【作者】 刘红；

【导师】 甄永苏；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2008， 博士

【摘要】 肺癌是世界上死亡率最高的恶性肿瘤之一,其中超过80%的是非小细胞肺癌。虽然近年来外科手术、放疗和化疗方面的进展在一定程度上提高了肺癌患者的生存时间和生存质量,但肺癌的5年生存率仍然只有14%,而且患者确诊为肺癌时多已发生远端转移。早期诊断率低、肺癌细胞对常用化疗药物的固有或继发性耐药和高的肿瘤转移率是导致治疗失败的重要原因。因此,临床上迫切需要高效低毒的新药。力达霉素（Lidamycin,LDM）是本研究所从一株放线菌（Streptomycesglobisporus C-1027）代谢产物中获得的对多种肿瘤细胞有强烈杀伤作用的大分子肽类抗肿瘤抗生素。大量研究表明,LDM对多数肿瘤细胞及移植瘤具有极强的抗肿瘤活性,这都源于LDM对DNA的强烈地切割作用。最近研究显示LDM可引起肿瘤细胞染色体畸变及端粒功能异常。目前正在进行临床Ⅱ期试验研究。本课题选用3种人非小细胞肺癌A549,H460及H157细胞株,探讨LDM体外体内对人非小细胞肺癌的影响及其作用机制。此外,本课题还初步研究了LDM联合gefitinib对表达不同水平EGFR的A431细胞及H460细胞的影响,并比较了两药对H460移植瘤的抑制作用,从而为LDM联合gefitinib的临床应用提供依据。一、LDM作用于非小细胞肺癌的体外实验研究1、MTT结果显示LDM显著抑制人表皮样癌A431、人肺腺癌A549、人大细胞肺癌H460、人肺鳞癌H157、人巨细胞性肺癌801D及人高转移性巨细胞肺癌PG等肿瘤细胞的增殖。LDM对这些细胞的IC₅₀值分别为1.12×10^-10±1.10×10^-10M、3.65×10^-12±2.94×10^-12M、2.41×10^-12±1.15×10^-12M、1.24×10^-10±1.14×10^-10M、5.66×10^-12±1.13×10^-12M、2.40×10^-11±1.16×10^-11M,细胞增殖抑制作用显著强于常用抗肿瘤化疗药。2、Hoechst33342荧光染色法和Annexin V-FITC/PI染色法结合流式细胞仪检测LDM对H460、H157、A549细胞凋亡的诱导作用。结果显示不同浓度LDM作用细胞48 h后,细胞凋亡率剂量依赖性地增高。H460细胞对LDM的凋亡诱导作用最敏感,0.5 nM的LDM可诱导43.30±4.26%的H460细胞发生早期凋亡,2 nM剂量下可以产生69.87±3.29%的凋亡细胞。LDM也可显著诱导H157细胞及A549细胞发生早期凋亡,但凋亡程度较H460弱。荧光显微镜下可见1 nM、5 nM LDM组的细胞出现染色质凝集、细胞核固缩、形成凋亡小体等典型的凋亡细胞形态学改变。TUNEL法检测结果也显示,LDM可剂量依赖地诱导H460及H157细胞凋亡。3、PI单染结合流式细胞仪检测LDM对细胞周期的影响。结果显示,小于1 nM的LDM主要将细胞阻滞于G2/M期,1 nM及2 nM LDM可同时引起S期和G2/M期阻滞。同时检测的SubG1细胞剂量依赖地增加也证实了LDM对凋亡的诱导作用。在1 nM、2nM剂量下,LDM可分别引起H460细胞39.45±3.75%及52.55±4.45%的细胞产生Sub-G1峰,同样剂量的LDM也显著诱导H157及A549细胞的凋亡,但凋亡的程度不如H460细胞的显著。4、Western bloting法检测了LDM对细胞周期及凋亡相关蛋白的影响。结果显示LDM剂量依赖地诱导3种非小细胞肺癌细胞Caspase-3、Caspase-7的活化及PARP的切割,显著下调凋亡抑制蛋白Bcl-2及NF-κB的水平,诱导细胞产生caspase介导的凋亡。同时LDM还可上调p53及p21蛋白的表达及下调CyclinB1蛋白的表达,引起G2/M期阻滞。5、利用Transwell实验观察LDM对非小细胞肺癌细胞的迁移和侵袭能力的影响。实验结果显示,LDM对3种非小细胞肺癌细胞的迁移和侵袭能力均有显著的抑制作用,并呈明显的剂量依赖关系。6、利用明胶酶谱法观察LDM对3种非小细胞肺癌细胞基质金属蛋白酶的影响。结果显示,LDM可剂量依赖地显著抑制各细胞MMP-9的分泌,并显著抑制H157细胞MMP-2的分泌及活化,而对H460及A549细胞的MMP-2无显著影响,进一步证实了LDM的抗侵袭迁移的能力。7、Western bloting法测定了LDM对侵袭和迁移相关靶点分子的影响。结果显示LDM可剂量依赖地下调3种非小细胞肺癌细胞KDR,VEGF,COX-2,及MMP-9、MMP-2的表达及活性,但下调的程度不尽相同。8、Western bloting法结果显示LDM可通过影响3种非小细胞肺癌细胞EGFR信号通路各信号分子的磷酸化水平从而起到抑制细胞增殖、诱导细胞凋亡、周期阻滞及抗侵袭的作用。虽然LDM对于3种细胞EGFR通路上多数信号分子产生不同的效应（可能与细胞的类型不同有关）,但经LDM处理后3种细胞最显著的相同之处为RAF/MEK/ERK通路的激活,即LDM剂量依赖地增强了MEK及ERK的磷酸化水平。9、Western bloting法及Annexin V-FITC/PI双染法结果显示MEK抑制剂U0126可部分消除LDM引起的ERK的活化,同时也可减轻LDM诱导凋亡的程度,说明ERK通路参与了LDM的凋亡诱导作用。二、LDM作用于非小细胞肺腺癌的体内实验研究利用人肺腺癌A549裸鼠移植瘤模型检测LDM的体内抗肿瘤作用。LDM剂量为0.02 mg/kg的抑瘤率为39.5%,0.04 mg/kg的抑瘤率为57.6%,与对照组相比有显著性差异。三、LDM与gefitinib联合作用的体外实验研究1、MTT法观察了gefitinib对A549、H460、H157及A431细胞的增殖抑制作用。Gefitinib对EGFR高表达A431细胞的IC₅₀值为0.28±0.03μM,远小于对EGFR表达适中的3株非小细胞肺癌细胞的IC₅₀值（16.04±2.96μM～19.57±6.6μM）。选择对gefitinib最不敏感的H460细胞及最敏感的A431细胞进行后续的联合作用的研究.2、MTT法比较了三种联合给药顺序对人H460细胞的联合抑制作用。结果显示先加LDM,8 h后加gefitinib联合效果较好。多数联合剂量有协同增殖抑制作用（CDI＜1）,其中0.1 nM LDM与5μM gefitinib联合作用时,对H460细胞协同增殖抑制作用非常显著（CDI＜0.70）。