力达霉素抗胰腺癌作用的分子机制研究
摘要
胰腺癌是一种预后极差的消化系统恶性肿瘤，其死亡率高，5年生存率低于5％，发病率呈逐年增加趋势。由于胰腺癌发病隐袭，临床表现多无特异性，病情进展迅速，早期缺乏敏感和特异的诊断指标，大多数患者在确诊时已达晚期或有远处转移，只有10％～15％的患者有手术切除的机会，其中能根治者仅5％～7.5％，因此药物治疗对于胰腺癌患者来说是必不可少的治疗手段。
力达霉素(Lidamycin，LDM)是本研究所从一株放线菌(Streptomyces globisporus 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。力达霉素分子由一个酸性的辅基蛋白和一个烯二炔发色团组成，其中发色团是力达霉素的主要活性部分，具有断裂DNA的作用，而辅基蛋白的主要作用是保护发色团。大量研究表明，力达霉素具有极强的抗肿瘤活性，目前正在进行临床Ⅱ期试验研究。本课题选用人胰腺癌PANC-1和SW1990细胞株，探讨力达霉素对人胰腺癌细胞的影响及其作用机制，并通过人胰腺癌细胞SW1990裸鼠移植瘤实验，观察力达霉素在体内的抗胰腺癌作用。此外，本课题还初步研究了力达霉素与健择(Gemcitabine，吉西他滨)联合作用对胰腺癌细胞的影响，并探讨其作用机制，从而为力达霉素的临床应用提供理论依据。
一、力达霉素作用于胰腺癌的体外实验研究
1．MTT法检测力达霉素(LDM)对人胰腺癌PANC-1和SW1990细胞的增殖抑制作用。不同浓度的阿霉素(ADM)、丝裂霉素(MMC)、紫杉醇(Taxol)、吉西他滨(Gemcitabine)和LDM作用细胞48 h后，利用MTT法检测各种药物对胰腺癌细胞的增殖抑制作用。MTT结果显示，与临床常用化疗药物相比，LDM抑制细胞的增殖作用更强，对PANC-1和SWl990细胞的IC_(50)值分别为0.955±0.414 nM和0.426±0.212 nM，低于其他药物的10~3～10~4倍。
2．Hoechst 33342荧光染色法和Annexin V-FITC／PI染色结合流式细胞仪检测细胞凋亡。不同浓度LDM作用细胞48h后，细胞凋亡比率逐渐增高，呈浓度依赖性，其中0.1nM LDM、1nM LDM、2 nM LDM和10 nM LDM作用于PANC-1细胞的细胞凋亡率分别为12.33％±0.66％、32.26％±1.58％、45.03％±3.16％和59.44％±4.04％，作用于SW1990细胞的细胞凋亡率分别为22.21％±0.28％、22.83％±0.26％、42.52％±0.67％和50.35％±0.56％，显著高于对照组的6.15％±1.47％和7.51％±0.45％(P＜0.001)。荧光显微镜下可见1 nM、2 nM和10 nM LDM作用组的细胞出现染色质凝集、细胞核固缩、核碎裂、形成凋亡小体等典型的凋亡细胞形态学改变。
3．利用PI单染并结合流式细胞仪检测LDM对细胞周期的影响。检测结果显示，1 nM LDM主要将细胞阻滞在G2／M期，2 nM LDM能引起S期和G2／M期阻滞，10 nM LDM则主要诱导S期阻滞。
4．利用Transwell实验观察LDM对胰腺癌细胞的迁移和侵袭能力的影响。实验结果显示，LDM对PANC-1细胞和SW1990细胞的迁移和侵袭能力均有显著的抑制作用，并且LDM的作用浓度越大，抑制作用越强，呈现明显的浓度依赖关系。
5．Western blot检测结果显示LDM能够减少基质金属蛋白酶-9(MMP-9)蛋白在胰腺癌细胞中的表达水平，并且随着LDM作用浓度的增加，抑制蛋白表达的作用亦随之加强，提示LDM通过降低细胞内MMP-9的含量而有效抑制细胞的迁移和侵袭能力，并呈剂量依赖性。
6．Western blot和RT-PCR结果表明，LDM能够明显抑制胰腺癌细胞中VEGF蛋白的表达，但不影响细胞中VEGF mRNA的表达水平，说明LDM是在转录后的翻译水平对VEGF的表达进行调节。
7．细胞中K-ras蛋白和mRNA表达水平，以及p-AKT和NF-κB蛋白含量都随LDM作用浓度的增高而降低，呈现一定的浓度依赖性。LDM对于细胞中的AKT蛋白含量没有影响，表明LDM能够抑制AKT蛋白的磷酸化过程。
二、力达霉素作用于胰腺癌的体内实验研究
1．利用人胰腺癌SW1990移植瘤裸鼠模型检测LDM的体内抗肿瘤作用。在瘤块接种后的第7天开始给药，每周1次，共2次。LDM通过尾静脉注射，给药剂量为0.02mg／kg和0.04 mg／kg；Gemcitabine通过腹腔注射，给药剂量为80mg／kg；对照组给予生理盐水。动物实验结果显示，LDM和Gemcitabine对SW1990细胞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。实验第31天处死动物，剥取各组动物的瘤块进行比较，计算抑瘤率，其中Gemcitabine组的抑瘤率为38.87％，LDM 0.02 mg／kg剂量组和0.04 mg／kg剂量组的抑瘤率分别为65.71％和78.27％，与对照组相比具有显著性差异。
