The action mechanism by which C1q/tumor necrosis factor-related protein-6 alleviates cerebral ischemia/reperfusion injury in diabetic mice

Studies have shown that C1q/tumor necrosis factor-related protein-6 (CTRP6) can alleviate renal ischemia/reperfusion injury in mice. However, its role in the brain remains poorly understood. To investigate the role of CTRP6 in cerebral ischemia/reperfusion injury associated with diabetes mellitus, a diabetes mellitus mouse model of cerebral ischemia/reperfusion injury was established by occlusion of the middle cerebral artery. To overexpress CTRP6 in the brain, an adeno-associated virus carrying CTRP6 was injected into the lateral ventricle. The result was that oxygen injury and inflammation in brain tissue were clearly attenuated, and the number of neurons was greatly reduced. In vitro experiments showed that CTRP6 knockout exacerbated oxidative damage, inflammatory reaction, and apoptosis in cerebral cortical neurons in high glucose hypoxia-simulated diabetic cerebral ischemia/reperfusion injury. CTRP6 overexpression enhanced the sirtuin-1 signaling pathway in diabetic brains after ischemia/reperfusion injury. To investigate the mechanism underlying these effects, we examined mice with depletion of brain tissue-specific sirtuin-1. CTRP6-like protection was achieved by activating the sirtuin-1 signaling pathway. Taken together, these results indicate that CTRP6 likely attenuates cerebral ischemia/reperfusion injury through activation of the sirtuin-1 signaling pathway.

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in glioma U87 cells

Studies have shown that tumor necrosis factor-related apoptosis-inducing ligand（TRAIL）exhibits strong induction of apoptosis in human glioma cells.It remains unclear whether the mitochondrion pathway,an important apoptosis signaling pathway,is involved in TRAIL-induced glioma cell apoptosis.In the present study,in vitro cultured human glioma U87 cells were treated with human recombinant soluble TRAIL.Apoptosis of glioma U87 cells,mitochondrial transmembrane potential（Δψm）,cytoplasmic cytochrome c concentration and changes in caspase-3,-8 and-9 activity following human recombinant soluble TRAIL treatment were investigated to determine the mechanism of glioma U87 cell apoptosis induced by TRAIL.Additionally,blocking caspase-8resulted in TRAIL-induced mitochondrion pathway activation,suggesting that TRAIL,through activating caspase-8,initiated a series of mitochondrial events and resulted in apoptosis of glioma U87 cells.

Polyphyllin Ⅰ enhances tumor necrosis factor-related apoptosis-inducing ligand-induced inhibition of human osteosarcoma cell growth via downregulating the Wnt/β-catenin pathway

OBJECTIVE:To investigate the synergistic effects of polyphyllin Ⅰ(PPⅠ)combined with tumor necrosis factorrelated apoptosis-inducing ligand(TRAIL)on the growth of osteosarcoma cells through downregulating the Wnt/β-catenin signaling pathway.METHODS:Cell viability,apoptosis and cell cycle distribution were examined using cell counting kit-8 and flow cytometry assays.The morphology of cancer cells was observed with inverted phase contrast microscope.The migration and invasion abilities were examined by xCELLigence real time cell analysis DP system and transwell assays.The expressions of poly(adenosine diphosphate-ribose)polymerase,C-Myc,Cyclin B1,cyclin-dependent kinases 1,N-cadherin,Vimentin,Active-β-catenin,β-catenin,p-glycogen synthase kinase 3β(GSK-3β)and GSK-3βwere determined by Western blotting assay.RESULTS:PPⅠ sensitized TRAIL-induced decrease of viability,migration and invasion,as well as increase of apoptosis and cell cycle arrest of MG-63 and U-2 OS osteosarcoma cells.The synergistic effect of PPⅠwith TRAIL in inhibiting the growth of osteosarcoma cells was at least partially realized through the inactivation of Wnt/β-catenin signaling pathway.CONCLUSION:The combination of PPⅠ and TRAIL is potentially a novel treatment strategy of osteosarcoma.

