Ⅰ型磷酸酶抑制亚基-1对人宫颈癌HeLa细胞增殖的抑制作用

PPI1(Inhibitor-1 ofprotein phosphatase 1)是I型磷酸酶的抑制亚基之一,其活化依赖于35位苏氨酸蛋白激酶(PKA)的磷酸化而发挥抑制作用.本研究目的在于探讨PPIl持续活化型突变体的表达对人宫颈癌细胞株增殖的影响及其可能的作用机制.利用PPI1野生型和活化型突变体表达质粒分别转染HeLa细胞,首先通过H~3TdR掺入实验、迁移实验观察PPI1基因对HeLa细胞增殖能力的影响,研究结果表明:在活化型突变体表达的HeLa细胞株中,细胞~3H掺入量和迁移能力明显受抑.其次通过流式细胞术、Giemsa染色法分析PPIl对HeLa细胞的细胞周期的影响;FACS分析表明HeLa细胞G2/M期比例明显升高;经脱氧胸苷同步化后,该组细胞进入有丝分裂期明显滞后.最后利用Western blot分析PPI1对MAPK信号转导通路的影响,Western blot分析显示该组细胞的ERK磷酸化水平明显下降.研究表明PPI1活化型突变体的表达可抑制人宫颈癌细胞株的增殖,这与其诱导HeLa细胞G2/M期停滞、有丝分裂的进入延缓有关,其中涉及到MAPK信号转导通路的活化受抑制.     

趋化因子CXCL5对宫颈癌HeLa细胞恶性表型的影响及机制

目的研究趋化因子CXCL5对宫颈癌HeLa细胞恶性表型的影响及其机制。方法通过基因转染构建过表达趋化因子CXCL5的宫颈癌HeLa细胞株,研究过表达CXCL5对宫颈癌HeLa细胞恶性行为和肿瘤相关基因表达的影响。结果 CCK-8、集落形成和划痕实验结果显示,过表达CXCL5可明显促进HeLa细胞的增殖和迁移能力;Western Blot实验结果表明,与空载体转染细胞株相比较,CXCL5过表达HeLa细胞株的ERK和p-ERK蛋白表达水平显著提高。结论趋化因子CXCL5通过ERK信号通路经自分泌途径促进宫颈癌细胞的增殖与迁移。     

阿霉素联合顺铂诱导宫颈癌细胞凋亡实验研究

目的:研究化疗药物阿霉素(ADM)联合顺铂(DDP)对宫颈癌CaSki细胞株的增殖及凋亡的影响,并探讨其可能的相互作用机制。方法:应用MTT比色法检测不同浓度阿霉素、顺铂单独和联合应用对宫颈癌CaSki细胞的增殖抑制作用;同时RT-PCR法在mRNA水平上检测Bcl-2和TNF-α基因表达量的变化。结果:两种药物单独应用均可抑制CaSki细胞的增殖,联合用药(<12μg/mL)时具有协同抑制作用并与各药物单一应用比较具有显著性差异(P<0.05);阿霉素、顺铂作用CaSki细胞后能上调TNF-α基因和下调Bcl-2基因的表达。结论:宫颈癌CaSki细胞在化疗药物阿霉素和顺铂两药联合作用下通过诱导TNF-α和Bcl-2 mRNA表达量的变化发挥协同抑制作用,同时TNF-α的高表达增强了化疗药物阿霉素和顺铂诱导肿瘤细胞凋亡的敏感性,其机制主要与凋亡诱导效应有关。     

野艾蒿挥发油对HeLa癌细胞形态与结构的影响

采用MTT法检测细胞活力,用倒置显微镜、荧光显微镜和扫描电子显微镜观察细胞形态与结构的变化,用激光共聚焦显微镜观察细胞微管的分布,从而研究了野艾蒿挥发油对HeLa人宫颈癌细胞形态与结构的影响。结果表明:(1)野艾蒿挥发油对HeLa癌细胞的增殖有明显的抑制作用,呈剂量和时间依赖性。(2)野艾蒿挥发油处理HeLa癌细胞24h后,100、200μg/mL实验组细胞体积缩小,核染色质凝集、微绒毛消失、细胞表面有泡状突起,微管解聚,呈现典型的凋亡特征;400μg/mL实验组细胞膜破裂、胞浆内含物外泄,呈明显的坏死特征。(3)野艾蒿挥发油具有抑制HeLa癌细胞增殖的作用,低、中浓度的野艾蒿挥发油诱导细胞凋亡,而高浓度的野艾蒿挥发油引起细胞坏死。     

