PTEN、PIK3CA在培美曲塞耐药的胰腺癌细胞株Patu8988中的表达及其意义的初步探讨

目的 探索胰腺癌细胞株Patu8988对培美曲塞产生获得性耐药的机制,分析PTEN、PIK3CA在获得耐药前后mRNA及蛋白水平变化及其对细胞迁移及侵袭能力的影响。方法 通过RT-PCR及Western blot技术分别检测正常胰腺癌细胞株Patu8988及培美曲塞耐药的胰腺癌细胞株Patu8988中PTEN及PIK3CA的mRNA及其蛋白产物表达水平变化。应用迁移及侵袭实验分别检测正常胰腺癌细胞株和耐药的胰腺癌细胞株不同生物学行为。结果 RT-PCR发现与正常胰腺癌细胞株Patu8988相比,培美曲塞耐药的胰腺癌细胞株Patu8988中,PTEN和PIK3CA在mRNA水平表达均显著增高;Western blot发现培美曲塞耐药的胰腺癌细胞株Patu8988中PTEN、PIK3CA在蛋白水平同样表达升高,PTEN和PIK3CA在耐药的胰腺癌细胞株中的表达较正常胰腺癌细胞株分别上升89%和76%;迁移及侵袭实验的结果显示,培美曲塞耐药的胰腺癌细胞株Patu8988迁移及侵袭能力较正常胰腺癌细胞株Patu8988均显著下降(P＜0.05)。结论 PTEN、PIK3CA在对培美曲塞耐药的胰腺癌细胞株Patu8988中表达均异常升高,而同时高表达PTEN和PIK3CA的胰腺癌耐药细胞株的迁移及侵袭能力较正常胰腺癌细胞株显著降低,提示PTEN和PIK3CA共同参与胰腺癌细胞对培美曲塞产生获得性耐药过程,且有可能改变细胞的生物学行为。     

胃癌中胸苷酸合成酶与培美曲塞／雷替曲塞敏感性的初步研究

目的 探讨胃癌组织中胸苷酸合成酶（TS）mRNA表达与培美曲塞或雷替曲塞药物敏感性之间的关系。方法 收集50例经病理确诊的新鲜人胃癌标本,采用三维微组织块培养法（HDRA）进行两种药物的体外敏感试验;实时荧光定量PCR检测对应胃癌石蜡组织中TS mRNA水平。结果 新鲜胃癌组织对培美曲塞或雷替曲塞的敏感性以及胃癌石蜡组织中TS mRNA水平均与临床病理特征无明显相关性。培美曲塞敏感组与耐药组的TS mRNA相对表达量分别为6.72 ±1.34和15.39 ± 2.43（P=0.002）;雷替曲塞敏感组与耐药组的TS mRNA相对表达量分别为7.96 ± 1.70和14.14 ± 2.37（P=0.028）。结论 胃癌石蜡组织TS mRNA水平与新鲜胃癌组织对培美曲塞或雷替曲塞的敏感性呈负相关,TS在胃癌应用新型抗叶酸代谢类药物个体化治疗中具有潜在的指导价值。     

TS基因表达与培美曲塞治疗老年晚期肺腺癌疗效关系的研究

目的 探讨老年晚期肺腺癌患者胸苷酸合成酶（TS）基因与培美曲塞疗效的关系。方法 收集37例老年晚期肺腺癌患者,随机分为培美曲塞联合卡铂方案组和紫杉醇联合卡铂方案组;RT-PCR检测肿瘤组织中TS mRNA的表达,Western blotting检测TS蛋白的表达。结果 两组疾病控制率差异无统计学意义。TS mRNA和蛋白的表达与年龄、性别及分期无关。培美曲塞联合卡铂组中获疾病控制者（CR+PR+SD）的TS mRNA和蛋白表达量为5.08±2.30和4.46±2.22,进展者（PD）为13.66±3.84和12.6±3.92,差异均有统计学意义（P＜0.05）。紫杉醇联合卡铂方案组中获疾病控制者与进展者的TS mRNA和蛋白表达差异均无统计学意义（P＞0.05）。结论 肺腺癌患者肿瘤组织中TS基因的表达与患者对培美曲塞的敏感性相关,TS基因高表达提示对培美曲塞敏感性降低。     