同样经过这种给药方案处理后,一定的联合剂量下两药对A431细胞有协同增殖抑制作用,其中0.01 nM LDM与0.1μM gefitinib联合对A431细胞具显著地协同增殖抑制作用（CDI＜0.70）。3、用Annexin V-FITC/PI双染结合流式细胞术检测LDM与gefitinib对H460及A431细胞的凋亡诱导作用。结果显示随着两药剂量的增加凋亡细胞的比率渐增。LDM显示了强大的凋亡诱导作用,2 nM的LDM可分别诱导69.87±3.29%（P＜0.001）的H460细胞、63.2±1.39%（P＜0.001）的A431细胞发生凋亡。而gefitinib对敏感细胞A431的凋亡诱导作用较强。1μM的gefitinib诱导30.70%±1.69%（P＜0.01）的A431细胞发生凋亡;而对不敏感细胞H460,20μM gefitinib仅引起13.60±1.37%（P＜0.05）的细胞发生凋亡。4、Annexin V-FITC/PI染色法观察两药联合对凋亡的诱导作用。结果显示在低剂量组合下,0.01 nM的LDM与0.1μM的gefitinib联合对A431的凋亡诱导率为31.87%,明显高于两药单独作用引起的凋亡率6.71%及12.79%;在高剂量组合下,0.1 nM的LDM与1μM的gefitinib联合对A431的凋亡诱导率为53.93%,明显高于两药单独作用引起的凋亡率21.45%及31.89%。Gefitinib单药对H460细胞的凋亡诱导作用较弱,但LDM可增强gefitinib对于凋亡的诱导作用。0.5 nM的LDM与20μM的gefitinib联合对H460细胞的凋亡诱导率为53.85%,高于两药单独作用引起的凋亡率42.88%及12.63%。以上结果说明联合用药对gefitinib敏感细胞的凋亡诱导率显著高于单独用药,不敏感细胞联合用药后显示略强于单独用药的凋亡诱导作用。5、Western bloting法测定单药及联合用药对PARP的切割作用及对凋亡抑制蛋白NF-κB的作用。在A431细胞中观察到gefitinib剂量依赖地增强了PARP的切割。两药联合引起PARP切割的显著增强。然而,在试验剂量下,gefitinib几乎不能引起H460细胞的PARP切割。两药联合作用后的效果与LDM单药相似,仅在10μMgefitinib与0.5 nM LDM联合作用时才显示略强于单药的作用。联合用药可显著降低两种细胞的NF-κB水平,从而增强了单药的凋亡诱导作用。6、Western bloting法检测了联合用药对H460及A431细胞EGFR通路信号分子的影响。结果显示0.01 nM的LDM与0.1μM、1μM gefitinib联合较单药显著地降低了A431细胞p-EGFR,p-ERK及p-Akt水平。对于H460细胞,在0.5 nM LDM与10μM gefitinib剂量组合下,联合作用较单药可略微降低p-EGFR的水平,但联合用药可显著抑制EGF引起的H460细胞EGFR及AKT磷酸化水平的增高。四、LDM与gefitinib对于大细胞肺癌H460裸鼠移植瘤的抑制作用利用人大细胞肺癌H460裸鼠移植瘤模型检测LDM的体内抗肿瘤作用。结果显示,LDM和gefitinib对H460移植瘤的生长均有抑制作用,并且LDM显示出更强的抑瘤效果。0.025 mg/kg、0.05 mg/kg的LDM对移植瘤的抑瘤率分别为52.8%及72.4%,与生理盐水对照组相比有显著性差异。50 mg/kg的gefitinib的抑瘤率为69.4%,与0.5%Tween 80溶剂对照组相比有显著性差异,与可耐受剂量0.05mg/kg LDM组抑制作用相当。更多还原

【Abstract】 Lung cancer is the leading cause of cancer deaths worldwide. Non-small cell lung cancer （NSCLC） accounts for 80% of lung cancer patients. In spite of new treatments, the overall five-year survival rate remains about 14% and most patients present with advanced disease. Treatment outcomes for NSCLC patients still are considered disappointing because of chemo-resistance and dose-accumulated toxicity.LDM showed extremely potent cytotoxicity toward culture cancer cells and markedly inhibited the growth of transplantable tumors in mice and human cancer xenografts in nude mice. The potent efficacy of LDM was ascribing to its DNA strand-scission activity. Recent study displayed the chromosomal aberrations and telomere dysfunction induced by LDM. LDM is currently being evaluated in phase II clinical trials as a potential chemotherapeutic agent in China.Studies on the molecular mechanisms of LDM alone and in combination with gefitinib against NSCLC were investigated.1. Effects of LDM on NSCLC cells in vitro1.1. Antiproliferative activities were measured by MTT assays. Remarkable growth inhibition effects of LDM were found in all tested NSCLC cell lines. The IC50 values of LDM for all the cell lines tested were much lower than