18­α­glycyrrhetinic acid induces apoptosis in gingival fibroblasts exposed to phenytoin.

Phenytoin (PHT)-induced gingival overgrowth is caused by the increased proliferation and reduced apoptosis of gingival fibroblasts in inflammatory gingiva. Licorice has long been used as a component of therapeutic preparations. It inhibits cell proliferation, induces cell apoptosis and has anti-inflammatory effects. 18-α-glycyrrhetinic acid (18α-GA), the active compound in licorice, promotes apoptosis in various types of cells. The present study determined whether 18α-GA affects apoptosis in gingival fibroblasts exposed to PHT. The present study aimed to establish a basis for the therapeutic application of 18α-GA to treat the gingival overgrowth induced by PHT. Human gingival fibroblasts from healthy donors were cultured to semi-confluence and then stimulated in serum-free DMEM containing PHT with or without 18α-GA for subsequent experiments. Apoptotic cells were detected by ELISA. Analysis of the distribution of cell cycle phases and the apoptotic cell population was performed by flow cytometry. The expression levels of mRNAs and proteins of apoptotic regulators were measured using reverse transcription-quantitative PCR and western blotting, respectively. Caspase (CASP) activities were assessed by an ELISA. Treatment with 18α-GA markedly increased the number of apoptotic cells, reduced BCL2 mRNA expression, increased CASP2 and receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1) domain containing adaptor with death domain, Fas (TNFRSF6)-associated via death domain, RIPK1, tumor necrosis factor receptor superfamily; member 1A, TNF receptor-associated factor 2, CASP2, CASP3 and CASP9 mRNA expression, and also upregulated the protein expression levels and activities of caspase-2, caspase-3 and caspase-9. These results demonstrated that 18α-GA induced apoptosis through the activation of the Fas and TNF pathways in the death receptor signaling pathway in gingival fibroblasts treated with PHT. 18α-GA exhibited therapeutic potential for the treatment of PHT-induced gingival overgrowth.

Regulation of anoikis by extrinsic death receptor pathways.

Metastatic cancer cells can develop anoikis resistance in the absence of substrate attachment and survive to fight tumors. Anoikis is mediated by endogenous mitochondria-dependent and exogenous death receptor pathways, and studies have shown that caspase-8-dependent external pathways appear to be more important than the activity of the intrinsic pathways. This paper reviews the regulation of anoikis by external pathways mediated by death receptors. Different death receptors bind to different ligands to activate downstream caspases. The possible mechanisms of Fas-associated death domain (FADD) recruitment by Fas and TNF receptor 1 associated-death domain (TRADD) recruitment by tumor necrosis factor receptor 1 (TNFR1), and DR4- and DR5-associated FADD to induce downstream caspase activation and regulate anoikis were reviewed. This review highlights the possible mechanism of the death receptor pathway mediation of anoikis and provides new insights and research directions for studying tumor metastasis mechanisms. Video Abstract.

Effects of polyphyllin Ⅵ on the proliferation and apoptosis of glioma cells and potential mechanism / 中国药房

OBJECTIVE To investigate the effects of polyphyllin Ⅵ（PPⅥ） on the proliferation and apoptosis of glioma cells and potential mechanism. METHODS Using human glioma LN229 cells as objects， MTT assay was used to detect the survival rate after treated with different concentrations of PPⅥ [0 （control group）， 1， 2， 4， 8， 16， 32， 64 μmol/L] for different time （24， 48， 72 h）. The clone formation experiments were adopted to detect the number of cell clones and clone formation rate after being treated with different concentrations of PPⅥ [0 （control group）， 2， 4， 8 μmol/L] for 14 days. The flow cytometry and Western blot assay were used to detect the apoptotic rate of cells， the expressions of apoptosis-related protein [B cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， cleaved caspase-3]， and the expressions of related proteins of Fas/Fas ligand （FasL） death receptor pathway and protein kinase B （Akt）/glycogen synthesis kinase-3β （GSK-3β） pathway after being treated with different concentrations of PPⅥ [0（control group）， 4， 8 μmol/L] for 24 h. RESULTS Compared with the control group， the survival rate of cells， the number of clones and clone formation rate， the protein expression of Bcl-2， and the phosphorylation levels of Akt and GSK-3β protein were decreased significantly in different concentration groups of PPⅥ （P＜0.05 or P＜0.01）. The apoptotic rate， the protein expressions of Bax， cleaved caspase-3， Fas， FasL and cleaved caspase-8 were increased significantly （P＜0.05 or P＜ 0.01）. CONCLUSIONS PPⅥ can inhibit the proliferation and induce the apoptosis of human glioma LN229 cells， which may be related to the activation of the Fas/FasL death receptor pathway and the inhibition of the Akt/GSK-3β pathway.

