在EBV阳性鼻咽癌细胞内SAHA联合GCV细胞毒作用的时间点分析

目的探讨伏立诺他（SAHA）联合GCV对EBV阳性鼻咽癌细胞的细胞毒作用有效时间点,为临床化疗提供依据。方法。利用Real-timePCR检测SAHA作用下EBV阳性鼻咽癌细胞C666TK的表达情况;通过MTI＂检测SAHA联合GCV的细胞毒性。结果SA—HA作用16h时TK的表达最多,并且MTr结果显示SAHA作用16h后联合GCV两者的细胞毒作用最强。结论SAHA作用16h后联合GCV抗鼻咽癌效果较好。     

重组adv5.oriP.HRP腺病毒载体构建及对EBV阳性鼻咽癌C666-1细胞株的靶向表达

目的:构建腺病毒载体adv5.oriP.HRP,观察其对EBV阳性鼻咽癌C666-1细胞株的靶向表达。方法:采用p△EIsp1A穿梭质粒系统,酶切、定向克隆方法构建含HRP自杀基因的p△EIsp1A/oriP-HRP重组质粒,在293细胞中进行重组腺病毒adv5.oriP.HRP包装、扩增、纯化与病毒滴度测定,体外转染EBV阳性鼻咽癌C666-1细胞株与EBV阴性鼻咽癌CNE-2Z细胞株,RT-PCR法检测不同细胞系中adv5.oriP.HRP的表达,Western blot法检测经不同浓度adv5.oriP.HRP转染后细胞中HRP蛋白的表达。结果:p△EIsp1A/oriP-HRP在293细胞中包装扩增后的病毒滴度为528@1012TCID50/L;体外转染鼻咽癌C666-1细胞株与CNE-2Z细胞株后,RT-PCR检测到C666-1细胞系1229bp处得明亮条带,而CNE-2Z细胞几乎未检测到重组基因mRNA的表达,Western blot可见随着MOI的增加C666-1细胞中HRP蛋白表达逐渐增多,而在CNE-2细胞中HRP蛋白的表达几乎呈阴性,两组间差别有统计学意义(P<0.05)。结论:重组腺病毒adv5.oriP.HRP能在EBV阳性的鼻咽癌C666-1细胞株内靶向性转染和表达。     

组织蛋白酶B在SAHA诱导乳腺癌MCF-7细胞凋亡过程中的调控作用

目的阐明组织蛋白酶B(cathepsin B,Cat B)在SAHA诱导的乳腺癌雌激素受体阳性细胞系MCF-7细胞凋亡中的调控作用。方法采用MTT法检测SAHA对乳腺癌MCF-7细胞生长状况的影响;应用ELISA方法测定SAHA作用于MCF-7细胞后相关蛋白表达变化的情况;通过Bio Station~(IM)活细胞工作站实时收集各种处理因素对乳腺癌MCF-7细胞增殖干预的形态学影响;并通过自动细胞分析仪Muse Cell Analyzer分析SAHA对MCF-7细胞活力和细胞凋亡的影响。结果 SAHA能明显抑制乳腺癌MCF-7细胞的增殖,其最佳作用浓度为10μmol·L~(- 1),最佳作用时间为24 h。ELISA结果表明,SAHA能诱导乳腺癌MCF-7细胞中Cat B的表达。实时活细胞工作站成像实验从形态学上证明,Cat B抑制剂Cystatin C和SAHA联合应用可有效阻止SAHA对MCF-7细胞生长的抑制作用。细胞学实验结果表明,SAHA能使MCF-7细胞活力下降,细胞凋亡发生率明显增高;然而经过Cystatin C处理后的MCF-7细胞活力增加,细胞凋亡发生率下降。结论 Cat B在SAHA诱导乳腺癌雌激素受体阳性细胞MCF-7凋亡过程中具有重要的调控作用。     

