诱骗寡核苷酸技术靶向阻断STAT3对卵巢癌生物学行为影响的实验研究
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摘要
研究背景
Janus蛋白酪氨酸激酶／信号转导和转录激活因子(Janus kinases/signal transducer and activator of transcription, JAK/STAT)通路是细胞因子信息从细胞外向细胞核传递的重要信号转导通路,其中信号转导和转录激活因子3(Signal Transducer and Activator of Transcription 3, STAT3)是该信号通路中一个至关重要的转录因子。正常信号转导中STAT3蛋白的激活快速而短暂,STAT3持续激活与细胞的恶性转化进程密切相关。目前,在多种肿瘤细胞和组织中检测到STAT3过度激活,如乳腺癌、卵巢癌、头颈部鳞状细胞癌、前列腺癌、恶性黑色素瘤、多发性骨髓瘤、淋巴瘤、脑瘤、非小细胞肺癌和各种白血病等。STAT3过度激活后诱导与细胞增殖、分化、生长、凋亡密切相关的基因的异常高表达,通过各种途径促进细胞增殖、恶性转化、阻碍细胞凋亡,表现出致癌作用。目前STAT3已被定义为癌基因,其在恶性肿瘤发生发展中的作用备受关注,成为肿瘤基因治疗的新靶点。
卵巢癌是妇科恶性肿瘤中发病率占第三位,死亡率占第一位的恶性疾病。卵巢癌起病隐匿,进展快,缺乏有效的早期诊断方法,一经发现往往已有盆腹腔的播散和转移,尤其盆腹腔的转移发生早,范围广,是卵巢癌患者预后不良的主要原因。卵巢癌的发生是一个多步骤、多因素参与的复杂的生物学过程,涉及多个癌基因的激活和抑癌基因的失活。因此,深入研究卵巢癌发生发展、侵袭转移的机制,从分子水平上寻找合理有效的治疗靶点,是卵巢癌基因治疗研究的目的之一。
以STAT3为靶点进行卵巢癌基因治疗的依据：(1)STAT3在卵巢癌细胞系及卵巢癌患者组织标本中存在过度激活现象,且STAT3在人类卵巢癌发生发展以及侵袭转移过程中发挥重要作用,STAT3过度激活与卵巢癌恶性进展及不良预后相关。(2)STAT3是多个致癌性通路汇聚的焦点,以STAT3为靶点治疗效应强大。(3)肿瘤细胞中持续激活的STAT3是导致肿瘤免疫逃逸的重大因素,以STAT3为靶点激活抗肿瘤免疫应答对肿瘤治疗起到事半功倍的作用。(4)转录因子诱骗技术是调控持续活化STAT3的有效方法。(5)腹腔注射或腹腔灌洗是STAT3诱骗寡核苷酸制剂(STAT3 decoy ODN)合理的给药途径。
由于诱骗寡核苷酸技术成本低,特异性高,合成的寡核苷酸片段相对较稳定,该技术已在人类多种肿瘤如肺癌、胰腺癌、头颈部鳞癌等研究中得到应用。然而STAT3 decoy ODN在卵巢癌中的研究较少,尤其是STAT3 decoy ODN在活体内生物学作用的研究更为少见。本课题主要探讨诱骗寡核苷酸技术靶向调控STAT3信号通路对人卵巢癌细胞生长增殖、侵袭活性的影响及相关机制,研究诱骗寡核苷酸技术靶向调控STAT3信号通路对人卵巢癌裸鼠皮下种植瘤治疗作用及其机制。
第一部分诱骗寡核苷酸靶向阻断STAT3对人卵巢癌细胞生长抑制作用及机制研究
研究目的：
1.检测阳离子脂质体将STAT3 decoy ODN转入卵巢癌细胞的转染效率。
2.探讨STAT3 decoy ODN对体外培养卵巢癌细胞系生长、凋亡及细胞周期的影响。
3.观察STAT3 decoy ODN对体外培养卵巢癌细胞系紫杉醇敏感性的影响。
研究方法：
1.应用阳离子脂质体LipofectamineTM 2000介导STAT3 decoy ODN的转染,流式细胞术及荧光显微镜检测FITC标记STAT3 decoy ODN在卵巢癌细胞中的转染效率。
2.采用四甲基偶氮唑盐(Dimethylthiazol-2-yl-diphenyl tetrazalium bromide, MTT)比色法,检测不同浓度STAT3 decoy ODN(0,12.5,25,50nmol/L)转染卵巢癌细胞SKOV3和OVCAR3后在不同时间点(24h,48h,72h)的生长抑制率(growth inhibition rate, GIR); MTT比色法检测STAT3 decoy ODN对SKOV3、OVCAR3细胞紫杉醇敏感性的影响。