TNF-α-mediated podocyte injury via the apoptotic death receptor pathway in a mouse model of IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide and it is characterized by mesangial IgA deposits. Proteinuria is a common clinical feature of IgAN, which has a critical connection to podocyte injury and has been used as a clinical prognostic factor for IgAN. Evidence has shown that TNF-α released from mesangial cells may lead to podocyte apoptosis. METHODS: Forty male BALB/c mouse were randomly divided into the control group and IgAN group. A mice model of IgAN was developed by oral administration of bovine serum albumin (BSA) combined with Staphylococcus Enterotoxin B (SEB) tail vein injection. Urinary protein concentrations, renal function, renal morphological, IgA deposition, apoptosis situation, and the mRNA and protein expression of nephrin, podocin, TNF-α, TNFR1, caspase-8 and caspase-3, were detected after 12 weeks. RESULTS: BSA and SEB can successfully establish an IgAN mouse model, and the main pathological changes are the IgA immune complex deposition in the mesangial area. The gene and protein expression levels of nephrin and podocin were found to be downregulated, and death receptor pathway-related indicators were upregulated, and they were involved in TNF-α-activated podocyte injury and apoptosis in IgAN mice. CONCLUSION: TNF-α may play an important role in the pathogenesis of podocyte apoptosis in IgAN, and its effects may be mediated through the apoptotic death receptor pathway.

Inhibitory regulation of purple sweet potato polysaccharide on the hepatotoxicity of tri-(2,3-dibromopropyl) isocyanate.

Tri-(2,3-dibromopropyl) isocyanate (TBC), a new emerged persistent organic pollutant, is widely used in fields of flame retardant, textile, rubber and plastic with strong hepatotoxicity. Purple Sweet Potato Polysaccharide (PSPP) has antioxidant and hepatoprotective effects. This study aims to answer the scientific question whether PSPP has a protective effect on TBC induced liver injury. The effect of PSPP on the apoptosis of HepG2 cells was detected by MTT assay, the morphological changes were observed by morphological observation, and the apoptosis rate was determined by flow cytometry. The apoptotic genes were detected by qPCR assay, the relevant protein express was detected by western blot. The correlation between proteins and genes in the apoptosis pathway of HepG2 cells was calculated. To further reveal the apoptosis mechanism of TBC hepatotoxicity in vivo, 19 target genes and 14 apoptotic related proteins of inhibiting apoptosis via death receptor and mitochondria were discussed, all the above results proved that PSPP had protective effect on liver injury induced by TBC. This study not only provided a scientific basis for clarifying the mechanism of TBC hepatotoxicity and the protective effect of PSPP, but also generated the new point and method in terms of the prevention in advance and early intervention of diseases caused by environmental pollution.

Targeted therapy for intervertebral disc degeneration: inhibiting apoptosis is a promising treatment strategy.

