1 案例资料 被检父和孩子母亲为给女儿和儿子办理户籍事宜,前来进行亲子鉴定.分别用FTA卡采集4人的指尖血备检. 分别采用HuaxiaTM Platinum PCR扩增试剂盒(美国ABI公司)和HEALTH Gene STRtyper-21G Plus扩增荧光检测试剂盒(海尔施基因科技公司),采用10μL扩增体系对4人的FTA血卡直接进行短串联重复序列复合PCR扩增,产物用3130XL基因分析仪(美国ABI公司)进行电泳检测及GeneMapper软件判读基因分型.

自噬是细胞内的重要生理机制之一,对细胞来说自噬是一把双刃剑,它曾一度被认为是机体的保护机制,但随着研究的深入,学者发现自噬也可促进肿瘤的生长.由于细胞核在真核生物中的重要性,相关自噬蛋白在核中的大量分布,核自噬在多种疾病的发生发展中起着非常重要的作用.为了全面了解疾病与自噬的关系,人们开始了核自噬方面的探索.本文对近年来核自噬的研究进展进行文献综述,总结了其研究历史和里程碑式的发现,为核自噬的研究提供了新的思路.

Resveratrol is a potential polyphenol drug used in cancer treatment. We examined the relationship between autophagy and apoptosis in RSV-treated non-small lung adenocarcinoma A549 cells. Resveratrol treatment increased autophagy and autophagy-mediated degradation of P62. Immunocytochemistry revealed P62 co-localized with Fas/Cav-1 complexes, known to induce apoptosis. However, siRNA-mediated P62 downregulation enhanced formation of Fas/Cav-1 complexes, suggesting that P62 inhibited Fas/Cav-1 complex formation. Fas/Cav-1 complexes triggered caspase-8 activation and cleavage of Beclin-1, releasing a C-terminal Beclin-1 peptide that translocated to the mitochondria and initiate apoptosis. Inhibition of autophagy by siRNA-mediated repression of Beclin-1 also blocked RSV-induced apoptosis, showing a dependence of apoptosis on autophagy. P62 knockdown by siRNA accelerated the activation of caspase-8 and initiate apoptosis, while Cav-1 knockdown inhibited apoptosis, but increased autophagy. Inhibition of autophagy by 3-MA prevented both P62 degradation and induction of apoptosis, whereas inhibition of apoptosis by z-IETD-FMK or z-DEVD-FMK enhanced both P62 induction and autophagic cell death. In conclusion, P62 links resveratrol-induced autophagy to apoptosis. P62 blocks apoptosis by inhibiting Fas/Cav-1 complex formation, but RSV-induced autophagic degradation of P62 enables formation of Fas/Cav-1 complexes which then activate caspase-8-mediated Beclin-1 cleavage, resulting in translocation of the Beclin-1 C-terminal fragment to the mitochondria to initiate apoptosis.

异常糖基化和细胞内质网应激与肿瘤的发生发展密切相关.证据表明,衣霉素通过表皮生长因子受体信号通路、蛋白激酶B/核转录因子-κB信号通路、信号转导及转录激活因子3、CD44分子等途径,抑制N-糖基化和诱导内质网应激,阻滞肿瘤发展,有可能逆转肿瘤细胞对紫杉醇、曲妥珠单抗、顺铂的耐药性,并增强多种肿瘤的治疗敏感性.衣霉素还可能诱导肿瘤细胞自噬,促进细胞凋亡.研究衣霉素的糖基化抑制和内质网应激的诱导作用,有助于揭示其抗肿瘤特性的潜在机制,为治疗难治性肿瘤提供新思路.本文就衣霉素抗肿瘤耐药的最新研究进展作一综述.

尽管二甲双胍为二型糖尿病的首选药物,可近年来其抗肿瘤作用越来越受到人们的关注,其中二甲双胍诱导细胞自噬尤为重要.二甲双胍通过不同途径诱发线粒体自噬和内质网自噬,清除机体内受损物质,从而达到抑制细胞增殖、侵袭迁移,同时还具有延缓细胞衰老等作用.二甲双胍诱导细胞自噬及其机制的研究具有重要理论价值和潜在的应用前景,二甲双胍有望成为新一代抗肿瘤药物或辅助药物.