腺病毒载体介导的HPV16E7基因沉默及其对宫颈癌CaSki细胞的影响

目的:构建利用RNA干扰技术沉默HPV16E7基因的腺病毒载体,并探讨其对人宫颈癌细胞系CaSki细胞增殖的影响,以及腺病毒载体在宫颈癌基因治疗中的可行性。方法:利用腺病毒载体介导的RNA干扰对CaSki细胞中HPV16E7蛋白的表达进行抑制。显微镜观察细胞病变情况。MTT实验用来检测腺病毒载体感染的CaSki细胞的增殖情况。结果:成功构建了用以介导CaSki细胞中HPV16E7基因沉默的腺病毒载体。腺病毒感染后的CaSki细胞发生典型病理变化,HPV16E7基因表达受到明显抑制,细胞分裂明显减慢。结论:腺病毒载体介导的RNA干扰能够特异性的沉默HPV16E7基因,抑制CaSki细胞的增殖,为腺病毒载体用于宫颈癌基因治疗的可行性提供了依据。     

鳞盖肉齿菌的抗肿瘤细胞增殖活性及抗肿瘤细胞增殖机制的研究

为了从鳞盖肉齿菌(Sarcodon scabrosuskarst)的二氯甲烷提取物中分离纯化得到Sarcodonin G,对Sarc-odonin G进行抗肿瘤细胞增殖活性及其抗肿瘤细胞增殖机制的研究。本文利用MTT实验法测定Sarcodonin G对HeLa细胞株增殖的抑制率,检测其抗肿瘤细胞增殖的效果;利用流式细胞术检测Sarcodonin G对HeLa细胞的凋亡的影响;利用电镜技术观察Sarcodonin G对HeLa细胞形态学改变。结果发现,Sarcodonin G对体外培养的HeLa细胞的增殖具有明显抑制作用,其IC50为7.19μmol/L,并有较好的剂量依赖关系;流式细胞学检查发现Sarcodonin G处理后的HeLa细胞出现凋亡现象;超微结构发现Sarcodonin G处理后的HeLa细胞的胞核染色质浓缩,边集,核固缩及形成新月体,线粒体肿胀,空泡样变,胞浆出现大量空泡等凋亡的形态学改变。结果表明,鳞盖肉齿菌的纯化物Sarcodonin G能抑制体外培养的HeLa细胞的增殖,并初步推测Sarcodonin G有可能是通过诱导HeLa细胞的凋亡来实现其抗肿瘤增殖作用的。     

羟基丹参酮ⅡA的合成及体外抗肿瘤作用

对丹参酮ⅡA进行结构修饰,合成了羟基丹参酮ⅡA。采用MTT法考察了羟基丹参酮ⅡA对人宫颈癌细胞株Hela细胞,人肝癌细胞株HepG-2细胞、人胃癌细胞株SGC-7901细胞的增殖抑制作用。结果表明:羟基丹参酮ⅡA对三种肿瘤细胞增殖都有很好的抑制作用,抑制作用呈剂量依赖性。羟基丹参酮ⅡA对SGC-7901细胞抑制作用最强,其IC_(50)为4.18μM;对HeLa细胞的抑制作用次之,其IC_(50)为6.08μM;对HepG-2细胞抑制作用较弱,其IC_(50)为10.20μM。而丹参酮ⅡA对SGC-7901细胞、HeLa细胞和HepG-2细胞的IC_(50)分别是17.15μM、27.28μM和46.34μM。羟基丹参酮ⅡA抑制肿瘤细胞增殖作用明显强于丹参酮ⅡA(P0.05)。     