Intervertebral disc (IVD) degeneration (IDD) is a multifactorial pathological process associated with low back pain (LBP). The pathogenesis is complicated, and the main pathological changes are IVD cell apoptosis and extracellular matrix (ECM) degradation. Apoptotic cell loss leads to ECM degradation, which plays an essential role in IDD pathogenesis. Apoptosis regulation may be a potential attractive therapeutic strategy for IDD. Previous studies have shown that IVD cell apoptosis is mainly induced by the death receptor pathway, mitochondrial pathway, and endoplasmic reticulum stress (ERS) pathway. This article mainly summarizes the factors that induce IDD and apoptosis, the relationship between the three apoptotic pathways and IDD, and potential therapeutic strategies. Preliminary animal and cell experiments show that targeting apoptotic pathway genes or drug inhibition can effectively inhibit IVD cell apoptosis and slow IDD progression. Targeted apoptotic pathway inhibition may be an effective strategy to alleviate IDD at the gene level. This manuscript provides new insights and ideas for IDD therapy.

Ginsenoside Rb1 protects from Staphylococcus aureus-induced oxidative damage and apoptosis through endoplasmic reticulum-stress and death receptor-mediated pathways.

Acute lung injury (ALI) is acute uncontrolled inflammation of lung tissue that leads to high fatality both in human and animals. Staphylococcus aureus (S. aureus) could be an opportunistic, versatile bacterial etiology of ALI. Ginsenoside Rb1 (Rb1) is extracted from the Panax ginseng, which displays a wide range of biological and pharmacological effects. However, protective effects of Rb1 in S. aureus-induced ALI though endoplasmic reticulum (ER) stress and death receptor-mediated pathways have not yet been reported. Therefore, present study was planned with the aims to investigate the antioxidant and anti-apoptotic properties of Rb1 through regulation of ER stress as well as death receptor-mediated pathways in ALI induced by S. aureus in mice. In this study, four groups of healthy Kunming mice (n = 48) were used. The S. aureus (80 µl; 1 ×107 CFU/10 µl) was administered intranasally to establish mice model of ALI. After 24 h of onset of S. aureus-induced ALI, the mice were injected thrice with Rb1 (40 mg/kg) intraperitoneally six hours apart. Histopathology, enzyme linked immunosorbent assay (ELISA), real time quantitative polymerase chain reaction (RT-qPCR), Immunohistochemistry and western blotting assay were employed in the current study. Our results suggested that Rb1 administration save lungs from pulmonary injury by reducing wet to dry (W/D) ratio, protein levels, total cells, neutrophilic count, reactive oxygen species (ROS), myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx)1 depletion. Meanwhile, Rb1 therapy ameliorated histopathology alteration of lung tissue and pro-inflammatory cytokines secretion. The gene expression of ER stress marker (PERK, AFT-6, IRE1 and CHOP) were upregulated markedly (P < .05) in S. aureus-instilled groups, which was reduced by Rb1 administration that is reveled from the result findings of the RT-qPCR and immunoblot assay. The results of immunohistochemistry for CHOP indicated the increased expression in S. aureus groups which in turn ameliorated by Rb1 treatment. The mRNA expression demonstrated that death receptor-associated genes (FasL, Fas, FADD and caspase-8) showed up-regulation in S. aureus group. The similar findings were observed for the protein expression of caspase-8, FADD and Fas. Rb1 treatment markedly (P < .05) reversed protein and mRNA expression levels of these death receptor-associated genes when compared to the S. aureus group. Taken together, Rb1 attenuated S. aureus-induced oxidative damage via the ER stress-mediated pathway and apoptosis through death receptor-mediated pathway. Conclusively, our findings provide an insight into preventive mechanism of Rb1 in ALI caused by S. aureus and hence proven a scientific baseline for the therapeutic application of Rb1.

Protective effect of Lycium barbarum polysaccharide on reproductive system injury of female rats induced by 2,4-dichlorophenoxyacetic acid / 环境与职业医学

