Role of P2X_7 receptors in neuronal death in the retina

Acknowledgments： I would like to express my appreciation to Professor Puro DG for leading me to this research topic during my stay as a research fellow in his laboratory at the University of Michigan in 2001, and also to Professor Ikeda T forgiving me the opportunity to study abroad and then to continue to investigate this topic in the Department of Ophthalmology at Osaka Medical College, lapan.

Immune-related adverse events induced by programmed death protein-1 inhibitors from the perspective of lymphoma immunotherapy

Lymphoma,which is highly malignant,stems from lymph nodes and lymphoid tissue.Lymphoma cells express programmed death-ligand 1/2(PD-L1/PD-L2),which binds with programmed cell death 1 protein(PD-1)to establish inhibitory signaling that impedes the normal function of T cells and allows tumor cells to escape immune system surveillance.Recently,immune checkpoint inhibitor immunotherapies such as PD-1 inhibitors(nivolumab and pembrolizumab)have been introduced into the lymphoma treatment algorithm and have shown remarkable clinical efficacy and greatly improve prognosis in lymphoma patients.Accordingly,the number of lymphoma patients who are seeking treatment with PD-1 inhibitors is growing annually,which results in an increasing number of patients developing immune-related adverse events(irAEs).The occurrence of irAEs inevitably affects the benefits provided by immunotherapy,particularly when PD-1 inhibitors are applied.However,the mechanisms and characteristics of irAEs induced by PD-1 inhibitors in lymphoma need further investigation.This review article summarizes the latest research advances in irAEs during treatment of lymphoma with PD-1 inhibitors.A comprehensive understanding of irAEs incurred in immunotherapy can help to achieve better efficacy with PD-1 inhibitors in lymphoma.

Programmed death 1 and programmed death ligand 1 expressions in patients with chronic hepatitis B

BACKGROUND: The role of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) in persistent HBV infection is controversial. Increasing PD-1 and PD-L1 expression has been found in hepatitis B patients during immune clearance phase, but not in HBV-tolerant patients. We investigated PD-1 and PD-L1 expression and inflammation in chronic hepatitis B. METHODS: Twenty patients with chronic hepatitis B participated in this study. Fifteen patients were in the immune clearance phase, and 5 were in the immune inactive phase. Circulating HBV-specific T cells were analyzed by flow cytometric detection of major histocompatibility complex (MHC) class I peptide complexes, known as pentamers. Intra-hepatic PD-1 and PD-L1 expressions were analyzed by immunostaining. RESULTS: The frequency of pentamers, including core 18-27 (1.88%±0.36%), env 335-343 (1.85%±0.37%), and pol 575-583 (1.56%±0.29%) was 8.30-, 7.71- and 8.48-fold greater during immune clearance phase than those during the immune inactive phase. In addition, more than 70% of circulating pentamers were PD-1 positive. During immune clearance phase, the numbers of intra-hepatic PD-1 and PD-L1 positive cells were 108±23/HPF and 97±20/HPF respectively, in contrast, there was a paucity of PD-1 and PD-L1 positive cells in the immune inactive phase. The numbers of intra-hepatic PD-1 and PD-L1 positive cells were positively correlated with serum alanine aminotransferase and the number of intra-hepatic CD8 + T cells. Immunofluorescence showed that almost all of the intra- hepatic CD8 + T cells were PD-1 and CCR6 positive. These cells aggregated around macrophage inflammatory protein-3 alpha (MIP3α) positive cells and mixed with PD-L1 positive cells.CONCLUSIONS: PD-1 and PD-L1 expressions were significantly correlated with inflammation. CCR6 and PD-1 co-expressed in the same cells; these cells were increased both in circulation and the inflamed liver and aggregated around MIP3α positive cells. The mixture of CCR6 and PD-1, MIP3α and PD-L1 positive cells created immune response compartments which played an important role in specific immune response in HBV immune clearance.