肉桂油成分分析及肉桂醛体外抗肿瘤活性研究

目的探讨肉桂醛对不同肿瘤细胞株的生长抑制作用。方法通过水蒸气蒸馏法提取肉桂油,用气相色谱—质谱联用仪进行肉桂油成分分析;采用噻唑蓝（MTT）比色法检测肉桂醛对体外培养的人宫颈癌细胞系HeLa细胞株、人肺癌细胞系A-549细胞株和人肝癌细胞系HepG2细胞株的生长抑制作用,计算半数抑制浓度（IC50）。结果肉桂油的收率为1.96%,分析了肉桂油中的10种成分,主要为肉桂醛,占总馏出峰面积的93.94%;肉桂醛能抑制人宫颈癌细胞系HeLa细胞、人肺癌细胞系A-549细胞和人肝癌细胞系HepG2细胞增殖,且呈剂量依赖性,IC50值分为0.20、0.36和0.73 mg/mL。结论肉桂醛具有体外抗肿瘤活性。     

BPA 激活 GPER-EGFR-ERK1 /2 信号通路诱导乳腺癌细胞增殖

目的:研究雌激素G蛋白偶联受体(G protein-coupled estrogen receptor,GPER)-表皮生长因子受体(epidermal growthfactor receptor,EGFR)-细胞外调节蛋白激酶1/2(extracellular regulated protein kinases,ERK)信号通路在双酚A促乳腺癌细胞SKBR-3增殖中的作用。方法:采用CCK8试剂盒检测SKBR-3细胞的增殖情况,利用Western Blot观察ERK1/2磷酸化变化。结果:与对照组相比,10-11~10-7 M浓度的双酚A具有促乳腺癌细胞增殖作用,10-9M浓度的双酚A可诱导细胞增殖最大效应(细胞活力高于对照组约38.84%,P<0.001);预孵GPER、EGFR、ERK1/2特异性抑制剂G15、AG-1478、PD98059后,双酚A诱导的乳腺癌细胞增殖明显减少,细胞活力与单纯双酚A(10-9 M)处理相比降低约14.27%、12.23%和17.98%(P<0.05);双酚A(10-9 M)处理细胞0.5、1、3小时后,发现磷酸化的细胞外信号调节激酶(p-ERK)的表达明显高于对照组,双酚A可快速激活ERK1/2;而阻断GPR30和EGFR后,ERK1/2的磷酸化表达减少。结论:双酚A诱导的乳腺癌细胞增殖可能与GPR30-EGFR-ERK1/2信号通路有关,但不是唯一通路。深入研究双酚A对促进乳腺癌细胞增殖机制,可能为乳腺癌的防治提供新的方向。     

姜半夏乙醇提取物对人胃癌SGC7901 细胞增殖和凋亡的影响

目的:观察姜半夏乙醇提取物对人胃癌SGC7901细胞增殖和凋亡的影响。方法:不同浓度姜半夏乙醇提取物(终浓度为1mg/ml,0.5mg/ml,0.25mg/ml,0.125mg/ml)处理SGC7901细胞后,倒置相差显微镜下观察细胞的形态学变化;通过甲基噻唑基四唑法检测细胞增殖状况、描绘生长曲线,使用紫外分光光度法观察药物干预后细胞ATP酶活力;AnnexinV-异硫氰酸荧光素(fluorescein isothiocyanate,FITC)/碘化丙啶(propidium iodide,PI)双标记法流式细胞术检测姜半夏乙醇提取物对SGC7901细胞诱导凋亡的情况。结果:不同浓度姜半夏乙醇提取物均能不同程度地抑制人胃癌SGC7901细胞的增殖;在姜半夏乙醇提取物诱导细胞后细胞发生了边缘毛刺、体积缩小等形态学变化,同时可见细胞折光度和贴壁能力下降;AnnexinV-FITC/PI双标记法检测显示姜半夏乙醇提取物可诱导细胞发生凋亡;细胞总ATP酶活力在药物干预72小时后出现明显下降;并且随着药物浓度增加细胞凋亡率、细胞形态异常改变以及ATP酶活力抑制作用均呈上升趋势。结论:姜半夏乙醇提取物可抑制人胃癌SGC7901细胞的增殖,促进其凋亡,抑制细胞ATP酶活力。     

Inhibin/activin-betaE subunit in normal and malignant human cervical tissue and cervical cancer cell lines

