatocellular/genetics* ; Cell Proliferation ; Hep G2 Cells ; Hepatitis B virus/metabolism* ; Humans ; Liver Neoplasms/genetics* ; Mice ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology* ; Proto-Oncogene Proteins c-bcl-2/pharmacology* ; RNA, Long Noncoding/metabolism* ; Staurosporine/pharmacology* ; Transforming Growth Factor beta/pharmacology* ; Tumor Suppressor Protein p53/pharmacology* ; bcl-2-Associated X Protein/pharmacology*

This study aims to investigate the effect of ethoxysanguinarine(Eth) on cisplatin(DDP)-resistant human gastric cancer cells and decipher the underlying mechanism. The human gastric cancer cell line SGC7901 and the DDP-resistant cell line SGC7901/DDP were used as the cell models. Western blot was employed to determine the expression levels of multidrug resistance-related proteins, and methyl thiazolyl tetrazolium(MTT) assay to detect the proliferation of SGC7901 and SGC7901/DDP cells exposed to DDP. After treatment with different concentrations of Eth, the proliferation of SGC7901 and SGC7901/DDP cells was detected by MTT assay, trypan blue exclusion assay, colony formation assay, and high-content imaging and analysis system. The apoptosis of SGC7901/DDP cells was detected by flow cytometry with Annexin V-FITC/PI staining. GFP-LC3 transfection was carried out to detect the effect of Eth on the autophagy of SGC7901/DDP cells. The expression levels of the multidrug resistance-related protein P-glycoprotein(P-gp), the apoptosis-related proteins [caspase-9, caspase-3, and poly(ADP-ribose) polymerase(PARP)], the autophagy-related protein light chain 3-Ⅱ(LC3-Ⅱ), the key effectors [mammalian target of rapamycin(mTOR), 70 kDa ribosomal protein S6 kinase(P70 S6 K), and 4 E binding protein 1(4 E-BP1)] of the mammalian target of rapamycin complex 1(mTORC1) signaling pathway, cancerous inhibitor of protein phosphatase 2A(CIP2A), and protein kinase B(Akt) were measured by Western blot. The mRNA level of CIP2A in the SGC7901/DDP cells exposed to Eth for 24 h was analyzed by RT-qPCR. After SGC7901/DDP cells were transfected with CIP2A expression vector pcDNA3.1-HA-CIP2A and treated with different concentrations of Eth, MTT assay was used to determine the prolife-ration of SGC7901/DDP cells and Western blot to detect the expression levels of related proteins. The interaction sites of Eth and CIP2A were predicted by molecular docking. The affinity between Eth and CIP2A was determined by drug affinity responsive target stability(DARTS) assay. The pharmacokinetic properties and drug-like activity of Eth were predicted by SwissADME. The results indicated that SGC7901/DDP cells were more sensitive to Eth than SGC7901 cells. Eth significantly inhibited proliferation and colony formation and changed the morphology, roundness, and area of SGC7901/DDP cells. Eth treatment caused the nucleus shrinking and significantly increased the apoptosis rate of the cells. Furthermore, Eth down-regulated the expression of caspase-9 and caspase-3 precursors and promoted the cleavage of PARP, which suggested that Eth induced the apoptosis of SGC7901/DDP cells. The GFP-LC3 in Eth-treated cells showed speckled aggregation. The up-regulated expression of LC3-Ⅱ by Eth indicated that Eth activated the autophagy of SGC7901/DDP cells. Eth down-regulated the expression of P-gp, the phosphorylation of mTOR, P70 S6K, and 4E-BP1, the expression of CIP2A, and the phosphorylation of Akt. Additionally, it increased the activity of PP2A, and had no significant effect on the expression of CIP2A in SGC7901/DDP cells. CIP2A overexpression antagonized the inhibition of cell proliferation and the activation of autophagy by Eth. Molecular docking suggested that Eth bound to CIP2A. The results of DARTS assay further proved the above binding effect. Eth has potential drug-like activity. The above results demonstrated that Eth inhibited the proliferation, induced the apoptosis, and activated the autophagy of SGC7901/DDP cells by targeting CIP2A and then down-regulating PP2A/mTORC1 signaling pathway. This study provided a new target for the treatment of cisplatin-resistant gastric cancer.
Humans ; Cisplatin/therapeutic use* ; Caspase 9/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Caspase 3/metabolism* ; Stomach Neoplasms/metabolism* ; Drug Resistance, Neoplasm ; Antineoplastic Agents/therapeutic use* ; Molecular Docking Simulation ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Autophagy ; Apoptosis ; Cell Proliferation ; Apoptosis Regulatory Proteins/metabolism* ; Mechanistic Target of Rapamycin Complex 1/metabolism* ; Cell Line, Tumor