2．肿瘤组织切片经HE染色，显微镜下可见，LDM作用组的肿瘤组织出现血管减少，肿瘤细胞坏死增多等病理学变化。
3．利用脱氧核糖核酸末端转移酶介导的缺口末端标记(TUNEL)技术，对裸鼠肿瘤组织中的凋亡细胞进行观察和比较。TUNEL实验结果显示，与对照组和Gemcitabine组相比，LDM治疗组中凋亡细胞明显增多。
4．免疫组化结果显示，LDM能够抑制肿瘤细胞中K-ras蛋白和CD31蛋白的表达，并呈现剂量效应关系，表明LDM具有抑制肿瘤新生血管生成的作用。
三、力达霉素与吉西他滨联合作用于胰腺癌的体外实验研究
目前，健择(Gemcitabine，吉西他滨)是临床上用于治疗胰腺癌的一线药物，健择与其他化疗药物合用治疗肿瘤亦得到广泛研究和应用，本课题对力达霉素联合健择对胰腺癌细胞的作用进行了研究，并探讨其主要作用机理。
1．MTT结果显示，1 nM LDM与500 nM Gemcitabine联合作用时，能够明显抑制细胞增殖，其CDI＜0.75，具有协同抗肿瘤作用。
2．TUNEL实验结果显示，与单独用药相比较，1 nM LDM联合500 nM Gemcitabine作用于PANC-1和SW1990细胞，能够明显提高凋亡细胞的比率。流式细胞仪检测结果显示，二者联合用药作用PANC-1和SW1990细胞的细胞凋亡率分别为59.44％±1.54％和54.68％±2.62％，显著高于Gemcitabine单独作用组的18.48％±0.94％和25.79％±2.06％(P＜0.05)。
3．RT-PCR结果显示，500 nM Gemcitabine和1 nM LDM联合作用能够显著降低PANC-1和SW1990细胞中K-ras mRNA的表达水平。
4．Western blot结果表明，LDM与Gemcitabine联合作用于PANC-1和SW1990细胞，能够减少细胞中K-ras蛋白的表达；500 nM Gemcitabine或1nM LDM单独作用时，使细胞中的NF-κB蛋白水平提高，但二者联用能够显著降低细胞中NF-κB的蛋白表达水平。
5．利用荧光探针JC-1负载，通过共聚焦激光扫描显微镜观察和测定细胞线粒体膜电位的改变，结果表明，LDM与Gemcitabine联合作用能够明显降低细胞的线粒体膜电位。
6．Caspase-3活性测定结果表明，LDM和Gemcitabine联合作用，能够导致PANC-1和SW1990细胞内的凋亡系统caspase-3蛋白酶激活；Western blot结果显示，LDM与Gemcitabine联合作用，能够显著降低PANC-1和SW1990细胞中Bcl-2的蛋白表达水平。
Pancreatic carcinoma is a highly malignant neoplasm characterized by locally advanced unresectable disease or metastasis at the time of diagnosis. Current therapies are largely ineffective in this disease and the 5-year survival rate remains dismal. Pancreatic cancer is now one of the most common causes of cancer death worldwide. Complete surgical resection remains the only potentially curative treatment modality for patients with pancreatic cancer. Unfortunately, the vast majority of patients present with unresectable or metastatic disease. Even among patients undergoing a potentially curative resection, many will develop metastatic disease. Therefore, the development of novel approaches for pancreatic cancer treatment is essential as tumor cells are highly resistant to most conventional chemotherapy drugs.