Background 2,4-Dichlorophenoxyacetic acid (2,4-D) is widely used as a broad-leaved herbicide and plant growth regulator. Related studies have shown that 2,4-D has neurotoxicity, ability to disrupt endocrine function, genotoxicity, carcinogenicity, and reproductive toxicity. Objective This experiment is conducted to investigate the effect of 2,4-D exposure on reproductive system of female rats, and to preliminarily explore the potential ameliorative effect of Lycium barbarum polysaccharide (LBP) and its possible mechanism. Methods Twenty-four SPF female SD rats with six rats in each group were randomly divided into a blank control group (deionized water 1 mL·d−1), an exposure group (75 mg·kg−1 2,4-D), an LBP control group (50 mg·kg−1 LBP), and an LBP intervention group (75 mg·kg−1 2,4-D + 50 mg·kg−1 LBP). The rats were given intragastric administration once a day for 28 consecutive days. Body weight was measured every two days. After exposure, ovary and uterus were weighed and organ coefficients were calculated; the pathological changes of ovary and uterus were detected by hematoxylin-eosin staining (HE); the level of estradiol (E2) in serum was detected by ELISA; the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in serum were measured by corresponding kits; the apoptosis of ovarian and uterine cells was detected by TUNEL fluorescence staining; and the protein expression levels of Fas, FasL, FADD, Pro-Caspase-8, Cleaved-Caspase-8, Pro-Caspase-3, and Cleaved-Caspase-3 in ovarian tissues were detected by Western blotting. Results Compared with the blank control group, the ovarian structure of the exposure group was abnormal, the number of follicles at different developmental stages decreased, morphological changes were observed, and the number of atretic follicles increased; the endometrium was incomplete, with different degrees of nuclear pseudostratification and decreased number of glands in lamina propria. Compared with the exposure group, the ovarian structure of the LBP intervention group was complete, and the follicles at different developmental stages increased in amount, remained intact, and were arranged closely; the uterine structure was relatively intact, showing decreased endometrial loss and nuclear pseudostratification. There were significant differences in the levels of SOD, GSH-Px, E2, and MDA among the four groups (F=86.1, 26.2, 43.3, and 22.3, all P<0.01). Compared with the blank control group, the levels of serum SOD, GSH-Px, and E2 decreased in the exposure group (P<0.01), while the concentration of MDA increased (P<0.01). Compared with the exposure group, the levels of serum SOD, GSH-Px, and E2 in the LBP intervention group increased (P<0.01), and the concentration of MDA decreased (P<0.01). There were significant differences in the apoptosis rates of ovarian and uterine cells among the four groups (F=64.8, 55.5, both P<0.01). Compared with the blank control group, the apoptosis rates of ovarian and uterine cells increased in the exposure group (P<0.01). Compared with the exposure group, the apoptosis rates of ovarian and uterine cells decreased in the LBP intervention group (P<0.01). There were significant differences in the expression levels of death receptor pathway-related proteins in ovarian tissues among the four groups (all P<0.05). Compared with the blank control group, the expression levels of Fas, FasL, FADD, Pro-Caspase-8, Cleaved-Caspase-8, Pro-caspase-3, and Cleaved-Caspase-3 were increased in the exposure group (P<0.05 or 0.01). Compared with the exposure group, the expression levels of above proteins were decreased in the LBP intervention group (P<0.05 or 0.01). Conclusion The study findings reveal that 2,4-D can induce oxidative stress and further mediate Fas-FasL pathway to induce apoptosis, resulting in reproductive system damage in female rats. LBP can reduce the oxidative stress level, down-regulate the expression of Fas-FasL pathway-related proteins, and reduce the apoptosis of germ cells, therefore protecting reproductive system of female rats.

Antitumor effects of curcumin on the proliferation, migration and apoptosis of human colorectal carcinoma HCT­116 cells.