those of the other chemotherapeutic drugs.1.2. Flow cytometry combined with FITC-Annexin V/PI staining showed that LDM induced apoptosis of NSCLC cells dose-dependently. H460 cell line was the greatest sensitive cells which consisted of 43.30%±4.26% （ P<0.001） apoptotic cells after exposure to 0.5 nM LDM and the apoptotic cells induced by 2nM LDM reached 69.87±3.29% （P<0.001）. LDM also induced significant apoptosis in H157 and A549 cells, but the extent of apoptosis was lower than that of H460 cells.The Hoechst 33342 staining was used to assess the change in nuclear morphology after the treatment of LDM. The nuclei of untreated cells and low concentration LDM treated cells were normal and exhibited diffused staining of the chromatin. After exposure to LDM for 48 h, most cells of the three cell lines treated with 1nM and 5nM LDM presented typical morphological changes of apoptosis such as chromatin condensation, nucleus shrink and the formation of apoptotic bodies.The ratios of apoptosis cells detected with TUNEL method were increased with the concentration of LDM increasing in H460 cells and H157 cells.1.3. The flow cytometric cell cycle analysis showed that LDM at the doses of below 1 nM induced G₂/M arrest, 1nM and 2nM LDM produced an S-phase block in addition to G2/M arrest.Sub 2N DNA formation was assessed for measuring apoptosis quantitatively. The peak effect was in dose dependent and achieve to the maximum percentage of 52.55±4.45 of apoptotic cells in H460 cells exposed to 2 nM of LDM. At 1 nM and 2 nM, LDM also induced significant apoptosis in H157 and A549 cells, but the extent of apoptosis was lower than that of H460 cells.1.4. Western blotting showed that LDM significantly increased caspase-3 and caspase-7 activities as well as PARP cleavage. The decreased Bcl-2 and NF-κB indicated that mitochondria-caspase cascade was responsible for LDM-induced apoptosis. The decreased CyclinB1 and the up-regulated of P53 and P21 were the potent evidence for G₂/M phase arrest.1.5. The effects of LDM on the migration and invasion of the NSCLC cells were examined with transwell chamber assay. A dose dependent reduction in migration or invasion of the three cell lines was found after exposure to various concentrations of LDM. Both of H157 and A549 cells have high invasion activities; however, H460 cells have the weakest migration and invasive activity. 