Antitumor effect of tumor necrosis factor-related apoptosis inducing ligand combined with mevastatin on a human glioma cell line SWO-38

BACKGROUND： Previous studies have reported that statins are less toxic to the human body and have greater antitumor activity; however, few studies have addressed the antitumor effect of statins combined with tumor necrosis factor-related apoptosis inducing ligand （TRAIL）. OBJECTIVE： To explore the effect of TRAIL combined with mevastatin on the proliferation and apoptotic cell death of a human glioma cell line SWO-38, and to study its mechanism of action. DESIGN, TIME AND SETTING： An in vitro control experiment was performed at the Central Laboratory of the Third Hospital Affiliated to Sun Yat-sen University, between January and April 2009. MATERIALS： The human SWO-38 cell line was provided by Cell Research, Department of Animal Experimental Center of Sun Yat-sen University; human recombinant soluble TRAIL by R＆D, USA; and mevastatin by Sigma, USA. METHODS： SWO-38 cells were separately incubated in TRAIL （100, 200, 300, 400, and 500 tJg/L） and mevastatin （5, 10, 20, 30, and 40 pmol/L） for 72 hours. In addition, SWO-38 cells were incubated in TRAIL （300 μg/L）, mevastatin （30 μmol/L）, and a solution containing both TRAIL and mevastatin for 12, 24, 48 and 72 hours. MAIN OUTCOME MEASURES： Cell proliferation was detected using methyl thiazolyl tetrazolium assay; cell apoptosis was observed using Hoechst 33258 staining and fluorescence microscopy and was measured using Annexin V/propidium iodide flow cytometry; TRAIL R1/DR4 and TRAIL R2/DR5 protein expressions levels were measured using indirect immunofluorescence staining combined with flow cytometry in the recombinant soluble TRAIL （rsTRAIL, 300 tJg/L）, mevastatin （30 IJmol/L） and combination groups; TRAIL R1/DR4 and TRAIL R2/DR5 mRNA expression was detected using real-time polymerase chain reaction. RESULTS： rsTRAIL, mevastatin and their combination inhibited tumor proliferation in a time- and dose-dependent manner. The proliferation inhibitory rate and apoptosis rate of human SWO-38 cells in the combined group were significantly greater than the rsTRAIL or mevastatin alone group （P 〈 0.01）. TRAIL R1/DR4 and TRAIL R2/DR5 protein and mRNA expressions were increased in the combination group compared with mevastatin or rsTRAIL alone after 72 hours （P 〈 0.01）. CONCLUSION： Both rsTRAIL and mevastatin inhibit the proliferation and apoptosis of the human glioma cell line SWO-38, while their combination enhances the anti-tumor effect. The mechanism of action possibly correlates to the upregulation of TRAIL R1/DR4 and TRAIL R2/DR5 mRNA expression by mevastatin, thereby enhancing the cell sensitivity to rsTRAIL.

TNF related apoptosis-inducing ligand and its receptors in ocular tumors

Most of the ocular tumors have poor prognosis, and they remain a difficult problem in the area of ophthalmology. With the rapid development of molecular biology and immunologic techniques and the deep research on ocular tumor related genes, it becomes possible to diagnose and treat malignant tumors from the molecular level. The tumor necrosis factor related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) super family, is a promising candidate, either alone or in combination with established cancer therapies, since it can initiate apoptosis through the activation of their death receptors. The ability of TRAIL to selectively induce apoptosis of transformed, virus-infected or tumor cells but not normal cells promotes the development of TRAIL-based cancer therapy. Here, we will review TRAIL and its receptors' structure, function, mechanism of action and application in ocular tumors therapy.

Expression of tumor necrosis factor related apoptosis inducing ligand receptor in glioblastoma