Curcumin is the main component of the Chinese herbal plant turmeric, which has been demonstrated to possess antitumor and other pharmacological properties. The aim of the present study was to investigate the effects of curcumin on the viability, migration and apoptosis of human colorectal carcinoma HCT­116 cells, and to explore the underlying molecular mechanisms. In addition, it was investigated whether the antitumor effect of curcumin on HCT­116 cells could match that of the chemotherapeutic drug 5­fluorouracil (5­FU). HCT­116 cells were treated with curcumin (10, 20 and 30 µM) and 5­FU (500 µM), and cell viability and proliferation were detected by Cell Counting Kit­8 and colony formation assays, respectively. The migration and invasion of treated cells were determined using Transwell and carboxyfluorescein succinimidyl amino ester fluorescent labeling assays. Cell cycle distribution and apoptosis rates were detected by flow cytometry. Furthermore, cell morphology changes associated with apoptosis were observed by fluorescence microscopy with acridine orange/ethidium bromide dual staining. To investigate the possible underlying molecular mechanisms, the gene and protein levels of Fas, Fas­associated via death domain (FADD), caspase­8, caspase­3, matrix metalloproteinase (MMP)­9, nuclear factor (NF)­κB, E­cadherin and claudin­3 were detected using quantitative PCR analysis, zymography and western blotting. The results revealed that curcumin markedly inhibited the viability and proliferation of HCT­116 cells in a dose­ and time­dependent manner. The migration, aggregation and invasion of HCT­116 cells into the lungs of mice were decreased by curcumin treatment in a dose­dependent manner. S­phase arrest and gradually increased apoptotic rates of HCT­116 cells were observed with increasing curcumin concentrations. Additionally, the mRNA and protein levels of apoptosis­associated proteins (Fas, FADD, caspase­8 and caspase­3) and E­cadherin in HCT­116 cells were upregulated following treatment with curcumin in a dose­dependent manner. By contrast, the expression of migration­associated proteins, including MMP­9, NF­κB and claudin­3, was downregulated with increasing curcumin concentrations. These data suggested that the inhibitory effect of curcumin on HCT­116 cells may match that of 5­FU. Therefore, curcumin induced cell apoptosis and inhibited tumor cell metastasis by regulating the NF­κB signaling pathway, and its therapeutic effect may be comparable to that of 5­FU.

The preparation of a cold-water soluble polysaccharide from Grifola frondosa and its inhibitory effects on MKN-45 cells.

In this study, a novel water soluble polysaccharide (named GFP-4) was extracted from Grifola frondosa at 4 oC, and its preliminary structure and inhibitory effects on human gastric carcinoma MKN-45 cells through the Fas/FasL death receptor apoptosis pathway were investigated. High-performance gel permeation chromatography (HPGPC), fourier-transform infrared spectroscopy (FT-IR), and ion chromatography (IC) results showed that GFP-4 was a 1.09 × 106 Da neutral hetero polysaccharide with pyranose rings, and α- and ß-type glycosidic linkages that contained galactose, glucose, and mannose at a molar ratio of 1.00:3.45:1.19. MTT results indicated that GFP-4 significantly inhibited the proliferation of MKN-45 cells in a concentration-dependent manner. The H&E staining and Hoechst 33342/PI double staining results showed that GFP-4-treated MKN-45 cells were subjected to underwent typical apoptotic morphologic changes such as nuclear pyknosis, chromatin condensation, and an increase of membrane permeability. Annexin V-FITC/PI double staining, cell cycle analysis, and western blot results revealed the GFP-4 induced MKN-45 cells apoptosis through the Fas/FasL-mediated death receptor pathway with cells arrested at the G0/G1 phase. These data indicate that GFP-4 is a promising candidate for treating gastric cancer and provide a theoretical basis for the future development and utilization of G. frondosa clinically.

Apoptotic effects of rhein through the mitochondrial pathways, two death receptor pathways, and reducing autophagy in human liver L02 cells.

