五步蛇毒AAVC-I对人口腔鳞癌细胞的增殖抑制作用

目的观察祁门五步蛇毒抑瘤组分I(Agkistrodon acutus venom component-I, AAVC-I)对人口腔鳞癌Tca8113细胞的增殖抑制及凋亡的影响。方法以离子交换和凝胶过滤法从安徽祁门五步蛇粗毒中分离纯化五步蛇毒AAVC-I。常规培养口腔鳞癌Tca8113细胞,观察正常细胞形态学。实验分为对照组(加入等量细胞培养液)和实验组(分别加入AAVC-I 2.5、5.0、10.0μg/ml)。采用MTT法检测不同浓度(2.5、5.0、10.0μg/ml)的AAVC-I对Tca8113细胞24、48及72 h的增殖抑制率,倒置显微镜观察不同浓度AAVC-I处理后的细胞形态变化,透射电镜及AO/EB双荧光染色观察AAVC-I对细胞凋亡的影响,并与对照组进行比较。结果与对照组比较,实验组AAVC-I对Tca8113细胞增殖具有明显抑制作用(P0.05);AAVC-I处理后,倒置显微镜下可见明显细胞凋亡现象,电镜下可见细胞核染色质、胞质浓缩及凋亡小体;AO/EB双荧光染色可见细胞膜完整,细胞核致密浓染,染成黄绿色荧光的早期凋亡细胞及细胞膜破损,细胞核浓聚及偏移,呈橘红色荧光的晚期凋亡细胞。结论祁门五步蛇毒AAVC-I能抑制人口腔鳞癌Tca8113细胞生长增殖,诱导其凋亡。     

MS-275联合平阳霉素对Tca-8113细胞生长和凋亡的影响

目的:观察组蛋白去乙酰化酶抑制剂(HDACIs)MS-275联合抗生素类化疗药物平阳霉素(PYM)对口腔鳞状癌细胞Tca-8113的生长及凋亡的影响。方法:以体外培养的口腔鳞状细胞癌Tca-8113细胞为研究对象,应用MTT法检测不同浓度(0、1、2、4、8μmol/L)MS-275、(0、0.05、0.1、0.2、0.4μmol/L)平阳霉素(PYM)单独和联合用药对Tca-8113细胞增殖活性的影响;Annexin-V-FITC/PI双染流式细胞术定量检测细胞的凋亡情况。结果:不同浓度(1、2、4、8μmol/L)MS-275和(0.05、0.1、0.2、0.4μmol/L)PYM均可显著抑制Tca-8113细胞的增殖,并促进其凋亡,且呈浓度依赖性(P0.05)。4μmol/L MS-275和0.2μmol/L PYM联合应用时,其抑制Tca-8113细胞增殖和促进其凋亡的作用均显著高于单独应用MS-275或PYM(P0.05)。结论:MS-275能有效增强口腔鳞状细胞癌Tca-8113细胞对平阳霉素(PYM)的敏感性,体外实验中呈现较好的抗肿瘤效应。     

miR-9-5p靶向Ambra1促进舌癌细胞增殖

miRNAs在肿瘤中异常表达,且与肿瘤的发生发展密切相关。目前发现,miR-9-5p在肿瘤中可能发挥原癌或抑癌效应,功能尚未完全阐述清楚。本文拟探讨miR-9-5p在舌癌中的作用。前期研究中收集10例舌癌组织及配对的癌旁组织,实时荧光定量PCR技术检测后发现,miR-9-5p在舌癌组织中的表达量显著高于癌旁组织,且其在舌癌细胞中的表达量也明显高于正常舌上皮细胞。此外,在舌癌细胞Tca8113中过表达miR-9-5p显著增加细胞的增殖能力。生物信息学预测及双荧光素酶报告基因实验证实,miR-9-5p可直接结合在自噬/苄氯素1调节因子1（activating molecule in beclin1-regulated autophagy, Ambra1）的 3′-UTR区域,靶向抑制Ambra1表达。Western印迹结果证实过表达miR-9-5p降低Ambra1的表达,反之亦然。Ambra1在舌癌细胞中的表达量显著低于正常舌上皮细胞。BrdU实验证实在舌癌细胞SCC-25中过表达Ambra1可显著抑制其增殖能力;相反,使用siRNA技术沉默Ambra1能够显著促进Tca8113细胞的增殖。在干预miR-9-5p的细胞中同时干预Ambra1的表达,结果发现Ambra1可显著逆转miR-9-5p对舌癌细胞增殖的促进作用。总之,miR-9-5p在舌癌中可能发挥原癌基因样作用,通过直接靶向抑制Ambra1表达进而促进舌癌细胞发生增殖。     