3. Annexin V-FITC双染法检测：STAT3 decoy ODN对SKOV3、OVCAR3细胞凋亡的影响；流式细胞术(FCM)检测：STAT3 decoy ODN对SKOV3、OVCAR3细胞周期的影响。
4. Western blot法检测：STAT3 decoy ODN对卵巢癌细胞SKOV3、OVCAR3中相关蛋白P-gp、pAkt、caspase-3表达的影响。
结果：
1. STAT3 decoy ODN在卵巢癌细胞中的转染效率：应用阳离子脂质体LipofectamineTM 2000介导的DNA转染技术,能够将STAT3 decoy ODN高效转导入人卵巢癌细胞中；当转染浓度为25nmol／L时,SKOV3和OVCAR3细胞的转染效率分别为81.6±3.66%和79.5±3.15%。
2. STAT3 decoy ODN对卵巢癌细胞生长抑制作用：STAT3 decoy ODN 25nmol/L转染24h、48h、72h,SKOV3细胞的生长抑制率分别为25.87±3.02%、35.78±2.98%、44.65±3.21%,OVCAR3细胞的生长抑制率分别为20.17±3.01%、34.78±3.35%、40.67±4.23%；在两种细胞系中,与空白对照组及阴性对照组相比,decoy组生长抑制率均显著增高,差异具有统计学意义(P<0.05)。
3. STAT3 decoy ODN对卵巢癌细胞凋亡的影响：STAT3 decoy ODN 25 nmol/L转染卵巢癌细胞24h,转染组与空白对照组SKOV3细胞凋亡比例分别为27.76±5.06%、5.32±0.98%,差异具有显著性(P<0.05)；转染组与空白对照组OVCAR3细胞凋亡比例分别为27.04±4.89%、6.34±1.20%,差异具有显著性(P＜0.05); STAT3 decoy ODN转染卵巢癌细胞后可使细胞凋亡比例显著增加,差异具有统计学意义(P<0.05)。
4. STAT3 decoy ODN对卵巢癌细胞周期的影响：STAT3 decoy ODN 25 nmol/L转染细胞后,转染组与空白对照组SKOV3细胞中G0／G1期细胞所占比例分别为64.38±3.10%、50.71±1.32%,差异具有统计学意义(P<0.05)；转染组与空白对照组SKOV3细胞中S期细胞所占比例分别为26.30±1.50%、37.85±2.31%,差异具有统计学意义(P<0.05)；转染组与空白对照组OVCAR3细胞中G0／G1期细胞所占比例分别为63.84±3.31%、47.23±1.32%,差异具有统计学意义(P<0.05)；转染组与空白对照组OVCAR3细胞中S期细胞所占比例分别为27.72±1.92%、39.62±1.62%,差异具有统计学意义(P<0.05)。统计学分析结果显示,STAT3 decoy ODN转染SKOV3、OVCAR3细胞后,G0／G1期细胞所占比例显著增加,S期细胞所占比例显著减少,差异具有统计学意义(P<0.05)。
5. STAT3 decoy ODN对卵巢癌细胞紫杉醇敏感性的影响：STAT3 decoy ODN25nmol／L和紫杉醇2μmol/L单独或联合作用于卵巢癌细胞24h。在SKOV3细胞中,STAT3 decoy ODN、紫杉醇单独作用时细胞生长抑制率分别为24.86±2.87%、37.80±3.64%,两者联合作用时细胞生长抑制率为63.45±3.82%。在OVCAR3细胞中,STAT3 decoy ODN、紫杉醇单独作用时细胞生长抑制率分别为34.67±3.12%、48.67±3.78%,两者联合作用时细胞生长抑制率为65.23±3.90%。统计学分析显示,STAT3 decoy ODN和紫杉醇联合作用产生的生长抑制率显著高于单独作用产生的抑制率(P<0.05)。
6. STAT3 decoy ODN对卵巢癌细胞中相关蛋白表达的影响：与空白对照组和阴性对照组相比,STAT3 decoy ODN转染组SKOV3和OVCAR3细胞中P-gp、磷酸化Akt(pAkt)的表达水平明显下调,凋亡相关蛋白caspase-3表达显著增加。
结论：
