顺铂联合人类白细胞介素-24基因对于宫颈癌Siha细胞增殖和侵袭力的影响

目的研究人类白细胞介素(hIL)-24基因联合顺铂对人宫颈癌Siha细胞的生长抑制作用以及hIL-24基因对Siha宫颈癌细胞侵袭、迁移能力的影响。方法构建重组质粒pDC316-hIL-24,使用脂质体携带pDC316-hIL-24质粒转染Siha细胞;聚合酶链反应(PCR)法检测hIL-24基因在宫颈癌细胞中的表达情况。Siha细胞在转染hIL-24基因后,添加不同剂量的顺铂,用CCK-8试剂检测hIL-24基因联合顺铂对Siha细胞增殖抑制效果;Transwell实验检测hIL-24基因对Siha细胞迁移侵袭能力的影响。结果重组质粒pDC316-hIL-24成功转染Siha宫颈癌细胞;hIL-24联合顺铂对Siha细胞的增殖抑制作用大于单独使用顺铂或hIL-24基因组(P<0.05);Transwell实验中,hIL-24干预组肿瘤细胞的穿膜细胞数最少(P<0.05)。结论 hIL-24基因的表达可增加Siha宫颈癌细胞对顺铂的敏感性;hIL-24基因可以明显抑制Siha宫颈癌细胞的迁移侵袭能力。     

SOX1对鼻咽癌细胞化疗敏感性的影响

目的 观察SOXl基因过表达对鼻咽癌细胞化疗敏感性的影响.方法 采用脂质体Lipofectamin2000介导的方法,将SOXl基因转入鼻咽癌细胞CNE2和HONE1中,RT-PCR和Western blot法检测转染空载体对照组及转染SOX1过表达组鼻咽癌细胞中SOX1基因的表达,并用MTT法检测两组鼻咽癌细胞对化疗药物的敏感性.结果 RT-PCR和Western blot法表明,转染过表达组鼻咽癌细胞的SOX1基因的mRNA及蛋白表达水平均较对照组明显增强,MTT法结果表明过表达SOX1的鼻咽癌细胞对化疗药物顺铂的敏感性显著提高（P＜0.05）.结论 转染外源性SOX1并使之在鼻咽癌细胞中过表达,能提高CNE2和HONE1细胞对化疗药物的敏感性.     

下调miR-374a通过靶向TCF21抑制宫颈癌细胞SiHa增殖、迁移、侵袭的分子机制

摘要：目的:阐明下调miR-374a靶向转录因子21（TCF21）调控宫颈癌细胞增殖、迁移、侵袭的分子机制。方法:采用Realtime PCR法检测微小RNA（miR）-374a在正常宫颈上皮细胞和宫颈癌细胞中的表达变化。在宫颈癌细胞Si Ha中转染miR-374a inhibitor,MTT检测细胞增殖,Transwell小室检测细胞侵袭以及迁移能力变化,Western blot检测细胞中基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）蛋白表达变化。在线靶基因预测软件预测miR-374a的靶基因可能为TCF21,荧光素酶报告系统鉴定靶向关系。在宫颈癌细胞Si Ha中共转染miR-374a inhibitor和TCF21 siRNA,检测细胞增殖、侵袭、迁移变化。结果:miR-374a在正常宫颈上皮细胞中的表达水平明显低于宫颈癌细胞。转染miR-374a inhibitor后的宫颈癌细胞Si Ha增殖、迁移和侵袭能力下降,细胞中MMP-2和MMP-9蛋白表达减少。miR-374a靶向负调控TCF21表达。TCF21 siRNA逆转miR-374a inhibitor对宫颈癌细胞Si Ha增殖、侵袭和迁移的抑制作用。结论:下调miR-374a通过靶向TCF21抑制宫颈癌细胞Si Ha增殖、迁移、侵袭。     