建国以来我国自建的一些细胞株或系(四) 人舌鳞状细胞癌Tca8113细胞系的建立及其生物学特性

Tca 8113系细胞,组织培养来自一例舌癌女病人(住院号217649)的原发灶,临床及病理诊断为舌鳞状细胞癌Ⅰ级(T_2N_1a Mo,Ⅱ期),未经任何放疗或化疗等处理,于1981年3月17日取手术标本,用含20％灭活小牛血清和适量     

miR-9-5p靶向抑制Ambra1促进舌癌细胞增殖

miRNAs在肿瘤中异常表达,且与肿瘤的发生发展密切相关。目前发现,miR-9-5p在肿瘤中可能发挥原癌或抑癌效应,功能尚未完全阐述清楚。本文拟探讨miR-9-5p在舌癌中的作用。前期研究中收集10例舌癌组织及配对的癌旁组织,实时荧光定量PCR技术检测后发现,miR-9-5p在舌癌组织中的表达量显著高于癌旁组织,且其在舌癌细胞中的表达量也明显高于正常舌上皮细胞。此外,在舌癌细胞Tca8113中过表达miR-9-5p显著增加细胞的增殖能力。生物信息学预测及双荧光素酶报告基因实验证实,miR-9-5p可直接结合在自噬/苄氯素1调节因子1(activating molecule in beclin1-regulated autophagy,Ambra1)的3'-UTR区域,靶向抑制Ambra1表达。Western印迹结果证实过表达miR-9-5p降低Ambra1的表达,反之亦然。Ambra1在舌癌细胞中的表达量显著低于正常舌上皮细胞。Brd U实验证实在舌癌细胞SCC-25中过表达Ambra1可显著抑制其增殖能力;相反,使用siRNA技术沉默Ambra1能够显著促进Tca8113细胞的增殖。在干预miR-9-5p的细胞中同时干预Ambra1的表达,结果发现Ambra1可显著逆转miR-9-5p对舌癌细胞增殖的促进作用。总之,miR-9-5p在舌癌中可能发挥原癌基因样作用,通过直接靶向抑制Ambra1表达进而促进舌癌细胞发生增殖。     

microRNA-21对人舌鳞癌细胞的增殖和凋亡的影响

目的:探讨micro RNA-21(mi R-21)对人舌鳞癌细胞增殖和凋亡的影响。方法:选取8例舌鳞癌组织和4例癌旁组织为研究材料,采用实时荧光定量聚合酶链式反应(q RT-PCR)法对舌鳞癌及癌旁组织中的mi R-21相对表达量进行检测,利用人工合成的mi R-21mimic对人舌鳞癌Tca8113细胞进行瞬时转染,采用q RT-PCR法对Tca8113细胞中mi R-21相对表达量进行检测,采用四唑盐比色法(MTT)法对Tca8113细胞增殖情况进行检测,采用流式细胞术对Tca8113细胞周期与凋亡情况进行检测。结果:舌鳞癌组织中mi R-21的相对表达量(3.502±0.674),高于癌旁组织(0.998±0.192),差异有统计学意义(P0.05)。mi R-21mimic导致了Tca8113细胞中的mi R-21相对表达量上调(6.864±1.324),明显高于对照scramble组[(0.997±0.187),P0.05],对Tca8113细胞的增殖发挥了促进作用(P0.05)。经mi R-21mimic转染之后,Tca8113细胞进入S期的细胞出现了明显的增加[(27.4±5.1)%vs(48.6±8.7)%,P0.05],处于G1期的细胞出现了显著的减少[(56.3±9.6)%vs(36.2±7.2)%,P0.05],细胞凋亡数量出现了显著减少[(9.4±2.3)%vs(18.6±3.9)%,P0.05]。结论:mi R-21在舌鳞癌组织中高表达,过表达mi R-21有效促进了Tca8113细胞的增殖,抑制细胞的凋亡,mi R-21在舌鳞癌诊断和治疗可能具有一定的新型靶点价值。     