1.阳离子脂质体LipofectamineTM 2000介导的STAT3 decoy ODN能够高效转导入人卵巢癌细胞中,STAT3 decoy ODN能够有效抑制人卵巢癌细胞的生长,诱导卵巢癌细胞凋亡,抑制细胞周期从G0／G1期向S转化,并增加卵巢癌细胞对紫杉醇的敏感性。
2. STAT3 decoy ODN发挥作用的机制与降调卵巢癌细胞中P-gp、pAkt的表达,以及增加细胞中凋亡相关caspase-3活性裂解片段的表达有关。
第二部分诱骗寡核苷酸靶向阻断STAT3对人卵巢癌细胞侵袭力影响的研究
研究目的：探讨STAT3 decoy ODN对卵巢癌细胞侵袭力的影响及其机制。
研究方法：
1.应用肿瘤细胞侵袭实验(Transwell侵袭实验)检测转染STAT3 decoy ODN对卵巢癌细胞SKOV3、OVCAR3体外侵袭力的影响。
2.应用Western blot法检测STAT3 decoy ODN对卵巢癌细胞SKOV3、OVCAR3
侵袭相关蛋白EMMPRIN、MMP-2、MMP-9表达水平的影响。
结果：
1. STAT3 decoy ODN对卵巢癌细胞侵袭力的影响：STAT3 decoy ODN 25nmol/L转染卵巢癌细胞SKOV3和OVCAR3 24h后,Transwell侵袭实验结果显示,在SKOV3细胞中,空白对照组、阴性对照组、STAT3 decoy ODN转染组细胞每高倍视野穿过人工基底膜的细胞数目分别为68±7.30个、70.2±5.0个、36±4.33个；在OVCAR3细胞中,空白对照组、阴性对照组、STAT3 decoy ODN转染组细胞每高倍视野穿过人工基底膜的细胞数目分别为73.2+7.4个、69.4±5.6个、39±4.2个。统计学分析显示,与空白对照组和阴性对照组相比,STAT3 decoy ODN转染组卵巢癌细胞穿过人工基底膜的数目显著减少,差异具有统计学意义(P<0.05)。
2. STAT3 decoy ODN对卵巢癌细胞侵袭相关蛋白表达水平的影响：Western blot检测结果显示,STAT3 decoy ODN转染卵巢癌细胞SKOV3、OVCAR3后,细胞中EMMPRIN、MMP-2、MMP-9蛋白的表达水平受抑制。
结论：
1. STAT3 decoy ODN能够明显抑制卵巢癌细胞的体外侵袭力。
2. STAT3 decoy ODN发挥作用的机制与降调卵巢癌细胞中侵袭相关蛋白EMMPRIN、MMP-2、MMP-9的表达有关。
第三部分诱骗寡核苷酸靶向阻断STAT3对人卵巢癌裸鼠种植瘤治疗作用的研究
研究目的：
1.建立人卵巢癌裸鼠皮下种植瘤模型,探讨STAT3 decoy ODN对荷瘤裸鼠的体内抗瘤效应。
2.探讨STAT3 decoy ODN体内抗瘤效应的机制及对裸鼠正常器官的影响。
研究方法：
1.皮下注射法将常规培养的SKOV3细胞(浓度：2×107／ml)接种到裸鼠右侧胸部皮下,裸鼠在无菌SPF级的动物培养设备中饲养。
2.接种后约12天,待皮下肿瘤结节明显触到,将裸鼠随机分成三组,分别给予STAT3 decoy ODN、STAT3 decoy ODN、PBS隔日瘤周注射,连续处理30天；通过监测肿瘤体积及治疗结束后瘤体重量,评价其对荷瘤裸鼠的抗瘤效应。
3.治疗结束后,取肿瘤组织及裸鼠心、肝、肾组织,制作石蜡切片,HE染色镜检。
4. TUNEL法检测：STAT3 decoy ODN在体内对人卵巢癌细胞的凋亡诱导作用。
5.应用Western blot法检测STAT3 decoy ODN对裸鼠肿瘤组织中凋亡相关蛋白及肿瘤转移相关蛋白表达水平的影响。
结果：
1. STAT3 decoy ODN对裸鼠体内肿瘤生长抑制作用：人卵巢癌细胞SKOV3可以在裸鼠体内成瘤,成瘤时间为10-12天。成瘤后将裸鼠随机分成三组,隔日进行瘤周注射,连续处理30天。治疗结束后,PBS组、scramble组、decoy组三组裸鼠肿瘤平均体积分别为620±68mm3、540±55mm3、226±35mm3。数据统计分析显示,decoy组的肿瘤体积与scramble、PBS两个对照组肿瘤体积有显著性差异(P<0.05),scramble组和PBS组之间的差异无统计学意义。治疗结束后,摘除并称量三组裸鼠肿瘤组织,计算每组裸鼠肿瘤组织的平均重量,结果显示,STAT3 decoy ODN治疗组肿瘤组织的重量比两个对照组肿瘤组织的重量明显降低(P<0.05)。
