Delivery of cytolethal distending toxin B induces cell cycle arrest and apoptosis in gingival squamous cell carcinoma in vitro

The cytolethal distending toxin (Cdt) from Actinobacillus actinomycetemcomitans consists of three proteins, CdtA, CdtB, and CdtC, which are responsible for cell cycle arrest and apoptosis. In the present study, local delivery systems of recombinant CdtB and CdtB-expressing plasmid were established using Ca9-22, human gingival squamous cell carcinoma cell line. When CdtB was delivered to Ca9-22 cells using a BioPORTER, a 32-kDa protein was detected by Western blotting, and G2 cell cycle arrest and apoptosis occurred. In addition, the CdtB delivered upregulated the expression of phosphorylated p53 and the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in Ca9-22 cells, suggesting that these intracellular molecules might contribute to the induction of G2 cell cycle arrest and apoptosis. When the CdtB-expressing plasmid was transfected into Ca9-22 cells by lipofection or electroporation, CdtB (32 kDa) was clearly detected. Further, TdT-mediated dUTP nick end labeling positive cells were observed after transfection of the CdtB-expressing plasmid. These findings indicated that delivery of the CdtB protein and transfection of the cdtB gene induced cell cycle arrest and apoptosis in Ca9-22 cells in vitro, and we conclude that it may be possible to induce apoptosis in human gingival squamous cell carcinoma by electroporation of the cdtB gene.     

槟榔碱诱导体外培养的血管内皮细胞凋亡研究

了解槟榔碱 (Arecoline)对培养的人脐静脉内皮细胞 (HumanUmbilicalVeinEndothelialCells ,HU VECs)凋亡的影响。方法 :不同浓度的槟榔碱处理体外培养的HUVECs ,用Hoechst3 3 2 5 8染色 ,在荧光显微镜下观察凋亡细胞形态学改变并计算凋亡百分率 ,琼脂糖凝胶电泳观察凋亡特征性”梯状”条带。结果 :①槟榔碱能以时间和浓度依赖方式诱导培养的HUVECs发生凋亡 ,其细胞凋亡率较正常对照组明显增高 (P <0 .0 1)。② 90ug/ml槟榔碱诱导VEC凋亡的DNA片段琼脂糖凝胶电泳可见典型的DNA“梯状”条带。结论 :槟榔碱浓度和时间依赖性诱导HUVECs凋亡 ,VEC凋亡异常可能是口腔黏膜下纤维性变的重要发病机理之一     

Sonicated extract of Enterococcus faecalis induces irreversible cell cycle arrest in phytohemagglutinin-activated human lymphocytes

The purpose of this study was to examine whether the sonicated extract of Enterococcus faecalis (SEF) alters the cell cycle transition of lymphocytes and thus regulates the fate of the arrested cells. Human lymphocytes were activated by phytohemagglutinin in the presence or absence of SEF, and cell cycle was assessed by flow cytometry. Seventy-two hours after activation with phytohemagglutinin, cells were activated from G0/G1 to S (6.1%) and G2/M (3.8%) phases of the cell cycle. In contrast, pretreatment with SEF resulted in 90.5% of cells remaining in G0/G1, and cell cycle progression to the S and G2/M phases was consequently inhibited. Caspase assay demonstrated that SEF-treated cells exhibited significantly increased apoptosis (56.7%) compared with phytohemagglutinin alone (28.1%). We propose that if this irreversible cell cycle arrest induced by E. faecalis occurs in vivo, it may result in local immunosuppression and contribute to the pathogenesis of endodontic failure. Our findings that E. faecalis can inhibit lymphocyte responses may be of particular relevance to the pathogenesis of endodontic failure. Although the immunologic mechanism involved in the pathogenesis of persistent periapical lesion is not clearly defined, it is reasonable to predict that the altered immune reaction may be linked to the immunosuppressive potential of E. faecalis or other oral bacteria.     