BACKGROUND： Receptors for tumor necrosis factor related apoptosis inducing ligand （TRAIL） include death receptor 4, death receptor 5, decoy receptor 1, and decoy receptor 2. Activation of death receptor 4 and 5 selectively kills tumor cells. OBJECTIVE： To detect TRAIL receptor expression in glioblastoma by immunohistochemistry and RT-PCR, and to compare this expression to that in normal brain tissue. DESIGN： Observational analysis. SETTING： Department of Pathology, the First Affiliated Hospital of Zhengzhou University; Henan Tumor Pathology Key Laboratory. PARTICIPANTS： Twenty-five patients （17 males and 8 females） who received glioblastoma resection were selected from the Fifth Affiliated Hospital of Zhengzhou University, between September 2003 to June 2004. All glioblastoma samples were diagnosed pathologically. Twenty patients （12 males and 8 females） with craniocerebral injury who received normal brain tissue resection were selected in the same time period. There were no significant differences in sex and age between glioblastoma patients or between craniocerebral injury patients （P 〉 0.05）. All patients and appropriate relatives provided informed consent, and this study was approved by the local research ethics committee. METHODS： Polyclonal antibody against TRAIL receptors and an immunohistochemical kit （batch number： 200502） were purchased from Boster Company, Wuhan. Immunohistochemistry： Expression of death receptor 4, death receptor 5, decoy receptor l, and decoy receptor 2 were observed in both glioblastoma and normal brain tissue. The experiment was performed according to the kit instructions, and positive staining was brown-yellow. Assessment： There were no positive signals （-）; weakly positive signals, positive cells 〈 25% （＋）; weakly positive signals, positive cells 25%-50% （＋＋）; strongly positive signals, positive cells 50%-75% （＋＋＋）; strongly positive signals, positive cells 〉 75% （＋＋＋＋）. Evaluation： Expression levels of TRAIL receptors were estimated in both normal brain tissue and glioblastoma. Expression of decoy receptor 1 and decoy receptor 2 mRNA in glioblastoma were detected by reverse transcription polymerase chain reaction, and expression of decoy receptor in glioblastoma was estimated. MAIN OUTCOME MEASURES： Comparison of death receptor and decoy receptor protein expression between glioblastoma and normal brain tissue; decoy receptor mRNA expression in glioblastoma. RESULTS： Death receptor protein expression was strongly positive （＋＋＋） in glioblastoma, while it was weakly positive （＋, ＋＋） in normal brain tissue. Therefore, expression rate of death receptor protein in the glioblastoma was significantly higher than that in the normal brain tissue （.~ 2 = 18.48, 23.03, P 〈 0.01）. Decoy receptor protein expression in the glioblastoma was significantly lower than that in the normal brain tissue （ x2 = 6.65, 18.76, P 〈 0.01）. The level of decoy receptor mRNA expression in glioblastoma was significantly higher than those of protein expression （ x 2 = 9.82, 10.09, P〈 0.01）. CONCLUSION： High expression of death receptor and low expression of decoy receptor are frequently observed in glioblastoma, suggesting that TRAIL receptor genes show an anti-tumor and expressive response during the initiation and development of the tumor. There are significant differences in decoy receptor expression between normal brain tissue and glioblastoma, suggesting that the restricted expression of decoy receptor in glioblastoma is regulated at the post-transcriptional level.

TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies

AIM:To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors,in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand(TRAIL),on overcoming TRAIL resistance in hepatocellular carcinoma(HCC)and to study the efficacy of agonistic TRAIL antibodies,as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS:Surface expression of TRAIL receptors (TRAIL-R1-4)and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL were analyzed by flow cytometry and Western blotting,respectively. Knock-down of MCL-1 and BCL-xL was performed by transfecting specific small interfering RNAs.HCC cellswere treated with kinase inhibitors and chemotherapeutic drugs.Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS:TRAIL-R1 and-R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However,treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates.Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002[inhibition of phosphoinositol- 3-kinase(PI3K)],AG1478(epidermal growth factor receptor kinase),PD98059(MEK1),rapamycin(mam- malian target of rapamycin)and the multi-kinase inhibitor Sorafenib.Furthermore,the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL play a major role in TRAIL resistance:knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells.Additionally, knock-down of MCL-1 and BCL-xL led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.CONCLUSION:Our data identify the blockage of survival kinases,combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.

Induction of apoptosis in osteogenic sarcoma cells by combination of tumor necrosis factor-related apoptosis inducing ligand and chemotherapeutic agents

Background Osteosarcoma is one of the most common primary malignant tumors of bone with poor prognosis. TNF-related apoptosis inducing ligand （TRAIL） is a member of the tumor necrosis factor （TNF） cytokine family. TRAIL induces apoptosis in various tumor cell lines but is not found to be cytotoxic to many normal cell types in vitro. We investigated the cytotoxic activity of TRAIL and chemotherapeutic agents, including methotrexate （MTX）, doxorubicin （DOX） and cisplatin （CDDP）, on established osteosarcoma cell line-OS-732. Methods OS-732 cells were incubated with chemotherapeutic agents MTX,DOX and CDDP at various peak plasma concentrations（PPC）, 0.1PPC,1PPC and 10PPC, alone or with 100 ng/ml of TRAIL for 24 hours or 48 hours. MTT was used to evaluate the cytotoxic activity of different agents on OS-732. The apoptosis proportion was assayed by flow cytometry. Cellular morphologic changes were observed by phase contrast microscope, scan electron microscope, and transmission electron microscope. Results The inhibitory rate was （24.438±3.414）% with TRAIL of 100 ng/ml for 24 hours. The cells were responsive to DOX and CDDP with a dose-effect relationship （P〈0.05）. In OS-732 cells, DOX and CDDP cooperated synergistically with TRAIL when incubated the cells with them for 24 hours （the combined inhibitory rate is （58.360±2.146）% and （54.101±-2.721）%, respectively）. TRAIL alone or drugs alone induced the apoptosis rate was less than 25% （P〈0.05）. However, the combination of TRAIL and MTX did not present synergistic effects on OS-732 cells （P〉0.05, compared with TRAIL alone）. Conclusions Osteosarcoma OS-732 cells were not responsive to TRAIL-induced apoptosis. DOX and CDDP sensitize osteosarcoma OS-732 cells to TRAIL-induced apoptosis. The combination of TRAIL and MTX presented no synergistic effects on killing OS-732 cells.