2. STAT3 decoy ODN能够诱导裸鼠体内肿瘤细胞凋亡：TUNEL法检测结果显示,decoy组每高倍视野中凋亡细胞数为43±7个,scramble对照组每高倍视野中凋亡细胞数为11±3个,PBS对照组每高倍视野中凋亡细胞数为13±4个。Decoy治疗组与另外两个对照组相比,凋亡细胞数目显著增多,差异具有统计学意义(P<0.05)。
3. STAT3 decoy ODN对肿瘤组织及裸鼠心、肝、肾组织的影响：裸鼠肿瘤组织切片HE染色显示,肿瘤细胞核大、深染,可见核分裂相；肿瘤组织内部有坏死灶。裸鼠心、肾组织HE染色未见异常；但在STAT3 decoy ODN治疗组的裸鼠中有1只裸鼠肝脏标本中出现部分肝小叶结构异常,存在炎症和坏死。
4. STAT3 decoy ODN在体内对肿瘤侵袭相关蛋白及凋亡相关蛋白表达水平的影响：Western blot检测结果显示,与PBS治疗组和STAT3 scramble decoy治疗组相比,STAT3 decoy ODN治疗组卵巢癌组织中侵袭转移相关蛋白MMP-2、MMP-9、抗凋亡蛋白Bcl-2的表达受抑制,而凋亡相关蛋白caspase-3活性片段的表达水平增加。
结论：
1.人卵巢癌细胞SKOV3可以在裸鼠体内成瘤,成瘤时间为10.12天。STAT3 decoy ODN可以显著抑制裸鼠体内种植瘤的生长,并对肿瘤细胞具有凋亡诱导作用。
2. STAT3 decoy ODN在体内发挥作用的机制与调控肿瘤侵袭转移相关蛋白及凋亡相关蛋白的表达有关。
Background
JAK/STAT (Janus kinases/signal transducer and activator of transcription) signaling pathway is an important pathway by which cytokines can transfer information from surface of the cell into nucleus. The signal transducers and activators of transcription 3 (STAT3) is a crucial member in the JAK/STAT signaling pathway. The activation of STAT3 is quickly and transient during the normal signal transduction. However, the constitutive activation of STAT3 is closely related to malignant transformation. Previous studies have found that STAT3 is constitutively activated in many different tumor cell lines and primary tumors, including breast, ovarian, head and neck cancer, prostate cancer, melanoma, leukemia, and so on. The functions of activated STAT3 proteins vary, including cell growth, differentiation, development, apoptosis, and angiogenesis. STAT3 has been recognized as an oncogene, and researchers have paid close attention to the effects of STAT3 on malignant tumor. Therefore, STAT3 may be a potential molecular target for cancer treatment.