汉黄芩素诱导人舌鳞状细胞癌HN-6细胞凋亡的研究

目的本实验旨在体外探讨汉黄芩素对人舌鳞状细胞癌(简称鳞癌)HN-6细胞的增殖、细胞凋亡及细胞周期的影响。方法采用不同浓度的汉黄芩素对HN-6细胞进行干预,应用CCK-8法检测汉黄芩素对HN-6细胞增殖的影响;通过DAPI染色,荧光显微镜下观察细胞核变化及细胞凋亡情况;Annexin V-FITC/PI双标染色,流式细胞仪检测细胞凋亡情况;PI染色,流式细胞仪检测细胞周期变化。结果随着汉黄芩素浓度和作用时间的增加,HN-6细胞生长明显受到抑制,呈现时间和剂量依赖性。与对照组比较,当不同浓度汉黄芩素作用48 h时,各实验组细胞生长抑制率分别是23.18%、45.80%、54.61%、68.98%、76.82%,P均<0.05,差别有统计学意义。加药组细胞可见明显的核固缩、核碎裂、核溶解及染色质凝聚等典型的凋亡特性。流式细胞仪检测结果显示汉黄芩素可诱导细胞凋亡,实验组细胞总凋亡率分别为7.00%、12.03%、32.19%,与对照组相比,差异有统计学意义(P<0.05),并使细胞阻滞于G0/G1期(实验组的G0/G1期细胞百分比分别是61.21%,63.65%,70.78%,与对照组相比,差异有统计学意义,P均<0.05)。结论汉黄芩素能够体外抑制人舌鳞癌HN-6细胞的增殖,促进细胞凋亡。     

Effects of fluorides on apoptosis and activation of human umbilical vein endothelial cells

Oral Diseases (2012) 18 , 280–284 Objective: To determine the effects of fluorides on endothelial functioning. Materials and methods: We analyzed expressions of adhesion molecules, ICAM‐1 and ICAM‐3, and annexin V, on the surface of human umbilical vein endothelial cells (HUVECs) exposed to various concentrations of NaF and SnF₂. We compared the effects of fluoride‐induced changes with those obtained when stimulating HUVECs with TNF‐α and verified whether N‐acetyl cysteine (NAC), well‐known antioxidant, can prevent both fluoride‐ and TNF‐α‐induced alterations. Results: The expressions of annexin V and ICAM‐1 increased significantly after adding NaF (5.0 or 7.5 mM) or Sn₂F (0.5 or 0.75 mM) to the culture medium. Pre‐incubating HUVECs with NAC prevented the effects induced by 5.0 mM of NaF and 0.5 mM of Sn₂F. Only the highest concentration of NaF (7.5 mM) triggered the expression of ICAM‐3. The expressions of all three molecules increased significantly upon stimulating the cultures with TNF‐α (20 ng ml^?1); these changes were not reversed by pre‐incubation with NAC. Conclusions: Fluorides induce oxidative stress, resulting in apoptosis and activation of HUVECs, manifested by an elevated expression of ICAM‐1. The oxidative stress resulting from a stimulation by the highest NaF concentration triggers ICAM‐3 expression on the HUVECs’ surface.     