EGFR inhibitors sensitize non-small cell lung cancer cells to TRAIL-induced apoptosis

Apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) can be regulated by the epidermal growth factor(EGF) signaling pathway.In this study,recombinant adenoviral vectors that encode TRAIL gene from the hTERT/RGD promoter(AdTRAIL) was combined with drugs including gefitinib,elotinib,and cetuximab that inhibit EGFR and the EGF signaling pathway in non-small cell lung cancer(NSCLC) cell lines to investigate their antitumor activity.In vitro,compared to single reagent,AdTRAIL combined with EGFR inhibitors reduced proliferation and enhanced apoptosis in H460,A549,and SW1573 cell lines.Western blot results suggested that these effects were relative to up-regulation of pro-apoptosis protein BAX and down-regulation of p-AKT.In vivo,AdTRAIL combined with cetuximab resulted in a significant growth reduction in H460 xenografts without damage to the main organs of nude mice.Histological examination and TUNEL analyses of xenografts showed that cetuximab enhanced cell apoptosis induced by AdTRAIL.These results indicate that EGFR inhibitors enhanced AdTRAIL anti-tumor activity in NSCLC cell lines and that inhibiting the AKT pathway played an important role in this enhancement.

Piperlongumine inhibits cell growth and enhances TRAIL-induced apoptosis in prostate cancer cells

Objective: To investigate whether piperlongumine can sensitize prostate cancer cells to tumor necrosis factor-related apoptosisinducing ligand(TRAIL) and trigger apoptosis in prostate cells. Methods: Human prostate cancer cell lines PC3, LNCa P, and VCa P were cultured with piperlongumine and TRAIL. Then, cell proliferation, migration, caspase activation, apoptotic protein expressions, and death receptor expressions were measured.Results: Piperlongumine inhibited cell proliferation at low doses(<10 μM) alone and in combination with TRAIL(25 ng/m L), induced apoptosis, and suppressed cyclooxygenase activation. Additionally, piperlongumine induced expression of death receptors which potentiated TRAIL-induced apoptosis in cancer cells but did not affect decoy receptors. Piperlongumine also downregulated tumor cell-survival pathways, inhibited colony formation and migration of cancer cells alone or in combination with TRAIL. The combination of piperlongumine with TRAIL was found to be synergistic. Conclusions: Our findings indicate that piperlongumine can sensitize cancer cells to TRAIL through the upregulation of death receptors and can trigger apoptosis with the downregulation of antiapoptotic proteins.

The flavonoid casticin enhances TRAIL-induced apoptosis of colon cancer cells through endoplasmic reticulum stress-mediated up-regulation of DR5

Objective： The aim of this study was to explore the mechanisms by which the flavonoid casticin enhances tumor necrosis factor-related apoptosis-inducing ligand （TRAIL）-induced apoptosis in colon cancer cells. Methods： Human colon cancer HT-29 cells were treated with TRAIL or casticin. Cytotoxicity was examined by MTT assay, and apoptosis determined by morphological observation and flow cytometric analysis. Death receptor 5 （DRS）, DR4, and endoplasmic reticulum （ER） stress response markers, including glucose regulating protein 78 （GRP78）, activating transcription factor 4 （ATF4） and CHOP （CCAAT/enhancer binding protein homologous protein）, were examined with western blot. Small interfering RNA （siRNA） transfection was employed to knock down CHOP. Results： HT-29 cells were resistance to TRAIL-induced apoptosis, but casticin, at subtoxic concentrations, potentiated HT-29 cells to TRAIL-induced apoptosis. Casticin up-regulated the expression of DR5 time-and dose-dependent manners, but had no effect on the expression of DR4. Also, casticin increased the levels of ER stress response markers （GRP78, ATF4 and CHOP） in a similar way to DR5. Knockdown of CHOP by specific siRNA, or salubrinal, an ER stress inhibitor, abolished the up-regulation of DR5 and enhancement of TRAIL-induced apoptosis by casticin. Conclusion： Casticin enhances TRAIL-induced apoptosis of colon cancer cells by ER stress-mediated up-regulation of DR5.