辛二酰苯胺异羟肟酸对人卵巢癌细胞恶性表型的抑制作用

目的 探讨辛二酰苯胺异羟肟酸(SAHA)对人卵巢癌细胞恶性表型的作用及其可能的分子作用机制。方法 （1）选取 2 组人卵巢癌细胞(SKOV3 和 SKOV3/DDP; HO8910 和 HO8910-PM)后, 设置对照组和终浓度为 1、 3、 5 及 7 μmol/L SAHA 组(SAHA 1~4 组), 采用 CCK-8 法分别检测 SAHA 对细胞增殖的影响。（2） 设置对照组和 SAHA 1、 2 组(2 和 5 μmol/L), Annexin V-FITC/PI 双标记流式细胞术分别检测 SAHA 对细胞凋亡及周期的影响。（3） RT-PCR 和 Western blot 检测 SAHA 1~4 组表型相关蛋白的表达水平。结果 （1）随着 SAHA 浓度的增加, SKOV3 细胞株 SKOV3/DDP 及 HO8910 细胞株 48 h 光密度 （OD） 值均呈逐渐降低趋势(均 P＜0.05)。（2） 48 h SAHA 1、 SAHA 2 组凋亡率高于对照组(均 P＜0.05); SAHA 作用 48 h, 细胞周期发生阻滞, 与对照组比较, SAHA 1 组和 SAHA 2 组 SKOV3 和 SKOV3/DDP 细胞 S 期和 G2/M 期比例增高, HO8910 和 HO8910-PM 细胞 G0/G1期比例增高(P＜0.05)。（3） SAHA 1、 2 组 CyclinB1 和 Cdc2(p34)的 mRNA 表达水平均低于对照组, Caspase-3、 p21 和 p53 的 mRNA 表达水平明显高于对照组(P < 0.05); SAHA 1~4 组 Ac-Histone H3 和 Ac-Histone H4 和 p53 蛋白的表达较对照组明显增强, CyclinB1、 Cdc2 (p34)蛋白的表达减弱。结论 SAHA 通过调节恶性表型相关蛋白 Caspase-3、 p53、 CyclinB1 和 Cdc2(p34)的表达水平, 促进组蛋白乙酰化, 抑制卵巢癌细胞增殖, 阻滞细胞周期并诱导细胞凋亡。     

SAHA-CTSV轴通过诱导过度自噬抑制乳腺癌MCF-7细胞的生长

目的研究SAHA-CTSV轴通过诱导过度自噬抑制乳腺癌MCF-7细胞生长的作用和机制。方法以人乳腺癌MCF-7细胞为研究目标,Muse细胞分析仪检测SAHA对细胞活力的作用;Real-time PCR和Western blot检测SAHA对CTSV的mRNA及蛋白表达的影响,同时检测利用siRNA干扰技术敲除CTSV后的细胞CTSV表达水平;细胞免疫荧光法检测SAHA对LC3II分子的作用;Real-time PCR和Western blot检测SAHA诱导自噬过程中相关ATG分子和LC3的mRNA和蛋白的变化;Bafilomycin A1抑制自噬,随后利用Western blot和Muse细胞分析仪分别检测p62蛋白水平和SAHA作用后的细胞活力。结果SAHA可以抑制MCF-7细胞的增殖,并促进CTSV的表达。CTSV-siRNA转染细胞后,CTSV的mRNA和蛋白水平均显著性降低。SAHA增加了LC3免疫荧光点的分布,与CTSV-siRNA共同作用可恢复由CTSV-siRNA敲减引起的细胞自噬减弱。SAHA在增强ATG4C和Beclin1表达的同时,LC3B也随之升高而mTOR则下降。抑制CTSV表达后也同时逆转了SAHA的效应。Bafilomycin A1阻断自噬后,被SAHA抑制的p62蛋白的表达和细胞活力显著增强。结论SAHA-CTSV轴通过诱导过度自噬抑制乳腺癌MCF-7细胞的生长。     