神经钙黏素表达下调对舌鳞状细胞癌细胞生物学能力的影响

目的 研究神经钙黏素(N-cadherin)表达下调对舌鳞状细胞癌Tca8113细胞增殖、细胞周期、凋亡以及迁移的影响,并探讨其可能的分子机制.方法 利用LipofectamineTM2000将神经钙黏素siRNA转染舌鳞状细胞癌Tca8113细胞,将细胞分为3组:①未处理组;②对照siRNA组;③神经钙黏素siRNA组.分别收集转染后48 h的细胞,采用新型的细胞计数试剂盒(cell counting kit,CCK)8试剂分析转染神经钙黏素siRNA后对细胞增殖能力的影响;运用流式细胞术检测下调神经钙黏素表达对Tca8113细胞周期及细胞凋亡的影响;采用Boyden 小室实验分析下调神经钙黏素对舌鳞状细胞癌细胞Tca8113细胞迁移能力的影响;进一步采用蛋白质印迹法检测神经钙黏素表达下调对细胞增殖、细胞周期以及细胞迁移相关基因表达的影响.结果 神经钙黏素siRNA能明显下调舌鳞状细胞癌Tca8113细胞中神经钙黏素蛋白的表达,并显著抑制Tca8113细胞的增殖(P<0.05).细胞周期结果显示,神经钙黏素siRNA组中在G0/G1的细胞比率[(65.41±0.92)%]明显高于未处理组[(41.59±1.43)%]和对照siRNA组[(43.70±2.08)%],差异有统计学意义(F=216.839,P＝0.000).神经钙黏素siRNA组中细胞凋亡的比率[(25.66±1.36)%]明显高于未处理组[(2.38±0.14)%]和对照siRNA组[(2.81±0.12)%],差异有统计学意义(F=850.364,P=0.000).Boyden 小室体外侵袭实验结果表明,与未处理组和对照siRNA组相比,神经钙黏素siRNA组中Tca8113细胞的迁移能力显著下降,差异有统计学意义(F=140.858,P=0.000).蛋白质印迹法结果表明,与未处理组和对照siRNA组相比,神经钙黏素siRNA组中的基质金属蛋白酶(matrix metalloproteinases,MMP)2和MMP-9明显下调,而p21明显上调,且差异有统计学意义(P<0.05).结论 神经钙黏素在舌鳞状细胞癌的发生发展中可能具有重要的作用.

Abstract：

Objective To investigate the effect of downregulation of N-cadherin expression on cell proliferation, cell cycle, cell apoptosis and cell migration in tongue squamous cell carcinoma cell line Tca8113 cells. Methods N-cadherin siRNA was transfected into tongue squamous cell carcinoma cell line Tca8113 cells and Tca8113 cells were divided into three groups: untreated group, control siRNA group and N-cadherin siRNA group. The cells were harvested 48 h after transfection with N-cadherin siRNA. Cell proliferation of Tca8113 cells was examined by cell counting kit(CCK)-8 after transfection with N-cadherin, and the effects of downregulation of N-cadherin on cell cycle and cell apoptosis of Tca8113 cells were investigated by flow cytometry.The effect of downregulation of N-cadherin expression on cell migration of Tca8113 cells was observed by Boyden chamber experiment, and further expression changes of gene-related cell proliferation, cell cycle and cell migration were detected by Western blotting. Results N-cadherin siRNA downregulated the N-cadherin expression and significantly inhibited cell proliferation of Tca8113 cells (P<0.05). The results of cell cycle revealed that the percentage of G0/G1 phase in N-cadherin group [(65.41±0.92) %] was significantly higher than that in untreated group [(41.59±1.43)%] or control siRNA group [(43.70±2.08)%], and there was significant difference among the three groups (F=216.839,P＝0.000).The percentage of cell apoptosis in N-cadherin group [(25.66±1.36)%] was significantly higher than that in untreated group [(2.38±0.14)%] or control siRNA group [(2.81±0.12)%], and there was significant difference among the three groups (F=850.364,P=0.000). The cell number migrated into memebrane in N-cadherin group was significantly lower than that in untreated group and control siRNA group, and there was significant difference among the three groups (F=140.858,P=0.000). Further, compared with untreated group and control siRNA group, the expressions of matrix metalloproteinase(MMP)-2 and MMP-9 proteins were significantly downregulated and expression of p21 protein was significantly upregulated (P<0.05). Conclusions N-cadherin may play a role in occurrence and development of tongue squamous cell carcinoma.     

沉默Icmt基因对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡及细胞周期的影响

目的:探讨异戊二烯基半胱氨酸羧基甲基转移酶(isoprenylcysteine carboxyl methyltransferase,Icmt)对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡和细胞周期的影响及其相关机制.方法:针对人Icmt基因序列设计并构建3条小干扰RNA(small interfering RNA,...     

