Cyclin D1反义寡核苷酸对胃癌细胞化疗敏感性的影响

目的 研究Cyclin D1反义寡核苷酸（ASODN）对胃癌细胞SGC-7901和HS-746T化疗敏感性的影响,并探讨其内在机制。方法 在给予Cyclin D1 ASODN和Cyclin D1反义寡核苷酸转染细胞24h后,分别给予梯度浓度的5-氟尿嘧啶（5-FU）、氨甲喋呤（MTX）、顺铂（CDDP）,观察各组的量效反应,计算IC50。Cyclin D1 ASODN转染细胞后24h、48h时应用RT—PCR分别检测各组细胞中胸苷酸合酶（TS）、胸腺嘧啶磷酸化酶（TP）、二氢叶酸还原酶（DHFR）的表达情况。结果 Cyclin D1 ASODN转染增加了两种胃癌细胞对5-FU、MTX、CDDP的敏感性,ASODN＋化疗组对各化疗药物的lc50与对照组相比均有显著下降。RT—PCR显示Cyclin D1 ASODN转染后24h时Cyclin D1、TS、DHFR mRNA表达较对照组下降,而TP mRNA表达较对照组升高,在48h时各种酶mRNA表达的改变更加明显。结论 Cyclin D1 ASODN能提高胃癌细胞对多种化疗药物的敏感性,可能是由于影响了化疗药物代谢相关酶表达的改变所致。     

结肠癌耐药细胞株LoVo/5-FU的建立及其差异表达基因的筛选

目的:建立结肠癌细胞耐药模型LoVo/5-FU并初步筛选可能的耐药相关基因.方法:采用5-FU浓度递增法建立人结肠癌细胞耐药模型LoVo/5-FU,观察其生长规律并绘制细胞生长曲线:用MTT法鉴定耐药细胞株耐药性并计算耐药指数(RI);用基因芯片技术检测耐药细胞株LoVo/5-FU与其亲本细胞株LoVo中的差异表达基因,从中筛选出可能的耐药相关基因;用半定量RT-PCR方法对筛选出的部分耐药相关基因在耐药细胞及其亲本细胞中的表达情况进行验证.结果:LoVo/5-FU细胞与LoVo细胞相比,生长缓慢,细胞体积增大.LoVo/5-FU对5-FU、ADM、MMC耐药,而对CDDP无耐药性(RI:7.69,2.78,1.43和0.96).通过基因芯片筛选出差异表达基因425个,可能的耐药相关基因包括CYP1B1,NAT2,RNF20,SNAI2,MAP3K2,其中CYP1B1在LoVo/5-FU与LoVo中的表达有明显差异(Cy5/Cv3=5.877).结论:LoVo/5-FU细胞株耐药性稳定,耐药机制可能与CYP1B1,NAT2等耐药相关基因有关.     

ZFX promotes tumorigenesis and confers chemotherapy resistance in esophageal squamous cell carcinoma

IntroductionZinc finger X-chromosomal protein (ZFX) has been shown to be essential for the development and progression of multiple types of human cancers. However, its potential roles in esophageal squamous cell carcinoma (ESCC) have not yet been elucidated.Materials and methodsEighty-three pairs of frozen ESCC samples and their para-cancer samples and 24 fresh ESCC samples were collected. In vitro chemosensitivity was tested using the histoculture drug response assay. Quantitative RT-PCR and western blotting were used to measure the expression of functional genes. The effects of ZFX on cell growth, cell apoptosis, and chemosensitivity of the esophageal cancer cells were assessed.ResultsWe found that ZFX was more upregulated in ESCC tissues than in the para-cancer tissues, and its high expression was correlated with inferior clinicopathological characteristics and overall survival. Multivariate analysis revealed that ZFX was an independent prognostic factor in ESCC patients. In ESCC cell lines, ZFX silencing suppressed cell growth and induced cell apoptosis. In addition, ZFX expression was negatively correlated with the sensitivity of fresh ESCC tissues to chemotherapeutic drugs, including cisplatin, docetaxel, fluorouracil, and irinotecan. Furthermore, the depletion of ZFX sensitized ESCC cells to cisplatin, and docetaxel treatment. Mechanistically, ZFX silencing resulted in the inactivation of the MEK/ERK pathway, which mediated the downregulation of P-glycoprotein expression.ConclusionOur study therefore indicates that ZFX possibly plays a critical role in ESCC tumorigenesis and chemotherapy resistance and could be a significant prognostic biomarker and therapeutic target for ESCC.     