Tamoxifen Induces Apoptosis of Mouse Microglia Cell Line BV-2 Cells via both Mitochondrial and Death Receptor Pathways

Little is known about whether tamoxifen (TAM) can affect resting state microglia apoptosis and about the cellular mechanism that may account for this. To explore this question, we incubated the microglia cell line BV-2 cells with TAM at different concentrations. Cell viability was assessed by the MTT assay, and flow cytometric analysis was performed to detect the cell apoptosis rate. Furthermore, mitochondrial membrane potential (Δψm) was tested by flow cytometry, and Bax, Bcl-2, Fas, and Fas-L expression was detected by Western blot. The results demonstrated that TAM decreased cell viability and induced apoptosis of BV-2 cells in a concentration- and time-dependent manner. In addition, disruption of Δψm was followed by up-regulated expression of pro-apoptotic Bax, Fas and Fas-L, and down-regulated expression of anti-apoptotic Bcl-2. These results indicate that TAM may induce apoptosis of BV-2 cells through both mitochondria- and death receptor-mediated pathways.

Antihepatoma peptide,scolopentide,derived from the centipede scolopendra subspinipes mutilans

BACKGROUND Centipedes have been used to treat tumors for hundreds of years in China.However,current studies focus on antimicrobial and anticoagulation agents rather than tumors.The molecular identities of antihepatoma bioactive components in centipedes have not yet been extensively investigated.It is a challenge to isolate and characterize the effective components of centipedes due to limited peptide purification technologies for animal-derived medicines.AIM To purify,characterize,and synthesize the bioactive components with the strongest antihepatoma activity from centipedes and determine the antihepatoma mechanism.METHODS An antihepatoma peptide(scolopentide)was isolated and identified from the centipede scolopendra subspinipes mutilans using a combination of enzymatic hydrolysis,a Sephadex G-25 column,and two steps of high-performance liquid chromatography(HPLC).Additionally,the CCK8 assay was used to select the extracted fraction with the strongest antihepatoma activity.The molecular weight of the extracted scolopentide was characterized by quadrupole time of flight mass spectrometry(QTOF MS),and the sequence was matched by using the Mascot search engine.Based on the sequence and molecular weight,scolopentide was synthesized using solid-phase peptide synthesis methods.The synthetic scolopentide was confirmed by MS and HPLC.The antineoplastic effect of extracted scolopentide was confirmed by CCK8 assay and morphological changes again in vitro.The antihepatoma effect of synthetic scolopentide was assessed by the CCK8 assay and Hoechst staining in vitro and tumor volume and tumor weight in vivo.In the tumor xenograft experiments,qualified model mice(male 5-week-old BALB/c nude mice)were randomly divided into 2 groups(n=6):The scolopentide group(0.15 mL/d,via intraperitoneal injection of synthetic scolopentide,500 mg/kg/d)and the vehicle group(0.15 mL/d,via intraperitoneal injection of normal saline).The mice were euthanized by cervical dislocation after 14 d of continuous treatment.Mechanistically,flow cytometry was conducted to evaluate the apoptosis rate of HepG2 cells after treatment with extracted scolopentide in vitro.A Hoechst staining assay was also used to observe apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro.CCK8 assays and morphological changes were used to compare the cytotoxicity of synthetic scolopentide to liver cancer cells and normal liver cells in vitro.Molecular docking was performed to clarify whether scolopentide tightly bound to death receptor 4(DR4)and DR5.qRT-PCR was used to measure the mRNA expression of DR4,DR5,fas-associated death domain protein(FADD),Caspase-8,Caspase-3,cytochrome c(Cyto-C),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),x-chromosome linked inhibitor-of-apoptosis protein and Cellular fas-associated death domain-like interleukin-1βconverting enzyme inhibitory protein in hepatocarcinoma subcutaneous xenograft tumors from mice.Western blot assays were used to measure the protein expression of DR4,DR5,FADD,Caspase-8,Caspase-3,and Cyto-C in the tumor tissues.The reactive oxygen species(ROS)of tumor tissues were tested.RESULTS In the process of purification,characterization and synthesis of scolopentide,the optimal enzymatic hydrolysis conditions(extract ratio:5.86%,IC_(50):0.310 mg/mL)were as follows:Trypsin at 0.1 g(300 U/g,centipede-trypsin ratio of 20:1),enzymolysis temperature of 46°C,and enzymolysis time of 4 h,which was superior to freeze-thawing with liquid nitrogen(IC_(50):3.07 mg/mL).A peptide with the strongest antihepatoma activity(scolopentide)was further purified through a Sephadex G-25 column(obtained A2)and two steps of HPLC(obtained B5 and C3).The molecular weight of the extracted scolopentide was 1018.997 Da,and the peptide sequence was RAQNHYCK,as characterized by QTOF MS and Mascot.Scolopentide was synthesized in vitro with a qualified molecular weight(1018.8 Da)and purity(98.014%),which was characterized by MS and HPLC.Extracted scolopentide still had an antineoplastic effect in vitro,which inhibited the proliferation of Eca-109(IC_(50):76.27μg/mL),HepG2(IC_(50):22.06μg/mL),and A549(IC_(50):35.13μg/mL)cells,especially HepG2 cells.Synthetic scolopentide inhibited the proliferation of HepG2 cells(treated 6,12,and 24 h)in a concentration-dependent manner in vitro,and the inhibitory effects were the strongest at 12 h(IC_(50):208.11μg/mL).Synthetic scolopentide also inhibited the tumor volume(Vehicle vs Scolopentide,P=0.0003)and weight(Vehicle vs Scolopentide,P=0.0022)in the tumor xenograft experiment.Mechanistically,flow cytometry suggested that the apoptosis ratios of HepG2 cells after treatment with extracted scolopentide were 5.01%(0μg/mL),12.13%(10μg/mL),16.52%(20μg/mL),and 23.20%(40μg/mL).Hoechst staining revealed apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro.The CCK8 assay and morphological changes indicated that synthetic scolopentide was cytotoxic and was significantly stronger in HepG2 cells than in L02 cells.Molecular docking suggested that scolopentide tightly bound to DR4 and DR5,and the binding free energies were-10.4 kcal/mol and-7.1 kcal/mol,respectively.In subcutaneous xenograft tumors from mice,quantitative real-time polymerase chain reaction and western blotting suggested that scolopentide activated DR4 and DR5 and induced apoptosis in SMMC-7721 Liver cancer cells by promoting the expression of FADD,caspase-8 and caspase-3 through a mitochondria-independent pathway.CONCLUSION Scolopentide,an antihepatoma peptide purified from centipedes,may inspire new antihepatoma agents.Scolopentide activates DR4 and DR5 and induces apoptosis in liver cancer cells through a mitochondria-independent pathway.