AIMS:Complicated mechanisms in cancer cells have been restricting the medicinal value of resveratrol (Res). The mechanisms by which Res exerts its anti-tumor activity in lung cancer cells have diverged among reports in recent years, whether cells choose to undergo autophagic cell death or apoptosis remains controversial. Yet, whether Res-induced autophagic cell death transforms into apoptosis is still unknown, and by which autophagy regulates programmed cell death is still undefined.MAIN METHODS:Here, A549 cells were treated with Res to investigate the mechanisms of autophagy and apoptosis using western blot, immunofluorescence staining for LC3B.KEY FINDINGS:Non-canonical autophagy was induced by Res-treatment in a Beclin-1- and ATG5-independent manner, with apoptosis being activated simultaneously. Autophagy induced by Res was activated by rapamycin with decreased apoptosis, suggesting that autophagy may serve as a protective pathway in cells. Mitophagy was found to be induced by Res using fluorescence co-localization of mitochondria with lysosomes. Subsequently, it was identified that mitophagy was mediated by LC3B/p62 interaction and could be inhibited by LC3B knockout and p62 knockdown following increased apoptosis.SIGNIFICANCE:In conclusion, the current results demonstrate that Res-induced non-canonical autophagy in A549 lung cancer cells with apoptosis activation simultaneously, while LC3B/p62-mediated mitophagy protects tumor cells against apoptosis, providing novel mechanisms about the critical role of mitophagy in regulating cell fate.

Zinc finger E-box binding homeobox 1 (ZEB1, also termed TCF8 and δEF1) is a crucial member of the zinc finger-homeodomain transcription factor family, originally identified as a binding protein of the lens-specific δ1-crystalline enhancer and is a pivotal transcription factor in the epithelial-mesenchymal transition (EMT) process. ZEB1 also plays a vital role in embryonic development and cancer progression, including breast cancer progression. Increasing evidence suggests that ZEB1 stimulates tumor cells with mesenchymal traits and promotes multidrug resistance, proliferation, and metastasis, indicating the importance of ZEB1-induced EMT in cancer development. ZEB1 expression is regulated by multiple signaling pathways and components, including TGF-β, β-catenin, miRNA and other factors. Here, we summarize the recent discoveries of the functions and mechanisms of ZEB1 to understand the role of ZEB1 in EMT regulation in breast cancer.

Background Oral squamous cell carcinoma (OSCC) is a solid tumor, which originates from squamous epithelium, with about 400,000 new-cases/year worldwidely. Presently, chemoradiotherapy is the most important adjuvant treatment for OSCC, mostly in advanced tumors. However, clinical resistance to chemotherapy still leads to poor prognosis of OSCC patients. Via high-throughput analysis of gene expression database of OSCC, we investigated the molecular mechanisms underlying cisplatin resistance in OSCC, analyzing the differentially expressed genes (DEGs) and their regulatory relationship, to clarify the molecular basis of OSCC chemotherapy resistance and provide a theoretical foundation for the treatment of patients with OSCC and individualized therapeutic targets accurately. Methods Datasets related to "OSCC" and "cisplatin resistance" (GSE111585 and GSE115119) were downloaded from the GEO database and analyzed by GEO2R. Venn diagram was used to obtain drug-resistance-related DEGs. Functional enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were performed on DEGs using The Database for Annotation, Visualization and Integrated Discovery (DAVID) software. Protein-protein interaction (PPI) network was constructed by STRING (search tool for recurring instances of neighbouring genes) database. Potential target genes of miRNA were predicted via miRDB, and cBioportal was used to analyze the function and survival of the potential functional genes. Results Forty-eight upregulated DEGs and 49 downregulated DEGs were obtained from the datasets, with cutoff as p < 0.01 and |log FC| > 1. The DEGs in OSCC mainly enriched in cell proliferation regulation, and chemokine activity. In PPI network with hub score > 300, the hub genes were identified as NOTCH1, JUN, CTNNB1, CEBPA, and ETS1. Among miRNA-mRNA targeting regulatory network, hsa-mir-200c-3p, hsa-mir-200b-3p, hsa-mir-429, and hsa-mir-139-5p were found to simultaneously regulate multiple hub genes. Survival analysis showed that patients with high CTNNB1 or low CEBPA expression had poor outcome. Conclusions In the OSCC cisplatin-resistant cell lines, NOTCH1, JUN, CTNNB1, CEBPA, and ETS1 were found as the hub genes involved in regulating the cisplatin resistance of OSCC. Members of the miR-200 family may reverse drug resistance of OSCC cells by regulating the hub genes, which can act as potential targets for the treatment of OSCC patients with cisplatin resistance.