Downregulation of cyclooxygenase-1 is involved in gastric mucosal apoptosis via death signaling in portal hypertensive rats

Portal hypertension （PHT） gastropathy is a frequent complication of fiver cirrhosis and one of the leading causes of death from cirrhosis. Apoptosis is widely considered to be an active energy-dependent mode of cell death and a distinct entity from necrotic cell death. It is unclear whether gastric mucosal apoptosis is involved in PHT gastropa- thy. Prostaglandins （PGs） produced through cyclooxygenase （COX） are thought to play a key role in protection of the gastrointestinal mucosa from injury and apoptosis. However, the role of COX in PHT gastropathy is still not clearly understood. The aims of this study were to investigate whether （1） gastric mucosal apoptosis is involved in PHT gas- tropathy and （2） downregulation of COX contributes to this apoptosis. In this study, we show that gastric mucosal apoptosis was remarkably increased while mucosal proliferation was inhibited in PHT rats. Gastric mucosal COX- 1 was significantly suppressed at both the mRNA and protein levels, and PGE2 was reduced in PHT rats. Further, PGE2 treatment suppressed gastric mucosal apoptosis in PHT rats. However, gastric mucosal COX-2 levels did not differ between sham-operated rats and PHT rats. Gastric mucosal levels of tumor necrosis factor-α （TNF-α） and Fas ligand, but not TNF-related apoptosis-inducing ligand, were increased, and activated caspase-8 and caspase-3 levels were upregulated in PHT rats. The release of cytochrome c from the mitochondria to the cytosol was not observed in PHT rats. Our data indicate that downregulation of COX-1 is involved in gastric mucosal apoptosis via death signal- ing-mediated type-I cell death in PHT rats.

COMBINATION OF γ-INTERFERON WITH TRAIL AND CISPLATIN OR ETOPOSIDE INDUCES APOPTOSIS IN HUMAN NEUROBLASTOMA CELL LINE SH-SY5Y

Objective To study the effect of γ-interferon （IFNγ）, tumor necrosis factor related apoptosis inducing ligand （TRAIL）, and cisplatin or etoposide induced apoptosis in human neuroblastoma cell line SH-SY5Y and its possible molecular mechanisms. Methods The expressions of Caspase 8 mRNA and protein were detected with RT-PCR and Western blot analysis. The effects of IFNγ, TRAIL, IFNγ ＋ TRAIL, IFNγ ＋ Caspase 8 inhibitor ＋ TRAIL, IFNγ ＋ cisplatin ＋ TRAIL, and IFNγ ＋ etoposide ＋ TRAIL on the growth and apoptosis of SH-SY5Y cells were detected with the methods of MTT and flow cytometry. The relative Caspase 8 activity was measured with colorimetric assay. Results Caspase 8 was undetectable in SH-SY5Y cells but an increased expression of Caspase 8 mRNA and protein was found after treatment with IFNγ. SH-SY5Y ceils themselves were not sensitive to TRAIL, but those expressing Caspase 8 after treatment with IFNγ were. The killing effect of TRAIL on SH-SY5Y cells expressing Caspase 8 was depressed by Caspase 8 inhibitor. Cisplatin and etoposide could enhance the sensitivity of TRAIL on SH-SY5Y cells. The relative Caspase 8 activity of SH-SY5Y cells in IFNγ ＋ TRAIL group was significantly higher than those of control group, IFNγ group, TRAIL group, and inhibitor group （ P 〈 0. 01 ）. There was no significant difference among IFNγ ＋ TRAIL group, IFNγ ＋ cisplatin ＋ TRAIL group, and IFNγ ＋ etoposide ＋ TRAIL group. Conclusions IFNγ could sensitize SH-SY5Y cells to TRAIL-induced apoptosis and this may be realized by the up-regulation of Caspase 8. Cisplatin and etoposide could enhance the killing effect of TRAIL on SH-SY5Y cells.