Epithelial ovarian cancer (EOC) is the most common malignant tumors in women. EOC has been described as a silent killer because when it is first diagnosed the disease always has aggravation, local invasion or distant metastasis. As a result, it is a disease with high mortality, and the survival rate within 5 years is only about 15%-30%. The development of ovarian cancer is multi-steps, multi-factors, and complicated biological progress, which involves many oncogenes activation and tumor suppressor genes inactivation. It is essential to make further study on mechanisms of carcinogenesis, development, and metastasis of ovarian cancer.
The advantages of targeting STAT3 for ovarian cancer treatment are as follows: (1) STAT3 plays important roles in carcinogenesis, development, and metastasis of ovarian cancer. Previous studies have found that STAT3 is constitutively activated in ovarian carcinoma cell lines and clinical specimens, and there is evidence indicating that STAT3 phosphorylation is associated with aggressive epithelial ovarian carcinomas. Recent studies reported that STAT3 activation in tumors may be associated with metastasis and poor prognosis. (2) STAT3 is the focus of multiple cancerigenic pathways, so there will be strong therapeutic effects by targeting STAT3. (3) Constitutively activated STAT3 inhibits the expression of mediators necessary for immune activation against tumour cells. Furthermore, STAT3 activity promotes the production of immunosuppressive factors that activate STAT3 in diverse immune-cell subsets, altering gene-expression programmes and, thereby, restraining anti-tumour immune responses. Consequently, STAT3 has emerged as a promising target for cancer immunotherapy. (4) The decoy oligodeoxynucleotides (decoy ODN) technology, which has high affinity for a target transcription factor (TF) and is a kind of "one-drug, multiple-target" strategy. This technology has advantages of specificity, simplicity, and effectiveness. (5) Intraperitoneal injection or peritoneal lavage is reasonable administration route of STAT3 decoy ODN.
The decoy ODN technology is a novel tool, which has advantages of low cost, specificity, simplicity, and effectiveness, moreover, the sequence of decoy ODN is relatively stable. This technology has been successfully used to inhibit STAT3 pathway activation in squamous cell carcinoma of the head and neck (SCCHN) pancreatic cancer, and in human lung cancer. However, the study of STAT3 decoy ODN on ovarian cancer is limited, especially the biological effects of STAT3 decoy ODN in vivo is limited, too. This research aims to study the inhibiting potency and mechanisms of STAT3 decoy ODN on ovarian cancer cells proliferation, growth, and invasive power; to evaluate the anti-tumor effects and study the mechanisms of STAT3 decoy ODN on xenografted nude mice.
Part I Growth Inhibition of Human Ovarian Cancer Cells by Blocking Activated STAT3 with Decoy ODN in Vitro
Objective:
1. To assay the transfection efficiency of STAT3 decoy ODN with LipofectamineTM 2000 in ovarian cancer cells.
2. To study the influence of STAT3 decoy ODN on cell proliferation, apoptosis and cell cycle of ovarian cancer cells in vitro.
3. To study the influence of STAT3 decoy ODN on chemotherapy sensitivity of ovarian cancer cells in vitro.
Methods:
1. STAT3 decoy ODN transfection was carried out with Lipofectamine TM 2000. Flow cytometry and fluorescence microscope were used to assay the transfection efficiency.
2. MTT assay were used to evaluate the cell growth inhibition rate (GIR) induced by different doses of STAT3 decoy ODN at different time in SKOV3 and OVCAR3 cells. MTT assay were used to evaluate chemotherapy sensitivity to paclitaxel in SKOV3/decoy and OVCAR3/decoy cells.
3. Annexin V/PI dual staining assay was used to examine cell apoptosis after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells. Flow cytometry (FCM) was used to investigate the cell cycle distribution after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.
4. Western blot analysis was used to examine the protein expression level of P-gp、pAkt、caspase-3 after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.