宫颈癌术前新辅助化疗Bcl-2和Bax基因表达与细胞凋亡及其临床意义

目的通过对细胞凋亡基因Bcl-2、Bax表达的研究,探讨宫颈癌术前新辅助化疗的作用机制及临床意义。方法采用免疫组织化学技术分别10例宫颈癌Ⅱa-Ⅱb期和相应的术前化疗标本的细胞凋亡基因Bcl-2、Bax表达的情况进行检测,并以10例正常宫颈鳞状上皮作对照。采用原位末端标记法（TUNEL）法检测各组细胞凋亡情况。结果Bcl-2在正常宫颈组织和术前化疗组中低表达,宫颈癌中表达明显增强（P〈0．05或〈0．01）;Bax在正常宫颈组织和宫颈癌中表达有明显差异（P〈0．05或〈0．01）;术前新辅助化疗组中Bcl-2／Bax比值较宫颈癌组织中明显减低而凋亡细胞数明显增多（P〈0．05或〈0．01）。结论术前新辅助化疗抑制肿瘤生长的机制可能与诱导肿瘤细胞凋亡有关,提示宫颈癌术前新辅助化疗对肿瘤预后有重要意义。     

宫颈癌组织中细胞周期相关激酶的表达功能与化疗药物敏感性的关系

目的：探究宫颈癌组织中细胞周期相关激酶（CCRK）的表达功能与化疗药物敏感性的关系。方法收集不同发展时期的90例宫颈癌样本并从中提取出 Caski 细胞系,通过 RT-PCR、Northern blot 鉴定筛选最有效的细胞系克隆,建立稳定沉默 CCRK 的宫颈癌细胞系模型,化疗药物顺铂、5-氟尿嘧啶处理 Caski 细胞系,分别检测化疗药物处理前后的 Caski 细胞系的 CCRK 表达功能。结果Ⅱ、Ⅲ、Ⅳ期的宫颈癌组织中 CCRK 蛋白表达量组间比较差异具有统计学意义（P ＜0．05）。随着宫颈癌临床分期的增加,CCRK 表达的阳性率逐渐升高,Ⅱ、Ⅲ、Ⅳ期的宫颈癌组织 CCRK 表达的阳性率组间比较差异具有统计学意义（P ＜0．05）;采用5-氟尿嘧啶处理的宫颈癌 Caski 细胞样本 CCRK 阳性化疗药物敏感度为49．2％,采用顺铂的 CCRK 阳性化疗药物敏感度为62．7％,采用5-氟尿嘧啶＋顺铂的 CCRK 阳性化疗药物敏感度为88．1％,三组间差异具有统计学意义（P ＜0．05）。采用5-氟尿嘧啶、顺铂及5-氟尿嘧啶＋顺铂的 CCRK 阴性化疗药物敏感度差异具有统计学意义（P ＜0．05）。结论宫颈癌组织中 CCRK的表达功能与化疗药物敏感性具有显著相关性,CCRK 的表达能够为宫颈癌的临床诊断提供参考,并能为宫颈癌的化疗治疗提供指导与参考。     

干扰c-myc基因表达增强人胰腺癌细胞对吉西他滨和卡铂的敏感性

目的　探讨干扰c-myc 基因表达对人胰腺癌细胞SW1990 对化疗药物敏感性的影响。方法　采用实时定量PCR和Western blot 检测人胰腺癌细胞系SW1990 中c-myc 基因的干扰效果;MTT 法检测癌细胞对化疗药物吉西他滨和卡铂的敏感性;流式细胞术检测吉西他滨诱导的细胞凋亡情况。结果　c-myc siRNA 显著下调人胰腺癌SW1990 细胞中c-myc 基因的mRNA 和蛋白表达水平。干扰c-myc 表达后,化疗药物吉西他滨和卡铂对SW1990 细胞的半数抑制浓度（IC50）均显著减小（P<0.05）,吉西他滨诱导的SW1990 细胞凋亡率显著升高（P<0.05）。结论　干扰c-myc 表达可增强人胰腺癌细胞株SW1990 对化疗药物吉西他滨和卡铂的敏感性。     

宫颈癌生存素的表达及其与化疗敏感性的关系

目的 研究宫颈癌生存素(suvivin)基因蛋白的表达及其与化疗敏感性的关系.方法 采用免疫组织化学法检测宫颈癌标本生存素的表达.对培养的宫颈癌细胞行1.0 mg/L的顺铂(DDP)化学药物处理,MTT法检测细胞活性.结果 生存素在宫颈癌中阳性表达率高达73.7%.宫颈癌细胞经过DDP处理后,生存素阳性表达组的细胞存活率为45.0%～65.0%,明显高于阴性组的15.0%～40.0%.结论 宫颈癌高表达生存素,表明生存素阳性癌细胞对DDP有一定耐受性,检测生存素有可能成为预测宫颈癌化疗敏感性指标之一.     