灵芝多糖诱导人肝癌细胞HepG2凋亡的研究

目的：研究灵芝多糖（GLPS）与化疗药氟尿嘧啶联合使用诱导人肝癌细胞HepG2凋亡作用。方法：MTT法测定细胞毒作用,流式细胞术检测细胞凋亡率,比色法测定Caspase-3、Caspase-8酶活性,Western—Blotting法检测细胞色素C、Caspase-3、Caspase-8蛋白表达情况。结果：灵芝多糖与氟尿嘧啶联合使用具有显著的细胞毒作用;流式细胞术检测,随着GLPS浓度增加,细胞凋亡率升高,Caspase-3、Caspase-8酶活性亦增强;Western-Blotting检测发现GLPS作用后细胞色素C、Caspase-3、Caspase-8蛋白表达不同程度增加。结论：灵芝多糖与氟尿嘧啶联合使用可通过引起细胞色素C的释放,活化Caspase诱导肝癌细胞凋亡。     

雄黄微生物提取液对K562／ADM细胞P-糖蛋白和多药耐药基因表达的影响

目的：研究雄黄微生物提取液（RBS）诱导K562／ADM细胞凋亡的作用,并探讨其对P-糖蛋白（P-gP）和多药耐药基因（MDRI）表达的影响。方法：采用“微生物浸出”法制备RBS,并以H3AsO3作为阳性对照,AnnexinV—PI双染法检测细胞凋亡;流式细胞仪检测细胞P—gP表达率;逆转录聚合酶链技术（RT-PCR）检测细胞MDRImRNA表达水平。结果：RBS可诱导多药耐药白血病细胞发生典型凋亡,抑制P-gp的表达,下调MDRI mRNA表达水平。在含砷量相同的条件下,RBS诱导效应明显强于H3AsO3。结论：诱导细胞凋亡、下调P-gp／MDRI蛋白的表达,可能是雄黄逆转白血病耐药的重要分子机制。     

EBV—DNA定量检测对鼻咽癌的诊断

目的通过对鼻咽癌患者经治疗后EBV—DNA含量变化的观察,为临床诊治陔病寻求最佳方法。方法血浆EBV—DNA检测采用荧光定量PCR法,选择确诊的145例鼻咽癌患者为研究对象,对其EBV-DNA进行定量检测,并以同期健康者140名的血浆情况进行对比研究。结果鼻咽癌组中有136例（93．79％）患者血浆EBV—DNA呈阳性：健康组中有10例（7．14％）血浆EBV—DNA呈阳性,鼻咽癌组患者出浆EBV—DNA阳性检出率明显高出健康组（P〈0．01）;血浆EBV-DNA呈阳性的患者在治疗过程中,血浆EBY—DNA含量呈逐渐下降趋势。结论给予鼻咽癌患者EBV—DNA定量检测,对诊断和指导治疗价值明显。     

姜黄素与紫杉醇联用对人子宫内膜癌细胞增殖的影响及其机制研究

目的探讨姜黄素（CCM）与紫杉醇（PIX）联用对人子宫内膜癌细胞（Ishikawa）增殖的影响及其机制。方法（1）设置对照组（不加药物）、CCM组（不同浓度的CCM）、PIX组、联合组（PIX联合不同浓度的CCM）;（2）采用MTT法检测不同浓度的CCM联合PIX对细胞活力的影响;（3）流式细胞术检测细胞凋亡率;（4）免疫组化法检测Caspase-3蛋白表达情况。结果CCM联用PIX能抑制人子宫内膜癌细胞的增殖（P〈0．05）,且随CCM浓度的增加,抑瘤作用增加;两种药物均可诱导细胞凋亡,且各组凋亡率与药物浓度呈正相关（P〈0．05）;免疫组化结果显示随着染毒浓度的增加和联合作用,Caspase-3蛋白的表达升高（P〈0．05）。两药联用抗瘤作用增强。结论CCM、PIX均可抑制人子宫内膜癌细胞的增殖、诱导细胞凋亡、上调细胞Caspase-3表达,呈剂量依赖性,且两者联用具有协同作用。     