Apoptosis and Expression of Protein TRAIL in Granulosa Cells of Rats with Polycystic Ovarian Syndrome

The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunhistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P<0.01, P<0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P>0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the con- trol rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regu- lating the apoptosis of granulosa cells in PCOS rats.

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.

Combination of oridonin and TRAIL induces apoptosis in uveal melanoma cells by upregulating DR5

AIM:To investigate the inhibitory effect of the combined use of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)and oridonin on choroidal melanoma cell lines,and to explore its underlying mechanism.METHODS:MUM-2B and C918 cells were treated with different concentrations of TRAIL and oridonin,and MTT assay used to evaluate the inhibition rate of the two compounds on cells.Then,the cell cycle distribution and apoptosis were detected by flow cytometry,and changes in apoptosis-related proteins such as death receptor 5(DR5),a-caspase-3,and x-linked inhibitor of apoptosis protein(XIAP)were detected by Western blot.MUM-2B cells were transfected with si-DR5,which interfered with the expression of the DR5 gene.MTT and Western blot assay were used to detect cell activity and apoptosis-related proteins.RESULTS:When TRAIL and oridonin were simultaneously administered to the MUM-2B cells,the apoptosis rate was significantly higher than that by the two drugs individually.However,the effect of combined use of TRAIL and oridonin on C918 cells was not significantly different from that used alone.Cell cycle analysis showed that TRAIL and oridonin could induce G2/M arrest in MUM-2B cells.The Western blot results showed that the protein expression levels of the DR5,a-caspase-3,and BAX increased,while the expression levels of the anti-apoptosis-related proteins XIAP and BCL-2 were suppressed when TRAIL and oridonin simultaneously administered to MUM-2B cells.Interfering the expression of DR5 gene in MUM-2B cells could reverse the inhibitory effect of oridonin and TRAIL on the proliferation and apoptosis induction of MUM-2B cells.CONCLUSION:The inhibitory effects of oridonin and TRAIL on MUM-2B cells are significantly enhanced when they were administered as a combined treatment,which may ascribe to up-regulation of DR5.

Antitumor and radiosensitization effect of 12C6+heavy-ion irradiation mediated by radiation-inducible gene therapy

Radio genetic therapy which combines gene therapy with radiotherapy has shown promising results in cancer treatment. In this study, an oncolytic adenovirusbased gene therapy system regulated by radiation was constructed to improve the cancer curative effect. This gene therapy system incorporated the radiation-inducible early growth response gene(Egr-1) promoter and the anticancer gene tumor necrosis factor-related apoptosis-inducing ligand(TRAIL). To confirm the antitumor effect of Ad-ET combined with^12C^(6+)tion irradiation, the survival and apoptosis fraction of tumor cells HT1080 and normal cells MRC-5 in combination treatment were detected by CCK-8 assay and FACS analysis. Then the expression levels of TRAIL gene and protein were tested by real-time PCR and western blotting. The results show that^12C^(6+)tion irradiation could induce cell growth inhibition and apoptosis by activating the TRAIL gene expression in tumor cells, while exhibiting no obvious toxicity to the normal lung cell line MRC-5. Theresults also demonstrate that use of an oncolytic adenovirusbased radiation-inducible gene therapy system together with^12C^(6+)tion irradiation could cause synergistic antitumor effect specifically in tumor cells but not in normal cells. The results indicate that the novel radio genetic therapy could potentiate radiation treatment by improving the safety and efficiency of monotherapy, and provide theoretical support for clinical application of combination treatment.