表皮生长因子受体(epidermal growth factor receptor,EGFR)这种酪氨酸激酶受体在许多类型实体肿瘤的发生及生长过程中起着主要作用,因而针对性地应用EGFR抑制剂已被批准作为多种类型肿瘤的治疗方法.然而,先天性或获得性耐药问题限制了这类药物的疗效.自噬作为细胞内废弃物降解的一种方式,被认为与肿瘤EGFR抑制剂耐药相关.EGFR抑制剂能诱导自噬,然而该作用是双向的.自噬既能减弱EGFR抑制剂的细胞毒性作用而介导肿瘤耐药,也会在过度自噬的情况下促进细胞死亡而逆转肿瘤耐药.因此,调节自噬水平有可能为克服肿瘤患者EGFR抑制剂耐药提供更多思路.

自噬即细胞自我"消化",不仅与肺癌的发生发展密切相关,还与癌细胞耐药的形成有关.细胞的自噬水平可由表皮生长因子受体(epidermal growth factor receptor,EGFR)等多种信号通路进行调控.靶向治疗药物表皮生长因子受体-酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitor,EGFR-TKI)可以有效阻断下游信号通路,并广泛应用于治疗晚期非小细胞肺癌(non-small cell lung cancer,NSCLC).EGFR-TKI疗效肯定但容易产生耐药,而且还能诱导肿瘤细胞自噬活性.最新研究表明,使用自噬诱导剂或抑制剂调节自噬水平,可延缓EGFR-TKI的耐药性,提高其治疗非小细胞肺癌的效果.

NAD(P)H:quinone oxidoreductase 1 (NQO1), a phase II flavoenzyme that catalyzes reduction reactions to protect cells against electrophiles and oxidants, is involved in tumorigenesis. Altered methylation of the NQO1 gene has been observed and is speculated to result in aberrant NQO1 expression in rat cells undergoing chemical carcinogenesis, although this has not been proven experimentally. In this study, we first investigated the potential epigenetic mechanisms underlying the phenomenon of NQO1 differential expression in individual subclones of rat arsenic-transformed lung epithelial cells (TLECs). NQO1 expression of TLEC subclones with or without 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), western blot analysis, and real-time PCR. Methylation status of the NQO1 promoter in TLEC subclones was analyzed by bisulfite sequencing. Transcriptional activity of NQO1 promoter in vitro methylated was determined by luciferase assay using a CpG-free luciferase reporter driven by the NQO1 promoter region (-435 to +229). We found that non-CpG island (non-CpGI) within the NQO1 promoter was hyper- or hypo-methylated in TLEC subclones and corresponded to low and high gene expressions, respectively. Following the treatment with 5-Aza-CdR, transcription of the NQO1 gene in the hypermethylated subclones was restored, accompanied by demethylation of the NQO1 promoter. In vitro promoter methylation almost completely silenced reporter activity in TLECs. These results indicate that DNA methylation of the non-CpGI promoter contributes to epigenetic silencing of NQO1 in rat TLECs.

BACKGROUND:All known mechanisms of apoptosis induced by resveratrol act through cell cycle arrest and changes in mitochondrial membrane potential. It is currently unknown whether resveratrol-induced apoptosis is associated with other physiological processes, such as autophagy.METHODS:Apoptosis-related markers involved in the intrinsic and extrinsic apoptotic pathways, and autophagic markers were detected by using western blotting and immunofluorescence. Mitochondrial membrane potential was assayed by flow cytometry. Pharmaceutical or genetic inhibition of autophagy involved were carried by 3- methyladenine or knockdown of autophagy-related (Atg) genes by siRNA. Differences between two values were tested by Student's unpaired t test.RESULTS:We show that resveratrol-induced apoptosis occurs through both the intrinsic and extrinsic apoptotic pathways. Mitochondrial membrane potential and apoptosis-related markers, such as an increased Bax/Bcl-2 ratio, and cleaved forms of caspase-8 and caspase-3, arise following resveratrol addition. Moreover, we find that resveratrol increases both the levels of microtubule-associated protein 1 light chain 3-II and the number of autophagosomes, and further demonstrate that resveratrol-induced autophagy depends on the LKB1-AMPK-mTOR pathway. We next reveal that some apoptosis-related markers induced by resveratrol are further attenuated by the inhibition of autophagy with 3-methyladenine or knockdown of autophagy-related (Atg) genes by siRNA.CONCLUSIONS:These results suggest that resveratrol induced apoptotic cell death of HL-60 cells depends on the autophagy activated through both the LKB1-AMPK and PI3K/AKT-regulated mTOR signaling pathways.