Inhibins are dimeric glycoproteins, composed of an alpha-subunit and one of two possible beta-subunits (betaA or betaB), with substantial roles in human reproduction and in endocrine-responsive tumours. Recently a novel beta subunit named betaE was described, although it is still unclear if normal or cancerous cervical epithelial cells as well as cervical cancer cell lines can synthesise the novel inhibin-betaE subunit. About 4 normal cervical tissue samples together with 10 specimens of well-differentiated squamous cervical cancer and adenocarcinoma of the cervix were immunohistochemical analyzed. Additionally, two cervical carcinoma cell lines (HeLa and CaSki) were analyzed by immunofluorescence and RT–PCR for the expression of this novel subunit. We demonstrated for the first time an immunolabelling of the inhibin-betaE subunit in normal and malignant cervical tissue, as well as cervical cancer cells. Although the physiological role is still quite unclear in cervical tissue, inhibin-βE might play important roles in carcinogenesis. Moreover, the synthesis of this subunit in cervical carcinoma cell lines of squamous and glandular epithelial origins also allows the use of these cell lines in elucidating its functions in cervical cancer pathogenesis. However, since the expression of the inhibin-βE is minimal in HeLa cells as assessed by immunofluorescence and RT–PCR, the CaSki cell line might be a better model for further functional experiments regarding cervical cancer pathogenesis.     

A free radical initiator, 2,2'-azobis (2-aminopropane) dihydrochloride enhances hyperthermia-induced apoptosis in human uterine cervical cancer cell lines

Hyperthermia-induced apoptosis and its enhancement in the presence of a temperature-dependent free radical initiator, 2,2'-azobis (2-aminopropane) dihydrochloride (AAPH) were examined in human uterine cervical cancer cell lines, CaSki and HeLa. When both cell lines were treated with hyperthermia at 44°C for 60 min, minimal apoptosis was observed. When combined with nontoxic AAPH (50 mM), significant enhancement of apoptosis was observed, where the initial rate of free radical formation was about twice as high than that at 37°C. Augmentation of the growth delay, lipid peroxidation (LPO), activation of caspase-3 and increase in [Ca₂₊]i were also observed after the combined treatment. A water-soluble vitamin E, Trolox, blocked the increase in [Ca₂₊]i and an intracellular Ca₂₊ chelator, BAPTA-AM, prevented the DNA fragmentation induced by the combination. Cytochrome c release was also revealed by fluorescence microscopy. However, no significant change in mitochondrial membrane potential and expression of Bax and Bcl-2 was observed. A slight increase in Fas expression was observed only in CaSki cells after the combined treatment. These results indicate that hyperthermia and AAPH induce enhanced apoptosis and subsequent cell killing via two pathways; a pathway dependent on increase in LPO and [Ca₂₊]i, and a pathway associated with cytochrome c release and subsequent caspase activation without changes of mitochondrial membrane potential and Bax/Bcl-2 expression in these cell lines. Since it is known that cancer cells are generally resistant to physical and chemical stress-induced apoptosis, free radical generators like AAPH appear to be a useful thermosensitizer for hyperthermic cancer therapy.     

Adenoviral p53 effects and cell-specific E7 protein-protein interactions of human cervical cancer cells

We investigated the time-course tumor growth suppression effects of recombinant adenovirus expressing p53 on human cervical cancer cells and cell-specific E7 protein-protein interactions in cell lysates using surface plasmon resonance (SPR) biosensor. Six HPV-infected human cervical cancer cell lines (HPV 16-positive cells, CaSki and SiHa cells; HPV 18-positive cells, HeLa and HeLaS3 cells; and HPV negative C33A and HT3 cells) were used. After infection with AdCMVp53, the cell-specific growth inhibition was studied in vitro and in vivo. Also, we produced the recombinant E7 oncoprotein of HPV 16 type and tested chip-based protein-protein interactions with each cell lysate. For each cervical cancer cell, differential cell growth inhibitions were shown via cell count assay and MTT assay. Note that the same trend in suppression levels was shown in CaSki, HeLa and in SiHa, HeLaS3, respectively. In contrast, infection with AdCMVLacZ showed increased cell growth in a manner similar to the negative control group. The levels of p53 protein were notably expressed in CaSki and HeLa more than in SiHa and HeLaS3 for 4 days. In contrast, p53 expression was continually maintained in C33A and HT3 for 6 days. After transfection AdCMVp53 into CaSki- and SiHa-xenografted nude mice, the size of tumor was remarkably decreased in SiHa cells as compared to AdCMVLacZ transfection. The SPR sensor surface was successfully modified with the recombinant E7 oncoprotein and showed cell-specific interactions between E7 and its target proteins from cell lysates. The anti-tumor effects were accomplished via differential role of p53-specific apoptotic cell death, which is dependent upon the cervical cancer cell line. Also, a molecular level understanding of cell-dependent protein interaction effects of recombinant E7 was shown.     