Survivin参与胰腺癌PaTu8988细胞对吉西他滨的耐药

目的:探讨凋亡抑制蛋白survivin在胰腺癌细胞株PaTu8988中的表达,及其与吉西他滨(gemcitabine,GEM)耐药的关系。方法:MTT法检测GEM(0.01、0.1、1.0、2.5、5.0、10.0μg/ml)对PaTu8988细胞生长的抑制作用,流式细胞术检测GEM作用后PaTu8988细胞的凋亡率,RT-PCR检测survivin mRNA在PaTu8988细胞中的表达。结果:高质量浓度GEM(≥1.0μg/ml)可显著抑制PaTu8988细胞的增殖、促进PaTu8988细胞的凋亡;而低质量浓度GEM(0.01、0.1μg/ml)作用不明显。低质量浓度GEM时间依赖性地上调PaTu8988细胞中survivin mRNA的表达;而质量高浓度GEM作用PaTu8988细胞后,sur-vivin mRNA的表达48 h时逐渐下降,而后逐渐上升。结论:胰腺癌PaTu8988细胞中survivin mRNA高表达,可能是其对GEM产生耐药的原因之一。     

PTEN及mTOR在人肺腺癌培美曲塞耐药株A549/PEM中的表达

目的:应用高浓度反复间歇法建立人肺腺癌A549培美曲塞耐药细胞株A549/PEM,探讨PTEN、mTOR在人肺腺癌亲本株A549及培美曲塞诱导人肺腺癌耐药株A549/PEM中的表达变化.方法:采用终浓度为500ng/ml的培美曲塞反复间歇冲击建立人肺腺癌A549培美曲塞耐药细胞株A549/PEM,MTT法检测细胞耐药性.RT-PCR、Western blot分别检测PTEN、mTOR在A549及A549/PEM细胞的mRNA及蛋白表达变化.结果:A549/PEM耐药指数为26.87±1.81,较亲本株A549升高约27倍.RT-PCR检测mRNA表达示A549/PEM的PTEN及mTOR基因表达与A549相比均表达上调(P=0.023;P ＜0.01);Western blot检测蛋白表达示A549/PEM的PTEN及mTOR蛋白表达与A549相比均表达上调(P＜0.01;P =0.04).结论:高浓度反复间歇法可成功建立人肺腺癌A549培美曲塞耐药细胞株模型;PTEN、mTOR表达变化可能与培美曲塞获得性耐药相关.     

培美曲塞和吉非替尼不同时序应用对人肺腺癌细胞的作用和机制

目的 探讨培美曲塞与吉非替尼不同时序应用对肺腺癌细胞A549和PC-9生长及凋亡的影响, 并阐述其可能机制。方法 MTT法检测各组细胞的增殖抑制情况,流式细胞仪检测各组细胞凋亡及细胞周期分布,Western印迹法检测对EGFR下游信号通路及TS酶蛋白水平表达的影响。结果 培美曲塞序贯吉非替尼、培美曲塞同步联合吉非替尼对PC-9和A549细胞增殖抑制率及凋亡率较单药组均提高（P<0.05）,培美曲塞可以提高EGFR、AKT 、ERK磷酸化水平,而吉非替尼表现为抑制作用, 同时吉非替尼降低TS酶表达。培美曲塞序贯吉非替尼,培美曲塞同步联合吉非替尼抑制EGFR、AKT 、ERK磷酸化水平较单药更强。吉非替尼主要将PC-9、A549细胞阻滞在G0/G1期;培美曲塞主要将细胞阻滞在S期。培美曲塞序贯吉非替尼、培美曲塞同步联合吉非替尼较其他组G2/M期细胞比例提高（P<0.05）。结论 培美曲赛序贯吉非替尼、培美曲赛同步联合吉非替尼在PC-9、A549细胞中均起到协同增效作用,且培美曲赛序贯吉非替尼协同作用更为显著,可能主要与培美曲赛诱导EGFR、AKT 、ERK磷酸化及吉非替尼降低TS酶作用有关。     

细胞因子IL-2和IL-6对胰腺癌细胞表达VEGF-D的调节

目的探讨细胞因子白细胞介素2（IL-2）和白细胞介素6（IL-6）对胰腺癌细胞表达VEGF-D的调节。方法以细胞因子IL-2或IL-6分别刺激胰腺癌细胞株SW1990和BXPC-3后用逆转录 聚合酶链式反应技术（RT-PCR）分析其VEGF-D基因的表达。结果IL-2使细胞株SW1990和BXPC-3产生VEGF-D mRNA减少;IL 6使细胞株SW1990产生VEGF-D mRNA增加,但对细胞株BXPC-3产生VEGF-D mRNA无明显影响。结论IL-2抑制胰腺癌细胞VEGF-D mRNA的表达,从而抑制胰腺癌淋巴结转移;IL-6促进某些胰腺癌细胞VEGF-D mRNA的表达,但对胰腺癌细胞生物学特性的影响有待于进一步深入研究。     

