Mechanism of Qiwei Guibao Granules in treatment of premature ovarian failure based on proteomics / 中国中药杂志

In this study, the underlying mechanism of Qiwei Guibao Granules(QWGB) in the treatment of premature ovarian fai-lure(POF) was explored by the proteomics technique. Firstly, the POF model was induced in mice by intragastric administration of Tripterygium wilfordii glycosides solution at 50 mg·kg~(-1) for 14 days. Ten days prior to the end of the modeling, the estrous cycle of mice was observed every day to evaluate the success of modeling. From the 1st day after modeling, the POF model mice were treated with QWGB by gavage every day and the treatment lasted four weeks. On the 2nd day after the end of the experiment, blood was collected from the eyeballs and the serum was separated by centrifugation. The ovaries and uterus were collected and the adipose tissues were carefully stripped. The organ indexes of the ovaries and uterus of each group were calculated. The serum estrogen(E_2) level of mice in each group was detected by ELISA. Protein samples were extracted from ovarian tissues of mice, and the differential proteins before and after QWGB intervention and before and after modeling were analyzed by quantitative proteomics using tandem mass tags(TMT). As revealed by the analysis of differential proteins, QWGB could regulate 26 differentially expressed proteins related to the POF model induced by T. wilfordii glycosides, including S100A4, STAR, adrenodoxin oxidoreductase, XAF1, and PBXIP1. GO enrichment results showed that the 26 differential proteins were mainly enriched in biological processes and cellular components. The results of KEGG enrichment showed that those differential proteins were involved in signaling pathways such as completion and coalescence cascades, focal adhesion, arginine biosynthesis, and terpenoid backbone biosynthesis. The complement and coalescence cascades signaling pathway was presumably the target pathway of QWGB in the treatment of POF. In this study, the proteomics technique was used to screen the differential proteins of QWGB in the treatment of POF in mice induced by T. wilfordii glycosides, and they were mainly involved in immune regulation, apoptosis regulation, complement and coagulation cascade reactions, cholesterol metabolism, and steroid hormone production, which may be the main mechanisms of QWGB in the treatment of POF.

Clinical significance of XAF1 expression in pancreatic cancer and its relationship with prognosis / 中华胰腺病杂志

Objective To investigate the expression ot XAF1 protein in pancreauc cancer,and its relationship with clinicopathological parameters and prognosis.Methods The tissue microarray was composed of 89 pancreatic cancer and 21 normal pancreatic tissues,and immunohistochemistry was used to examine the expression of XAF1 protein.The correlation between XAF1 expression and clinicopathological parameters was analyzed.The 89 patients were followed,and the survival was shown in Kaplan Meier curve.Cox proportional hazards regression was used to identify risk factors for the survival of pancreatic cancer patients.Results The negative expression rate of XAF1 was 44.9％ (40/89),and it was 9.5％ (2/21) in normal tissue,and the difference between the two groups was statistically significant (P ＜0.05).The expression of XAF1 was negatively associated with tumor staging (P ＜ 0.05),but it was not associated with age,gender,lymph node metastasis,distant metastasis,pathological grading,surgical margins,vascular and neural invasion.Patients with negative expression of XAF1 had much shorter survival than patients with positive expression.Univariate analysis showed that negative expression of XAF1,pathological grading,lymph node metastasis,distant metastasis was associated with the survival (P ＜ 0.05),and the multivariate analysis indicated that negative expression of XAF1 was an independent prognostic factor for survival of pancreatic cancer.Conclusions XAF1 may be involved in the development and growth of pancreatic cancer,and it is related with patient's prognosis.

XAF1 gene mediated by recombinant adenovirus induces apoptosis in pancreatic cell line BxPC-3 / 中华胰腺病杂志

Objective To investigate whether expression of XAF1 mediated by edenovirus vector AdS/F35 could induce apoptosis in pancreatic cancer cell line BxPC-3 and its possible mechanisms.Methods Preconstructed recombinant Ad5/F35-XAF1 virus and negative control Ad5/F35-Null was tranfected into BxPC3:the expression of XAF1 mRNA and protein before and after tranfection was,analyzed by semi-quantitative RT-PCR and Western blot.The expressions of proteins including Caspase-3,PARP,Caspase-8 and bcl-2 were detected by Western blots.Cell apoptosis was assessed by Annexin-v/PI and TUNEL staining.Results After Ad5/F35-XAF1 tranfection,XAF1 mRNA and protein expression significantly increased,and the difference was statistically significant when compared with control group and Ad5/F35-Null group(P＜0.05).The apoptosis rate was(19.90±3.09)%and(9.29±2.13)%,which was significantly different(P＜0.01)from those in the Ad5/F35-Null group[(6.72±0.7)6%,(2.73±0.51)%]or in the control group[(7.22±1.53)%,(1.56±0.47)%].The expression of Caspase-3,PARP and Caspase-8 significantly increased,but the expression of bcl-2 decreased.Conclusions XAF1 plays a major role in the apoptosis of pancreatic cancer that acts thriugh the activation of death receptor pathway and mitochondrial pathway of apoptosis.