Increased expression of CEA and MHC class I in colorectal cancer cell lines exposed to chemotherapy drugs

PURPOSE: Cancer-specific immunotherapy holds great promise as an emerging treatment for advanced colorectal cancer and may be combined with standard chemotherapy to provide a synergistic inhibitory action against tumor cells. To examine the interrelationship between the immune system and chemotherapy, we studied the induction of both CEA, a tumor-associated antigen, and MHC class I, a major component of the antigen presenting system, in response to a number of chemotherapeutic agents. METHODS: The effect of a selection of standard chemotherapeutics on MHC class I and CEA expression in human colorectal cancer cell lines was determined by flow cytometry and semi-quantitative RT-PCR. In addition, studies using mice bearing tumors derived from an injected murine colon cancer cell line were performed to determine if alteration in MHC class I expression occurs in vivo following continuous infusion of chemotherapeutic agents into the peritoneal cavity, as well as to facilitate correlations between expression of this factor and therapeutic effectiveness. RESULTS: All anti-cancer drugs examined, when given at IC50 values, induced expression of MHC class I protein in the human colon cancer cell line, COLO201. However, expression of CEA mRNA was only induced upon exposure to 5-FU, in contrast to obscure induction following CDDP and SN-38 treatment. Combined treatment with 5-FU and CDDP gave additional effect on CEA expression in COLO201 cells. Regarding the in vivo studies in mice, the size of the murine colon cancer cell-derived tumors was reduced only in response to treatment with CDDP, which also mediated the highest induction of MHC class I expression. CONCLUSION: These results suggest that chemotherapeutic agents trigger the immune system and cancer-specific immunotherapy may be effective when used in combination with systemic chemotherapy.     

细小病毒H-1感染对胃癌细胞凋亡相关基因表达的影响

背景:细小病毒H鄄1对肿瘤细胞和转化细胞具有选择性杀伤和抑制生长的作用,是很好的基因治疗用载体。但肿瘤细胞对细小病毒H鄄1杀伤作用敏感或耐受的分子机制目前尚不十分清楚。目的:从mRNA水平观察细小病毒H鄄1感染对胃癌细胞凋亡相关基因表达的影响,探讨细小病毒H鄄1对胃癌细胞细胞毒作用的相关机制。方法:选取对细小病毒H鄄1敏感的人胃癌细胞株HGC鄄27和不敏感的人胃癌细胞株BGC鄄823,于细小病毒H鄄1感染48h后提取细胞总RNA。应用不同荧光染料标记的dUTP逆转录制备荧光探针,与含有8000点人类体细胞基因序列的基因表达谱芯片杂交,再经计算机荧光扫描以分析敏感细胞株HGC鄄27凋亡相关基因表达谱的改变。采用逆转录聚合酶链反应(RT鄄PCR)对敏感细胞株HGC鄄27部分凋亡相关基因,如SARP1、BCL鄄10、CL鄄20、NCDRP和RAIDD等的表达作进一步检证,并比较其与不敏感细胞株BGC鄄823相应凋亡相关基因的表达差异。结果:基因表达谱芯片检测结果显示,HGC鄄27细胞感染细小病毒H鄄148h后,所检测的64对凋亡相关基因中有15对基因表达水平下调至原水平的50%以下,仅有1对基因表达水平上调至2倍以上。RT鄄PCR扩增结果显示,感染细小病毒H鄄148h后,敏感细胞株HGC鄄27的SARP1、BCL鄄10、CL鄄20和NCDRP基因表达明显下调,RAIDD基因     