肝素和阿霉素对人鼻咽癌CNE2细胞增殖与凋亡的协同作用

目的:观察肝素联合阿霉素对人鼻咽癌细胞的=增殖与凋亡的影响及其可能的分子机制.方法:用MTT法、流式细胞术观察肝素联合阿霉素对人鼻咽癌细胞增殖及细胞周期的影响;而用TUNEL、琼脂糖凝胶电泳、Westem blot等方法观察肝素与阿霉素联合应用对人鼻咽癌细胞凋亡的作用.结果:肝素与阿霉素联合应用后对鼻咽癌细胞的生长抑制及其凋亡具有显著增强作用,同阿霉素单独应用于诱导细胞凋亡相比,低剂量的肝素与阿霉素联合应用后发现:TUNEL阳性细胞从12.6％±1.1％增加到65.7％±1.3％;G_0/G_1期细胞阻止,S期细胞明显减少,DNA“梯形”变化更加明显;Bax/Bcl-2的比率及p~(53)和p~(21)基因的表达明显升高.结论:肝素与阿霉素对人鼻咽癌细胞在抗增殖及促凋亡方面有协同作用,这可能与Bax/Bcx-2比率的升高及p~(53)和p~(21)的表达上调有关.     

TPM1在鼻咽癌中的表达及其临床意义

目的观察TPM1基因的表达与鼻咽癌（NPC）的关系。方法采用实时定量rt-PCR技术检测TPM1基因在NPC细胞株（CNE1、CNE2、58F）和鼻咽上皮细胞株（NP69）中的表达;采用组织芯片技术通过免疫组化检测TPM1在124例恶性鼻咽癌肿瘤、31例对应的癌旁正常组织和12例正常鼻咽黏膜组织的表达。结果 TPM1 mRNA在NP69中表达水平较高,而在NPC细胞株中表达水平显著下降。正常鼻咽黏膜及癌旁组织中TPM1蛋白染色阳性表达率约为65.1%（28/43）;而在鼻咽癌组织中阳性表达率约为34.7%（43/124）;TPM1蛋白表达水平与肿瘤的侵犯范围（T）呈负相关（P〈0.05）,与颈淋巴结转移（N）无关（P〉0.05）。结论 TPM1基因表达的抑制可能参与鼻咽癌的发生。     

Fucoxanthin treatment inhibits nasopharyngeal carcinoma cell proliferation through induction of autophagy mechanism

Nasopharyngeal carcinoma (NPC) arises from the epithelium of the nasopharyngeal mucosa. Elderly people above the age of 65 years are more susceptible to NPC. Nasopharyngectomy is the renowned treatment procedure to NPC; however, it is too risky due to its complicated surgical procedure. Other treatment methods also reported with serious side effects such brain injury; hence, the alternative anticancer drug without any side effects was needed. Fucoxanthin is a carotenoid derived from marine algae with the numerous pharmacological functions. This study aims to examine the inhibitory potential in NPC cell proliferation via apoptosis and autophagy. The cytotoxicity of fucoxanthin on C666‐1 cells was observed by the MTT assay. The expression of autophagy‐linked proteins was assessed with immunoblotting analysis. The expression of autophagy protein LC3 was estimated using immunocytochemical analysis in C666‐1 and GFP‐LC3 transfected cells. Furthermore, the fucoxanthin‐treated C666‐1 cells were analyzed with TUNEL assay. The apoptotic level in the fucoxanthin‐treated C666‐1 cells was evaluated using acridine orange staining. Fucoxanthin significantly increased the expression of autophagy‐linked proteins which is clearly depicted in the immunoblotting analysis and immunocytochemical analysis of GFP‐tagged LC3 protein. The results of TUNEL assay of fucoxanthin‐treated C666‐1 in the presence autophagy inhibitors demonstrated the induction of autophagy by fucoxanthin. Acridine orange staining results of C666‐1 confirmed fucoxanthin decreases the expression of autophagy‐linked proteins during stressed condition thereby causes apoptosis. Our overall results authentically conclude that fucoxanthin induces autophagy and apoptosis in NPC cell line, and it can be ideal agent to treat nasopharyngeal cancer in future with further investigations.     