A Free Radical Initiator, 2,2′-azobis (2-aminopropane) Dihydrochloride Enhances Hyperthermia-induced Apoptosis in Human Uterine Cervical Cancer Cell Lines

Hyperthermia-induced apoptosis and its enhancement in the presence of a temperature-dependent free radical initiator, 2,2′-azobis (2-aminopropane) dihydrochloride (AAPH) were examined in human uterine cervical cancer cell lines, CaSki and HeLa. When both cell lines were treated with hyperthermia at 44°C for 60?min, minimal apoptosis was observed. When combined with nontoxic AAPH (50?mM), significant enhancement of apoptosis was observed, where the initial rate of free radical formation was about twice as high than that at 37°C. Augmentation of the growth delay, lipid peroxidation (LPO), activation of caspase-3 and increase in [Ca²⁺]i were also observed after the combined treatment. A water-soluble vitamin E, Trolox, blocked the increase in [Ca²⁺]i and an intracellular Ca²⁺ chelator, BAPTA-AM, prevented the DNA fragmentation induced by the combination. Cytochrome c release was also revealed by fluorescence microscopy. However, no significant change in mitochondrial membrane potential and expression of Bax and Bcl-2 was observed. A slight increase in Fas expression was observed only in CaSki cells after the combined treatment. These results indicate that hyperthermia and AAPH induce enhanced apoptosis and subsequent cell killing via two pathways; a pathway dependent on increase in LPO and [Ca²⁺]i, and a pathway associated with cytochrome c release and subsequent caspase activation without changes of mitochondrial membrane potential and Bax/Bcl-2 expression in these cell lines. Since it is known that cancer cells are generally resistant to physical and chemical stress-induced apoptosis, free radical generators like AAPH appear to be a useful thermosensitizer for hyperthermic cancer therapy.     

Role of E-cadherin in the induction of apoptosis of HPV16-positive CaSki cervical cancer cells during multicellular tumor spheroid formation

Multicellular tumor spheroids (MCTS) are three dimensional cell culture systems induced by suspension culture. MCTS are widely used in cancer research because of their similarity to solid tumors. CaSki cells are derived from a metastatic cervical cancer containing human papillomavirus 16 (HPV16). Cell death of CaSki cells in MCTS has been previously reported, and our model is used to better characterize the mechanisms of cell death of HPV16-positive keratinocytes. In this study, we found that apoptosis of CaSki cells was induced by suspension culture along with the formation of MCTS after 24 h of incubation. In suspended CaSki cells, monoclonal antibodies blocking E-cadherin function inhibited MCTS formation and suppressed suspension-induced apoptosis in a dose-dependent manner. Western blot for E-cadherin detected upregulation of the authentic 120 kDa band from MCTS of CaSki cells as well as a shorter 100 kDa band. Addition of EGF, whose receptor is known to form a complex with E-cadherin, abrogated apoptosis of suspended CaSki cells in a dose-dependent manner. These findings suggest that E-cadherin-dependent cell–cell contact, directly or indirectly, mediates the signal to undergo apoptosis of CaSki cells during MCTS formation, and thus provides new information on the role of E-cadherin in cervical cancer cell apoptosis.     

High-mobility group box 2 promoted proliferation of cervical cancer cells by activating AKT signaling pathway