腺病毒介导的小鼠过氧化物酶体增殖剂活化受体γ基因过度表达对Raw264.7细胞小窝蛋白-1基因和蛋白表达影响的实验研究

目的比较腺病毒介导的小鼠过氧化物酶体增殖剂活化受体γ（PPARγ）基因过度表达与配体活化对小鼠Raw264.7巨噬细胞小窝蛋白-1（CAV-1）基因和蛋白表达的影响,探讨PPARγ基因对巨噬细胞CAV-1的调控作用及机制.方法首先构建表达小鼠PPARγ1基因的复制缺陷型腺病毒表达载体;将Raw264.7细胞随机分成对照组、PPARγ基因过度表达组、PPARγ活化剂曲格列酮干预组以及PPARγ基因过度表达和曲格列酮共刺激组进行干预;观察各组Raw264.7细胞PPARγ和CAV-1基因和蛋白的表达变化.结果RT-PCR检测对照组Raw264.7细胞有CAV-1基因的表达,免疫印迹法未发现CAV-1蛋白表达,但免疫细胞化学证实胞膜和核膜上均有少量CAV-1表达;经RT-PCR、免疫细胞化学和免疫印迹检测发现PPARγ基因过度表达组、曲格列酮干预组和二者共刺激组Raw264.7细胞CAV-1基因和蛋白表达明显增加（且共刺激组＞PPARγ基因过度表达组＞曲格列酮干预组,P〈0.05）.对照组Raw264.7胞浆内PPARγ表达量较低,而PPARγ基因过度表达组和共刺激组PPARγ表达均明显增加（P〈0.05）,而曲格列酮干预组无明显变化.结论PPARγ基因活化或过度表达均能上调Raw264.7细胞CAV-1基因和蛋白表达.曲格列酮活化PPARγ基因,增加CAV-1基因和蛋白表达,但不增加PPARγ基因和蛋白表达水平.与单一作用比较,同时活化和过度表达PPARγ基因对CAV-1基因和蛋白的表达有累积效应,说明PPARγ的这一作用在配体活化下增强.推测曲格列酮上调CAV-1表达依赖于PPARγ,非本身药理特性所致.     

Establishment of a human hepatoma multidrug resistant cell line in vitro

AIM:To establish a multidrug-resistant hepatoma cell line(SK-Hep-1),and to investigate its biological characteristics.METHODS:A highly invasive SK-Hep-1 cell line of human hepatocellular carcinoma,also known as malignant hepatoma was incubated with a high concentration of cisplatin(CDDP) to establish a CDDP-resistant cell subline(SK-Hep-1/CDDP).The 50% inhibitory dose(IC50) values and the resistance indexes [(IC50 SK-Hep-1/CDDP)/(IC50 SK-Hep-1)] for other chemotherapeutic agents and the growth curve of cell...     

Zinc protoporphyrin IX enhances chemotherapeutic response of hepatoma cells to cisplatin

AIM: To investigate the effect of zinc protoporphyrin IX on the response of hepatoma cells to cisplatin and the possible mechanism involved.METHODS: Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by a flow cytometric assay. Western blotting was used to measure protein expression. Heme oxygenase (HO)-1 activity was measured by determining the level of bilirubin generated in isolated microsomes. Reactive oxygen species (ROS) production was monitored by flow cytometry. Caspase-3 activity was measured with a colorimetric assay kit. Mice were inoculated with 1 × 10⁷ tumor cells subcutaneously into the right flanks. All mice were sacrificed 6 wk after the first treatment and tumors were weighed and measured.RESULTS: Overexpression of HO-1 in HepG2 cell line was associated with increased chemoresistance to cis-diaminedichloroplatinum (cisplatin; CDDP) compared to other cell lines in vitro. Inhibition of HO-1 expression or activity by zinc protoporphyrin IX (ZnPP IX) markedly augmented CDDP-mediated cytotoxicity towards all liver cancer cell lines in vitro and in vivo. In contrast, induction of HO-1 with hemin increased resistance of tumor cells to CDDP-mediated cytotoxicity in vitro and in vivo. Furthermore, cells treated with ZnPP IX plus CDDP exhibited marked production of intracellular ROS and caspase-3 activity, which paralleled the incidence of cell apoptosis, whereas hemin decreased cellular ROS and caspase-3 activity induced by CDDP.CONCLUSION: ZnPP IX increases cellular sensitivity and susceptibility of liver cancer cell lines to CDDP and this may represent a mechanism of increasing ROS.     