铜绿假单胞菌L型诱导血管内皮细胞凋亡机制的探讨

目的探讨铜绿假单胞菌L型诱导血管内皮细胞凋亡机制。方法 Annexin V-FITC/PI双染荧光法和流式细胞术法检测铜绿假单胞菌L型感染血管内皮细胞8 h时的凋亡率,分光光度法检测细胞Caspase-9活化情况。结果铜绿假单胞菌L型和血管内皮细胞共培养8 h时细胞凋亡率、Caspase-9活化程度较对照组增高（P〈0.05）。结论铜绿假单胞菌L型可通过活化Caspase-9的线粒体途径诱导血管内皮细胞凋亡。     

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡及其机理研究

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡从而抑制其细胞生长.为了研究该过程的作用机理,我们研究了丝裂原活化蛋白激酶(MAPKs),包括胞外信号调节激酶(ERK1/2),c-jun氨基末端蛋白激酶(JNK)和p38丝裂原活化蛋白激酶(MAPK),在该过程中的磷酸化特征与动态变化.结果表明,柴胡提取物显著的增加了p38丝裂原活化蛋白激酶和胞外信号调节激酶(ERK1/2)的磷酸化作用,其增加值在测试范围内与测试剂量和作用时间成正相关,但在柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡过程中,没有发现对氨基末端蛋白激酶(JNK)表现出磷酸化活性.柴胡提取物诱导白血病HL-60的细胞凋亡部分归结于对p38丝裂原活化蛋白激酶的上调节作用,这种上调节作用能够受到p38 MAPK特异性的抑制剂SB203580的部分逆转,而MEK的抑制剂U0126则对柴胡提取物诱导HL-60细胞凋亡过程中的胞外信号调节激酶(ERK1/2)的磷酸化具有显著的协同效应.这是首次报道柴胡提取物在诱导人白血病细胞HL-60细胞凋亡过程中参与p38丝裂原活化蛋白激酶的磷酸化,同时柴胡提取物作为胞外信号调节激酶(ERK1/2)抑制剂的协同作用物具有相应的药物学功能.     

骨形成蛋白Ⅱ型突变受体基因转染对口腔鳞癌细胞增殖和凋亡的影响

目的探讨骨形成蛋白（BMPs）与口腔鳞癌的发生、发展的可能关系。方法将含有BMP-Ⅱ突变受体的真核表达载体转染入Tea8113舌癌细胞,筛选和鉴定后,构建稳定表达BMP-Ⅱ突变受体的细胞株tBRⅡ-Tea8113。对tBRⅡ—Tca8113细胞和Tca8113细胞分别进行MTT检测,流式细胞仪（FCM）分析、BrdU标记检测细胞的增殖活性及DNA合成;检测tBRⅡ-Tca8113细胞和Tea8113细胞的凋亡及细胞周期相关因子（CyclinD1,CDK-4,p27,p57）的表达。结果Tea8113细胞和tBRⅡ-Tea8113细胞的增殖指数MTT检测为0．47±0．01和0．35±0．008（t=22．953,P=0．000）,BrdU检测为12．0±3．4和23．0±1．9（f=6．918,P=0．000）,FCM检测为6.3和7．9;两组的凋亡指数为3．7±1．2和8．7±1．6（t=29．583,P=0．000）;细胞周期因子在Tca8113和tBRⅡ-ca8113细胞中的平均灰度测量值为CyclinD1（186．5±2．4和145．6±3．9,t=28．244,P=0．000）,CDK4（169．9±2．9和129．5±3．2,t=29．583,P=0．000）,p27（110．1±1．1和167．34-1．8,f=85．754,P=0．000）,p57（107．9±2．1和156．8±2．2,t=50．844,P=0．000）。结论BMPs及其受体可能在口腔上皮组织的恶变过程中有重要作用,研究结果为探讨BMPs信号在上皮性肿瘤中的作用提供了重要依据。tBRⅡ-Tea8113细胞的建立,进一步表明了BMPs及其受体在口腔上皮组织的恶变过程中有重要作用,并为进一步探讨BMP信号在上皮性肿瘤的恶变过程中的作用奠定了基础。     