Environmental hypoxia induces apoptosis in large yellow croaker Larimichthys crocea via both intrinsic and extrinsic pathways

Hypoxia has become an unfavorable factor affecting the sustainable development of the large yellow croaker Larimichthys crocea,an economically important mariculture fish in China.Apoptosis is a consequence of hypoxia on fish.However,the effects of hypoxia stress on apoptosis in L.crocea remain largely unknown.We investigated the effect of environmental hypoxia on apoptosis in L.crocea.Results show that hypoxia induced apoptosis in L.crocea both in vivo and in vitro.The mitochondrial membrane potential was significantly reduced in large yellow croaker fry(LYCF)cells.The expression levels of Bcell lymphoma/leukemia-2(Bcl-2)m RNA and protein were also significantly decreased in the liver and LYCF cells during 96 h and 48 h of hypoxia stress,respectively,whereas the expression level of Bcl-2 associated X(Bax)mRNA,Casp3 mRNA,and activity of caspase-3/7/9 were significantly increased,indicating that hypoxia induced caspase-dependent intrinsic apoptosis in L.crocea.The expression level of the apoptosis-inducing factor(AIF)protein was significantly increased in the liver and LYCF cells.The level of AIF protein was significantly decreased in the cytoplasm but increased in the nuclei of L.crocea,demonstrating that hypoxia induced the AIF-mediated caspase-independent intrinsic apoptosis.In addition,the activity of caspase-8 was significantly increased,indicating that hypoxia stress induced extrinsic apoptosis in L.crocea.Therefore,hypoxia induced apoptosis in L.crocea through both the intrinsic and extrinsic pathways.The present study accumulated basic biological information to help elucidate the mechanism of hypoxia response in marine fish.

Executionary pathway for apoptosis:lessons from mutant mice

Apoptosis or programmed cell death (PCD) is an evolutionarily conserved cellular process that is essential for normal development and homeostasis of multicellular organisms. Defects in the apoptosis signaling result in many diseases including autoimmune diseases and cancer. The apoptosis signaling pathway was first described genetically in the nematode Caenorhabditis elegans which serves as a framework for the more complex apoptotic pathways that exist in mammals. In this review, we will discuss the apoptotic pathways that are emerging in mammals as elucidated by studies of gene-targeted mutant mice.