注意缺陷多动障碍患儿血清ACE2,TWEAK和CCL5水平检测及诊断价值研究

目的 探究血清血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2),细胞因子肿瘤坏死因子样细胞凋亡弱诱导因子(tumor necrosis factor-like weak inducer of apoptosis,TWEAK)和CC趋化因子配体5(CC motif chemokine ligand 5,CCL5)水平在注意缺陷多动障碍(attention deficit hyperactivity disorder,ADHD)患儿诊断、病情严重程度评估中的价值。方法 选取2022年10月~2023年10月在河北省儿童医院就诊的125例ADHD患儿记为ADHD组,另选105例在河北省妇幼保健中心体检的健康儿童为对照组,根据临床总体印象严重程度量表(CGI-S)将患儿分为轻中度组(n=83)和重度组(n=42)。ELISA方法检测血清ACE2,TWEAK,CCL5,促黄体生成素(LH)和催乳素(PRL)水平,联合型瑞文测验(CRT)和Conners父母症状问卷(PSQ)对患儿认知和行为状况进行评分。Spearman相关性分析重度组血清ACE2,TWEAK,CCL5与CGI-S,CRT,PSQ评分的相关性;受试者工作特征(ROC)曲线分析ACE2,TWEAK,CCL5对ADHD及严重程度的诊断价值。结果 ADHD患儿血清ACE2(284.35±92.34 pg/ml),TWEAK(2.56±0.76 pg/ml)水平低于对照组(379.23±106.28 pg/ml,3.52±1.12 pg/ml),CCL5水平(7.36±2.37ng/ml)高于对照组(5.24±1.63 ng/ml),差异具有统计学意义(t=7.244,7.703,7.753,均P<0.05);重度组患儿CRT,血清ACE2,TWEAK水平低于轻中度组(t=5.318,6.686,6.490),而PSQ,CCL5水平较高于轻中度组(t=5.220,6.134),差异具有统计学意义(均P<0.05);Spearman相关性分析结果显示,重度组患儿血清ACE2,TWEAK水平与CGI-S,PSQ评分负相关(r=-0.432,-0.453;-0.421,-0.426,均P<0.001),与CRT评分呈正相关(r=0.427,0.418,均P<0.001);CCL5与CGI-S,PSQ评分呈正相关(r=0.421,0.433,均P<0.001),与CRT评分呈负相关(r=-0.446,P<0.001)。血清ACE2,TWEAK和CCL5诊断ADHD发生的AUC分别为0.814,0.803和0.807,三者联合诊断的AUC为0.945,优于各自单独诊断(Z=5.439,4.258,5.576,均P<0.001);血清ACE2,TWEAK,CCL5诊断重度ADHD的AUC分别为0.853,0.796和0.805,三者联合诊断的AUC为0.930,优于各自单独诊断(Z=2.604,3.851,3.567,均P<0.001)。结论 血清ACE2,TWEAK在ADHD患儿血清中低表达,而CCL5高表达,三者表达水平具有相关性,并且诊断ADHD发生和严重程度具有较高的价值。

肺源性心脏病并发肺动脉高压患者血清β-NGF和TRAIL水平检测在临床诊断及预后评估中的意义

目的探讨肺源性心脏病(pulmonary heart disease,PHD)并发肺动脉高压(pulmonary heart disease,PAH)患者血清β-神经生长因子(β-nerve growth factor,β-NGF)、肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)表达水平及其在临床诊断及预后评估中的意义。方法采用1∶1病例-对照研究设计选取2019年1月~2022年6月北京市大兴区人民医院86例并发PAH的PHD患者为病例组,86例单纯PHD患者为对照组,进行回顾性分析。将病例组根据肺动脉收缩压(pulmonary arterial systolic pressure,PASP)分为轻度PAH组(n=39)、中度PAH组(n=25)和重度PAH组(n=22),根据出院后一年的结果分为预后良好组(n=75)和预后不良组(n=11)。收集研究对象人口学资料和实验室检查指标,采用酶联免疫吸附(ELISA)法检测血清β-NGF,TRAIL水平。Pearson积矩相关分析β-NGF,TRAIL与PASP的关系,Logistic回归分析PHD患者PAH影响因素,ROC曲线评估β-NGF,TRAIL对PAH的诊断价值,COX比例风险回归分析β-NGF,TRAIL与PHD并发PAH患者预后不良的关系,ROC曲线评估其对预后不良的预测价值。结果与对照组比较,病例组PHD病程长(8.63±1.27年vs 5.49±1.15年),血清β-NGF(26.97±8.25 ng/ml vs 22.14±7.32 ng/ml)和TRAIL(2.83±0.76 ng/ml vs 1.71±0.68 ng/ml)水平升高,差异具有统计学意义(t=17.006,4.064,10.183,均P<0.05)。血清β-NGF,TRAIL对PHD患者PAH有诊断价值,AUC分别为0.842,0.838,二者联合诊断的AUC为0.920,诊断价值高于单一指标(Z=3.416,3.508,均P<0.05)。轻度PAH组、中度PAH组和重度PAH组血清β-NGF(23.26±5.13 ng/ml,27.83±5.57 ng/ml,32.57±6.02 ng/ml),TRAIL(2.24±0.65 ng/ml,2.89±0.71 ng/ml,3.81±0.90 ng/ml)水平依次升高,差异具有统计学意义(F=20.624,31.972,均P<0.05)。病例组血清β-NGF,TRAIL与PASP呈正相关(r=0.673,0.659,均P<0.05)。预后不良组血清β-NGF(36.34±8.05 ng/ml),TRAIL(3.49±1.01 ng/ml)水平高于预后良好组(25.59±7.28 ng/ml,2.73±0.89 ng/ml),差异具有统计学意义(t=4.516,2.604,均P<0.05)。Logistic回归分析结果显示,PHD病程[OR(95%CI):1.784(1.135~2.806)]、β-NGF[OR(95%CI):1.976(1.108~3.523)],TRAIL[OR(95%CI):1.866(1.123~3.101)]是PHD患者发生PAH的独立危险因素(均P<0.05)。多因素COX比例风险回归结果显示,PHD病程[OR(95%CI):1.167(1.082~1.364]、β-NGF[OR(95%CI):1.322(1.134~1.649)],TRAIL[OR(95%CI):1.259(1.087~1.590)]是PHD并发PAH患者预后不良的独立危险因素(均P<0.05)。血清β-NGF,TRAIL可预测PHD并发PAH患者预后不良发生风险,AUC分别为0.863,0.881,二者联合检测的AUC为0.907,诊断价值高于单一指标检测(Z=2.905,3.128,均P<0.05)。结论血清β-NGF和TRAIL升高是PHD患者PAH独立危险因素,并与PAH严重程度有关,早期联合β-NGF和TRAIL检测可提高对PAH的诊断价值及对患者预后不良的预测效果。