TUSC3基因是一种抑癌基因,位于染色体8p22,其沉默还会导致精神发育异常,而新近的研究发现它在大肠癌中表达上调,表明其可能具有促癌作用.本文对近年来国内外学者在TUSC3基因的功能多样性,作用机制及其应用前景等方面的研究新进展进行综述,为揭示TUSC3基因的作用定基础.

Signal Transducer and Activators of Transcription (STAT) is a set of transcription factors, involved in diverse cellular functions. Evidences from cell lines, mouse models and human tissues implicate these transcription factors in the oncogenesis of breast cancer. However, the diverse expression patterns and prognostic values of 7 STATs remain to be elucidated. In the current study, we mined the transcriptional and survival data of STATs in patients with breast carcinoma (BC) through ONCOMINE, bc-GenExMiner, Kaplan-Meier Plotter and cBioPortal. It was found that STAT1/2 were up-regulated, whereas STAT3/4/5A/5B were down-regulated in BC patients compared with the normal tissues. The expressions of STAT5A/5B/6 were correlated with decreased levels of histological differentiation. In survival analyses through the Kaplan-Meier plotter database, high transcription levels of STAT2/4/5A/5B/6 were associated with better relapse-free survival (RFS) in all BC patients. Conversely, high STAT3 predicted shorter RFS in BC patients, suggesting that STAT3 is potential targets for precision therapy to BC patients. These data also provided STAT5A/5B/6 as new biomarker for BC prognosis.

Follicular lymphoma and diffuse large B cell lymphomas comprise the main entities of adult B cell malignancies. Although multiple disease driving gene aberrations have been identified by gene expression and genomic studies, only a few studies focused at the protein level. We applied 2 dimensional gel electrophoresis to compare seven GC B cell non Hodgkin lymphoma (NHL) cell lines with a lymphoblastoid cell line (LCL). An average of 130 spots were at least two folds different in intensity between NHL cell lines and the LCL. We selected approximately 38 protein spots per NHL cell line and linked them to 145 unique spots based on the location in the gel. 34 spots that were found altered in at least three NHL cell lines when compared to LCL, were submitted for LC-MS/MS. This resulted in 28 unique proteins, a substantial proportion of these proteins were involved in cell motility and cell metabolism. Loss of expression of B2M, and gain of expression of PRDX1 and PPIA was confirmed in the cell lines and primary lymphoma tissue. Moreover, inhibition of PPIA with cyclosporine A blocked cell growth of the cell lines, the effect size was associated with the PPIA expression levels. In conclusion, we identified multiple differentially expressed proteins by 2-D proteomics, and showed that some of these proteins might play a role in the pathogenesis of NHL.

Arsenic and benzo[α]pyrene (B[a]P) are common contaminants in developing countries. Many studies have investigated the consequences of arsenic and/or B[a]P-induced cellular transformation, including altered metabolism. In the present study, we show that, in addition to elevated glycolysis, B[a]P/arsenic-induced transformation also stimulates oxidative phosphorylation (OXPHOS). Proteomic data and immunoblot studies demonstrated that enzymatic activities, involved in both glycolysis and OXPHOS, are upregulated in the primary transformed rat lung epithelial cell (TLEC) culture, as well as in subcloned TLEC cell lines (TMCs), indicating that OXPHOS was active and still contributed to energy production. LEC expression, of the glycolytic enzyme phosphoglycerate mutase (PGAM) and the TCA cycle enzyme alpha-ketoglutarate dehydrogenase (OGDH), revealed an alternating cyclic pattern of glycolysis and OXPHOS during cell transformation. We also found that the expression levels of hypoxia-inducible factor-1a were consistent with the pattern of glycolysis during the course of transformation. Low doses of an ATP synthase inhibitor depleted endogenous ATP levels to a greater extent in TLECs, compared to parental LECs, indicating greater sensitivity of B[a]P/arsenic-transformed cells to ATP depletion. However, TLEC cells exhibited better survival under hypoxia, possibly due to further induction of anaerobic glycolysis. Collectively, our data indicate that B[a]P/arsenic-transformed cells can maintain energy production through upregulation of both glycolysis and OXPHOS. Selective inhibition of metabolic pathways may serve as a therapeutic option for cancer therapy.