TESTIN在鼻咽癌中的表达及对鼻咽癌细胞系增殖、上皮-间质转化和侵袭转移的影响

目的 观察TESTIN在鼻咽癌组织和细胞中的表达,分析TESTIN过表达对鼻咽癌细胞增殖、上皮-间质转化(EMT)和侵袭转移的影响.方法 采用免疫组化、Real time-PCR、Western-blot检测TESTIN在鼻咽癌组织和细胞系的表达状况.利用脂质体LipofectamineTM2000转染鼻咽癌5-8F细胞,Real time-PCR、Western-blot验证转染效率.MTT、细胞划痕实验、细胞侵袭实验检测TESTIN过表达对5-8F细胞增殖、EMT和侵袭转移的影响.Western-blot检测TESTIN过表达对侵袭转移相关基因表达的影响.结果 TESTIN在鼻咽癌组织的阳性表达率和表达水平明显低于正常鼻咽部组织,差异有统计学意义(P<0.05).与人正常永生化鼻咽上皮细胞系NP69比较,鼻咽癌细胞系CNE1、CNE2、C666-1、5-8F、6-10B中TESTIN表达水平显著降低,差异有统计学意义(P<0.05).转染pcDNA3.1/TESTIN的5-8F细胞中TESTIN表达显著上调(P<0.05).TESTIN过表达显著抑制5-8F细胞增殖、EMT和侵袭转移,同时上调E-cadherin表达,下调N-cadherin、Vimentin、MMP-2、MMP-9表达(P<0.05).结论 鼻咽癌组织和细胞系中TESTIN表达显著降低,TESTIN过表达可抑制鼻咽癌5-8F细胞增殖、EMT和侵袭转移.     

EB病毒NAl一IgA和VCA—IgA抗体与鼻咽癌的诊断和疗效的关系

目的探讨EB病毒NAl一IgA和VCA—IgA抗体与鼻咽癌的诊断和治疗效果的相关性。方法使用酶联免疫吸附试验（ELISA）测定124例治疗前和治疗后鼻咽癌患者以及173例正常体检人群血清EB病毒NAl一IgA和VCA—IgA抗体。结果单项检测时EB病毒检测的敏感性和准确性分别为：NAl一IgA88．7％、91．2％,VCA—IgA75．8％、84．8％,两者联合检测的敏感性和准确性分别为97．6％、93．3％。鼻咽癌患者血清EBNAl一IgA抗体rA值和阳性率在临床各期（I期阳性率除外）治疗前后均无统计学差异（P〉0．05）,而鼻咽癌患者血清EBVCA—IgA抗体rA值和阳性率在临床各期（Ⅳ期除外）治疗后均比治疗前有显著下降（P〈O．05）。结论两项指标联合检测可提高早期鼻咽癌诊断的敏感性和准确性。血清EB病毒NAl一IgA抗体水平和阳性率基本与治疗效果无关．VCA—IgA抗体水平和阳性率与治疗呈明显的相关性,可作为鼻咽癌疗效判断以及监测病情和预后的重要指标。     

Preferential cytotoxicity of the type I ribosome inactivating protein alpha-momorcharin on human nasopharyngeal carcinoma cells under normoxia and hypoxia