To investigate the inhibitory effects and mechanism of Cistanche tubulosa ethanol extract( CTEE) against oxygen-glucose deprivation/reperfusion( OGD/R)-induced PC12 cells neuronal injury. In this study,OGD/R-induced PC12 cells were used to explore the neuroprotective effects of CTEE( 12. 5,25,50 mg·L-1) by detecting cell viability with MTT assay,apoptosis with AO/EB and Hoechst 33258,mitochondrial membrane potential changes with JC-1 staining,mitochondrial oxidative stress with MitoSOX staining,as well as the apoptosis-related protein expression( PARP,cleaved PARP,caspase-3,cleaved caspase-3,Bax,Bcl-2) with Western blot. RESULTS:: showed that CTEE effectively protected OGD/R-induced neuronal injury and increased the survival rate of PC12 cells.AO/EB and Hoechst 33258 staining showed that CTEE could effectively inhibit apoptosis. Moreover,JC-1 and MitoSOX staining results showed that CTEE decreased mitochondrial stress and mitochondrial membrane potential imbalance in PC12 cells in a concentration-dependent manner. Meanwhile,CTEE could obviously suppress the activation of key proteins in mitochondrial apoptosis pathway such as caspase-3 and PARP,and significantly inhibit the rise of Bax and down-regulation of Bcl-2. In conclusion,CTEE has obvious protective effects on OGD/R-induced PC12 cells neuronal injury,potentially via inhibiting mitochondrial oxidative stress and apoptosis-related signaling pathway.
Animals ; Apoptosis ; Caspase 3 ; metabolism ; Cistanche ; chemistry ; Ethanol ; Glucose ; Neuroprotective Agents ; pharmacology ; Oxidative Stress ; Oxygen ; PC12 Cells ; Plant Extracts ; pharmacology ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; bcl-2-Associated X Protein ; metabolism

Viperin is an IFN-stimulated gene (ISG)-encoded protein that was identified in human primary macrophages treated with IFN-γ and in human primary fibroblasts infected with cytomegalovirus (CMV). This protein plays multiple roles in various cell types. It inhibits viral replication, mediates signaling pathways, and regulates cellular metabolism. Recent studies have shown that viperin inhibits IFN expression in macrophages, while it enhances TLR7 and TLR9-mediated IFN production in plasmacytoid dendritic cells, suggesting that viperin can play different roles in activation of the same pathway in different cell types. Viperin also controls induction of ISGs in macrophages. However, the effect of viperin on induction of ISGs in cell types other than macrophages is unknown. Here, we show that viperin differentially induces ISGs in 2 distinct cell types, macrophages and fibroblasts isolated from wild type and viperin knockout mice. Unlike in bone marrow-derived macrophages (BMDMs), viperin downregulates the expression levels of ISGs such as bone marrow stromal cell antigen-2, Isg15, Isg54, myxovirus resistance dynamin like GTPase 2, and guanylate binding protein 2 in murine embryonic fibroblasts (MEFs) treated with type I or II IFN. However, viperin upregulates expression of these ISGs in both BMDMs and MEFs stimulated with polyinosinic-polycytidylic acid or CpG DNA and infected with murine CMV. The efficiency of viral entry is inversely proportional to the expression levels of ISGs in both cell types. The data indicate that viperin differentially regulates induction of ISGs in a cell type-dependent manner, which might provide different innate immune responses in distinct cell types against infections.
Animals ; Carrier Proteins ; Cytomegalovirus ; Dendritic Cells ; DNA ; Dynamins ; Fibroblasts ; GTP Phosphohydrolases ; Humans ; Immunity, Innate ; Interferons ; Macrophages ; Mesenchymal Stromal Cells ; Metabolism ; Mice ; Mice, Knockout ; Orthomyxoviridae ; Poly I-C