环状RNA circ_MTHFD2对mircoRNA-124的调控在肺癌培美曲塞耐药中的作用

目的 探讨circ_MTHFD2调控microRNA 124（miR 124）的表达在肺癌培美曲塞耐药发生过程中的作用。方法 采用高浓度反复间歇诱导法建立肺癌培美曲塞耐药细胞株A549／PEM。通过瘤内注射建立培美曲塞耐药的裸鼠移植瘤模型;采用Lipofectamine脂质体法将miR-124模拟物、miR-124抑制剂及阴性对照转染至A549和A549／PEM细胞,并分为miR-124上调组、miR-124抑制组及阴性对照组;采用CCK-8 法和流式细胞术检测各组细胞的增殖及凋亡情况,比较各组细胞对培美曲塞的敏感性;采用实时荧光定量PCR（QPCR）和基因芯片检测miR 124和环状RNA的表达;通过测序技术检测耐药细胞及耐药裸鼠的肿瘤组织与对照组间环状RNA的表达,预测环状RNA上的miR-124结合位点。结果 亲本细胞A549和耐药细胞的半数抑制浓度（IC50）分别为 （30.78±1.97）μmol／L和（913.53±14.1）μmol／L,最终获得耐药指数（RI）为29.69±1.49的培美曲塞耐药细胞,命名为A549／PEM。耐药细胞A549／PEM中miR 124的表达显著降低,相比A549细胞,下降约145.952倍;下调miR-124表达的A549细胞对培美曲塞敏感性降低,miR-124抑制组及阴性对照组细胞的IC50分别为（80.45±3.50）μmol／L和（9.94±1.90）μmol／L,差异有统计学意义（P＜0.05）;流式细胞术检测显示miR 124表达上调可以显著诱导A549、A549／PEM细胞的凋亡;通过高通量测序提示在耐药的细胞及组织中环状RNA表达明显上调,表达量上调的circ_MTHFD2可与miR-124结合。结论 circ_MTHFD2可能通过调控miR-124的表达,参与肺癌培美曲塞耐药的发生发展。     

hTERT启动子驱动的HSV-TK基因对人胰腺癌细胞patu8988的杀伤作用

[目的]探讨hTERT启动子驱动的HSV-TK基因对人胰腺癌细胞patu8988的杀伤作用。[方法]应用分子生物学方法构建hTERT启动子调控下的TK基因真核表达载体。经同源重组产生重组腺病毒PSG-TP-TK。同法制备PSU-CMV-TK。利用重组腺病毒PSG-TP-TK和PSU-CMV-TK感染人胰腺癌细胞株patu8988和正常人原代成纤维细胞．加入GCV。用MTT和流式细胞仪检测PSG-TP-TK对各种细胞的杀伤作用;应用RT—PCR检测转染腺病毒后肿瘤细胞和正常细胞中TK基国的表达情况。[结果]PSG-TP-TK对端粒酶阳性的人胰腺癌细胞有明显的杀伤作用．而对端粒酶阴性的正常细胞则无杀伤作用（P〈0．05）。hTERT启动子诱导人胰腺癌细胞株patu8988凋亡的效能与CMV启动子相似。两者差异无显著性（P〉0．05）。PSU-CMV-TK转染后．在patu8988细胞和正常人原代成纤维细胞中均可检测到TK基因;而PSG-TP-TK转染后只有patu8988细胞中有TK基因表达．正常人原代成纤维中则无。[结论]hTERT启动子驱动HSV-TK基因治疗是一种新的胰腺癌靶向治疗方法．具有更高的靶向性和高效性。     

吉非替尼联合培美曲塞对EGFR-TKI获得性耐药的非小细胞肺癌细胞的协同效应

目的:探讨培美曲塞联合吉非替尼对体外诱导的表皮生长因子受体-酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitor,EGFR-TKI)获得性耐药的人非小细胞肺癌PC9/吉非替尼耐药(gei tinib resistance,GR)细胞株的效应及其可能的机制。方法:吉非替尼和培美曲塞单药或联合作用于PC9/GR细胞后,MTT法检测各药物处理组细胞的增殖抑制率及药物的联合指数(combination index,CI),FCM法检测各组细胞的凋亡率,蛋白质印迹法检测各组细胞磷酸化AKT和Bcl-2蛋白的表达水平。结果:吉非替尼和培美曲塞联合应用对PC9/GR细胞的增殖抑制作用和促凋亡作用明显强于各单药组(P0.05);吉非替尼和培美曲塞的CI值1,表现出明显的协同效应。与未进行药物处理的对照组比较,培美曲塞联合吉非替尼可明显下调PC9/GR细胞中磷酸化AKT和Bcl-2蛋白的表达水平(P0.01)。结论:培美曲塞联合吉非替尼对PC9/GR细胞具有较好的协同作用,这协同作用可能与诱导细胞凋亡和下调磷酸化AKT蛋白表达有关。     