血清骨硬化蛋白、OPG、OPG/TRAIL比值对高血压伴慢性心力衰竭患者发生主要不良心血管事件的预测价值

目的探讨血清骨硬化蛋白、骨保护素(OPG)/肿瘤坏死因子相关凋亡诱导配体(TRAIL)比值对高血压伴慢性心力衰竭(CHF)患者发生主要不良心血管事件(MACE)的预测价值。方法选取2019年10月至2022年10月于该院就诊的134例高血压伴CHF患者作为研究对象,根据患者治疗1年内是否发生MACE将其分为MACE组(46例)和无MACE组(88例)。另选取同期于该院进行体检的74例健康者作为对照组。采用酶联免疫吸附实验检测3组血清骨硬化蛋白、OPG和TRAIL水平。收集所有患者的临床资料(包括冠心病史、糖尿病史、收缩压、舒张压)。检测3组血清胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6和IL-10水平。采用Pearson相关分析高血压伴CHF患者血清骨硬化蛋白、OPG和TRAIL水平与相关实验室指标水平的关系。采用多因素Logistic回归分析高血压伴CHF患者发生MACE的危险因素。绘制受试者工作特征(ROC)曲线评估血清骨硬化蛋白、OPG/TRAIL比值单独及2项指标联合检测对MACE的预测价值。结果无MACE组和MACE组血清骨硬化蛋白、OPG水平及OPG/TRAIL比值均明显高于对照组,TRAIL水平明显低于对照组,差异均有统计学意义(P<0.05)。MACE组血清骨硬化蛋白、OPG水平及OPG/TRAIL比值均明显高于无MACE组,TRAIL水平明显低于无MACE组,差异均有统计学意义(P<0.05)。MACE组TNF-α、IL-10、IL-6水平均高于无MACE组,差异均有统计学意义(P<0.05)。MACE组和无MACE组冠心病史和糖尿病史患者比例、收缩压、舒张压以及TC、TG、HDL-C、LDL-C水平比较,差异均无统计学意义(P>0.05)。高血压伴CHF患者血清骨硬化蛋白、OPG水平与TNF-α、IL-10、IL-6水平均呈正相关(P<0.05),TRAIL水平与TNF-α、IL-10、IL-6水平均呈负相关(P<0.05)。多因素Logistic回归分析结果显示,骨硬化蛋白水平升高、OPG水平升高均为高血压伴CHF患者发生MACE的危险因素(P<0.05),而TRAIL水平升高为高血压伴CHF患者发生MACE的保护因素(P<0.05)。血清骨硬化蛋白、OPG/TRAIL比值单独及2项指标联合检测预测高血压伴CHF患者发生MACE的曲线下面积(AUC)分别为0.847、0.803、0.927,且2项指标联合检测的AUC优于血清骨硬化蛋白、OPG/TRAIL比值单独检测的AUC(Z=2.350、2.824,P<0.05)。结论高血压伴CHF患者血清骨硬化蛋白、OPG/TRAIL比值联合检测患者预后发生MACE的预测价值较高。