mdr1反义寡核苷酸增强多药耐药肝癌细胞HepG2/ADM化疗敏感性的实验研究

目的：探讨在体外实验条件下mdr1反义寡核苷酸对多药耐药肝癌细胞株化疗敏感性的影响。方法以肝癌细胞HepG2/ADM为研究对象,设立mdr1反义寡核苷酸组和空白试剂组作对照,利用脂质体包载肿瘤耐药基因mdr1的反义寡核苷酸进行细胞转染,通过反转录聚合酶链反应（RT-PCR）、免疫印迹实验（Western blotting）分别检测mdr1基因mRNA和P-gp蛋白表达,通过MTT实验检测细胞转染前后对阿霉素（ADM）、顺铂（DDP）和5-氟尿嘧啶（5-FU）的化疗敏感性。结果 HepG2/AMD肝癌细胞经反义寡核苷酸处理后,mdr1 mRNA、P-gp蛋白表达水平均明显降低,对ADM、DDP 和5-FU的化疗敏感性明显增强。结论反义寡核苷酸能在体外有效增加肝癌细胞HepG2/ADM对化疗药物的敏感性。     

p53基因蛋白在宫颈上皮内瘤样变的表达及意义的研究

目的探讨p53基因蛋白在宫颈上皮内瘤样变（CIN）的表达及意义。方法随机选取2010年3月。2012年9月在本院治疗的100例患者的手术标本为研究对象,实验组60份宫颈上皮内瘤样变标本采用免疫组化染色检测,依据CIN的级别分成3组,对照组40份标本采用TUNEL法检测各组细胞的凋亡情况,分成阳性组（宫颈癌）和阴性组（正常宫颈鳞状上皮）;分析5组间的p53阳性表达率。结果正常宫颈鳞状上皮的p53蛋白表达率为0,而宫颈癌p53蛋白表达率为40％,且CIN分级越高,p53阳性表达率也越高,且十＋＋的概率也越高;宫颈癌的细胞分级越高,p53蛋白表达率也越高,即癌细胞的分化率越差,p53蛋白表达率越高,CINI与CINⅡ、Ⅲ比较,差异有统计学意义（P〈0．05）。结论p53基因蛋白表达与CIN的发生、发展有密切关系,是宫颈癌发生的早期事件。     

ERCC-1在晚期结肠癌中的表达及与奥沙利铂化疗敏感性的关系

目的探讨核苷酸切除修复交错互补基因（ERCC1）在晚期人结肠癌中的表达水平,分析其与铂类药物化疗敏感性及生存时间的关系。方法以江西省肿瘤医院行结肠癌剖腹探查术患者44例的标本为材料,采用免疫组织化学SP法检测其ERCC1表达水平,所有入组患者随访2年。结果肿瘤组织中ERCC1的阳性率为50.00%,ERCC1阴性者铂类药物化疗有效率为59.1%（13/22）,而阳性者则为27.3%（6/22）,两者之间具有统计学差异（P=0.033）;Cox回归分析显示ERCC1表达为结肠癌患者独立预后因子（P=0.002）。结论ERCC1表达可作为预测结肠癌患者对铂类药物敏感性及预后判断的指标之一。     

Enhancement of platinum-drug cytotoxicity in a human head and neck squamous cell carcinoma line and its platinum-resistant variant by liposomal amphotericin B and phospholipase A2-II.