Enediyne antibiotics have been focused on their potent antitumor activity due to their unique ability to damage the DNA of tumor cells by inducing single strand and/or double strand breaks through free radical attacks on the deoxyribose moieties in DNA. Lidamycin (LDM, also named as C-1027) is a member of the enediyne antibiotic family, which was produced by a Streptomyces globisporus C-1027 strain isolated in China. LDM molecule contains an enediyne chromophore (MW 843 Da) responsible for the extremely potent bioactivity and a noncovalently bound apoprotein (MW 10.5 kDa), which formed a hydrophobic pocket for protecting the chromophore. Lidamycin is currently being evaluated in phaseⅡclinical trials as a potential chemotherapeutic agent in China. As reported, LDM shows extremely potent cytotoxicity, anti-angiogenic activity and marked growth inhibition of transplantable tumors in mice. To determine the action of lidamycin on pancreatic cancer cells and to reveal its possible mechanism, we investigated the effects of lidamycin on two established pancreatic cell lines, PANC-1 and SW1990.
1. Effects of lidamycin on PANC-1 and SW1990 cells cultured in vitro
1.1 Inhibition of cell proliferation was measured by the MTT assays. A remarkable difference in chemosensitivity to lidamycin, mitomycin, adriamycin, taxol, and gemcitabine was found in both cell lines. The IC_(50) value of lidamycin for PANC-1 or SW1990 cells was 0.955±0.414nM or 0.426±0.212nM, and much lower than that of other drugs.
1.2 Flow cytometry combined with FITC-Annexin V/PI staining showed that LDM at 1nM induced earlier apoptosis in both cell lines (the apoptotic ratio were 32.26%±1.58% and 22.83%±0.26% in PANC-1 and SW1990 cells, respectively). The rate of apoptosis was significant enhanced when cells were incubated with 2nM or 10nM LDM for 48h (45.03%±3.16% and 59.44%±4.04% in PANC-1 cells, 42.52%±0.67% and 50.35%±0.56% in SW1990 cells, respectively). After exposure to LDM for 48h, most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken nucleus by Hoechst33342 staining. Some condensed nuclei were observed when cells exposed to higher concentrations of LDM.
1.3 Flow cytometric cell cycle analysis showed that LDM at 1nM induced G2/M arrest and at 2nM induced S and G2/M arrest. The accumulation of cells in S phase was induced by 10nM of LDM.
1.4 The effect of LDM on the migration or invasion of PANC-1 and SW1990 cells through an extracellular matrix was examined using a transwell cell migration chamber assay. A dose-dependent reduction in cell migration or invasion in both cell lines was found after exposure to LDM.
1.5 The expression of MMP-9 was determined by Western blot analysis. PANC-1 and SW1990 cells treated with LDM showed a dose-dependent decrease in the expression of MMP-9. The level of MMP-9 was markedly reduced by 1nM of LDM.
1.6 The decrease of VEGF protein expression was observed in PANC-1 and SW1990 cells after exposure to LDM for 48h, Whereas the VEGF mRNA level was unaffected. The results indicated that LDM could affect VEGF on the translation phase.
