口腔鳞癌细胞系NTCR中侧群细胞的相关研究

目的:口腔鳞癌是口腔颌面部常见的恶性肿瘤之一,本研究以侧群细胞为肿瘤干细胞突破口,通过检测、分选口腔鳞癌细胞系NTCR中侧群细胞(side population,SP)细胞亚群,深入研究不同细胞亚群的体内、外相关生物学特性,寻找口腔鳞癌中肿瘤干细胞存在的证据。方法:选取口腔鳞癌细胞系NTCR作为研究对象,Hoechst 33342染色后行流式细胞仪检测,分选口腔鳞状细胞癌中的SP细胞和非SP细胞,进行体外培养、长期分化和体内成瘤实验,对2种亚群细胞的体内和体外生物学特性进行检测和比较。结果:口腔鳞状细胞癌细胞系NTCR中含有9.3%SP细胞,其SP细胞在细胞的增殖能力、自我更新能力及裸鼠体内成瘤能力等方面与干细胞特性相似。结论:SP细胞可以认为是肿瘤干细胞的富集。进一步深入研究,有可能作为口腔鳞癌诊断、治疗和预后的靶标。     

人肺腺癌细胞A—549和正常细胞HBE的蛋白质组差异分析

为了研究人肺腺癌细胞A 5 49和正常细胞HBE的蛋白质组差异 ,用固相pH梯度双向凝胶电泳分离人肺腺癌细胞系A 5 49和正常细胞HBE的总蛋白质 ,银染显色 ,PDQuest 2 DE软件分析 ,对部分差异蛋白质点进行基质辅助激光解吸电离飞行时间质谱 (MALDI TOF MS)测定其胶内酶解后的肽质指纹图谱 ,用PeptIdent软件查询SWISS PROT数据库。结果获得了分辨率和重复性均较好的双向电泳银染图谱 ,图象分析探测到A 5 492 DE图谱的平均蛋白质点数为 (890± 38)个 ,HBE的平均蛋白质点数为 (75 7± 2 7)个 ,不同胶间蛋白质点的位置偏差在IEF方向为 (2 .85± 0 .48)mm ,在SDS PAGE方向为 (2 .6 9± 0 .37)mm。差异表达分析发现A 5 49和HBE图谱有5 35个蛋白质点相互匹配 ,其中A 5 49有 35 5个未被匹配 ,HBE中有 2 2 2个未被匹配 ;对A 5 49和HBE中的 18个差异蛋白质点分别进行肽质指纹分析 ,经数据库查询 ,初步鉴定为一些与物质代谢、细胞因子、信号转导有关的蛋白质。提示人肺腺癌细胞A 5 49和正常细胞HBE的蛋白质组具有差异 ,这种蛋白质组的差异分析有助于进一步研究肺腺癌的相关蛋白质及分子标记物     

Decitabine Rescues Cisplatin Resistance in Head and Neck Squamous Cell Carcinoma

Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) reduces survival. In this study we hypothesized that methylation of key genes mediates cisplatin resistance. We determined whether a demethylating drug, decitabine, could augment the anti-proliferative and apoptotic effects of cisplatin on SCC-25/CP, a cisplatin-resistant tongue SCC cell line. We showed that decitabine treatment restored cisplatin sensitivity in SCC-25/CP and significantly reduced the cisplatin dose required to induce apoptosis. We then created a xenograft model with SCC-25/CP and determined that decitabine and cisplatin combination treatment resulted in significantly reduced tumor growth and mechanical allodynia compared to control. To establish a gene classifier we quantified methylation in cancer tissue of cisplatin-sensitive and cisplatin-resistant HNSCC patients. Cisplatin-sensitive and cisplatin-resistant patient tumors had distinct methylation profiles. When we quantified methylation and expression of genes in the classifier in HNSCC cells in vitro, we showed that decitabine treatment of cisplatin-resistant HNSCC cells reversed methylation and gene expression toward a cisplatin-sensitive profile. The study provides direct evidence that decitabine restores cisplatin sensitivity in in vitro and in vivo models of HNSCC. Combination treatment of cisplatin and decitabine significantly reduces HNSCC growth and HNSCC pain. Furthermore, gene methylation could be used as a biomarker of cisplatin-resistance.     