Strategies for improving the efflcacy of immunotherapy in hepatocellular carcinoma

Primary liver cancer,mainly hepatocellular carcinoma(HCC),is the sixth most diagnosed cancer and third leading cause of cancer-related death globally.Recently,immunotherapies such as immune checkpoint inhibitors(ICIs)have made great progress in the systemic treatment of HCC.However,anti-PD-1 therapy with pembrolizumab or nivolumab as a single agent did not meet their predefined end points of overall survival in the KEYNOTE-240 and Check Mate 459 trials.It is urgent to understand the immunological rationale and explore novel ways to improve the efflcacy of immunotherapy.The combination of ICIs with other therapies,such as tyrosine kinase inhibitors(TKIs),monoclonal antibodies,or local therapy,has been demonstrated to improve overall response rate and survival.In addition,modulating tumor microenvironment is a potential way to overcome the primary and secondary resistance to immunotherapies.In this review,we summarized the latest findings in the immune microenvironment,the mechanisms of their synergistic effects when combined with anti-VEGF agents or TKIs,as well as other kinds of immune treatment.

Mechanisms involved in the cytotoxic effects of berberine on human colon cancer HCT-8 cells

Berberine,a constituent of some traditional Chinese medicinal plants,has been reported to have cytotoxicity effects on different human cancer cell lines.There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8.In this paper,the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay,fluorescence microscopy and flow cytometry analysis.Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose-and time-dependent manner.Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining.The concentrations of lactate dehydrogenase and both acid and alkaline phos-phatases were significantly increased in cell supernatants after berberine treatment,suggesting cell death.Furthermore,flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner.To further investigate the apoptotic molecular mechanism,reverse transcription-poly-merase chain reaction(RT-PCR)and western blotting methods were used.The up-regulated mRNA and/or protein expressions of Fas,FasL,TNF-α,caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine.Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis.We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin(PHB),and decreased vimentin expression.These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.

CANSTATIN, A ENDOGENOUS INHIBITOR OF ANGIOGENESIS AND TUMOR GROWTH

Canstatin is a novel inhibitor of angiogenesis and tumor growth, derived from the C-terminal globular non-collageneous (NCl) domain of the a2 chain of type IV collagen. It inhibits endothelial cell proliferation and migration in a dose-dependent manner, and induces endothelial cell apoptosis. In vivo experiments show that canstatin significantly inhibits solid tumor growth. The canstatin mediated inhibition of tumor is related to apoptosis. Canstatin- induced apoptosis is associated with phosphatidylinositol 3-kinase/Akt inhibition and is dependend upon signaling events transduced trough membrane death receptor.

EGCG Enhances TRAIL-mediated Apoptosis in Human Melanoma A375 Cell Line

Tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） is a promising anti-cancer agent. Epigallocatechin-3-gallate （EGCG） is a polyphenolic constituent of green tea. In this study, inhibitory effect of combined use of EGCG and TRAIL on human melanoma A375 cells was examined and the possible mechanism investigated. The cells were divided into 4 groups： control group, EGCG group （EGCG： 10, 20 μg/mL）, TRAIL group （TRAIL： 25 ng/mL） and EGCG＋TRAIL group （combined group）. The growth inhibition was measured in the A375 cells treated with different concentrations of TRAIL （（25, 50, 75, 100, 125, 150 ng/mL） by MTT assay. The apoptosis was assessed by flow cytometry. The expressions of DR4 and DR5 were detected by flow cytometry and western blotting. The activities of caspase-8 and caspase-3 were determined by colorimetric assay. The results showed that TRAIL could dose-dependently inhibit the growth of A375 cells and the IC50 of TRAIL was 150 ng/mL. The apoptosis rate was 11.8% in the TRAIL group, 5%–7% in the EGCG group and 48.9%–59.1% in the combined group. Significant difference was found in the apoptosis rate between the combined group and the EGCG or TRAIL group （P〈0.05 for each）. The expression of DR4 instead of DR5 was significantly increased in the EGCG group. The activity of caspase-3 rather than caspase-8 was substantially enhanced in the EGCG group. These results suggest that EGCG is useful for the TRAIL-based treatment for melanoma.