1.7 The result of RT-PCR showed that the levels of K-ras mRNA were decreased after treated with LDM at 2nM in PANC-1 cells and 1nM in SW1990 cells. Meanwhile, the levels of K-ras, phospho-Akt and NF-κB proteins decreased in a dose-dependent manner in either cell line, whereas the level of Akt protein was unaffected by LDM.
2. In vivo antitumor activity of lidamycin
2.1 Athymic female BALB/c (nu/nu) mice (20±2g, obtained from the Institute for Experimental Animals, Chinese Academy of Medical Sciences) at the age of 4-6 weeks were used for SW1990 human pancreatic tumor xenografts. Animals bearing s.c. tumors received by i.v. injection (200μl) either PBS (vehicle control) or LDM once a week for 2 weeks. The doses of LDM were 0.02mg/kg and 0.04mg/kg respectively. Animals received by i.p. injection (＜300μl) of gemcitabine at a dose of 80mg/kg as the positive control. The mice were weighed and tumor sizes were measured with a vernier caliper and recorded every other day. Tumor volume was calculated using the formula: length×width~2×0.5. Growth of the established s.c. tumors in nude mice was decreased significantly when treated with LDM over a period of 3 weeks compared with control PBS-treated animals. Treatment with LDM at the dose of 0.02mg/kg and 0.04mg/kg inhibited the growth of pancreatic carcinoma SW1990 xenografts by 66% and 72%, respectively. The antitumor activity of LDM is more remarkable than that of gemcitabine. The body weights of animals showed no significant differences between control and treated groups.
2.2 By hematoxylin and eosin staining, the pathologic changes of tumor tissues from SW1990 xenografted nude mice were observed. There were a decrease in tumor angiogenesis and an increase in necrosis of tumor cells in LDM-treated groups.
2.3 The induction of apoptosis in the pancreatic tumors was evaluated by TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) assay. In tumors from control-treated mice, the median number of apoptotic cells was minimal. The number of apoptotic cells in tumors of LDM-treated mice increased.
2.4 Vessel density in the tumors was determined by immunohistochemical staining with antibodies against CD31. The number of CD31-positive cells was significantly decreased in tumors from LDM-treated groups. Moreover, the level of K-ras protein in LDM-treated tumors showed a decreased trend by immunohistochemical examination.
3. Effects on pancreatic carcinoma cells by the combination of lidamycin with gemcitabine
Gemcitabine (2', 2'-difluorodeoxycytidine) is currently considered the optimal treatment for patients with newly diagnosed metastatic pancreatic cancer. Preclinical studies suggested that the combination of gemcitabine and other therapeutic agents may have greater activity against pancreatic cancer. In the present study, we investigated the effects on PANC-1 and SW1990 cells by the combination of lidamycin and gemcitabine.
3.1 The effects of LDM alone or in combination with gemcitabine on the viability of pancreatic cancer cells were determined by the MTT assays. A significant reduction in proliferation was observed in PANC-1 and SW1990 cells treated with the combination of 1nM LDM with 500nM gemcitabine.
3.2 Induction of apoptosis by lidamycin, gemcitabine, and the combination was measured by TUNEL assay and flow cytometry combined with FITC-Annexin V/PI staining. There was a significant potentiation of apoptosis observed in PANC-1 and SW1990 cells treated with gemcitabine and lidamycin compared with cells treated with gemcitabine alone.
3.3 The level of mRNA encoding K-ras in PANC-1 and SW1990 cells treated with gemcitabine (500nM), lidamycin (1nM) and the combination was determined by reverse transcription-PCR analysis. The result showed that the K-ras mRNA level was significantly reduced by the combination of gemcitabine and lidamycin.
3.4 In the PACN-1 and SW1990 cells, the combination of gemcitabine and lidamycin resulted in significant down-regulation of the K-ras and Bcl-2 compared with cells treated with gemcitabine or lidamycin alone. Gemcitabine treatment enhanced NF-κB level. However, treatment with lidamycin prevented the gemcitabine-induced NF-κB enhancement.