COX-2, MMP-7 expression in oral lichen planus and oral squamous cell carcinoma

ObjectiveTo observe cyclooxygenase (COX)-2 expression in normal oral mucosa (NOM), oral lichen planus (OLP) and oral squamous cell carcinoma (OSCC) and explore its significance in the incidence of oral cancer.MethodsThe immunohistochemical method and RT-PCR method were applied to detect the expression of COX-2 and MMP-7 in 10 cases with NOM, 33 cases of with OLP and 38 cases with OSCC.ResultsThe expression of COX-2 mRNA in OSCC tissues (68.4%, 26/38) was significantly higher than in the OLP (24.2%, 8/33) and NOM (0.0%, 0/10) (P<0.01). The expression of MMP-7 mRNA in OSCC tissues (65.8%, 25/38) was significantly higher than in the OLP (30.3%, 10/33) and NOM (0.0%, 0/10) (P<0.01). The expression of MMP-7 in OLP was significantly higher than in the NOM (P<0.05). There was no significant expression of COX-2 protein in NOM, and the positive rate was 42.4% (14/33) and 89.5% (34/38) in OLP and OSCC group, respectively. The COX-2 expression in cancer tissues was significantly higher than in NOM and OLP (P<0.05). The MMP-7 protein expression in cancer tissues (84.2%, 32/38) was significantly higher than in NOM (10.0%, 1/10) and in OLP (42.4%, 14/33), and the positive rate in OLP was significantly higher than in NOM (P< 0.01). The COX-2 expression was associated with clinical stage (P<0.05), the MMP-7 expression was associated with clinical stage and lymph node metastasis (P<0.05). The expressions of COX-2 and MMP-7 mRNA were positively correlated with OSCC.ConclusionsThe abnormal expressions of COX-2 and MMP-7 are closely related to the biological behavior of OSCC, the MMP-7 may be induced by COX-2, and further lead to the invasion and metastasis of OSCC.     

高温对SGC7901/ADM细胞多药耐药蛋白表达的影响和意义

目的:研究高温43℃对多药耐药基因表达产物P-gp、MRP、LRP在蛋白水平上的影响及其生物学意义,更深入地了解热效应逆转耐药的机制.方法:采用免疫细胞化学染色法、RT-PCR以及Western blot方法,检测在不同温度和不同药物作用下,人胃癌耐药细胞株SGC7901/ADM和对照的人胃癌敏感细胞SGC7901株中,与人胃癌多药耐药相关的分子MDR1、P-gp、MRP、LRP在蛋白水平上的表达差异.结果:采用免疫细胞化学染色法检测人胃癌耐药株SGC7901/ADM细胞,发现高温43℃60 min处理可使P-gp蛋白表达下调率(down-regulated rate,DRR)31.78%(P=0.016),而MRP表达DRR为20.22%(P=0.037),差异有统计学意义,LRP未表达.采用Western blot法在SGC7901细胞中未检测出P-gp蛋白表达,而SGC7901/ADM细胞中P-gp高表达;在ADM和CDDP处理组细胞中,高温43℃时P-gp的表达量较37℃时DRR分别为45.65%(P=0.007)、17.95%(P=0.021),差异有显著学意义;TAX处理组DRR为11.90%,差异无显著学意义(P=0.065).结论:高温43℃的短期处理对人胃癌SGC7901/ADM细胞中P-gp和MRP多药耐药蛋白的表达有一定的抑制作用,可能是逆转耐药的机制之一.     