Identification of carbonic anhydrase 9 as a contributor to pingyangmycin-induced drug resistance in human tongue cancer cells

Drug resistance is the major obstacle to successful cancer treatment. To understand the mechanisms responsible for drug resistance in tongue cancer, Tca8113 cells derived from moderately differentiated human tongue squamous cell carcinoma were exposed to stepwise escalated concentrations of pingyangmycin (PYM) to develop the resistant cell line called Tca8113/PYM, which showed over 18.78-fold increased resistance to PYM as compared with Tca8113 cells, and cross-resistance to cisplatin, pirarubicin, paclitaxel, adriamycin, and mitomycin. We found that the resistance was not associated with multidrug resistance transporter 1 (p170, p-gp), multidrug resistance-associated protein 1 and breast cancer resistance protein overexpression, so we hypothesized that Tca8113/PYM cells must have some other resistance mechanism selected by PYM. To test this hypothesis, the global gene expression profiles between Tca8113 and Tca8113/PYM cells were compared by cDNA microarray. Eighty-nine genes and thirteen expressed sequence tags with differential expression levels between the two cell lines were identified. Some differential expression levels were validated with real-time PCR and western blot. Furthermore, the functional validation showed that both carbonic anhydrase (CA) inhibitor acetazolamide application and CA9 silencing with CA9 antisense oligonucleotides contribute to the medium pH increase of Tca8113/PYM cells and enhanced PYM chemosensitivity. Moreover, both acetazolamide and CA9 antisense oligonucleotides significantly increased PYM-induced caspase 3 activation in Tca8113/PYM cells. Thus, our study suggests that the resistance of Tca8113/PYM cells is probably associated with CA9 and other differential expression molecules, and that CA9 may be an important marker for prediction of PYM responsiveness in tongue cancer chemotherapy.     

Cloning and functional characterization of TCRP1, a novel gene mediating resistance to cisplatin in an oral squamous cell carcinoma cell line

To explore the mechanisms of chemotherapy resistance, we previously established a multi-drug resistant cell line, Tca8113/Pingyangmycin (Tca8113/PYM) and identified differential expression in known genes and ESTs using microarray analysis. From among those ESTs we have now identified a novel gene producing an mRNA of 1834 nucleotides translated into a protein having 235 amino acids. This gene was denominated as tongue cancer resistance-associated protein 1 gene (TCRP1, accession number: EF363480). We further determined its functional characteristics. The results demonstrate that TCRP1 mediates a specific resistance to cisplatin in Tca8113 cells by reducing the cisplatin-induced apoptosis. This suggests that TCRP1 might be a novel molecular target to develop agents to reverse cisplatin-induced chemoresistance.     

Fat-1 基因真核表达载体的构建及其对人口腔鳞癌细胞 脂肪酸含量的影响

目的:构建真核表达载体pcDNA3.1-Fat1,线性化稳定转染人口腔鳞癌细胞株Tca8113,检测其细胞内脂肪酸含量变化。方法:通过重叠延伸PCR方法人工合成利于真核表达的Fat-1基因,用基因重组技术构建真核表达载体pcDNA3.1-Fat-1,用脂质体转染真核细胞的方法转染人口腔鳞癌细胞株Tca8113,用气相色谱仪检测脂肪酸的变化情况。结果:测序及酶切鉴定成功合成真核偏好表达的Fat-1基因。与对照组相比,转染Fat-1基因的口腔癌细胞的n-3脂肪酸明显增多,n-6/n-3明显下降。结论:成功构建真核表达载体pcDNA3.1-Fat1,并对口腔鳞癌细胞内脂肪酸含量产生明显影响,为进一步研究Fat-1基因在口腔鳞癌中的生物学功能奠定了基础。     