Downregulation of HIPK2 Increases Resistance of Bladder Cancer Cell to Cisplatin by Regulating Wip1

Cisplatin-based combination chemotherapy regimen is a reasonable alternative to cystectomy in advanced/metastatic bladder cancer, but acquisition of cisplatin resistance is common in patients with bladder cancer. Previous studies showed that loss of homeodomain-interacting protein kinase-2 (HIPK2) contributes to cell proliferation and tumorigenesis. However, the role of HIPK2 in regulating chemoresistance of cancer cell is not fully understood. In the present study, we found that HIPK2 mRNA and protein levels are significantly decreased in cisplatin-resistant bladder cancer cell in vivo and in vitro. Downregulation of HIPK2 increases the cell viability in a dose- and time-dependent manner during cisplatin treatment, whereas overexpression of HIPK2 reduces the cell viability. HIPK2 overexpression partially overcomes cisplatin resistance in RT4-CisR cell. Furthermore, we showed that Wip1 (wild-type p53-induced phosphatase 1) expression is upregulated in RT4-CisR cell compared with RT4 cell, and HIPK2 negatively regulates Wip1 expression in bladder cancer cell. HIPK2 and Wip1 expression is also negatively correlated after cisplatin-based combination chemotherapy in vivo. Finally, we demonstrated that overexpression of HIPK2 sensitizes chemoresistant bladder cancer cell to cisplatin by regulating Wip1 expression.

Conclusions

These data suggest that HIPK2/Wip1 signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of bladder cancer.     

融合细胞株Eahy926与亲本人肺腺癌细胞株A549差异表达蛋白的蛋白质组学分析

融合细胞株Eahy926是人肺腺癌细胞株A549和人脐静脉内皮细胞杂交而成的永生化细胞株,具有血管内皮细胞的特性,已广泛用于内皮细胞相关研究. 本研究应用蛋白质组学技术分析融合细胞株Eahy926与亲本人肺腺癌细胞株A549的蛋白质差异表达,探讨融合细胞生物学特性变化及其机制. 对Eahy926和亲本A549的细胞总蛋白质进行双向凝胶电泳,在PDQuest软件辅助下找出差异表达蛋白质点,经肽质量指纹图谱(peptide mass fingerprinting, PMF) 和串级质谱(tandem mass spectrometry,TMS) 分析,SWISS-PROT数据库检索,成功鉴定出28个差异蛋白,如CATB、CK8、CK18、annexin A2、GRP78、HSP90、HSP60、vimentin等一些与分子伴侣、氧化应激、能量代谢、信号转导等有关,并与肿瘤细胞分裂增殖、分化凋亡、侵袭转移、免疫逃逸以及肿瘤血管生长密切关联的蛋白质. 研究发现,融合细胞株Eahy926和人肺腺癌细胞株A549的蛋白质组表达谱存在明显差异,这将有助于今后进一步探讨肿瘤细胞与内皮细胞的相互作用机制及其融合细胞的特性,筛选肿瘤增殖和转移相关蛋白质及分子标志物,亦可为肿瘤的抗血管生成治疗提供新思路.     

肺腺癌A549/DDP细胞周期变化及其多药耐药性

用Fura 2 /AM标记药物敏感的肺腺癌细胞A54 9和抗顺铂药物的肺腺癌细胞A54 9/DDP两种细胞胞内游离Ca2 ,用碘化丙锭 (PI)标记细胞DNA ,检测其胞内Ca2 的变化及两种细胞增殖能力和细胞周期 .实验结果表明 ,抗药性细胞株A54 9/DDP胞浆内游离Ca2 的浓度仅为药物敏感细胞株A54 9的 1/ 3左右 ,同时前者的细胞增殖能力较后者明显增强 ,而且细胞周期也明显缩短 .当用BAPTA AM和EGTA或A2 3187和Thapsigargin处理细胞以降低或升高其胞内自由Ca2 浓度时可改变细胞的生长周期 ,二者也呈现明显差别 .这些结果表明 ,对顺铂产生耐药性的人肺腺癌A54 9/DDP细胞胞内Ca2 浓度的降低 ,可能影响细胞的增殖 ,缩短细胞的生长周期 ,特别是影响起决定作用的G1期 ,从而有利于肿瘤细胞多药耐药特性的维持     

Interleukin-6-Mediated Autocrine Growth Promotion in Human Glioblastoma Multiforme Cell Line U87MG