Significance of thymidylate synthase for resistance to pemetrexed in lung cancer

Pemetrexed (MTA) is a multitargeted antifolate with promising clinical activity in lung cancer. We exposed the small cell lung cancer cell line PC6 to stepwise-increasing pemetrexed concentrations of 0.4, 1.6, and 4.0 μ m , and established three pemetrexed-resistant lung cancer cell lines: PC6/MTA-0.4, PC6/MTA-1.6, and PC6/MTA-4.0 cells. To investigate the mechanisms of acquired resistance to pemetrexed, we measured the expression levels of the thymidylate synthase ( TS ), reduced folate carrier ( RFC ), and folylpoly-gamma-glutamate synthetase ( FPGS ) genes. TS gene expression was significantly increased in PC6/MTA-1.6 and PC6/MTA-4.0 cells relative to parental cells in a pemetrexed dose-dependent manner. In contrast, the levels of RFC gene expression in PC6/MTA-0.4 cells and FPGS in PC6/MTA-1.6 cells were significantly decreased, whereas the levels of both genes were restored in PC6/MTA-4.0 cells. Knockdown of TS expression using siRNA enhanced pemetrexed cytotoxicity in PC6/MTA-4.0 cells. The expression level of the TS gene was significantly correlated with the concentration of pemetrexed for 50% cell survival (IC₅₀) in 11 non-small cell lung cancer cell lines. These results suggest that the alteration of molecular pharmacological factors in relation with pemetrexed resistance is dose-dependent, and that up-regulation of the expression of the TS gene may have an important role in the acquired resistance to pemetrexed. In addition, TS may be a predictive marker for pemetrexed sensitivity in lung cancer. ( Cancer Sci 2009)     

Cytotoxic effects of pemetrexed in gastric cancer cells

Pemetrexed is a newly developed multitargeted antifolate with promising clinical activity in many solid tumors including gastric cancer. The aim of the present study was to evaluate the cytotoxicity of pemetrexed and its mode of interaction with cisplatin in gastric cancer cell lines, and to identify genes associated with sensitivity to pemetrexed. The cytotoxic activity of pemetrexed was assessed by tetrazolium-based colorimetric assay (MTT assay) and the interaction between pemetrexed and cisplatin was evaluated by the isobologram method. Western immunoblotting and real time RT-PCR analysis of thymidylate synthase (TS), folylpoly-gamma-glutamate synthetase (FPGS) and reduced folate carrier (RFC1) were performed in order to determine whether sensitivity to pemetrexed would be predictable by protein or mRNA expression levels. Pemetrexed was more cytotoxic than 5-fluorouracil, with IC50 between 17 and 310 nM in most of the gastric cancer cell lines examined and the pemetrexed/cisplatin combination resulted in additive or synergistic interaction. The protein expressions of TS, FPGS, and RFC1 were significantly associated with IC50 for 5-fluorouracil, but no such association was found for pemetrexed chemosensitivity. The mRNA expressions of RFC1, FPGS and other target and resistance related genes revealed no significant association with pemetrexed sensitivity. In conclusion, pemetrexed is active against gastric cancer cell lines and the pemetrexed/cisplatin combination showed a synergistic or additive interaction, supporting its clinical use in gastric cancer. Drug sensitivity toward pemetrexed could not be predicted by the expressions of TS, RFC1, or FPGS and we suggest that it is determined by interactions between multiple genes.     

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method.

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of beta-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR x FPGH)/(RFC x FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.     

Loss of reduced folate carrier function and folate depletion result in enhanced pemetrexed inhibition of purine synthesis.