Results:
1. The transfection efficiency of STAT3 decoy ODN:STAT3 decoy ODN could be efficiently transfected into ovarian cancer cells LipofectamineTM 2000. Flow cytometry assay showed the ODN was efficiently introduced into SKOV3 and OVCAR3 cells in a dose-dependent manner. The transfection rate was 81.6±3.66% for SKOV3 and 79.5±3.15% for OVCAR3 at the dosage of 25nmol/L.
2. The growth inhibition of STAT3 decoy ODN on ovarian cancer cells:With transfection of 25nM STAT3 decoy ODN for 24h,48h, and 72h, the growth inhibition rate (GIR) was respectively 25.87±3.02%,35.78±2.98%, and 44.65±3.21% in SKOV3 cells; and GIR was respectively 20.17±3.01%, 34.78±3.35%, and 40.67±4.23% in OVCAR3 cells. GIR induced by STAT3 decoy ODN was significantly increased, and the difference had statistic significance (P＜0.05).
3. STAT3 decoy ODN could induce apoptosis of SKOV3 and OVCAR3 cells:With transfection of 25nM STAT3 decoy ODN, the percentages of early apoptotic cells and later apoptotic cells were both increased. The percentage of apoptotic cells was significantly increased from 5.32%±0.98% to 27.76%±5.06% (P＜0.05) in SKOV3 cells. The percentage of apoptotic cells was also significantly increased from 6.34%±1.20% to 27.04%±4.89%(P＜0.05) in SKOV3 cells. However, the difference of apoptotic percentage induced by STAT3 scramble ODN had no statistic significance.
4. STAT3 decoy ODN could induce cell cycle arrest in SKOV3 and OVCAR3 cells: With transfection of 25nM STAT3 decoy ODN, the percentage of GO/G1 phase in SKOV3 cells was increased from 50.71±1.32% to 64.38±3.10%, and the percentage of S phase in SKOV3 cells was decreased from 37.85±2.31% to 26.30±1.50%. With transfection of 25nM STAT3 decoy ODN, the percentage of GO/G1 phase in OVCAR3 cells was increased from 47.23±0.95% to 63.84±3.31%, and the percentage of S phase in OVCAR3 cells was decreased from 39.62±1.62% to 27.72±1.92%. Results showed that with transfection of STAT3 decoy ODN, the percentages of G0/G1 phase were significantly increased and the percentages of S phase were significantly decreased, the difference had statistic significance (P＜0.05).
5. STAT3 decoy ODN could enhance the chemotherapy sensitivity of ovarian cancer cells:In SKOV3 cells, the growth inhibit rate (GIR) was respectively 24.86±2.87% and 37.80±3.64% with single treatment of STAT3 decoy ODN (25nmol/L) or paclitaxel (2μmol/L), while the GIR was 63.45±3.82% with combined treatment of STAT3 decoy ODN and paclitaxel. In 0VCAR3 cells, the growth inhibit rate (GIR) was respectively 34.67±3.12% and 48.67±3.78% with single treatment of STAT3 decoy ODN (25nmol/L) or paclitaxel (2μmol/L), while the GIR was 65.23±3.90% with combined treatment of STAT3 decoy ODN and paclitaxel. Results showed that GIR caused by combined treatment of STAT3 decoy ODN and paclitaxel was significantly higher than single treatment, the difference had statistic significance (P＜O.05).
6. STAT3 decoy ODN could regulate the expression of proteins:In contrast to vacant and scramble groups, the expression of protein P-gp and pAkt in decoy groups of cells were decreased, and the expression of protein caspase-3 was up-regulated.
Conclusions:
1. STAT3 decoy ODN can be efficiently transfected into ovarian cancer cells by using LipofectamineTM 2000. STAT3 decoy ODN can inhibit cell growth, induce cell apoptosis and cell cycle arrest, and enhance the sensitivity to paclitaxel for ovarian cancer cells.
2. The mechanism of STAT3 decoy ODN is to down-regulate the protein expression of P-gp, pAkt, and up-regulate the expression of caspase-3 in ovarian cancer cells.
