【作者】 刘永萍；

【导师】 刘宝瑞； 凌扬；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 [目的]探讨铂类化疗药物相关基因在胃癌含奥沙利铂辅助化疗中的疗效预测价值以及重楼复方(Chong Lou Fu Fang,CLFF)与胃癌常用化疗药物体外协同抗肿瘤作用。[方法]荧光定量RT-PCR法测定化疗药物相关基因表达水平；采用TaqMan探针real-timePCR方法和直接测序法对ERCC1第118位密码子,XRCC1第399位密码子,XPD第751位密码子和GSTP1第105位密码子进行多态性分析；分析ERCC1基因表达水平及各基因型与接受含奥沙利铂方案辅助化疗的胃癌患者生存时间之间的关系。MTT法检测细胞增殖能力；基于中位效应原则的联合指数法评估重楼复方与各化疗药物对胃癌细胞系SGC-7901和BGC-823的交互作用；Annexin-V-FITC和PI双染色法检测细胞凋亡率。[结果]1、75例胃癌患者术后新鲜组织ERCC1基因表达水平检测结果显示中位值为7.32(0.50-147.03)；75例胃癌患者中52例接受术后改良FOLFOX4方案辅助化疗至少6周期,23例只接受手术治疗。单因素分析提示在辅助化疗组中,ERCC1低表达患者较高表达患者RFS和OS均显著延长(中位RFS分别为18和7月,P=0.001;中位OS分别为27和11月,P=0.001),在单纯手术组中,ERCC1高表达患者较低表达患者RFS和OS均显著延长(中位RFS分别为33和12月,P=0.038；中位OS分别为43和21月,P=0.004)；多因素Cox风险模型分析提示在含奥沙利铂辅助化疗组中,肿瘤组织ERCC1高表达可能是RFS和OS的不良预后因素,但在单纯手术组中,肿瘤组织ERCC1高表达却可能是OS的良好预后因素。2、126例接受术后改良FOLFOX4方案辅助化疗的胃癌患者基因型分析显示野生纯合子,杂合子和突变纯合子频率在ERCC1-118中为64.29%,28.57%和7.14%；在XRCC1-399中为56.35%,38.89%,和4.76%；在XPD-751中为84.92%,15.08%和0,以及在GSTP1-105中为68.25%,30.60%和3.97%。单因素分析显示ERCC1-118,XRCC1-399和GSTP1-105单核苷酸多态性对患者无复发生存时间和总生存时间均有预测价值。就无复发生存时间而言，ERCC1-118T/T和C/T基因型患者中位生存时间为5个月,C/C基因型患者为45个月；XRCC1-399 A/A和A/G基因型患者M-RFS为47个月,G/G基因型患者为8个月;GSTP1-105 G/G和A/G基因型患者M-RFS为47个月,A/A基因型患者为12个月。就总生存时间而言,ERCC1-118T/T和C/T基因型患者中位生存时间(MST)为15个月,C/C基因型患者中位生存时间不能确定；XRCC1-399A/A和A/G基因型患者中位生存时间不能确定,G/G基因型患者MST为18个月；GSTP1-105 G/G和A/G基因型患者MST尚不能确定,A/A基因型患者为21个月。多因素Cox风险模型分析提示ERCC1-118基因型对无复发生存时间(P<0.001,HR=2.362；CI95%：1.458-3.827)和总生存时间(P=0.001；HR=2.388；CI 95%：1.448-3.937)均具有预测价值,而XRCC1-399基因型仅对无复发生存时间有预测价值,XRCC1-399 A/A和A/G基因型患者疾病复发风险显著降低(P=0.031；HR=0.569；CI 95%：0.341-0.949)。3、重楼复方与5-FU在较宽抑制率范围内产生协同抗肿瘤作用(在SGC-7901细胞系中为20-95%抑制率范围,在BGC-823细胞中为5-65%抑制率范围),而与奥沙利铂和多西紫杉醇均在低浓度范围内产生协同抗肿瘤作用(两株细胞中均在<50%的抑制率范围内),联合指数<1。4、凋亡分析显示重楼复方与各药以协同作用方式诱导肿瘤细胞凋亡。CLFF,5-氟尿嘧啶,奥沙利铂和多西紫杉醇单药作用48h后细胞凋亡率在SGC-7901细胞中分别为(8.4±6.8),(24.6±4.6),(9.7±3.7)和(29.6±5.9)%,在BGC-823细胞中分别为(6.7±1.7),(12.6±3.8),(11.3±1.3)和(6.6±1.2)%。CLFF与5-氟尿嘧啶,奥沙利铂和多西紫杉醇联合作用48h后细胞凋亡率在SGC-7901细胞中分别为(80.5±12.9),(32.5±5.7)和(63.2±6.2)%,在BGC-823细胞中分别为(66.1±6.4),(48.6±3.1)和(33.5±9.6)%。5、重楼复方单独或分别与5-FU、奥沙利铂和多西紫杉醇联合作用后,SGC-7901和BGC-823细胞中化疗药物相关基因TS、ERCC1 (Excision repair cross-complementation 1)、β-tublinⅢ和Tau mRNA表达水平均显著下调,而化疗药单药不能下调化疗相关基因表达水平。重楼复方单独作用24小时后,上述各基因表达水平在SGC-7901细胞系中下调至(0.30±0.03)、(0.32±0.02)、(0.31±0.03)和(0.28±0.02)倍,在BGC-823细胞中下调至(0.46±0.03)、(0.46±0.02)、(0.44±0.04)和(0.56±0.07)倍。重楼复方分别联合5-FU、奥沙利铂和多西紫杉醇作用24h后,上述各基因表达水平在SGC-7901细胞系中下调至(0.55±0.02)、(0.48±0.03)、(0.59±0.04)和(0.43±0.02)倍,在BGC-823细胞中下调至(0.64±0.04)、(0.55±0.03)、(0.51±0.04)和(0.62±0.06)倍。[结论]①肿瘤组织ERCC1低表达胃癌患者接受含奥沙利铂方案辅助化疗生存可能获益,但是ERCC1高表达可能是单纯手术患者的良好预后因子。②外周血ERCC1-118C/C基因型(野生型)和XRCC1-399A/G或G/G基因型(突变型)胃癌患者接受含奥沙利铂方案辅助化疗生存可能获益；③XRCC1-399和GSTP1-105基因多态性可能与含奥沙利铂方案化疗毒副反应相关；④重楼复方与5-氟尿嘧啶,奥沙利铂和多西紫杉醇有较好的体外协同抗肿瘤作用,可能与该复方与各化疗药协同诱导肿瘤细胞凋亡以及下调化疗相关基因表达水平有关。更多还原

【Abstract】 [Objective] To explore the predictive values of platinum-related genes in gastric cancer treated with oxaliplatin-based chemotherapy and to explore the potentially synergistic effects of "Chong Lou Fu Fang" (CLFF) with chemotherapeutic agents commonly used in gastric cancer therapy in vitro.