Pemetrexed is a novel antifolate with polyglutamate derivatives that are potent inhibitors of thymidylate synthase (TS) and to a lesser extent glycinamide ribonucleotide formyltransferase (GARFT). Conditions that might modulate relative suppression of these sites were assessed by the pattern of hypoxanthine and thymidine protection. When grown with 25 nmol/L racemic 5-formyltetrahydrofolate, thymidine alone fully protected wild-type HeLa cells to at least 1 micromol/L pemetrexed, but protection of a reduced folate carrier (RFC)-null subline required both thymidine and hypoxanthine above a concentration of 30 nmol/L pemetrexed. As medium 5-formyltetrahydrofolate was decreased, protection by thymidine alone decreased, and was further diminished when HeLa cells were grown in dialyzed serum. There was little protection by thymidine of RFC-null HeLa cells under the latter conditions. Thymidine alone was not protective, and hypoxanthine alone produced only a small (2-fold) increase in IC(50), in a HeLa-derived line 8-fold resistant to pemetrexed due to a modest increase in TS. Finally, in MCF-7 breast cancer cells there was greater protection with thymidine alone than in HeLa cells when cells were grown in medium containing a low concentration of 5-formyltetrahydrofolate. These observations indicate that as intracellular folates decrease in HeLa cells, due to decreased extracellular reduced folate, or loss of RFC function, pemetrexed inhibition of GARFT increases. These data support the concept that the contribution to pemetrexed activity by inhibition of GARFT, particularly at low folate levels, is a contributing factor to drug activity but relative inhibition of TS and GARFT may vary among human tumors and cell lines.     

Resistance to methotrexate in SKOV-3 cell lines after chronic exposure to carbamazepine is associated with a decreased expression of folate receptor

The detrimental effect of chronic administration of carbamazepine (CBZ) on serum and erythrocyte folates of patients has been extensively analyzed. However, at present, no data have been reported on the effect of CBZ on the intracellular content and activity of antimetabolite cytotoxic agents that can be used together with CBZ in the treatment of cancer patients. To investigate this issue, we chronically exposed in vitro the human ovarian cancer cell line SKOV-3 (grown under physiological folate concentrations) to CBZ, thus selecting SKOV-CBZ clones (SKOV-CBZ-50-2, SKOV-CBZ-50-5 and SKOV-CBZ-100-2) able to grow in the continuous presence of the antiepileptic drug. All of the SKOV-CBZ clones were more resistant to methotrexate (MTX) than the parental cells. MTX resistance index, as determined by the ratio between MTX concentrations inhibiting cell growth by 50% (MTX IC(50)) in SKOV-CBZ clones and in the parental cells, ranged between 3- and 5-fold. This resistance was related to a reduced intracellular content of MTX. No alteration activity of the cellular enzymes directly involved in MTX cytotoxicity (dihydrofolate reductase, thymidylate synthase [TS] and folylpolyglutamate synthetase) was observed. SKOV-CBZ clones were cross-resistant to the TS inhibitors tomudex and CB3717, but not to the TS inhibitor 5-fluoro-deoxy uridine and other antineoplastic drugs (cisplatin, doxorubicin, vincristine and taxol), whose cellular uptake is derived from transmembrane transport mechanisms different from folate receptor alpha (FRalpha) or reduced folate carrier (RFC). FRalpha mRNA and protein levels were significantly lower in SKOV-CBZ clones than in the parental cells. The reduced level of FRalpha in SKOV-CBZ clones was associated with a decreased binding capacity of folic acid. No variation of mRNA RFC expression in the SKOV-CBZ clones as compared to the parental cells was observed. Thus, after chronic exposure to CBZ, SKOV-CBZ clones develop resistance towards MTX due to defective MTX uptake.     

Predicting the chemosensitivity of pancreatic cancer cells by quantifying the expression levels of genes associated with the metabolism of gemcitabine and 5-fluorouracil

Gemcitabine (GEM) is the standard treatment for advanced/metastatic pancreatic cancer. However, there is a substantial subset of patients in whom the efficacy of GEM, when used as a single agent, is inadequate. Recently, the 5-fluorouracil (5-FU) prodrugs capecitabine and S-1 have been used as an alternative, either alone or in combination with GEM. The aim of the present study was to investigate the expression pattern of genes that render pancreatic cancer cells sensitive to GEM and 5-FU, and to identify markers for individualized chemotherapy, even in patients who have developed resistance. We investigated the correlation between the expression of genes associated with the metabolism of GEM and 5-FU, and sensitivity to these drugs in 15 human pancreatic cancer cell lines. We also established GEM- and 5-FU-resistant pancreatic cancer cell lines to investigate changes in the expression levels of these genes and the effects of one drug on cells resistant to the other. We found no correlation between pancreatic cancer cell sensitivity to either GEM- or 5-FU. GEM-resistant cells did not become resistant to 5-FU and vice versa. High expression of RRM1 (P=0.048) and TS x DPD (P=0.035) correlated significantly with sensitivity to GEM and 5-FU, respectively. 5-FU-resistant cells expressed significantly higher levels of TP than parental cells (P<0.05). In conclusion, pancreatic cancer cells showed no cross-resistance to GEM and 5-FU. Quantitative analyses of RRM1, TP, DPD and TS mRNA levels in pancreatic cancer cells may be useful for predicting their sensitivity to GEM and 5-FU.