PartⅡSTAT3 decoy ODN Inhibits the Invasive Power of Ovarian Cancer Cells in vitro
Objective:
1. To study the influence of STAT3 decoy ODN on invasive power of ovarian cancer cells in vitro. To study the mechanisms of STAT3 decoy ODN influencing the invasive power of ovarian cancer cells.
Methods:
1. The invasive power was tested by transwell matrigel invasion assays after SKOV3 and OVCAR3 were transfected with STAT3 decoy ODN.
2. Western blot analysis was used to examine the protein expression level of EMMPRIN、MMP-2、MMP-9 after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.
Results:
1. The influence of STAT3 decoy ODN on invasive power of ovarian cancer cells: With transfection of 25nM STAT3 decoy ODN, the percentages of SKOV3 cells invading the membrane were reduced from 68±7.30 to 36±4.33 (P＜0.05), and the percentages of OVCAR3 cells invading the membrane were reduced from 69.4±5.6 to 39±4.2 (P＜0.05). The difference between vacant and scramble control groups had no statistic significance.
2. Western blot analysis showed the protein expression levels of EMMPRIN、MMP-2、MMP-9 were decreased with transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.
Conclusions:
1. STAT3 decoy ODN can significantly inhibit the invasive power of ovarian cancer cells.
2. The mechanisms of STAT3 decoy ODN on cancer cell invasion involve the down-regulation of protein EMMPRIN、MMP-2、MMP-9 in ovarian cancer cells.
Part III Therapeutic Effects of STAT3 decoy ODN on Human Ovarian Cancer Cells in vivo
Objective:
1. Establish subcutaneous xenografts of human ovarian cancer cells SKOV3 in nude mice, and evaluate the anti-tumor effects of STAT3 decoy ODN on xenografted nude mice.
2. To study the mechanisms of STAT3 decoy ODN on anti-tumor effects in vivo.
Methods:
1. SKOV3 cells (2×107/ml) were subcutaneously injected into the right chest of nude mice. The nude mice were cultured according to the regulation of SPF grade.
2. STAT3 decoy ODN was peritumorally injected after the tumor formation (about 13 days after SKOV3 cells injection). Evaluate the anti-tumor effects of STAT3 decoy ODN by monitoring the tumor volume and measuring the tumor weight in the end.
3. After the treatment was finished, make paraffin sections for tissues of tumor, heart, liver and kidney.
4. The effects on apoptotic induction of STAT3 decoy ODN in vivo was detected by TUNEL.
5. Western blot analysis was used to examine the expression of associated proteins in the tumor tissues of nude mice.
Results:
1. SKOV3 cells could form xenografts in the nude mice, and the average time was about 10-12 days. The xenografted nude mice were divided into 3 groups randomly. STAT3 decoy ODN was peritumorally injected into the xenografts every other day. In the end of the treatment, the average volume of xenografts was as follows:620±68mm3 in PBS group,540±55mm3 in scramble group, 226±35mm3 in decoy group. The average volume of xenografts in decoy group was decreased significantly contrast to PBS and scramble groups (P＜0.05). However, the difference between PBS and scramble groups had no statistically significance. The average weight of tumor tissues in decoy group was significantly lower than that in the other control groups (P<0.05).
2. STAT3 decoy ODN could induce ovarian cancer cells apoptosis in vivo. TUNEL analysis showed that there were 43±7 positive cells per high power field in the group treated with STAT3 decoy ODN, while 11±3 positive cells in scramble group and 13±4 positive cells in PBS group. The difference between decoy group and the other two groups had statistically significance (P＜0.05).
3. The biological effects of STAT3 decoy ODN on tissues of xenograft, heart, liver and kidney in nude mice. The sections of xenograft tissues (HE staining) showed karyomegaly, anachromasis, and karyokinesis in cancer cells. There was necrosis partly in the xenograft tissues. The sections of heart and liver tissues had no significant abnormality. However, one in five nude mice treated with STAT3 decoy ODN had abnormality, inflammation and necrosis in part of hepatic lobule.
4. The expression of MMP2, MMP9, Bcl-2, and caspase-3 in the xenograft tissues of nude mice:Compared with PBS and scramble treatment group, the protein expression level of MMP2, MMP9, Bcl-2 in STAT3 decoy ODN treatment group decreased significantly, while the protein expression level of caspase-3 increased significantly.