1.6. Zymography analysis showed that LDM strongly inhibited the secretion of MMP-9 in all the tested cells and the secretion and activation of MMP-2 in HT-1080 cells and H157 cells dose dependently. However, the MMP-2 levels in H460 cells and A549 cells were little affected by LDM.1.7. Western blot analysis showed that LDM downregulated the levels of KDR, VEGF, COX-2, MMP-9 and MMP-2 dose dependently with different extent in the three cell lines.1.8. Western blot analysis showed that LDM exhibited potent effects of antiproliferation, apoptosis induction, cell cycle arrest and anti-invasion by regulating the activities of EGFR signaling pathway targets, although to the various extents in different cell lines. The most significant coincidence was the activation of RAF/MEK/ERK signal pathway in the three cell lines.1.9. Western blot analysis and FITC-Annexin V/PI staining showed that the up-regulations of p-ERK1/2 levels in the three cells treated with LDM were ablated by U0126 （MEK1/2 inhibitor）. Meanwhile, U0126 attenuated apoptosis induced by LDM. The results indicated a correlation between LDM-mediated apoptosis and ERK activation.2. In vivo antitumor activity of LDM on A549 xenograftsTreatment with LDM at the doses of 0.02 mg/kg and 0.04 mg/kg inhibited the growth of human adenocarcinoma A549 xenografts by 39.5% and 57.6%, respectively. The body weights of animals showed no significant differences between control and treated groups.3. Effects on H460 cells and A431 cells by the combination of LDM with gefitinib3.1. MTT assay showed that LDM was much more potent than gefitinib since the IC₅₀ values of LDM for cell lines tested were lower than those of gefitinib. The levels of EGFR were associated with the sensitivity to gefitinib, since potent inhibition (IC₅₀: 0.28±0.03μM) was observed in highly EGFR-expressing A431 cell line whereas the moderate EGFR-expressing NSCLC cells were relatively resistant （16.04±2.96μM～19.57±6.6μM）.3.2. To determine whether a potentiation of the antiproliferative activity could be obtained by the combination of LDM and gefitinib, a series of experiments were performed on H460 and A431 cell lines treated with different doses of each drug. Slight synergistic growth inhibitory effects at most combinations （CDK1） were found in the two cell lines. The CDI was<0.70 at the combination dose [gefitinib （μM）/LDM （nM）:5/0.1] in H460 cells, [gefitinib （μM）/LDM （nM）:0.1/0.01] in A431 cells. The synergistic effects were achieved at lower gefitinib concentration in A431 cells than in H460 cells.3.3. Induction of apoptosis by LDM and gefitinib in H460 and A431 cell lines were measured by flow cytometry combined with FITC-Annexin V/PI staining. The results showed that the percentage of Annexin V-positive cells increased dose dependently in the two cell lines after treated by the two drugs. LDM showed similar potent apoptosis induction effects on H460 cells and A431 cells. However, A431 cell line was more sensitive than H460 cell line to gefitinib; there occurred 30.70%±1.69% （ P<0.01） apoptotic cells as A431 cells exposed to 1μM gefitinib whereas only 13.60±1.37% （P<0.05） apoptotic cells induced by 20μM gefitinib in H460 cells.3.4. FITC-Annexin V/PI