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is a major component of many medicinal herbs such as Rheum palmatum L. and Polygonum multiflorum. Despite being widely used, intoxication cases associated with rhein-containing herbs are often reported. Currently, there are no available reports addressing the effects of rhein on apoptosis in human liver L02 cells. Thus, the aim of this study is to determine the cytotoxic effects and the underlying mechanism of rhein on human normal liver L02 cells. In the present study, the methyl thiazolyl tetrazolium assay demonstrated that rhein decreased the viability of L02 cells in dose-dependent and time-dependent ways. Rhein was found to trigger apoptosis in L02 cells as shown by Annexin V-fluoresceine isothiocyanate (FITC) apoptosis detection kit and cell mitochondrial membrane potential (MMP) assay, with nuclear morphological changes demonstrated by Hoechst 33258 staining. Detection of intracellular superoxide dismutase activity, lipid oxidation (malondialdehyde) content, and reactive oxygen species (ROS) levels showed that apoptosis was associated with oxidative stress. Moreover, it was observed that the mechanism implicated in rhein-induced apoptosis was presumably via the death receptor pathway and the mitochondrial pathway, as illustrated by upregulation of TNF-α, TNFR1, TRADD, and cleaved caspase-3, and downregulation of procaspase-8, and it is suggested that rhein may increase hepatocyte apoptosis by activating the increase of TNF-α level. Meanwhile, rhein upregulates the expression of Bax and downregulates the expression of procaspase-9 and -3, and it is suggested that the mitochondrial pathway is activated and rhein-induced apoptosis may be involved. In addition, we also want to explore whether rhein-induced apoptosis is related to the autophagic changes induced by rhein. The results showed that rhein treatment increased P62 and decreased LC3-II and beclin-1, which means that autophagy was weakened. The results of our studies indicated that rhein induced caspase-dependent apoptosis via both the Fas death pathway and the mitochondrial pathway by generating ROS, and meanwhile the autophagy tended to weaken.

Signaling pathways involved in regulating apoptosis induction in host cells upon PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of porcine reproductive and respiratory syndrome (PRRS), a devastating disease of swine that poses a serious threat to the swine industry worldwide. The induction of apoptosis in host cells is suggested to be the key cellular mechanism that contributes to the pathogenesis of PRRS. Various signaling pathways have been identified to be involved in regulating PRRSV-induced apoptosis. In this review, we summarize the potential signaling pathways that contribute to PRRSV-induced apoptosis, and propose the issues that need to be addressed in future studies for a better understanding of the molecular basis underlying the pathogenesis of PRRS.

Diterpene pekinenal from euphorbia pekinensis radix induced IEC-6 cells apoptosis mediated by mitochondria and death receptors.

Pekinenal, a diterpenoid from the roots of Euphorbia pekinensis Rupr., can cause serious intestinal toxicity. However, its toxic mechanism hasn't been comprehensively understood. This present study aims to clarify its toxic effects and investigate the potential mechanism. In vitro effects of pekinenal on cell proliferation, cell cycle and apoptosis were examined by performing experiments on rat intestinal crypt epithelial cells (IEC-6). Proteins and enzymes involved in cell apoptotic pathways were detected by Western blot and enzyme-linked immunosorbent assay (ELISA), and related mRNAs were detected by RT-PCR. The results showed that the cell cycle was arrested in G0/G1 phase, and apoptotic morphology changes in pekinenal-treated cells. Furthermore, pekinenal up-regulated the expression level of apoptotic protein including Bax, AIF, Apaf-1 and the expression level of mRNA such as Fas, FasL, TNFR1 and NF-κB, while down-regulated the expression level of Bcl-2, ultimately triggering the apoptosis of caspase dependence. In conclusion, the above data confirmed that pekinenal inhibited the proliferation of IEC-6 cells and induced cells apoptosis by modulating mitochondrial and death receptor pathways.

Licochalcone A from licorice root, an inhibitor of human hepatoma cell growth via induction of cell apoptosis and cell cycle arrest.

We investigated the anti-cancer activity of Licochalcone A (LCA), extracted from licorice root. LCA inhibited the proliferation of HepG2 cells with IC50 (65.96 µM) for 24 h and IC50 (44.13 µM) for 48 h and caused significant morphological changes and also led to intracellular ROS generation. LCA affected HepG2 cell growth by terminating cell cycle development at G2/M transition and further induced the apoptosis process. The mRNA expression of genes involved in cell cycles such as Survivin, Cyclin B1, and CDK1 were reduced; while, Weel, P21, Cyclin D1, and JNK1 showed increased mRNA expression. Two pathways consisting of internal and external factors were responsible for LCA -induced apoptosis. The anti-cancer action involved increased mRNA expression of DR3, DR5, caspases-3, caspases-8, caspases-10, Fas, Bad, Bax, Bcl-2, Bak, and PUMA; besides, decreased level of PKCε, p70S6K, and Akt. This study provides mechanistic explanation for anti-cancer activity of LCA and also suggests its potential role in the treatment of hepatoma cancer.

Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma.

Pomegranate (Punica granatum L.) fruit has been demonstrated to have the inhibitory activities to various tumors. In this study, we try to uncover the molecular mechanism underlying the inhibitory capability of Taiwanese local pomegranate fruit to urinary bladder urothelial carcinoma. The results collected from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated that the ethanol extract of pomegranate peel exhibited better inhibitory activity to human urinary bladder urothelial carcinoma T24 and J82 cells than that of pulp. Furthermore, the ethylacetate layer of peel ethanol extract was observed to have the best inhibitory activity against urinary bladder urothelial carcinoma cells. One of the eight fractions (PEPE2 fraction) collected from the ethylacetate layer with Diaion HP-20 column chromatography demonstrated the highest inhibitory activity in urinary bladder urothelial carcinoma cells. The results of the flow cytometry and apoptotic pathway studies suggested that the inhibitory activity of PEPE2 fraction were attributed to the UBUC cell apoptosis. To confirm the above results, our results of xenograft-induced bladder tumor in nude mice showed that the oral consumption of the ethylacetate layer (2, 5, 10 and 100 mg/kg) could decrease the volume and weight of T24 tumors and caused the apoptosis in the xenografted tumors, which was observed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. This study provided the likelihood that the traditionally non-edible pomegranate peel waste is re-utilized to make an affordable and promising chemopreventive product to prevent UBUC incidence or recurrence.

Molecular mechanism and therapy application of necrosis during myocardial injury.

Necrosis is an ancient topic which gains new attraction in the research area these years. There is no doubt that some necrosis can be regulated by genetic manipulation other than an accidental cell death resulting from physical or chemical stimuli. Recent advances in the molecular mechanism underlying the programmed necrosis show a fine regulation network which indicates new therapy targets in human diseases. Heart diseases seriously endanger our health and have high fatality rates in the patients. Cell death of cardiac myocytes is believed to be critical in the pathogenesis of heart diseases. Although necrosis is likely to play a more important role in cardiac cell death than apoptosis, apoptosis has been paid much attention in the past 30 years because it used to be considered as the only form of programmed cell death. However, recent findings of programmed necrosis and the related signalling pathways have broadened our horizon in the field of programmed cell death and promote new pharmacological application in the treatment of heart diseases. In this review, we summarize the advanced progress in these signalling pathways and discuss the pathos-physiological relevance and therapeutic implication of targeting necrosis in heart diseases treatment.

Hypoxia-induced apoptosis of mouse spermatocytes is mediated by HIF-1α through a death receptor pathway and a mitochondrial pathway.

Hypoxia in vivo induces oligozoospermia, azoospermia, and degeneration of the germinal epithelium, but the underlying molecular mechanism of this induction is not fully clarified. The aim of this study was to investigate the role of the death receptor pathway and the mitochondrial pathway in hypoxia-induced apoptosis of mouse GC-2spd (GC-2) cells and the relationship between HIF-1α and apoptosis of GC-2 cells induced by hypoxia. GC-2 cells were subjected to 1% oxygen for 48 hr. Apoptosis was detected by flow cytometry, TUNEL staining, LDH, caspase-3/8/9 in the absence and presence of HIF-1α siRNA. The protein levels of apoptosis-related markers were determined by Western blot in the presence and absence of HIF-1α siRNA. Mitochondrial transmembrane potential change was observed by in situ JC-1 staining. Cell viability was assessed upon treatment of caspase-8 and 9 inhibitors. The results indicated that hypoxia at 1% oxygen for 48 hr induced apoptosis of GC-2 cells. A prolonged exposure of GC-2 cells to hypoxic conditions caused downregulation of c-FLIP, Dc R2 and Bcl-2 and upregulation of DR5 , TRAIL, Fas, p53, and Bax, with an overproduction of caspase-3/8/9. Moreover, hypoxia at this level had an effect on mitochondrial depolarization. In addition, specific inhibitors of caspase-8/9 partially suppressed hypoxia-induced GC-2 cell apoptosis, and the anti-apoptotic effects of the caspase inhibitors were additive. Of note, HIF-1α knockdown attenuated hypoxia and induced apoptosis of GC-2 cells. In conclusion, our data suggest that the death receptor pathway and mitochondrial pathway, which are likely mediated by HIF-1α, contribute to hypoxia-induced GC-2 cell apoptosis.