Platinum drugs comprise one of the main classes of chemotherapy drugs that can induce remissions in various solid tumors. Although tumors often regress on treatment with cis-diamminedichloroplatinum II (cisplatin) or cis-diammine-1,1-cyclobutane dicarboxylate platinum II (carboplatin), they usually relapse as a drug-resistant tumor. Most mechanisms of platinum resistance could be overcome by increasing the amount of drug that is accumulated by tumor cells. Amphotericin B (Amph B) is efficient at increasing platinum drug uptake, but because of nephrotoxicity associated with extended usage, and the potential for synergistic nephrotoxicity when used with platinum drugs, Amph B has not been used clinically for this purpose. A liposomal preparation of Amph B (LipoAmph B), which is substantially less nephrotoxic, was studied for its ability to enhance platinum-drug toxicity to a human oral squamous cell carcinoma line, HN-5a, and its carboplatin-resistant variant, 5a/carbo-15a, in which cisplatin accumulation was reduced by approximately 40%. Amph B at 10 microg/ml enhanced cisplatin accumulation by approximately 100% in both cell lines, enhancing cytotoxicity of the drugs by 35 to 60%, and completely reversed resistance to both cisplatin and carboplatin. LipoAmph B in the presence of phospholipase A(2)-II (PLA2-II) was able to enhance cisplatin and carboplatin cytotoxicity as effectively as free Amph B in both cell lines. At optimal concentrations, LipoAmph B plus PLA2-II enhanced drug uptake sufficiently to abolish resistance in the platinum-resistant line. Because PLA2-II is elevated in some tumor microenvironments and in plasma of ill patients, LipoAmph B has potential clinical usefulness as a modulator of platinum-drug efficacy.     

Relevance of drug uptake and efflux for cisplatin sensitivity of tumor cells

Platinum sensitivity and platinum resistance may involve altered activity of transport proteins. In order to assess the role of drug uptake and efflux in this phenomenon, we compared the expression of three copper transporters, intracellular platinum accumulation, DNA platination and cytotoxicity of cisplatin in two cisplatin-sensitive and -resistant tumor cell line pairs (ovarian A2780/A2780cis and cervical HeLa/HeLaCK cells). Gene expression of importer CTR1, and ATP7A and ATP7B efflux transporters (with and without cisplatin treatment) was investigated using quantitative real-time PCR and platinum concentrations were determined by flameless atomic absorption spectrometry. After incubation with cisplatin, DNA platination was significantly lower in the resistant variants compared to the respective sensitive cell lines, whereas no obvious difference in DNA repair was found. Accordingly, the resistant variants exhibited lower intracellular platinum concentrations than their respective parental cells (2.5- and 2.9-fold lower in A2780cis and HeLaCK cells, respectively). No differences in efflux were observed. Resistant cells expressed lower levels of CTR1 (1.5-1.8-fold) than their sensitive counterparts. Expression differences of ATP7A and ATP7B between resistant and sensitive cells were cell type-specific. The results highlight the relevance of CTR1 for cisplatin sensitivity as there is a clear relationship between lower CTR1 expression, intracellular concentration, DNA platination and cytotoxicity of cisplatin in both resistant cell lines. Our data provide the basis for a quantitative understanding of alterations in uptake and efflux processes leading to cisplatin resistance and might hence facilitate the development of ex vivo assays that can predict cisplatin sensitivity in tumor specimens of patients.     

常用化疗药物对不同组织类型非小细胞肺癌体外药敏实验

目的探讨三种组织类型的非小细胞肺癌（NSCLC）对5种临床常用化疗药物的敏感性。方法28例临床肺部肿瘤标本制备成组织细胞悬液,在体外（96孔细胞培养板内）培养,24h后加入化疗药物。应用四氮唑蓝比色（MTT）法检测各孔的吸光度值并计算出肿瘤细胞对所试药物的敏感率。结果肺腺癌对异环磷酰胺、丝裂霉素、顺铂的敏感率分别为75％、83％和75％;鳞癌对异环磷酰胺、丝裂霉素、顺铂的敏感率均达到80G。肺腺癌、鳞癌对紫杉醇的敏感率不高,而腺鳞癌对该药的敏感率可达100％。肺腺癌对阿霉素的敏感率仅为33％,肺鳞癌对该药也无明显反应性。结论不同组织类型非小细胞肺癌对5种化疗药物的敏感性存在显著差异。     

Transporter-Mediated Interaction Between Platinum Drugs and Sorafenib at the Cellular Level