[Methods] Expression of chemotherapeutic agents associated genes was measured by real time quantitative PCR. SNPs in ERCC1 Asp118Asp, XRCC1 Arg399Gln, XPD Lys751Gln and GSTP1 Ile105Val were assessed by 5’nuclease allelic discrimination assay (TaqMan) using real-time PCR and direct sequencing, The expression levels of ERCC1 and the genotypes of ERCC1 Asp118Asp, XRCC1 Arg399Gln, XPD Lys751Gln and GSTP1 Ile105Val were tested for an association with survivals in gastric cancer patients treated with oxaliplatin-based adjuvant chemotherapy regimen.The cell proliferation capacity was determined by the MTT assay. Interaction between CLFF and chemotherapeutic agents in human gastric cancer SGC-7901 and BGC-823 cell lines was evaluated using the combination index (CI) method based on the median-effect principle. Apoptosis rates of cancer cells were evaluated by means of flow cytometry.[Results]1. The ERCC1 expression analysis in all 75 patients indicated that the median value for ERCC1 was 7.32(range 0.50-147.03).Among 75 patients,52 cases received surgery followed by at least 6 cycles modified FOLFOX4 adjuvant chemotherapy, and 23 cases received surgery alone. Univariate analysis suggested that low ERCC1 levels had longer RFS and OS than those with high ERCC1 levels (median RFS,18 versus 7 months, P=0.001; MST,27 versus 11 months, P =0.001) in group received adjuvant chemotherapy, and high ERCC1 levels had longer RFS and OS than those with low ERCC1 levels (median RFS,33 versus 12 months, P=0.038; MST,43 versus 21 months, P=0.004) in group received surgery alone. Cox proportional hazards regression model multivariable analysis suggested that high ERCC1 expression is an independent prognostic marker of poor RFS (hazard ratio 2.16,95% confidence interval 1.09-4.25, P=0.026) and OS (hazard ratio 2.21,95% confidence interval 1.07-4.55, P=0.031) in oxaliplatin-based adjuvant settings, but remained significantly positive prognostic marker for OS (hazard ratio 0.17,95% confidence interval 0.04-0.71, P=0.015) in patients receiving surgery alone.2.The genotype analysis in 126 gaseric cancer patients who received at least 6 cycles modified FOLFOX4 adjuvant chemotherapy indicated the frequencies for the homozygous wildtype allele, heterozygous and homozygous polymorphic variant:64.29%,28.57%, and 7.14% for ERCC1-118; 56.35%,38.89%, and 4.76% for XRCC1-399; 84.92%,15.08%,and 0 for XPD-751; and 68.25%,30.60%,and 3.97% for GSTP1-105. Univariate analysis indicated that the ERCC1-118, the XRCC1-399 and the GSTP1-105 SNPs showed the predictive values for RFS and OS. With regard to RFS, for ERCC1-118, the median RFS was 5 months for T/T and C/T cases and 45 months for C/C patients (P<0.001). For XRCC1-399, the median RFS was 47 months for A/A and A/G cases and 8 months for G/G patients (P=0.001), and For GSTP1-105, the median RFS was 47 months for G/G and A/G cases and 12 months for A/A patients (P<0.001). As for OS, the ERCC1-118, the XRCC1-399 and the GSTP1-105 SNPs also remained significant predictive value. For ERCC1-118, the MST was 15 months for T/T and C/T patients and no defined for C/C cases (P<0.001). For the XRCC1-399, the MST was 18 months for A/A patients and no defined for A/A and A/G patients P<0.001), and For GSTP1-105, the MST was no defined for G/G and A/G cases and 21 months for A/A patients (P=0.019). Cox proportional hazards regression model multivariable analysis suggested ERCC1-118 remained significant predictive