Abstract: Human glioblastoma multiforme cell lines, brain tumor biopsy tissue, and normal human fetal brain synthesize interleukin (IL)-6 and IL-6 receptor (IL-6R). Neither of these is expressed in human neurons or neuroblastoma cell lines in culture. Astrocytes from fetal brain grown in culture retain the ability to synthesize IL-6 but do not express IL-6R as inferred from RT-PCR and Southern blot studies. Coexpression of IL-6 and IL-6R in the glioblastoma cell line U87MG is confirmed by immunofluorescence staining. Both specific monoclonal antibodies against IL-6 and IL-6R and antisense oligonucleotide to IL-6 mRNA inhibit the growth of U87MG cells in culture, suggesting the existence of a functional autocrine growth loop. Anti-IL-6 antibodies also inhibit the growth of glioblastoma cell lines U373 and U118. The expression of IL-6 by human fetal astrocytes in culture is highly suggestive of its role as an oncofetal protein responsible for rapid proliferation of fetal and tumor cells but not cells of adult brain.     

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.     

Galectin-1-Induced Autophagy Facilitates Cisplatin Resistance of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers in Taiwan. Although chemotherapy is the primary treatment for HCC patients, drug resistance often leads to clinical failure. Galectin-1 is a beta-galactoside binding lectin which is up-regulated in HCC patients and promotes tumor growth by mediating cancer cell adhesion, migration and proliferation, but its role in chemoresistance of HCC is poorly understood. In this study we found that galectin-1 is able to lead to chemoresistance against cisplatin treatment, and subsequent inhibition has reversed the effect of cell death in HCC cells. Moreover, galectin-1 was found to induce autophagic flux in HCC cells. Inhibition of autophagy by inhibitors or knockdown of Atg5 cancels galectin-1-induced cisplatin resistance in HCC cells. Increase of mitophagy triggered by galectin-1 was found to reduce the mitochondrial potential loss and apoptosis induced by cisplatin treatment. Finally, using an in situ hepatoma mouse model, we clearly demonstrated that inhibition of galectin-1 by thiodigalactoside could significantly augment the anti-HCC effect of cisplatin. Taken together, our findings offer a new insight into the chemoresistance galectin-1 causes against cisplatin treatment, and points to a potential approach to improve the efficacy of cisplatin in the treatment of HCC patients.     

Ru-486、安宫黄体酮、环孢菌素、维拉帕米对非P-g介导的卵巢癌耐顺铂细胞株COC 1/DDP耐药性的逆转

目的 在同一实验系统中比较Ru-486、MPA、VP、CsA对非P-g介导的卵巢癌耐顺铂细胞株COC1/DDP耐药性的逆转作用并对其作用机制进行探讨,为临床克服耐药提供参考依据.方法 采用MTT法从细胞代谢,DNA凝胶电泳从细胞凋亡方面系统观察比较DDP、Ru-486、MPA、VP、CsA 5种药物对卵巢癌多药耐药细胞系COC1/DDP的影响及相互关系,检测上述5种药物与COC1/DDP作用时细胞内GSH-ST和GSH-Px活性的变化,并采用westernblotting以Bcl-2抗体为分子探针对上述5种药物与COC1/DDP作用时细胞内Bcl-2基因的表达进行测定对其逆转机制进行探讨.结果 1)Ku-486、CsA、MPA对COC1/DDP有直接抑制作用,其抑制率随浓度增高进行性升高,、而VP对COC1/DD,P没有直接抑制作用.和DDP联用时,Ru-486、MPA、VP、CsA都能显著增强COC1/DDP对DDP的敏感性,达到一定浓度后,可以完全逆转COC1/DDP对DDP的耐药性.这种逆转是通过抑制细胞代谢、诱导细胞凋亡的结果.2)单纯2.5μg/ml的DDP能使GSH-Px及GST活力上升,除VP外,Ru-486等药物可降低GSH-Px和GST活力,与DDP联用时亦如此.提示,Ru-486、CsA、MPA可以通过抑制GSH转移酶系统阻止对细胞损伤的修复而达到对耐药性的逆转.3)单独的DDP、Ru-486等药物非但对Bcl-2的表达没有抑制作用,反而促进了其表达,以MPA最甚.与DDP联用时,却出现了一个有趣的现象,即可以抑制Bcl-2的表达,Ru-486与DDP联用时Bcl-2的表达几乎完全被阻截.结论 :VP、Ru-486、CsA和MPA对卵巢癌多药耐药细胞系COC1/DDP的耐药性有确切逆转作用.其逆转机制可能与降低GSH转移酶系统活性有关.当与DDP联用时可以通过降低Bcl-2蛋白表达来逆转耐药.     