analysis showed that the combination treatment induced more intensive apoptosis when compared to that by each agent alone in A431 cells. The lower dose combination, 0.1μM gefitinib plus 0.01 nM LDM, induced apoptosis in 31.87% of cells, whereas the same doses of gefitinib and LDM given alone resulted in apoptosis in only 12.79% and 6.71% of cells, respectively. Likewise, at the higher dose level （1μM gefitinib plus 0.1 nM LDM） the apoptotic rate was 53.93%, clearly superior to the 31.89% and 21.45% of apoptosis with single-agent gefitinib and LDM, respectively. The percentage of apoptotic cells was much lower in H460 cells even in higher dose of gefitinib. However, LDM could slightly potentiate the apoptotic effect of gefitinib at certain doses. 20μM gefitinib plus 0.5 nM LDM induced apoptosis in 53.85% of cells, whereas the same dose of gefitinib and LDM given alone resulted in only 12.63% and 42.88% of apoptotic cells, respectively.3.5. Western blot analysis showed that a dose-dependent increase in PARP cleavage was observed in A431 cells exposed to gefitinib. More significant increase in PARP cleavage was observed after combined treatment. However, gefitinib barely increase the PARP cleavage in H460 cells at the doses tested. 10μM gefitinib plus 0.5 nM LDM introduced slight increase in PARP cleavage. Furthermore, greater down-regulating of the anti-apoptotic molecule NF-κB in the two cell lines also related to enhancement of apoptosis by combined treatment.3.6. Western blot analysis evaluated the effects of gefitinib and LDM on EGFR signaling pathway molecules in H460 and A431 cells. Phosphorylation of EGFR, ERK and Akt decreased in gefitinib-treated A431 cells and a more pronounced decrease in the levels of phosphorylation of each marker was observed after the combined treatment. 10μM gefitinib plus 0.5 nM LDM slightly decreased the level of p-EGFR but not of p-ERK and p-Akt as compared to each single agent in H460 cells. Pretreatment with gefitinib inhibited EGF-induced phosphorylation of EGFR and Akt in H460 cells. Combined treatment markedly inhibited the increment of EGFR and Akt phsphorylation induced by EGF. However, the inhibition of the activation of ERK by combination was similar to that of gefitinib alone.4. Comparison in vivo antitumor activity of LDM and gefitinib on H460 xenograftsTreatment with LDM at the dose of 0.025 mg/kg and 0.05 mg/kg inhibited the growth of human large cell lung carcinoma H460 xenografts by 52.8% and 72.4%, respectively. Oral administration of gefitinib at the dose of 50 mg/kg inhibited the growth of H460 xenografts by 69.4%. The inhibitory effect was comparable to the tolerable dose of LDM of 0.05 mg/kg. The body weights of the animals showed no significant differences between the control and treated groups.更多还原