3.5 Confocal microscopy analyses of cells stained with JC-1 clearly showed that PANC-1 and SW1990 cells have significantly reduced mitochondrial membrane potential (Δ_(ψm)) after treated with the combination of gemcitabine and lidamycin. Moreover, the caspase-3 activity significantly increased when cells were treated with the combination of gemcitabine and lidamycin.
力达霉素(Lidamycin，LDM)是本研究所从一株放线菌(Streptomyces globisporus 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。力达霉素分子由一个酸性的辅基蛋白和一个烯二炔发色团组成，其中发色团是力达霉素的主要活性部分，具有断裂DNA的作用，而辅基蛋白的主要作用是保护发色团。大量研究表明，力达霉素具有极强的抗肿瘤活性，目前正在进行临床Ⅱ期试验研究。本课题选用人胰腺癌PANC-1和SW1990细胞株，探讨力达霉素对人胰腺癌细胞的影响及其作用机制，并通过人胰腺癌细胞SW1990裸鼠移植瘤实验，观察力达霉素在体内的抗胰腺癌作用。此外，本课题还初步研究了力达霉素与健择(Gemcitabine，吉西他滨)联合作用对胰腺癌细胞的影响，并探讨其作用机制，从而为力达霉素的临床应用提供理论依据。
一、力达霉素作用于胰腺癌的体外实验研究
1．MTT法检测力达霉素(LDM)对人胰腺癌PANC-1和SW1990细胞的增殖抑制作用。不同浓度的阿霉素(ADM)、丝裂霉素(MMC)、紫杉醇(Taxol)、吉西他滨(Gemcitabine)和LDM作用细胞48 h后，利用MTT法检测各种药物对胰腺癌细胞的增殖抑制作用。MTT结果显示，与临床常用化疗药物相比，LDM抑制细胞的增殖作用更强，对PANC-1和SWl990细胞的IC_(50)值分别为0.955±0.414 nM和0.426±0.212 nM，低于其他药物的10~3～10~4倍。
2．Hoechst 33342荧光染色法和Annexin V-FITC／PI染色结合流式细胞仪检测细胞凋亡。不同浓度LDM作用细胞48h后，细胞凋亡比率逐渐增高，呈浓度依赖性，其中0.1nM LDM、1nM LDM、2 nM LDM和10 nM LDM作用于PANC-1细胞的细胞凋亡率分别为12.33％±0.66％、32.26％±1.58％、45.03％±3.16％和59.44％±4.04％，作用于SW1990细胞的细胞凋亡率分别为22.21％±0.28％、22.83％±0.26％、42.52％±0.67％和50.35％±0.56％，显著高于对照组的6.15％±1.47％和7.51％±0.45％(P＜0.001)。荧光显微镜下可见1 nM、2 nM和10 nM LDM作用组的细胞出现染色质凝集、细胞核固缩、核碎裂、形成凋亡小体等典型的凋亡细胞形态学改变。
3．利用PI单染并结合流式细胞仪检测LDM对细胞周期的影响。检测结果显示，1 nM LDM主要将细胞阻滞在G2／M期，2 nM LDM能引起S期和G2／M期阻滞，10 nM LDM则主要诱导S期阻滞。
4．利用Transwell实验观察LDM对胰腺癌细胞的迁移和侵袭能力的影响。实验结果显示，LDM对PANC-1细胞和SW1990细胞的迁移和侵袭能力均有显著的抑制作用，并且LDM的作用浓度越大，抑制作用越强，呈现明显的浓度依赖关系。
5．Western blot检测结果显示LDM能够减少基质金属蛋白酶-9(MMP-9)蛋白在胰腺癌细胞中的表达水平，并且随着LDM作用浓度的增加，抑制蛋白表达的作用亦随之加强，提示LDM通过降低细胞内MMP-9的含量而有效抑制细胞的迁移和侵袭能力，并呈剂量依赖性。
6．Western blot和RT-PCR结果表明，LDM能够明显抑制胰腺癌细胞中VEGF蛋白的表达，但不影响细胞中VEGF mRNA的表达水平，说明LDM是在转录后的翻译水平对VEGF的表达进行调节。
7．细胞中K-ras蛋白和mRNA表达水平，以及p-AKT和NF-κB蛋白含量都随LDM作用浓度的增高而降低，呈现一定的浓度依赖性。LDM对于细胞中的AKT蛋白含量没有影响，表明LDM能够抑制AKT蛋白的磷酸化过程。
二、力达霉素作用于胰腺癌的体内实验研究