[Mechanisms of the three pathways regulating the apoptosis of testicular germ cells].

Cell apoptosis is an active process of the self-destruction of biological cells, which is an important mechanism of multicellular organisms regulating the development of the body, controlling cell senescence, and maintaining the stability of the internal and external environment of the body. As a basic biological process of keeping the balance in the body, cell apoptosis also plays an important role in cell differentiation and sperm maturation and survival in the testis. In spermatogenesis, any change may affect cell apoptosis and consequently upset the balance between the survival and death of germ cells. Current studies have shown that the mitochondrial pathway, cell death receptor pathway and endoplasmic reticulum pathway are essential for the apoptosis of germ cells in the testis and the factors in the three pathways interact with one another, forming a complex network for the regulation of cell apoptosis. This review focuses on the mechanisms of the three pathways regulating the apoptosis of testicular germ cells.

Xanthotoxin induces apoptosis in SGC-7901 cells through death receptor pathway / 中草药·英文版

Objective: To investigate the effects of xanthotoxin from Apiaceae medicinal plants on cell proliferation and apoptosis, and explore its mechanism of action against human gastric carcinoma SGC-7901 cells in vitro. Methods: SGC-7901, HepG-2, MCF-7, and A549 cells were treated with different concentrations of xanthotoxin (10, 20, 60, 80, 100, 120, 140, and 160 µg/mL) for 48 h, and the cell viability (IC50) was determined by MTT assay; Xanthotoxin-induced apoptosis in cells was observed by using Hoechst 33258 Staining Kit and Annexin V-FITC Apoptosis Detection Kit; Flow cytometry was used to detect apoptosis related proteins of Fas/FasL, Bid, and DR5/TRAIL proteins in human gastric carcinoma SGC-7901 cells after being treated by xanthotoxin; The influence of xanthotoxin on Caspase-8 protein expression in the cells was determined by Flouormetric Assay Kit. Results: Xanthotoxin obviously inhibited SGC-7901, HepG-2, MCF-7, and A549 cells proliferation, and its inhibition was in a concentration-dependent manner; flow cytometry results showed that in a certain concentration range, xanthotoxin can increase the expression levels of Fas/FasL and DR5/TRAIL proteins in a concentration-dependence manner. The content of Bid protein in cells was increased, and it showed concentration-dependence. Conclusion: Xanthotoxin may induce SGC-7901 cells apoptosis in a certain concentration range through the Fas/FasL protein mediated death receptor pathway, or by DR5/TRAIL mediated death receptor pathway, and increase the expression level of death receptor protein, activation Caspase-8, activating downstream effect factor, inducing cell apoptosis, or activate Caspase-8 cutting activate protein Bid, and then enter the mitochondrial pathway, induction of apoptosis.

Study on apoptosis effect of human breast cancer cell MCF-7 induced by lycorine hydrochloride via death receptor pathway.

As research was conducted on the early apoptosis of human breast cancer cell MCF-7 caused by lycorine hydrochloride and the expression of the related apoptosis proteins. The early-period apoptosis rate of human breast cancer cell MCF-7 was tested with the AnnexinV/PI double staining and flow cytometry. The Western Blotting method was also used to detect the protein expression conditions of Fas, FasL, Caspase-8 and Bid. The results showed that the higher the dose, the higher the rate of apoptosis and that the rate of apoptosis was dependent on the dose; the relative protein activity of Fas, FasL, Caspase-8 and bid gradually rose with the increase of lycorine dosage and the activities revealed certain dose-independence. Results showed that lycorine hydrochloride could induce the apoptosis of human breast cancer cell MCF-7 through the death receptor pathway.