Combining the multikinase inhibitor sorafenib with the platinum-based chemotherapy of solid tumors was expected to improve treatment outcome. However, in many clinical trials, no benefit from sorafenib addition to the platinum-containing regimen could be demonstrated. Moreover, in some studies, decreased survival of ovarian cancer patients as well as non-small cell lung cancer patients with squamous cell histology was observed. The aim of this study was to investigate the cellular mechanisms of the pharmacological interaction between platinum drugs and sorafenib in different cancer cell lines. The interaction was characterized by combination index analysis, platinum accumulation and DNA platination were determined using flameless atomic absorption spectrometry, and protein expression was assessed with Western blot. In the sensitive A2780 ovarian carcinoma and H520 squamous cell lung carcinoma cell lines, sorafenib induced downregulation of Na⁺,K⁺-ATPase. In A2780 cells, the kinase inhibitor also decreased the expression of copper transporter 1 (CTR1). As a result, sorafenib treatment led to a diminished cellular accumulation of cisplatin and carboplatin and to a decrease in DNA platination in these cell lines. This was not the case in the cisplatin-resistant A2780cis ovarian carcinoma and H522 lung adenocarcinoma cell lines featuring lower basal expression of the above-mentioned transporters. In all cell lines studied, an antagonistic interaction between platinum drugs and sorafenib was found. Our results suggest that sorafenib impairs cisplatin and carboplatin uptake through downregulation of CTR1 and/or Na⁺,K⁺-ATPase resulting in reduction of DNA platination. This effect is not observed in cancer cells with defects in platinum accumulation.     

On the significance of telomerase activity in human malignant glioma cells

Telomerase is critical for tumor cell immortalization and is a novel target for cancer chemotherapy. Here, we examined whether telomerase is expressed in glioma cell lines, whether telomerase activity is regulated by bcl-2 or p53, and whether telomerase activity predicts response to chemotherapy. Further, we characterized the effects of a candidate telomerase inhibitor, penclomedine, in glioma cells. All 12 human malignant glioma cell lines examined were telomerase positive. Telomerase activity was not modulated during cell cycle progression, did not correlate with p53 status or bcl-2 family protein expression, and did not predict drug sensitivity, except for an association with resistance to carmustine. Ectopic bcl-2 expression did not enhance telomerase activity. Wild-type p53 reduced telomerase activity in cell lines retaining p53 activity but not in p53-mutant cell lines. Penclomedine killed glioma cells via an apoptotic, but death receptor-, bcl-2- and caspase-independent pathway, but did not inhibit telomerase and did not act synergistically with cytotoxic drugs. We conclude that telomerase activity does not account for the differential chemosensitivity of human glioma cells and that penclomedine kills glioma cells via a telomerase-independent pathway.     

Cellular pharmacology of cisplatin in relation to the expression of human copper transporter CTR1 in different pairs of cisplatin-sensitive and -resistant cells

The molecular mechanism of cisplatin uptake remains poorly defined and impaired drug accumulation may be implicated in the acquisition of resistance to cisplatin. Thus, we used cell lines of different tumor types (ovarian carcinoma A2780 and IGROV-1, osteosarcoma U2-OS, cervix squamous cell carcinoma A431) and stable cisplatin-resistant sublines, exhibiting variable levels of resistance (between 2.5 and 18.4), to investigate the mechanisms of cellular accumulation of cisplatin. Among the resistant lines we found that reduced cisplatin uptake was a common feature and ranged between 23 and 76%. In an attempt to examine the role of human copper transporter 1 (CTR1) in cisplatin accumulation by human cells, we selected the well characterized A431 cell line and the resistant variant A431/Pt. As compared with A431/Pt cells, A431/Pt transfectants overexpressing CTR1 (3.4-fold) exhibited increased uptake of copper, thereby supporting the expression of a functional transporter. However, no changes in cisplatin uptake and cellular sensitivity to drug were observed. Also overexpression of CTR1 in A431 cells did not produce modulation of cisplatin accumulation. An analysis of the expression of other factors that could affect drug accumulation indicated that A431/Pt cells displayed increased expression of ATPase, Cu(2+) transporting, alfa polypeptide. In conclusion, our results indicate that the overexpression of a functional CTR1 in a human cell line characterized by impaired cisplatin uptake fails (a) to restore cellular drug accumulation to the level of the parental cell line and (b) to modulate cisplatin sensitivity. Our data are consistent with the interpretation that the defects in cellular accumulation by resistant cells are not mediated by expression of CTR1, that plays a marginal role, if any, in cisplatin transport.     