1．利用人胰腺癌SW1990移植瘤裸鼠模型检测LDM的体内抗肿瘤作用。在瘤块接种后的第7天开始给药，每周1次，共2次。LDM通过尾静脉注射，给药剂量为0.02mg／kg和0.04 mg／kg；Gemcitabine通过腹腔注射，给药剂量为80mg／kg；对照组给予生理盐水。动物实验结果显示，LDM和Gemcitabine对SW1990细胞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。实验第31天处死动物，剥取各组动物的瘤块进行比较，计算抑瘤率，其中Gemcitabine组的抑瘤率为38.87％，LDM 0.02 mg／kg剂量组和0.04 mg／kg剂量组的抑瘤率分别为65.71％和78.27％，与对照组相比具有显著性差异。
2．肿瘤组织切片经HE染色，显微镜下可见，LDM作用组的肿瘤组织出现血管减少，肿瘤细胞坏死增多等病理学变化。
3．利用脱氧核糖核酸末端转移酶介导的缺口末端标记(TUNEL)技术，对裸鼠肿瘤组织中的凋亡细胞进行观察和比较。TUNEL实验结果显示，与对照组和Gemcitabine组相比，LDM治疗组中凋亡细胞明显增多。
4．免疫组化结果显示，LDM能够抑制肿瘤细胞中K-ras蛋白和CD31蛋白的表达，并呈现剂量效应关系，表明LDM具有抑制肿瘤新生血管生成的作用。
三、力达霉素与吉西他滨联合作用于胰腺癌的体外实验研究
目前，健择(Gemcitabine，吉西他滨)是临床上用于治疗胰腺癌的一线药物，健择与其他化疗药物合用治疗肿瘤亦得到广泛研究和应用，本课题对力达霉素联合健择对胰腺癌细胞的作用进行了研究，并探讨其主要作用机理。
1．MTT结果显示，1 nM LDM与500 nM Gemcitabine联合作用时，能够明显抑制细胞增殖，其CDI＜0.75，具有协同抗肿瘤作用。
2．TUNEL实验结果显示，与单独用药相比较，1 nM LDM联合500 nM Gemcitabine作用于PANC-1和SW1990细胞，能够明显提高凋亡细胞的比率。流式细胞仪检测结果显示，二者联合用药作用PANC-1和SW1990细胞的细胞凋亡率分别为59.44％±1.54％和54.68％±2.62％，显著高于Gemcitabine单独作用组的18.48％±0.94％和25.79％±2.06％(P＜0.05)。
3．RT-PCR结果显示，500 nM Gemcitabine和1 nM LDM联合作用能够显著降低PANC-1和SW1990细胞中K-ras mRNA的表达水平。
4．Western blot结果表明，LDM与Gemcitabine联合作用于PANC-1和SW1990细胞，能够减少细胞中K-ras蛋白的表达；500 nM Gemcitabine或1nM LDM单独作用时，使细胞中的NF-κB蛋白水平提高，但二者联用能够显著降低细胞中NF-κB的蛋白表达水平。
5．利用荧光探针JC-1负载，通过共聚焦激光扫描显微镜观察和测定细胞线粒体膜电位的改变，结果表明，LDM与Gemcitabine联合作用能够明显降低细胞的线粒体膜电位。
6．Caspase-3活性测定结果表明，LDM和Gemcitabine联合作用，能够导致PANC-1和SW1990细胞内的凋亡系统caspase-3蛋白酶激活；Western blot结果显示，LDM与Gemcitabine联合作用，能够显著降低PANC-1和SW1990细胞中Bcl-2的蛋白表达水平。
Pancreatic carcinoma is a highly malignant neoplasm characterized by locally advanced unresectable disease or metastasis at the time of diagnosis. Current therapies are largely ineffective in this disease and the 5-year survival rate remains dismal. Pancreatic cancer is now one of the most common causes of cancer death worldwide. Complete surgical resection remains the only potentially curative treatment modality for patients with pancreatic cancer. Unfortunately, the vast majority of patients present with unresectable or metastatic disease. Even among patients undergoing a potentially curative resection, many will develop metastatic disease. Therefore, the development of novel approaches for pancreatic cancer treatment is essential as tumor cells are highly resistant to most conventional chemotherapy drugs.