Frequent epigenetic inactivation of XAF1 (XIAP-associated factor 1), a candidate tumor suppressor, by aberrant promoter hypermethylation in human cervical carcinomas / 대한산부인과학회지

OBJECTIVE: X-linked inhibitor of apoptosis (XIAP) is the most potent member of IAP family that exerts antiapoptotic effects by interfering with activities of caspases. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. We explore the candidacy of XAF1, Smac/DIABLO and HtrA2 as a tumor suppressor in cervical carcinogenesis and determine the mechanisms of altered XAF1 expression. METHODS: We investigated the expression and mutation status of the genes in 64 cervical cancer tissues, 5 cervical cancer cell lines and 10 normal cervical tissues. RESULTS: XAF1 transcript was not expressed or extremely low levels in 40% (2/5) of cancer cell line and in 31% (20/64) of primary carcinomas whereas Smac/DIABLO and HtrA2 are normally expressed in all cells. As somatic mutations of the gene was not detected, expression of XAF1 transcript was reactivated in all nonexpressor cell lines after 5-aza-2-deoxycytidine treatment. Bisulfite DNA analysis for CpG sites in the promoter region revealed a strong association between CpG sites hypermethylation and gene silencing. CONCLUSION: XAF1 undergoes epigenetic silencing in a considerable proportion of cervical carcinomas by aberrant promoter hypermethylation rather than genetic alterations, and closely associated with reduced gene expression. Although additional studies are required to determine the biological significance of XAF1 inactivation, it will be valuable to examine the expression status of XAF1 could be a clinically useful marker for cancer treatment.

Frequent Epigenetic Inactivation of XAF1 by Promotor Hypermethylation in Human Colon Cancers / 대한소화기학회지

BACKGROUND/AIMS: X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family that exerts antiapoptotic effects. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. We explored the candidacy of XAF1, Smac/DIABLO and HtrA2 as a tumor suppressor in colonic carcinogenesis. METHODS: Expression and mutation status of the genes in 10 colorectal carcinoma cell lines and 40 primary tumors were examined by quantitative PCR analysis. RESULTS: XAF1 transcript was not expressed or present at extremely low levels in 60% (6/10) of cancer cell lines whereas Smac/DIABLO and HtrA2 are normally expressed in all cell lines examined. Tumor-specific loss or reduction of XAF1 was also found in 35% (14/40) of matched tissue sets obtained from the same patients. XAF1 transcript was reactivated in all the low expressor cell lines by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Moreover, bisulfite DNA sequencing analysis for 34 CpG sites in the promoter region revealed a strong association between hypermethylation and gene silencing. Restoration of XAF1 expression resulted in enhanced apoptotic response to etoposide and 5-flurouracil, whereas knockdown of XAF1 expression by siRNA transfection significantly inhibited chemotherapeutic drug-induced apoptosis. CONCLUSIONS: XAF1 undergoes epigenetic gene silencing in a considerable proportion of human colon cancers by aberrant promoter hypermethylation, suggesting that XAF1 inactivation might be implicated in colonic tumorigenesis.

Xaf1 regulates TNFR signal pathway and induces apoptosis / 中国病理生理杂志

AIM:Xaf1-Saos inducible cell lines,which contain "gene switch" system were used to detect the effect of Xaf1 on tumor necrosis factor receptor(TNFR) signal pathway and to investigate the mechanism of cooperation between Xaf1 and TNF-? in inducing cell apoptosis.METHODS:Xaf1 on TNFR1 expression was measured by RT-PCR and Western blotting.The effect of NF-?B on Xaf1 induced apoptosis was detected by DNA content flow cytometry after co-transfection.DNA binding activity of NF-?B was identified by gel mobility shift assay and transcription activity of NF-?B was analyzed by luciferase assay and RT-PCR.SAPK/JNK activity was checked by SAPK/JNK assay.RESULTS:Xaf1 did not modulate TNFR1 at protein and mRNA levels.Increased NF-?B activity in cells inhibited Xaf1 induced apoptosis.Expression of Xaf1 impaired modestly TNF-? induced NF-?B DNA binding activation and transcription activation,also modestly reduced SAPK/JNK activity.CONCLUSION:Xaf1 inhibits TNFR signal pathway,partly contributing to cooperation with TNF-? to induce apoptosis.

Identification of a high affinity IRF-1 binding element in XAF1 promoter of tumor suppressing gene / 解放军医学杂志

Objective To identify a high affinity IRF-1 binding element in the translational start site of XAF1 promoter. Methods By the bioinformatics analysis, a putative IFN regulatory factor 1 binding element (IRF-E), named as IRFE-XAF1, was identified from -30nt to -38nt of the XAF1 gene, with 76.2% homogeneity with the synonymous IRF-E sequence. Electrophoresis mobility shift assay (EMSA) was performed to confirm the binding capacity of IRFE-XAF1. Two site-directed mutations were made, one mutation site was outside of the IRF-E region (-28nt) and another located at the center of IRF-E (-34nt). The promoter of XAF1 after mutation was examined, including its binding activity and response to IFN-?. Results It was found by EMSA assay that the doublestranded oligonucleotide DNA probe, containing IRFE-XAF1 and labeled with 32P, may be connected to the nuclear protein, and blocked by the unlabeled synonymous IRF-1 probe (cold probe). The binding capacity of IRF-E was lost after site-directed mutation. The XAF1 promoter containing IRFE-XAF1 site had the priming activity, and could be induced by IFN-?. The priming activity declined markedly after site-directed mutation of IRFE-XAF1, and -34 site mutation completely eliminated the effect of IFN-?. Conclusion A high affinity of IRF-E is found in -30nt to -38nt region upstream of ATG initiator codon of XAF1 gene. The code sequence is -38nt-GAAACGAAA--30nt. The present study suggests that XAF1 is one of the genes with which IFN-? may induce the differentiation of cancer cells.