洛贝林逆转胃癌细胞SGC7901/VCR多药耐药的作用

目的:探讨哌啶类生物碱洛贝林(Lobeline)能否逆转人胃癌多药耐药细胞株SGC7901/VCR的多药耐药,并进一步探讨洛贝林逆转其多药耐药的机制,评估其有效性.方法:以人胃癌多药耐药细胞株SGC7901/VCR为研究对象,用四氮唑蓝(MTT)法分别测定在无和有洛贝林(细胞无毒浓度10 mol/L)作用下多药耐药细胞株SGC7901/VCR对VCR及5-Fu的半数生长抑制率,并由此求其耐药逆转的倍数;RT-PCR分别检测在无和不同终浓度洛贝林(5、10、20、50、100 mol/L)作用下多药耐药细胞株SGC7901/VCR的多药耐药基因MDR1 mRNA表达情况;Western blot分别检测在无和不同终浓度洛贝林(5、10、20、50、100 mol/L)作用下多药耐药细胞株SGC7901/VCR的P-gp蛋白表达.结果:在洛贝林无毒作用浓度(10 mol/L)作用下,多药耐药细胞株SGC7901/VCR对化疗药的敏感性增加,VCR对耐药细胞株的IC50由原来的16.55 g/L±0.13 g/L变成7.27g/L±0.65 g/L,逆转指数约为2.28;5-Fu对耐药细胞株的IC50由原来的11.01 g/L±0.43 g/L变成9.53 g/L±0.79 g/L,逆转指数约为1.16;RT-PCR检测示多药耐药细胞株SGC7901/VCR呈现MDR1 mRNA高度表达,随洛贝林终作用浓度的增大,MDR1 mRNA表达逐渐下降,各组间差距有统计学意义(P<0.05);Western blot检测表明多药耐药细胞株SGC7901/VCR高度表达P-gp蛋白,随洛贝林作用终浓度依次增加P-gp蛋白表达依次减弱,组间呈浓度依赖性,差别有统计学意义(P<0.05).结论:无细胞毒作用浓度的洛贝林可以逆转胃癌多药耐药细胞株SGC7901/VCR的多药耐药,增强VCR和5-Fu的化疗敏感性.洛贝林对SGC7901/VCR细胞多药耐药的逆转可能机制为抑制P-gp蛋白的表达.     

Activation of STAT3 signaling in human stomach adenocarcinoma drug-resistant cell line and its relationship with expression of vascular endothelial growth factor

AIM: To investigate the difference in activation of STAT3 signaling between two human stomach adenocarcinoma cell lines: 5-fluorouracil resistant cell line and its parental cell line, and to evaluate its relationship with the expression of vascular endothelial growth factor (VEGF). METHODS: Western blot and electrophoretic mobility shift assay (EMSA) were used to detect the expression of phospho-STAT3 protein and constitutive activation of STAT3 in two human stomach adenocarcinoma cell lines, 5-fluorouracil resistant cell line SGC7901/R and its parental cell line SGC7901, respectively. The mRNA expression of VEGF was analysed by semi-quantitative RT-PCR. The expressive intensity of VEGF protein was measured by immunocytochemistry. RESULTS: The expressions of phospho-STATS protein and constitutive activation of STAT3 between two human stomach adenocarcinoma cell lines were different. Compared with the parental cell line SGC7901, the STAT3DNA binding activity and the expressive intensity of phospho-STAT3 protein were lower in the drug-resistant cell line SGC7901/R. The expression levels of VEGF mRNA and its encoded protein were also decreased in drugresistant cell line. CONCLUSION: Over-expression of VEGF may be correlated with elevated STAT3 activation in parental cell line. Lower VEGF expression may be correlated with decreased STAT3 activation in resistant cell line, which may have resulted from negative feedback regulation of STAT signaling.     

Elucidation of the relationship of BNIP3 expression to gemcitabine chemosensitivity and prognosis

AIM: To evaluate the significance of BNIP3 in the pathogenesis of pancreatic cancer, we analyzed the relationship between the expression of BNIP3 and survival rate of the patients with pancreatic cancer, or chemosensitivities in pancreatic cancer cell lines, particularly for gemcitabine, the first-line anti-tumor drug for pancreatic cancer. METHODS: To compare the expression level of BNIP3 with the resistance to gemcitabine, eight pancreatic cancer cell lines were subjected to gemcitabine treatment and the quantitative real-time RT-PCR method was used to evaluate BNIP3 expression. Immunohistochemical analysis was also performed using 22 pancreatic cancer specimens to study relationship between BNIP3 expression and survival rate. RESULTS: Although no significantly positive association between BNIP3 mRNA level and gemcitabine chemosensitivity was observed, pancreatic cancer cell lines that were sensitive to gemcitabine treatment tended to show high levels of BNIP3 expression. The converse, an absence of BNIP3 expression, was not correlated with gemcitabine resistance. We further compared the BNIP3 expression profiles of resected primary pancreaticcancer specimens with the prognosis of the patients, and found a tendency of favorable prognosis and low BNIP3 expression. CONCLUSION: High levels of BNIP3 expression cannot be used as one of the predicting factors for gemcitabine chemosensitivity, and some yet to be known factors will have to fill the gap for the accurate prediction of pancreatic cancer chemosensitivity to gemcitabine. However, BNIP3 expression may have an impact on prediction of prognosis of patients with pancreatic cancer.