Enediyne antibiotics have been focused on their potent antitumor activity due to their unique ability to damage the DNA of tumor cells by inducing single strand and/or double strand breaks through free radical attacks on the deoxyribose moieties in DNA. Lidamycin (LDM, also named as C-1027) is a member of the enediyne antibiotic family, which was produced by a Streptomyces globisporus C-1027 strain isolated in China. LDM molecule contains an enediyne chromophore (MW 843 Da) responsible for the extremely potent bioactivity and a noncovalently bound apoprotein (MW 10.5 kDa), which formed a hydrophobic pocket for protecting the chromophore. Lidamycin is currently being evaluated in phaseⅡclinical trials as a potential chemotherapeutic agent in China. As reported, LDM shows extremely potent cytotoxicity, anti-angiogenic activity and marked growth inhibition of transplantable tumors in mice. To determine the action of lidamycin on pancreatic cancer cells and to reveal its possible mechanism, we investigated the effects of lidamycin on two established pancreatic cell lines, PANC-1 and SW1990.
1. Effects of lidamycin on PANC-1 and SW1990 cells cultured in vitro
1.1 Inhibition of cell proliferation was measured by the MTT assays. A remarkable difference in chemosensitivity to lidamycin, mitomycin, adriamycin, taxol, and gemcitabine was found in both cell lines. The IC_(50) value of lidamycin for PANC-1 or SW1990 cells was 0.955±0.414nM or 0.426±0.212nM, and much lower than that of other drugs.
1.2 Flow cytometry combined with FITC-Annexin V/PI staining showed that LDM at 1nM induced earlier apoptosis in both cell lines (the apoptotic ratio were 32.26%±1.58% and 22.83%±0.26% in PANC-1 and SW1990 cells, respectively). The rate of apoptosis was significant enhanced when cells were incubated with 2nM or 10nM LDM for 48h (45.03%±3.16% and 59.44%±4.04% in PANC-1 cells, 42.52%±0.67% and 50.35%±0.56% in SW1990 cells, respectively). After exposure to LDM for 48h, most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken nucleus by Hoechst33342 staining. Some condensed nuclei were observed when cells exposed to higher concentrations of LDM.
1.3 Flow cytometric cell cycle analysis showed that LDM at 1nM induced G2/M arrest and at 2nM induced S and G2/M arrest. The accumulation of cells in S phase was induced by 10nM of LDM.
1.4 The effect of LDM on the migration or invasion of PANC-1 and SW1990 cells through an extracellular matrix was examined using a transwell cell migration chamber assay. A dose-dependent reduction in cell migration or invasion in both cell lines was found after exposure to LDM.
1.5 The expression of MMP-9 was determined by Western blot analysis. PANC-1 and SW1990 cells treated with LDM showed a dose-dependent decrease in the expression of MMP-9. The level of MMP-9 was markedly reduced by 1nM of LDM.
1.6 The decrease of VEGF protein expression was observed in PANC-1 and SW1990 cells after exposure to LDM for 48h, Whereas the VEGF mRNA level was unaffected. The results indicated that LDM could affect VEGF on the translation phase.
1.7 The result of RT-PCR showed that the levels of K-ras mRNA were decreased after treated with LDM at 2nM in PANC-1 cells and 1nM in SW1990 cells. Meanwhile, the levels of K-ras, phospho-Akt and NF-κB proteins decreased in a dose-dependent manner in either cell line, whereas the level of Akt protein was unaffected by LDM.