Mechanisms adopted by cancer cells to escape apoptosis–A review

Inactivation of apoptosis is the prime phenomenon in cancer development and cancer treatments.Mutations in the apoptotic pathway not only exert resistance to apoptosis and provide a survival advantage to cancer cells but also confer resistance to cancer therapies.Escaping apoptosis is the“hallmark”of cancer cells.Cancer cells can withstand many apoptotic stimuli,such as DNA damage,unfavorable environments,and cytotoxic therapies.Substantial research has been carried out and is in good progress on the various mechanisms adopted by cancer cells to evade apoptosis.This article reviews the apoptosis escape mechanisms by cancer cells,viz.apoptotic gene alterations(in few essential and accessory apoptotic genes),post-translational modifications(phosphorylation and ubiquitination of apoptotic proteins),metabolic alterations,mitochondrial alterations,immunity escape,epigenetics,cancer cell dormancy,cancer clonal theory,and reversibility of apoptosis.The review reveals that there is a wide scope for further research to address the various challenges in realizing successful cancer therapies that involve reversing the apoptotic resistance and/or inducing apoptosis in tumor cells.

Immunotherapy:A new standard in the treatment of metastatic clear cell renal cell carcinoma

Renal cell cancer(RCC)represents 2%-3% of all adulthood cancers and is the most common malignant neoplasm of the kidney(90%).In the mid-nineties of the last century,the standard of treatment for patients with metastatic RCC was cytokines.Sunititib and pazopanib were registered in 2007 and 2009,respectively,and have since been the standard first-line treatment for metastatic clear cell RCC(mccRCC).Renal cell cancer is a highly immunogenic tumor with tumor infiltrating cells,including CD8+T lymphocytes,dendritic cells,natural killer cells(NK)and macrophages.This observation led to the design of new clinical trials in which patients were treated with immunotherapy.With the growing evidence that proangiogenic factors can have immunomodulatory effects on the host’s immune system,the idea of combining angiogenic drugs with immunotherapy has emerged,and new clinical trials have been designed.In the last few years,several therapeutic options have been approved[immunotherapy and immunotherapy/tyrosine kinase inhibitors(TKI)]for the first-line treatment of mccRCC.Nivolumab/ipilimumab is approved for the treatment of patients with intermediate and poor prognoses.Several checkpoint inhibitors(pembrolizumab,nivolumab,avelumab)in combination with TKI(axitinib,lenvatinib,cabozantinib)are approved for the treatment of patients regardless of their International mRCC Database Consortium prognostic group and PD-L1 expression.There is no specific and ideal biomarker that could help in selecting the ideal patient for the appropriate first-line treatment.

Targeting androgen receptor and trail： a novel treatment paradigm for breast cancer

OBJECTIVE TNF-related apoptosis-inducing ligand(TRAIL)is a promising cancer therapeutic agent due to its minimal toxicity to normal tissues and remarkable apoptotic activity in tumors.However,most breast cancer cells are resistant to TRAIL-induced apoptosis.Our objectives are to investigate the underlying molecular mechanisms and to develop strategies to overcome such resistance.METHODS To identify modulators of TRAIL-induced apoptosis,we carried out a genome wide si RNA screen.To validate the screening result,we either silenced or overexpressed the identified genes in various breast cancer cells and changes in growth and TRAIL-induced cell apoptosis were determined in vitro and in an orthotopic xenograft mouse model.Finally,we investigated whether small molecules targeting the identified genes improve the effectiveness of TRAIL-therapy.RESULTS We unexpectedly identified androgen receptor(AR)to be responsible for TRAIL resistance.While AR is classically viewed as the key factor in prostate cancer progression,we found that AR expression levels were markedly elevated in human invasive breast cancer specimens including triple-negative breast cancers(TNBC)that are highly aggressive with poor prognosis.Importantly,breast cancer cell lines express different levels of AR that correlated with their TRAIL resistance.AR overexpression in MDA-MB-231 and MDA-MB-436 cells suppressed the TRAIL sensitivity whereas knockdown of AR rendered MCF-7 and MDA-MB-453 cells sensitive to TRAIL-induced apoptosis.AR overexpression also induced TRAIL resistance in breast tumors in vivo.Further,we observed an upregulation of the TRAIL receptor,death receptor 5(DR5)in breast cancer cells,following the removal or inhibition of AR by its antagonists Casodex and MDV3100.Treatment with AR antagonists also enhanced TRAIL-induced breast cancer cell apoptosis.CONCLUSION AR signaling suppresses TRAIL-induced breast cancer cell apoptosis,in part,by suppressing DR5 expression,and a combination of AR antagonists together with TRAIL may be a novel and effective therapy for TNBC.