value for RFS (P<0.001, HR=2.362; CI 95%:1.458-3.827) and OS (P=0.001; HR=2.388;CI 95%:1.448-3.937), and XRCC1-399 only remained significant predictive value for RFS, and XRCC1-399 (A/A+A/G) genotype could significantly decrease the recurrence hazard of the patients (P<0.001, HR=0.569; CI 95%:0.341-0.949).3.The synergistic analysis indicated that CLFF had a synergistic effect on the cytotoxicity of 5-fluorouracil (5-FU) in a relative broad dose inhibition range (20-95% fraction affected in SGC-7901 cell lines and 5-65% fraction affected in BGC-823 cell lines), while the synergistic interaction between CLFF and oxaliplatin or docetaxel only existed in a low dose inhibition range (<50% fraction affected in both cell lines), CIs<1.4. The apoptosis analysis indicated the combination of CLFF and 5-fluorouracil, oxaliplatin or docetaxel could also induce apoptosis in a synergistic manner. The percentages of the apoptotic cells (early apoptosis plus late apoptosis) induced by CLFF and 5-fluorouracil、oxaliplatin and docetaxel were (8.4+6.8)%, (24.6+4.6)%(9.7+3.7)%and (29.5+5.9)%in SGC-7901 cell lines and (6.7+1.4)%, (12.6+3.8)%(11.6+1.3)%, (6.6+1.2)%in BGC-823 cell lines, while the percentage of them induced by drug combination in both cell lines were increased to (80.5+12.9)%, (32.5+5.7)%and (63.2+6.2)%in SGC-7901 cells, respectively, and (66.1+6.4)%, (48.6+3.1)%and (33.5+9.6)%in BGC-823 cells, respectively.5.The expression levels of ERCC1, TS, b-tubulinⅢand tau were significantly down-regulated after treatment with CLFF alone or CLFF combined with chemotherapeutic agents in SGC-7901 cell lines and BGC-823 cell lines. However, any of chemotherapeutic agents alone did not down-regulate their respective drug resistance-associated genes. TS、ERCC1、β-tublinⅢand Tau mRNA levels were markedly suppressed at (0.32±0.02)、(0.30±0.03)、(0.31±0.03)and (0.28±0.02) fold in SGC-7901 cell lines, respectively, and 0.46±0.03)、(0.46±0.02)、(0.44±0.04) and (0.56±0.07)fold in BGC-823 cell lines, respectively, by the presentation of CLFF for 24 h.At the same time, we found that the combination of CLFF and 5-FU, oxaliplatin and docetaxel for 24 h could downregulat the TS、ERCC1、β-tublinⅢand Tau mRNA levels to (0.55±0.02)、(0.48±0.03)、(0.59±0.04) and (0.43±0.02) fold in SGC-7901 cell lines, respectively, and (0.64±0.04)、(0.55±0.03)、(0.51±0.04) and (0.62±0.06) fold in BGC-823 cell lines, respectively.[Conclusion] It seemed that gastric cancer patients with low levels of ERCC1 expression showed a great benefit from oxaliplatin-based adjuvant chemotherapy, while high levels of ERCC1 expression might be a positive prognostic marker for patients receiving surgery alone.On the other hand, gastric cancer patients harboring ERCC1-118C/C genotype and XRCC1-399A/G or G/G genotypes may better receive oxaliplatin-based adjuvant chemotherapy. XRCC1-399 and GSTP1-105 polymorph-isms may be associated with the toxicities of the oxaliplatin-based chemotherapy regimen.The combination of CLFF and 5-fluorouracil, oxaliplatin or docetaxel showed significant synergistic antitumor activity in both two gastric cancer cell lines. The possible mechanisms might be the synergistic apoptotic effect and the down-regulation of chemotherapeutic agents associated genes.更多还原