HuR stabilizes lnc-Sox5 mRNA to promote tongue carcinogenesis

Long noncoding RNAs (lncRNAs) have been recently regarded as systemic regulators in multiple biological processes including tumorigenesis. In this study, we report an ultra-highly expressed lncRNA, lnc-Sox5, in tongue tumor tissues. The results imply that lnc-Sox5 may play vital role in tongue carcinoma progression. We observed that the growth of Tca8113 cells was suppressed by lnc-Sox5 downregulation. Additionally, lnc-Sox5 knockdown simultaneously increased Tca8113 cell apoptosis, but the cell cycle was arrested. RNA immunoprecipitation suggested that HuR directly bound to and stabilized lnc-Sox5 RNA. Consistently, HuR knockdown reduced the level of lnc-Sox5 in Tca8113 cells. However, overexpression of HuR induced more lnc-Sox5 in Tca8113 cells. Both lnc-Sox5 knockdown and HuR knockdown suppressed Tca8113 cell tumorigenesis in xenograft models. These results suggest that lnc-Sox5, which was stabilized by HuR, could regulate carcinogenesis of tongue cancer and may serve as a predicted target for tongue carcinoma therapies.     

Fat-1基因真核表达载体的构建及其对人口腔鳞癌细胞脂肪酸含量的影响

目的：构建真核表达载体pcDNA3．1-Fat1,线性化稳定转染人口腔鳞癌细胞株Tca8113,检测其细胞内脂肪酸含量变化。方法：通过重叠延伸PCR方法人工合成利于真核表达的Fat-1基因,用基因重组技术构建真核表达载体pcDNA3．1-Fat—1,用脂质体转染真核细胞的方法转染人1：／腔鳞癌细胞株Tca8113,用气相色谱仪检测脂肪酸的变化情况。结果：测序及酶切鉴定成功合成真核偏好表达的Fat-1基因。与对照组相比,转染Fat—1基N的口腔癌细胞的n-3脂肪酸明显增多,n-6／n-3明显下降。结论：成功构建真核表达载体pcDNA3．1-Fat1,并对口腔鳞癌细胞内脂肪酸含量产生明显影响,为进一步研究Fat-1基因在口腔鳞癌中的生物学功能奠定了基础。     

辣椒素对舌鳞癌细胞增殖和凋亡影响的初步研究

目的:研究辣椒素对人舌鳞癌细胞增殖和存活的影响。方法:不同浓度辣椒素处理TSCCA和Tca8113细胞24,48和72h,MTS法检测细胞增殖,Western Blot法检测细胞周期调控和细胞凋亡相关分子的表达。结果:随着辣椒素浓度的增加,p53,p21和p27表达上调,Bcl-2表达未受到明显影响,Bcl-XL和Mcl-1表达抑制,caspase7和PARP剪切体表达上调。结论:辣椒素呈剂量依赖和时间依赖性抑制TSCCA和Tca8113细胞增殖,其机制可能与细胞周期和细胞凋亡相关分子表达的改变相关。     