Nicotine inhibits apoptosis induced by cisplatin in human oral cancer cells

Oral cancer demonstrates a strong epidemiological association with smoking, but little is known about the effect of nicotine on oral cancer cell apoptosis. Nicotine, a major component of cigarette smoke, can regulate cell proliferation and angiogenesis and suppress apoptosis induced by chemotherapeutic drugs. The main aim of this study was to investigate the effects of nicotine on apoptosis induced by cisplatin, which is commonly used to treat advanced oral cancers, in the human oral cancer cell line Tca8113. The cells were stimulated with nicotine in the presence or absence of cisplatin, and apoptosis was assayed. The results showed that nicotine inhibited apoptosis induced by cisplatin. It was also observed that survivin played a role in the inhibitory effect of nicotine on apoptosis. Depletion of survivin reduced the protective effect of nicotine against cisplatin-induced apoptosis. Akt, a physiological survivin kinase, is activated by nicotine. Treatment of Tca8113 cells with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked nicotine-induced survivin expression and enhanced cell apoptosis. These studies suggest that exposure to nicotine might negatively impact on the apoptotic potential of chemotherapeutic drugs, and that survivin plays a key role in the anti-apoptotic effect of nicotine. The Akt pathway may be required for nicotine function.     

Adhesive resin induces apoptosis and cell-cycle arrest of pulp cells

The application of an adhesive resin near or directly over the pulp was shown to induce pulp inflammation and lack of dentin regeneration. We hypothesize that the absence of dentin bridging is due to adhesive-resin-induced apoptosis of cells responsible for pulp healing and dentin regeneration. Mouse odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD-21), or macrophages (RAW 264.7) were exposed to SingleBond polymerized for 0-40 seconds. Annexin V and propidium iodide assays demonstrated that SingleBond induced apoptosis of MDPC-23, OD-21, and macrophages. The proportion of apoptotic cells was dependent on the degree of adhesive resin polymerization. Adhesive-resin-induced death of pulp cells was associated with activation of the pro-apoptotic cysteine protease Caspase-3. Interestingly, most cells exposed to adhesive resin that did not undergo apoptosis showed cell-cycle arrest. We conclude that an adhesive resin induces apoptosis and cell-cycle arrest of cells involved in the regeneration of the dentin-pulp complex in vitro.     

丹参对槟榔碱诱导血管内皮细胞凋亡的保护作用

目的:探讨丹参对槟榔碱诱导血管内皮细胞凋亡的保护作用。方法:体外培养人脐静脉内皮细胞(Vein Endothelial Cells,VEC)为模型,槟榔碱作为致伤因素,丹参作为保护因素,用Hoechst 33258染色,在荧光显微镜下观察凋亡细胞核形态学改变并计算凋亡百分率。结果:正常培养的血管内皮细胞凋亡较少,槟榔碱刺激后可见到明显的细胞凋亡,丹参预处理后则凋亡细胞明显减少,凋亡率与槟榔碱组相比有显著性差异(P<0.05)。结论:丹参对槟榔碱诱导的VEC凋亡有一定的保护作用。     

Assessment of the relative cytotoxicity of Porphyromonas gingivalis cells, products, and components on human epithelial cell lines.

Established human cell lines derived from a transitional cell carcinoma (J82), a squamous carcinoma (SCaBER), and a normal urothelium (HCV-29) were used to assess the relative cytotoxicity and tissue specificity of putative virulence determinants of P. gingivalis W83. Intact cells of W83 had no effect on any of the cell lines, whereas disrupted cells caused extensive cytotoxicity particularly to monolayers of HCV-29 and J82. The purified cysteine proteinase, gingivain, caused marked disruption of the basement membrane of the SCaBER monolayers but had no cytotoxic effects. Use of the thiol-inhibitor, 2,2'-dipyridyl disulphide revealed that the effects observed with the vesicles and the culture supernatant were due to the presence of the cysteine proteinase. The attachment of vesicles to the SCaBER cells was evident in electron micrographs. Short-chain volatile fatty acids added in concentrations equivalent to those present in the culture supernatant had no effect on any of the cell lines tested. Culture supernatants obtained from high speed centrifugation (150,000 x g) showed no cytotoxic effects. This was in marked contrast to the supernatant obtained by lower sedimentation (18,000 x g), which damaged all monolayers tested. These results suggest that these cell lines are potentially useful for assessing putative virulence determinants of P. gingivalis and other periodontal pathogens.     