2. In vivo antitumor activity of lidamycin
2.1 Athymic female BALB/c (nu/nu) mice (20±2g, obtained from the Institute for Experimental Animals, Chinese Academy of Medical Sciences) at the age of 4-6 weeks were used for SW1990 human pancreatic tumor xenografts. Animals bearing s.c. tumors received by i.v. injection (200μl) either PBS (vehicle control) or LDM once a week for 2 weeks. The doses of LDM were 0.02mg/kg and 0.04mg/kg respectively. Animals received by i.p. injection (＜300μl) of gemcitabine at a dose of 80mg/kg as the positive control. The mice were weighed and tumor sizes were measured with a vernier caliper and recorded every other day. Tumor volume was calculated using the formula: length×width~2×0.5. Growth of the established s.c. tumors in nude mice was decreased significantly when treated with LDM over a period of 3 weeks compared with control PBS-treated animals. Treatment with LDM at the dose of 0.02mg/kg and 0.04mg/kg inhibited the growth of pancreatic carcinoma SW1990 xenografts by 66% and 72%, respectively. The antitumor activity of LDM is more remarkable than that of gemcitabine. The body weights of animals showed no significant differences between control and treated groups.
2.2 By hematoxylin and eosin staining, the pathologic changes of tumor tissues from SW1990 xenografted nude mice were observed. There were a decrease in tumor angiogenesis and an increase in necrosis of tumor cells in LDM-treated groups.
2.3 The induction of apoptosis in the pancreatic tumors was evaluated by TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) assay. In tumors from control-treated mice, the median number of apoptotic cells was minimal. The number of apoptotic cells in tumors of LDM-treated mice increased.
2.4 Vessel density in the tumors was determined by immunohistochemical staining with antibodies against CD31. The number of CD31-positive cells was significantly decreased in tumors from LDM-treated groups. Moreover, the level of K-ras protein in LDM-treated tumors showed a decreased trend by immunohistochemical examination.
3. Effects on pancreatic carcinoma cells by the combination of lidamycin with gemcitabine
Gemcitabine (2', 2'-difluorodeoxycytidine) is currently considered the optimal treatment for patients with newly diagnosed metastatic pancreatic cancer. Preclinical studies suggested that the combination of gemcitabine and other therapeutic agents may have greater activity against pancreatic cancer. In the present study, we investigated the effects on PANC-1 and SW1990 cells by the combination of lidamycin and gemcitabine.
3.1 The effects of LDM alone or in combination with gemcitabine on the viability of pancreatic cancer cells were determined by the MTT assays. A significant reduction in proliferation was observed in PANC-1 and SW1990 cells treated with the combination of 1nM LDM with 500nM gemcitabine.
3.2 Induction of apoptosis by lidamycin, gemcitabine, and the combination was measured by TUNEL assay and flow cytometry combined with FITC-Annexin V/PI staining. There was a significant potentiation of apoptosis observed in PANC-1 and SW1990 cells treated with gemcitabine and lidamycin compared with cells treated with gemcitabine alone.
3.3 The level of mRNA encoding K-ras in PANC-1 and SW1990 cells treated with gemcitabine (500nM), lidamycin (1nM) and the combination was determined by reverse transcription-PCR analysis. The result showed that the K-ras mRNA level was significantly reduced by the combination of gemcitabine and lidamycin.
3.4 In the PACN-1 and SW1990 cells, the combination of gemcitabine and lidamycin resulted in significant down-regulation of the K-ras and Bcl-2 compared with cells treated with gemcitabine or lidamycin alone. Gemcitabine treatment enhanced NF-κB level. However, treatment with lidamycin prevented the gemcitabine-induced NF-κB enhancement.
3.5 Confocal microscopy analyses of cells stained with JC-1 clearly showed that PANC-1 and SW1990 cells have significantly reduced mitochondrial membrane potential (Δ_(ψm)) after treated with the combination of gemcitabine and lidamycin. Moreover, the caspase-3 activity significantly increased when cells were treated with the combination of gemcitabine and lidamycin.
引文
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