Intrinsic drug resistance in a human lung carcinoma xenograft is associated with overexpression of multidrug-resistance DNA-sequences and of plasma membrane glycoproteins

The purpose of this study was to examine whether multidrug resistance can be detected in human lung tumors not previously treated with chemotherapy. Therefore, the intrinsic sensitivity or resistance of 8 human epidermoid lung cancer xenografts grown in nude mice has been examined. The tumor lines responded differently to vincristine and the other cytostatic agents. When the effects of vincristine on the 8 tumor lines are correlated with the effect of dactinomycin (actinomycin D), a close relationship could be found (r = 0.96). These results demonstrate that xenografts derived from human lung tumors not previously treated with chemotherapy exhibited a similar pattern of cross-resistance as is observed in sublines of murine and human tumors which were resistant to various drugs following repeated exposure of the cells to a sublethal concentration of these drugs. The resistant lung tumor HXL 54 also shows an overexpression of the P170-membrane glycoprotein (detected by Mab 265/F4). To determine whether multidrug genes were expressed in resistant lung tumors, slot blots and Northern blots were performed using the probes pDR 7.8 and pcDR 1.5. The level of expression can be correlated with the degree of drug resistance.     

小檗碱抗妇科肿瘤作用机制的研究进展

妇科肿瘤严重威胁女性的生命健康,目前妇科肿瘤的治疗仍以手术和放疗为主,并使用化疗药预防复发和转移。小檗碱为季铵生物碱,具有广泛的生物活性。小檗碱能够抑制肿瘤细胞生长、增殖,抑制肿瘤细胞迁移、侵袭,诱导肿瘤细胞凋亡,并增加化疗药敏感性。总结了小檗碱抗乳腺癌、卵巢癌、宫颈癌、子宫内膜癌等妇科肿瘤作用机制的研究进展,以期将小檗碱开发为防治妇科肿瘤的新药提供参考。     

Inhibition of MDR1 expression by retinol treatment increases sensitivity to etoposide (VP16) in human neoplasic cell line

Multidrug resistance (MDR) is the major obstacle to cancer chemotherapy. MDR phenotype is mainly related to the over-expression of MDR1 gene, being responsible for tumor resistance to several chemotherapeutic drugs. It has been suggested that MDR1 expression is redox-regulated and we have recently described a pro-oxidative effect of retinol. Here we tested the therapeutic use of retinol as a modulator of MDR1 gene expression in tumor cell lines, and verified in situ the enhancement of anticancer drug efficacy. Two human colorectal adenocarcinoma cell lines (HT29, SW620) with different degrees of MDR1 expression were used. Cells were pre-treated with a sublethal dose of retinol and then challenged with the etoposide (VP16) drug. The drug GI50 was assessed by SRB method and levels of MDR1 expression were determined by semi-quantitative rtPCR. Retinol treatment caused a 40% decrease in MDR1 expression and increased VP16 toxicity. MDR1 expression and drug sensitivity were restored to control values when mannitol (a hydroxyl radical scavenger) was co-administrated. Our data point a role to the use of retinol as an adjuvant in the treatment of tumors with MDR phenotype.     

JWA蛋白在食管鳞癌中的表达及意义

目的 探讨JWA蛋白在食管鳞癌组织和细胞株中的表达及意义.方法 采用蛋白免疫印迹技术检测食管鳞癌组织及癌旁正常组织、人食管上皮细胞株（HET-1A）及食管鳞癌细胞株（EC109和KYSE510）中JWA蛋白的表达情况.结果 食管鳞癌组织中JWA蛋白表达水平低于癌旁正常组织;JWA蛋白在3种细胞系中均有表达,其表达水平依次为HET-1A＞KYSE510＞EC 109,表达量随食管鳞癌细胞转移潜能增高而递减.结论 JWA蛋白可能与食管鳞癌的发生、发展及转移密切相关,可能成为基因治疗的一个潜在靶点.