Aldehyde dehydrogenase 1 (ALDH1) consists of a family of intracellular enzymes, highly expressed in stem cells populations of leukemia and some solid tumors. Up to now, 6 isoforms of ALDH1 have been reported. However, the expression patterns and the identity of ALDH1 isoenzymes contributing to ALDH1 activity, as well as the prognostic values of ALDH1 isoenzymes in cancers all remain to be elucidated. Here, we studied the expressions of ALDH1 transcripts in gastric cancer (GC) compared with the normal controls using the ONCOMINE database. Through the Kaplan-Meier plotter database, which contains updated gene expression data and survival information of 876 GC patients, we also investigated the prognostic values of ALDH1 isoenzymes in GC patients. It was found that when compared with normal tissues, ALDH1A1 mRNA expression was downregulated, whereas ALDH1A3 and ALDH1B1 were upregulated in GC patients. In survival analyses, high ALDH1A1 and ALDH1B1 expressions were associated with better overall survival (OS) in all GC patients. In addition, high transcription activity of ALDH1A1 predicted better OS in gastric intestinal type adenocarcinoma, but not in diffuse gastric adenocarcinoma. GC patients with high mRNA level of ALDH1B1 showed better OS in gastric intestinal type, and worse OS in diffuse type. Oppositely, high transcription activities of ALDH1A2, ALDH1A3 and ALDH1L1 predicted worsen overall survival in GC patients, suggesting that these isoenzymes might be responsible mainly for the ALDH1 activities in GC. These data provides ALDH1A2, ALDH1A3 and ALDH1L1 as excellent potential targets for individualized treatment of GC patients.

自噬曾被认为是细胞的自我保护系统.证据表明,自噬也能促进肿瘤细胞死亡.凋亡是细胞在受到刺激后,为维持内环境稳态而发生的有序自发性死亡过程.它们都与细胞生长,肿瘤发生发展密切相关.研究自噬和凋亡调节通路将有助于揭示肿瘤发生和肿瘤细胞死亡的潜在机制,为癌症的预防和治疗提供新思路.本文就细胞自噬和凋亡相关调节机制的最新研究进展作一综述.

microRNA regulation network is important for the cancer genetic heterogeneity. Relative to the increasing numbers of microRNA's targets identified, upstream regulatory mechanisms that control functional microRNAs are less well-documented. Here, we investigated the function of miR-31, a pleiotropically-acting microRNA, in esophageal squamous cell cancer (ESCC). We demonstrated that miR-31 only exerted tumor-suppressive effects in TE-7 ESCC cells, but not in TE-1 ESCC cells, although both of these cell lines harbor inactive p53. Interestingly, TE-1 cells highly expressed p21, while p21 levels were virtually undetectable in TE-7 cells, suggesting a p21-dependent mechanism of miR-31-mediated tumor suppression. Accordingly, knockdown of p21 in TE-1 cells reversed the tumor suppressive actions of miR-31. In patient ESCC specimens, real-time RT-PCR analysis revealed that expression of E2F2 and STK40, two known miR-31 target oncogenes, was negatively correlated with the expression of miR-31 in a p21-dependent manner, supporting the conclusion that miR-31 only downregulates its target oncogenes when p21 levels are low. Collectively, these data suggest a novel mechanism through which the tumor-suppressive effect of miR-31 is p21-dependent. In addition, we speculate that delivery of miR-31 could provide therapeutic benefit in the personalized management of a subgroup of ESCC patients with p21-deficient tumors.

Resveratrol has many biological effects, including anti-tumor, antiviral activities, and vascular protection. Recent studies have suggested that resveratrol exert its antitumor effects through induction of autophagy by an unknown mechanism. In this study, we investigated the involvement of autophagy in resveratrol-induced cell death and its potential molecular mechanisms in A549 human lung adnocarcinoma cells. Resveratrol-induced growth inhibition and cell death was assessed by MTT and clonogenic assays. Activation of autophagy was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Western blot analysis was used to study the cell signals involved in the mechanisms of autophagic death. Intracellular free calcium was detected with Fura2-AM staining. Our results indicated that resveratrol induced A549 cell death was mediated by autophagy. 3-methyladenine, an inhibitor of autophagy, suppressed resveratrol-induced autophagic cell death, and knockdown of autophagy-related genes Atg5 and Beclin-1 with siRNAs reversed RSV-induced cell death. Intracellular free calcium accumulated immediately following resveratrol addition, which led to the activation of phospho-AMPK and phospho-Raptor, and a reduction in the amount of phospho-p70S6K. These effects could be reversed by the AMPK inhibitor compound C, and the calcium ion-chelating agent EGTA. In conclusion, we demonstrate that resveratrol-induced A549 cell death was mediated by the process of autophagic cell death via Ca2+/AMPK-mTOR signaling pathway.