Prognostic role of plasma levels ofγ-glutamyl transpeptidase in patients with advanced gastric cancer treated with anti-PD-1 immunotherapy

Objective Antibodies targeting programmed cell death protein 1(PD-1)have become the mainstay of treatment for chemotherapy-refractory gastric cancer,characterized by high levels of programmed cell death ligand-1(PDL-1)expression.However,the routine clinical implementation of PDL-1 testing is currently limited by the lack of robust detection methods.In this regard,the role of plasmaγ-glutamyl transpeptidase(GGT),an N-terminal nucleophilic hydrolase,as an independent predictor of the efficacy of anti-PD-1 therapy remains unknown.In this study,we aimed to assessed the prognostic role of changes in plasma GGT levels(6 weeks vs.baseline)in patients with advanced gastric cancer treated with anti-PD-1 immunotherapy.Methods We retrospectively analyzed data from 57 patients with gastric cancer treated with anti-PD-1 antibodies(camrelizumab,sintilimab,nivolumab,tislelizumab,and toripalimab)at the Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan,China,from July 2018 to February 2021.Results We found that after 6 weeks of treatment,there were significant differences between responders and non-responders with respect to plasma GGT levels(P<0.001).Multivariate logistic regression analysis revealed that the continuous value of the 6-week difference in GGT levels(OR=1.437,95%CI=1.116-1.849,P=0.005)and 6-week difference in GGT≥0 or<0(OR=53.675,95%CI=6.379-451.669,P<0.001)were independent predictors of disease control.Survival analysis indicated that a reduction in plasma GGT6 levels during treatment was significantly associated with a favorable progression-free survival(PFS)and overall survival(P<0.001).Consistently,univariate and multivariate Cox regression analyses revealed that a reduction in plasma GGT6 levels during treatment was an independent predictor of PFS(HR=1.033,95%CI=1.013-1.053,P=0.001).Conclusion Alterations in plasma GGT levels during treatment can be used as a predictor of disease progression and survival in patients with advanced gastric cancer undergoing treatment with anti-PD-1 antibodies.

Xanthotoxin induces apoptosis in SGC-7901 cells through death receptor pathway

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.

Induction of Apoptosis in Human Hep3B Hepatoma Cells by Norcantharidin through a p53 Independent Pathway via TRAIL/DR5 Signal Transduction

Objective： To investigate the inhibitory activities of norcantharidin （NCTD）, a demethylated analogue of cantharidin, on Hep3B cells （a human hepatoma cell line） with deficiency of p53. Methods： The survival rate of the Hep3B cells after treating with NCTD was measured by MTT assay. Cell cycle of treated cells was analyzed by flow cytometry, and DNA fragmentation was observed by electrophoresis. The influence of inhibitors for various caspases and anti-death receptors antibodies on the NCTD-induced apoptosis in the cells was determined. Results： NCTD treatment resulted in growth inhibition of Hep3B cells in a dose- and time-dependent manner. Cell cycle analysis of the cells after treatment with NCTD for 48 h shows that NCTD induced G2M phase arrest occursat low concentration （≤25 μ mol/L） but G0G1 phase arrest at high concentration （50 μ mol/L）. The addition of both caspase-3 and caspase-10 inhibitors completely inhibited DNA fragmentation. Addition of anti-TRAIL/DR5 antibody significantly inhibited DNA fragmentation. Conclusion： NCTD may inhibit the proliferation of Hep3B cells by arresting cell cycle at G2M or G0G1 phase, and induce cells apoptosis via TRAIL/DR5 signal transduction through activation of caspase-3 and caspase-10 by a p53-independent pathway,

Iso-suillin from Suillus flavus Induces Apoptosis in Human Small Cell Lung Cancer H446 Cell Line