Proteomic Analyses of Sirt1-Mediated Cisplatin Resistance in OSCC Cell Line

Oral squamous cell carcinoma (OSCC) accounts for about 90% of malignant oral lesions, and is recognized as the third most common cancer in developing nations and the sixth most common cancer worldwide. While chemotherapy remains the primary treatment for both resectable and advanced OSCC, most OSCC are naturally resistant to anticancer drugs, rendering new therapeutic avenues in dire need. Sirt1, a class III histone deacetylase, was linked to cisplatin resistance in several cancer types; however, the underlying mechanism is still unclear. Here, we demonstrated that overexpression of Sirt1 survived OSCC cell line Tca8113 under cisplatin treatment. Notably, BML-210, a chemical inhibitor of class III histone deacetylase, significantly abolished Sirt1-mediated cisplatin resistance in Tca8113 cells. Further, inactivation of endogenic Sirt1 by nicotinamide markedly increased chemo-sensitivity in cisplatin resistant sub-cell line Tca8113/CDDP. Proteomic strategy was applied to profile the differentially expressed proteins between pcDNA3.1-Sirt1- and mock vector-treated Tca8113 cells. Among 54 spots identified, 31 proteins were up-regulated and 23 proteins were down-regulated upon Sirt1 expression. Expression of four proteins with most significant alteration, including Annexin A4, Stathmin, SOD2 and thioredoxin, were validated by both RT-PCR and Western blot. Finally, we showed that Sirt1 could prevent cisplatin-induced ROS accumulation in Tca8113 cells. Our findings are considered as a significant step toward a better understanding of Sirt1-mediated cisplatin resistance.     

Purification and biochemical characterization of a novel protein-tongue cancer chemotherapy resistance-associated protein1 (TCRP1)

Multidrug resistance is a major obstacle to successful treatment of oral squamous cell carcinoma (OSCC). Lately, we found a novel human gene named tongue cancer chemotherapy resistance-associated protein1 (TCRP1) in the tongue cancer multi-drug resistance cell line (Tca8113/PYM) established by us. In this study, we focus on recombinant expression, purification, and biochemical characterization of TCRP1. After molecular cloning and purification of the gene encoding the 24-kDa protein, a mouse polyclonal antibody against TCRP1 was prepared, and the specialty of the antibody was confirmed by Western blot. The cell proliferation was evaluated by MTS assay and DNA damage was determined by comet assay, the results indicated that this protein especially mediated the cell's resistance to cisplatin; it was associated with its role of providing protection against DNA damage. We also found that TCRP1 expression was increased in cisplatin-resistant carcinoma cell lines (Tca/PYM and A549/DDP), but not in cisplatin-sensitive MDR cell lines (MCF-7/5-Fu), compared with their parental counterparts by Western blot analysis. Immunofluorescence and immunohistochemical analysis showed TCRP1 is mainly expression in cytoplasmic, the Mann-Whitney U test exhibited that TCRP1 positive patients predicted the worst sensitive with cisplatin of OSCC patients. All these findings suggest that TCRP1 is a novel cisplatin-resistant protein which is mainly localized in the cytoplasm and can mediate cisplatin resistance against DNA damage; the expression level of TCRP1 in patients with OSCC may be useful as an indicator of therapeutic efficacy of the sensitivity to cisplatin.     

CCN5 inhibits proliferation and promotes apoptosis of oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) is a common cancer with poor prognosis and high mortality. The role of CCN5 has attracted a great focus on the regulation of cancer progression. However, the biological function and mechanism of CCN5 in OSCC are still not well elucidated. The current study was designed to determine the effects of CCN5 on OSCC cell proliferation and apoptosis using two OSCC cell lines. Further, LY294002, a PI3K/AKT antagonist, was employed to explore the mechanism underlying the effects of CCN5 in the regulation of OSCC. The results showed that overexpression of CCN5 in TSCCa cells significantly reduced viable cell number, arrested cell cycle, and suppressed cell‐cycle regulators (cyclin D1, cyclin E, and CDK2). CCN5 overexpression increased the apoptotic ratio and Hoechst‐positive cell number, and altered the apoptotic‐related proteins (caspase‐3/9, Bax, and Bcl‐2). However, CCN5 silencing induced opposite effects on cell proliferation and apoptosis in Tca‐8113 cells. In addition, we observed that CCN5 knockdown increased the expression levels of PI3K (p85α and p110α) and phosphorylated AKT at serine 473 (p‐AKT Ser473) in Tca‐8113 cells. Inhibiting PI3K/AKT signaling with LY294002 rescued the apoptotic process in CCN5‐silenced OSCC cells. Finally, xenograft analysis showed that CCN5 represses tumorigenesis of OSCC cells. These findings together suggest that CCN5 functions as a tumor suppressor for OSCC cell development through inactivation of PI3K/AKT signaling pathway, providing a potential candidate for OSCC therapy.