4,5,7-三羟基异黄酮和阿霉素诱导人舌癌细胞株Tca8113凋亡的研究

目的观察4,5,7-三羟基异黄酮(Genistein)和阿霉素(ADM)单用和联用对诱导体外培养的人舌癌细胞株Tca8113凋亡的影响。方法以体外培养的人舌癌细胞株Tca8113为实验模型,Genistein与ADM联合作用于癌细胞,倒置相差显微镜下观察药物处理后癌细胞的细胞形态学变化,四甲基偶氮唑蓝比色法分析药物对癌细胞的增殖抑制作用,流式细胞仪测定细胞凋亡率和细胞周期分布。结果Genistein和ADM两者单用对癌细胞均有生长抑制作用,且随剂量升高抑制趋势增强(P<0.05)。两药联用对癌细胞的抑制作用优于两者的单纯相加(Q>1)。联合用药使肿瘤细胞S期延长,G2/G1值缩小,凋亡率明显升高(P<0.05)。结论Genistein与ADM联用对Tca8113细胞具有协同抑制作用,其作用机理可能是联合用药促使肿瘤细胞S期延长,G2/G1值缩小,提高了ADM的诱导凋亡效应。     

4，5，7-三羟基异黄酮和阿霉素诱导人舌癌细胞株Tea8113凋亡的研究

目的观察4,5,7-三羟基异黄酮（Genistein）和阿霉素（ADM）单用和联用对诱导体外培养的人舌癌细胞株rrca8113凋亡的影响。方法以体外培养的人舌癌细胞株Tca8113为实验模型,Genistein与ADM联合作用于癌细胞,倒置相差显微镜下观察药物处理后癌细胞的细胞形态学变化,四甲基偶氮唑蓝比色法分析药物对癌细胞的增殖抑制作用,流式细胞仪测定细胞凋亡率和细胞周期分布。结果Genistein和ADM两者单用对癌细胞均有生长抑制作用,且随剂量升高抑制趋势增强（P〈0．05）。两药联用对癌细胞的抑制作用优于两者的单纯相加（Q〉1）。联合用药使肿瘤细胞S期延长,G2／G1值缩小,凋亡率明显升高（P〈0．05）。结论Genistein与ADM联用对Tca8113细胞具有协同抑制作用,其作用机理可能是联合用药促使肿瘤细胞S期延长,G2／G1值缩小,提高了ADM的诱导凋亡效应。     

甲状旁腺激素对人牙乳头间充质细胞DNA合成、细胞周期和超微结构的影响

目的：观察和分析体外培养的人牙乳头间充质细胞经甲状旁腺激素（ｐａｒａｔｈｙｒｏｉｄ ｈｏｒｍｏｎｅ,ＰＴＨ） 作用后,ＤＮＡ合成、细胞周期和超微结构的变化情况,探讨ＰＴＨ 对人牙乳头间充质细胞生长和分化的生物学特性的影响。方法：流式细胞仪（ＦＣＭ） 分析、３Ｈ－ ＴｄＲ 掺入试验和透射电镜（ＴＥＭ） 观察。结果：ＰＴＨ 不影响体外培养的人牙乳头间充质细胞ＤＮＡ合成和细胞增殖;ＰＴＨ 能促进人牙乳头间充质细胞向分化成熟、功能活跃的方向发展,且有浓度依赖性。结论：ＰＴＨ 可能不影响体外培养的人牙乳头间充质细胞增殖,而仅促进细胞的分化和功能活跃。