All primary nasopharyngeal carcinoma (NPC) tumors contain hypoxic regions which are implicated in decreased local control and increased distant metastases, as well as resistance to chemotherapy in advanced NPC patients. One of the promising therapeutic approaches for NPC is to use drugs that can target hypoxic factors in tumors. In the present investigation, the type I ribosome inactivating protein α-momorcharin (α-MMC), isolated from seeds of the bitter gourd Momordica charantia, reduced cell viability and inhibited clonogenic formation of human NPC CNE2 and HONE1 cells under normoxia and cobalt chloride-induced hypoxia. By comparison, α-MMC exhibited only slight cytotoxicity on human nasopharyngeal epithelial NP69 cells under normoxia. Interestingly, α-MMC suppressed the expression levels of hypoxia-inducible factor 1-alpha (HIF1α) and vascular endothelial growth factor (VEGF) in hypoxic NPC, as well as the growth of human umbilical vein endothelial cells. Further study disclosed that α-MMC targeted endoplasmic reticulum and down-regulated unfolded protein response (UPR) in NPC cells. Moreover, α-MMC induced apoptosis in NPC cells in a dose- and time-dependent manner. It initiated mitochondrial- and death receptor-mediated apoptotic signaling in CNE2 cells, but there was hardly any effect on HONE1 cells. In addition, α-MMC brought about G0/G1 phase cell cycle arrest in CNE2 cells and S phase arrest in HONE1 cells. Collectively, α-MMC preferentially exhibited inhibitory effect on normoxic and hypoxic NPC cells partly by blocking survival signaling (e.g. HIF1α, VEGF and UPR), and triggering apoptotic pathways mediated by mitochondria or death receptor. These observations indicate the potential utility of α-MMC for prophylaxis and therapy of NPC.     

Enhanced suppression of tumor growth by concomitant treatment of human lung cancer cells with suberoylanilide hydroxamic acid and arsenic trioxide

The efficacy of arsenic trioxide (ATO) against acute promyelocytic leukemia (APL) and relapsed APL has been well documented. ATO may cause DNA damage by generating reactive oxygen intermediates. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, modulates gene and protein expression via histone-dependent or -independent pathways that may result in chromatin decondensation, cell cycle arrest, differentiation, and apoptosis. We investigated whether ATO and SAHA act synergistically to enhance the death of cancer cells. Our current findings showed that combined treatment with ATO and SAHA resulted in enhanced suppression of non-small-cell lung carcinoma in vitro in H1299 cells and in vivo in a xenograft mouse model. Flow cytometric analysis of annexin V+ cells showed that apoptotic cell death was significantly enhanced after combined treatment with ATO and SAHA. At the doses used, ATO did not interfere with cell cycle progression, but SAHA induced p21 expression and led to G1 arrest. A Comet assay demonstrated that ATO, but not SAHA, induced DNA strand breaks in H1299 cells; however, co-treatment with SAHA significantly increased ATO-induced DNA damage. Moreover, SAHA enhanced acetylation of histone H3 and sensitized genomic DNA to DNase I digestion. Our results suggest that SAHA may cause chromatin relaxation and increase cellular susceptibility to ATO-induced DNA damage. Combined administration of SAHA and ATO may be an effective approach to the treatment of lung cancer.     

Antitumor effect and enhancement of cytotoxic drug activity by cetuximab in nasopharyngeal carcinoma cells

BACKGROUND: Nasopharyngeal carcinoma (NPC) is the most common head and neck cancer in southern China and South East Asia. Epidermal growth factor receptor (EGFR) has been proposed as a new target for anticancer therapy. EGFR was over-expressed in 85% of NPC tissues and was associated with poor prognosis. MATERIALS AND METHODS: EGFR protein expression in four NPC cell lines, CNE-2, HONE-1, HK1 and C666-1, was examined by Western immunoblotting. The antitumor effect of cetuximab was studied in the cell lines, either alone or in combination with cisplatin or paclitaxel. RESULTS: EGFR protein expression was highest in the HK1 cell line, moderate in CNE-2 and HONE-1, and lowest in C666-1. Single agent cetuximab demonstrated significant antitumor effect in the HK1 and HONE-1 cell lines, but minimal activity in CNE-2 and C666-1 cells. When cetuximab was combined with cisplatin or paclitaxel in the HK1 and HONE-1 cell lines, an additive enhancement of cytotoxic drug activity was demonstrated. CONCLUSION: Cetuximab demonstrated single agent activity selectively in NPC cell lines with moderate to high EGFR protein expression. Cetuximab could also additively enhance the antitumor effects of cisplatin or paclitaxel in these NPC cell lines. These results support the rationale of combining cetuximab with current standard chemotherapy to further improve the therapeutic ratio in the treatment of NPC. Future studies should aim at defining the predictive markers for response to cetuximab in order to select the responsive tumor for the correctly targeted agents.