Phenotypic Expression of rkn1-Mediated Meloidogyne incognita Resistance in Gossypium hirsutum Populations

The root-knot nematode Meloidogyne incognita is a damaging pest of cotton (Gossypium hirsutum) worldwide. A major gene (rkn1) conferring resistance to M. incognita was previously identified on linkage group A03 in G. hirsutum cv. Acala NemX. To determine the patterns of segregation and phenotypic expression of rkn1, F₁, F₂, F_2:3, BC₁F₁ and F_2:7 recombinant inbred lines (RIL) from intraspecific crosses between Acala NemX and a closely related susceptible cultivar Acala SJ-2 were inoculated in greenhouse tests with M. incognita race 3. The resistance phenotype was determined by the extent of nematode-induced root galling and nematode egg production on roots. Suppression of root galling and egg production was highly correlated among individuals in all tests. Root galling and egg production on heterozygous plants did not differ from the susceptible parent phenotype 125 d or more after inoculation, but were slightly suppressed with shorter screening (60 d), indicating that rkn1 behaved as a recessive gene or an incompletely recessive gene, depending on the screening condition. In the RIL, rkn1 segregated in an expected 1 resistant: 1 susceptible ratio for a major resistance gene. However, within the resistant class, 21 out of 34 RIL were more resistant than the resistant parent Acala NemX, indicating transgressive segregation. These results suggest that rkn1-based resistance in G. hirsutum can be enhanced in progenies of crosses with susceptible genotypes. Allelism tests and molecular genetic analysis are needed to determine the relationship of rkn1 to other M. incognita resistance sources in cotton.     

O-Glycomic and Proteomic Signatures of Spontaneous and Butyrate-Stimulated Colorectal Cancer Cell Line Differentiation

Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano–liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell proteome. It was found that the fully differentiated butyrate-stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O-glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these patterns in homeostasis and dysbiosis at the gut–microbiota interface.     

Sirt7 inhibits Sirt1-mediated activation of Suv39h1

Sirtuins regulate a variety of cellular processes through protein deacetylation. The best-known member of mammalian sirtuin family, Sirt1, plays important roles in the maintenance of cellular homeostasis by regulating cell metabolism, differentiation and stress responses, among others. Sirt1 activity requires tight regulation to meet specific cellular requirements, which is achieved at different levels and by specific mechanisms. Recently, a regulatory loop between Sirt1 and another sirtuin, Sirt7, was identified. Sirt7 inhibits Sirt1 autodeacetylation at K230 and activation thereby preventing Sirt1-mediated repression of adipocyte differentiation by inhibition of the PPARγ gene. Here, we extend the regulatory complexity of Sirt7-dependent restriction of Sirt1 activity by demonstrating that Sirt7 reduces activation of a previously described prominent Sirt1 target, the histone methyltransferase Suv39h1. We show that removal of the acetyl-group at K230 in Sirt1 due to the absence of Sirt7 leads to hyperactivation of Sirt1 and thereby to constantly increased activity of Suv39h1.     

Proteomic and phosphoproteomic landscape of salivary extracellular vesicles to assess OSCC therapeutical outcomes

Circulating extracellular vesicles (EVs) have emerged as an appealing source for surrogates to evaluate the disease status. Herein, we present a novel proteomic strategy to identify proteins and phosphoproteins from salivary EVs to distinguish oral squamous cell carcinoma (OSCC) patients from healthy individuals and explore the feasibility to evaluate therapeutical outcomes. Bi-functionalized magnetic beads (BiMBs) with Ti (IV) ions and a lipid analog, 1,2-Distearoyl-3-sn-glycerophosphoethanolamine (DSPE) are developed to efficiently isolate EVs from small volume of saliva. In the discovery stage, label-free proteomics and phosphoproteomics quantification showed 315 upregulated proteins and 132 upregulated phosphoproteins in OSCC patients among more than 2500 EV proteins and 1000 EV phosphoproteins, respectively. We further applied targeted proteomics by coupling parallel reaction monitoring with parallel accumulation-serial fragmentation (prm-PASEF) to measure panels of proteins and phosphoproteins from salivary EVs collected before and after surgical resection. A panel of three total proteins and three phosphoproteins, most of which have previously been associated with OSCC and other cancer types, show sensitive response to the therapy in individual patients. Our study presents a novel strategy to the discovery of effective biomarkers for non-invasive assessment of OSCC surgical outcomes with small amount of saliva.