PURPOSE: Tripartite-motif-containing protein 56 (TRIM56) has been found to exhibit a broad antiviral activity, depending upon E3 ligase activity. Here, we attempted to evaluate the function of TRIM56 in multiple myeloma (MM) and its underlying molecular basis. MATERIALS AND METHODS: TRIM56 expression at the mRNA and protein level was measured by qRT PCR and western blot analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry analysis was performed to investigate the effect of TRIM56 on MM cell proliferation and apoptosis. The concentrations of interferon (IFN)-β, interleukin (IL)-6, and tumor necrosis factor-α in MM cell culture supernatants were detected with respective commercial ELISA kits. Western blot was employed to determine the effect of TRIM56 on toll-like receptor 3 (TLR3)/toll-IL-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) signaling pathway. RESULTS: TRIM56 expression was prominently decreased in MM cells. Poly (dA:dT)-induced TRIM56 overexpression in U266 cells suppressed proliferation, induced apoptosis, and enhanced inflammatory cytokine production, while TRIM56 knockdown improved growth, diminished apoptosis, and inhibited inflammatory cytokine secretion in RPMI8226 cells. Moreover, TRIM56 knockdown blocked TLR3 signaling pathway. Furthermore, poly (I:C), a TLR3 agonist, markedly abolished TRIM56 depletion-induced increase of proliferation, decrease of apoptosis, and reduction of inflammatory factor in MM cells. CONCLUSION: TRIM56 may act as a tumor suppressor in MM through activation of TLR3/TRIF signaling pathway, contributing to a better understanding of the molecular mechanism of TRIM56 involvement in MM pathogenesis and providing a promising therapy strategy for patients with MM.
Adaptor Proteins, Vesicular Transport/metabolism ; Apoptosis/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cytokines/secretion ; Disease Progression ; Down-Regulation/drug effects ; Gene Knockdown Techniques ; Humans ; Multiple Myeloma/metabolism ; Multiple Myeloma/pathology ; Poly I-C/pharmacology ; Signal Transduction/drug effects ; Toll-Like Receptor 3/metabolism ; Tripartite Motif Proteins/deficiency ; Tripartite Motif Proteins/metabolism ; Ubiquitin-Protein Ligases/deficiency ; Ubiquitin-Protein Ligases/metabolism

OBJECTIVE: The fallopian tubes play a critical role in the early events of fertilization. The rapid innate immune defense is an important part of the fallopian tubes. Toll-like receptor 3 (TLR3), as a part of the innate immune system, plays an important role in detecting viral infections. In this basic and experimental study, the effect of sex hormones on the function of TLR3 in the OE-E6/E7 cell line was investigated. METHODS: The functionality of TLR3 in this cell line was evaluated by cytokine measurements (interleukin [IL]-6 and IL-1b) and the effects of sex hormones on TLR3 were tested by an enzyme-linked immunosorbent assay kit. Additionally, TLR3 small interfering RNA (siRNA) and a TLR3 function-blocking antibody were used to confirm our findings. RESULTS: The production of IL-6 significantly increased in the presence of polyinosinic-polycytidylic acid (poly(I:C)) as the TLR3 ligand. Using a TLR3-siRNA-ransfected OE-E6/E7 cell line and function-blocking antibody confirmed that cytokine production was due to TLR3. In addition, 17-β estradiol and progesterone suppressed the production of IL-6 in the presence and absence of poly(I:C). CONCLUSION: These results imply that sex hormones exerted a suppressive effect on the function of TLR3 in the fallopian tube cell line when different concentrations of sex hormones were present. The current results also suggest that estrogen receptor beta and nuclear progesterone receptor B are likely to mediate the hormonal regulation of TLR3, as these two receptors are the main estrogen and progesterone receptors in OE-E6/E7 cell line.
Cell Line ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells* ; Estradiol ; Estrogen Receptor beta ; Estrogens ; Fallopian Tubes* ; Female ; Fertilization ; Gonadal Steroid Hormones* ; Immune System ; Immunity, Innate ; Interleukin-6 ; Poly I-C ; Progesterone ; Receptors, Progesterone ; RNA, Small Interfering ; Toll-Like Receptor 3* ; Toll-Like Receptors*

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*