Background： The suillin isoform iso-suillin is a natural substance isolated from a petroleum ether extract of the fruiting bodies of the mushroom Suillus.flavus. Previous studies have found its inhibition effect on some cancer cells, and we aimed to study its effects on human small cell lung cancer H446 cell line. Methods： Cell viability was measured by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide assay. Cellular morphological changes （apoptosis and necrosis） were evaluated using an electron microscope and Hoechst 33258 staining detected by the inverted microscope. Flow cytometry was used to detect cell apoptosis, cell cycle distribution, and mitochondrial membrane potential. Protein expression was determined by Western blotting analysis. Results： Here, we describe the ability of iso-suillin to inhibit the growth of H446 cells in time- and dose-dependent way. Iso-suillin had no obvious impact on normal human lymphocyte proliferation at low concentrations （9.09, 18.17, or 36.35 μmol/L） but promoted lymphocyte proliferation at a high concentration （72.70 μmol/L）. After treatment of different concentrations of iso-suillin （6.82, 13.63, or 20.45 μmol/L）, the apoptosis rate of H446 cells increased with increasing concentrations of iso-suillin （16.70%, 35.54%, and 49.20%, respectively, all P 〈 0.05 compared with the control）, and the expression of related apoptotic proteins in the mitochondrial pathway including cytochrome c and caspase-9 were up-regulated compared with the control （all P 〈 0.05）. On the contrary, Bcl-2/Bax ratio was down-regulated compared with the control. Besides, the expression of pro-apoptotic proteins in the death receptor apoptosis pathway, including Fas-associating protein with a novel death domain and caspase-8, and the expression of caspase-3, a downstream regulatory protein of apoptosis, were also increased compared with the control （all P 〈 0.05）. lnhibitors of caspase-9 and caspase-8 reversed the apoptosis process in H446 cells to varying degrees. Conclusions： These results suggest that iso-suillin could induce H446 cell apoptosis through the mitochondrial pathway and the death-receptor pathway. Therefore, iso-suillin might have a potential application as a novel drug for lung cancer treatment.

Characterization of a Novel Anti-DR5 Monoclonal Antibody WD1 with the Potential to Induce Tumor Cell Apoptosis

TNF-related apoptosis-inducing ligand （TRAIL） is a TNF family member capable of inducing apoptosis. Death receptor 5 （DR 5） is a key receptor of TRAIL and plays an important role in TRAIL-induced apoptosis. To prepare monoclonal antibodies （mAbs） against DR5, cDNA encoding soluble DR5 （sDR5） was firstly amplified by reverse transcriptase-polymerase chain reaction （RT-PCR） with specific primers, and then inserted into a prokaryotic expression vector pET-30a. The recombinant plasmid was expressed in Escherichia coli strain BL21 （DE3）, and sDR5 was purified by nickel affinity chromatography. As an antigen, sDR5 was used to immunize mice. Hybridomas secreting antibodies against sDR5 were identified. One positive done was selected to produce antibody, WD1. ELISA and immunofluorescence demonstrated that WD1 could bind recombinant sDR5 and membranebound DR5 （mDR5） on Jurkat and Molt-4 cells. ATPLite assays showed that Jurkat and Molt-4 cells were sensitive to the antibody in a dose dependent manner. The Annexin V/PI assays and Giemsa＇s staining both showed that WD1 could induce Jurkat cell apoptosis efficiently. Transient transfection of 293T cells and indirect immunofluorescence assay demonstrated that mAb （WD1） couldn＇t cross-react with DR4. Our findings indicated that the novel antibody, WD1 could act as a direct agonist, bind DR5 characteristically, and initiate efficient apoptotic signaling and tumor regression. Thus, WD1 would be a leading candidate for potential cancer therapeutics. Cellular ＆ Molecular Immunology.

Induction of Tumor Cell Apoptosis via Fas/DR5

The apoptosis inducing effects on tumor cell lines MGC803, BEL7402 and HL60 by Fas ligand and anti-human DR5 monoclonal antibodies （anti-DR5 mAb） and the underlying mechanism was studied. Fas/DR5 mRNA was detected by RT-PCR. Cytotoxicity exerted by FasL/anti-DR5 mAb on tumor cell lines was measured by MTT assay and the induced apoptosis was determined by agarose gel electrophoresis. Flow cytometry was employed to analyze the mode of cell death. The mRNA expression of DR5 in MGC803 and BEL7402 cells after giving anti-DR5 mAb was up-regulated compared with control group, while it was down-regulated in HL60 cells in the same condition. The mRNA expression of Fas in HL60 was higher after giving FasL compared with control group, while it was lower in MGC803 and BEL7402. MGC803 and BEL7402 were sensitive to anti-DR5 mAb but partially to FasL, and HL60 was sensitive to FasL but less sensitive to anti-DR5 mAb. Apoptosis induced by Fas ligand and anti-DR5 mAb vary among tumor cell lines. The underlying mechanism may be relevant to Fas/DR5 mRNA expression, which was presented as the release of caspase-8 and Bcl-2.