Cervical cancer is one of the leading killers for female worldwide. Nevertheless, the less knowledge of molecular mechanism for cervical cancer limited the improvement of treatment effects. High-mobility group box 2 (HMGB2) belongs to the HMGB family, which could play diverse roles in cell proliferation. This work mainly aimed to study the functions of HMGB2 on cervical cancer cells proliferation. HMGB2 was highly expressed in cervical cancer tissue. The results of real-time polymerase chain reaction and Western blot analysis showed that HMGB2 was expressed in all the five cervical cancer cells (HeLa, CaSki, SiHa, C-33A, and C4-1 cells). In addition, HMGB2 overexpression obviously improved cell viability and promoted cell cycle progression, which suggested that HMGB2 could promote proliferation of cervical cancer cells. Moreover, HMGB2 overexpression increased the level of p-AKT and reduced the levels of p21 and p27. However, HMGB2 downregulation had contrary influences on cell proliferation, cell cycle distribution and the levels of p-AKT, p21, and p27. Notably, LY294002, as an inhibitor of AKT signaling pathway, could significantly weaken the effects of HMGB2 overexpression, which indicated that HMGB2 might promote cell proliferation by activating AKT signaling pathway. Therefore, HMGB2 was hopeful to be a candidate as a new biomarker and therapy target for cervical cancer.     

Identification using phage display of peptides promoting targeting and internalization into HPV-transformed cell lines

'High-risk' human papilloma viruses (HPVs) cause cervical tumours. In order to treat these tumours therapeutic approaches must be developed that efficiently target the tumour cells. Using phage display, we selected tumour-targeting peptides from a library of constrained nonamer peptides presented multivalently on pVIII of M13. Three different consensus peptide sequences were isolated by biopanning on HPV16-transformed SiHa cells. The corresponding phage-peptides targeted and were internalized in HPV16 transformed SiHa and CaSki cells as well as in HPV18-transformed HeLa cells, but failed to bind a panel of normal or transformed cell lines. Two of the three selected peptides targeted cells only when presented on phage particles in a constrained conformation. However, all three peptides retained their targeting capacity when presented on the reporter protein enhanced green fluorescent protein (EGFP) in a monovalent form. These peptides may be useful for the design of drug or gene delivery vectors for the treatment of cervical cancer.     

The coxsackie adenovirus receptor inhibits cancer cell migration

The coxsackie and adenovirus receptor (CAR) is a key factor in adenoviral cancer gene therapy. Reduced expression of CAR during progression of prostate and bladder cancer has been reported. In embryonic development and tissue differentiation, CAR is also differentially expressed. This study suggests a role of CAR expression in cell adhesion and cell motility of human cancer cells. Stable CAR-expressing clones from E-cadherin-deficient A2780 ovarian and CaSki cervical cancer cells with originally low and high CAR expression levels, respectively, were established. CAR reexpression in otherwise singularly growing A2780 parental cells resulted in formation of cell-cell contacts and aggregation in cell clusters. CAR overexpression in cell adhesion-forming CaSki cells did not result in morphological changes. Migration of the A2780 CAR clones was strongly reduced as characterized by using spread-off assays. Using migration chambers, formation of satellite colonies was reduced by 97% in CAR-expressing A2780 cell clones and by 23% in CAR-expressing CaSki cell clones. Parental A2780 and CaSki cells selected for high migratory ability by using migration chambers expressed endogenous CAR on lower levels associated with lower adenoviral transduction efficiency. Our data suggest CAR as a new inhibitory factor for cancer cell migration.     

Suppressor of Cytokine Signaling (SOCS) Genes Are Silenced by DNA Hypermethylation and Histone Deacetylation and Regulate Response to Radiotherapy in Cervical Cancer Cells

Suppressor of cytokine signaling (SOCS) family is an important negative regulator of cytokine signaling and deregulation of SOCS has been involved in many types of cancer. All cervical cancer cell lines tested showed lower expression of SOCS1, SOCS3, and SOCS5 than normal tissue or cell lines. The immunohistochemistry result for SOCS proteins in human cervical tissue also confirmed that normal tissue expressed higher level of SOCS proteins than neighboring tumor. Similar to the regulation of SOCS in other types of cancer, DNA methylation contributed to SOCS1 downregulation in CaSki, ME-180, and HeLa cells. However, the expression of SOCS3 or SOCS5 was not recovered by the inhibition of DNA methylation. Histone deacetylation may be another regulatory mechanism involved in SOCS1 and SOCS3 expression, however, SOCS5 expression was neither affected by DNA methylation nor histone deacetylation. Ectopic expression of SOCS1 or SOCS3 conferred radioresistance to HeLa cells, which implied SOCS signaling regulates the response to radiation in cervical cancer. In this study, we have shown that SOCS expression repressed by, in part, epigenetically and altered SOCS1 and SOCS3 expression could contribute to the radiosensitive phenotype in cervical cancer.