Conclusions:
1. SKOV3 cells can form xenografts in the nude mice. STAT3 decoy ODN can inhibit the growth of ovarian cancer cells and induce apoptosis of ovarian cancer cells in vivo.
2. STAT3 decoy ODN can down-regulate the protein expression level of MMP2, MMP9, Bcl-2, and up-regulate the protein expression level of caspase-3 in vivo.
Janus蛋白酪氨酸激酶／信号转导和转录激活因子(Janus kinases/signal transducer and activator of transcription, JAK/STAT)通路是细胞因子信息从细胞外向细胞核传递的重要信号转导通路,其中信号转导和转录激活因子3(Signal Transducer and Activator of Transcription 3, STAT3)是该信号通路中一个至关重要的转录因子。正常信号转导中STAT3蛋白的激活快速而短暂,STAT3持续激活与细胞的恶性转化进程密切相关。目前,在多种肿瘤细胞和组织中检测到STAT3过度激活,如乳腺癌、卵巢癌、头颈部鳞状细胞癌、前列腺癌、恶性黑色素瘤、多发性骨髓瘤、淋巴瘤、脑瘤、非小细胞肺癌和各种白血病等。STAT3过度激活后诱导与细胞增殖、分化、生长、凋亡密切相关的基因的异常高表达,通过各种途径促进细胞增殖、恶性转化、阻碍细胞凋亡,表现出致癌作用。目前STAT3已被定义为癌基因,其在恶性肿瘤发生发展中的作用备受关注,成为肿瘤基因治疗的新靶点。
卵巢癌是妇科恶性肿瘤中发病率占第三位,死亡率占第一位的恶性疾病。卵巢癌起病隐匿,进展快,缺乏有效的早期诊断方法,一经发现往往已有盆腹腔的播散和转移,尤其盆腹腔的转移发生早,范围广,是卵巢癌患者预后不良的主要原因。卵巢癌的发生是一个多步骤、多因素参与的复杂的生物学过程,涉及多个癌基因的激活和抑癌基因的失活。因此,深入研究卵巢癌发生发展、侵袭转移的机制,从分子水平上寻找合理有效的治疗靶点,是卵巢癌基因治疗研究的目的之一。
以STAT3为靶点进行卵巢癌基因治疗的依据：(1)STAT3在卵巢癌细胞系及卵巢癌患者组织标本中存在过度激活现象,且STAT3在人类卵巢癌发生发展以及侵袭转移过程中发挥重要作用,STAT3过度激活与卵巢癌恶性进展及不良预后相关。(2)STAT3是多个致癌性通路汇聚的焦点,以STAT3为靶点治疗效应强大。(3)肿瘤细胞中持续激活的STAT3是导致肿瘤免疫逃逸的重大因素,以STAT3为靶点激活抗肿瘤免疫应答对肿瘤治疗起到事半功倍的作用。(4)转录因子诱骗技术是调控持续活化STAT3的有效方法。(5)腹腔注射或腹腔灌洗是STAT3诱骗寡核苷酸制剂(STAT3 decoy ODN)合理的给药途径。
由于诱骗寡核苷酸技术成本低,特异性高,合成的寡核苷酸片段相对较稳定,该技术已在人类多种肿瘤如肺癌、胰腺癌、头颈部鳞癌等研究中得到应用。然而STAT3 decoy ODN在卵巢癌中的研究较少,尤其是STAT3 decoy ODN在活体内生物学作用的研究更为少见。本课题主要探讨诱骗寡核苷酸技术靶向调控STAT3信号通路对人卵巢癌细胞生长增殖、侵袭活性的影响及相关机制,研究诱骗寡核苷酸技术靶向调控STAT3信号通路对人卵巢癌裸鼠皮下种植瘤治疗作用及其机制。
第一部分诱骗寡核苷酸靶向阻断STAT3对人卵巢癌细胞生长抑制作用及机制研究
研究目的：
1.检测阳离子脂质体将STAT3 decoy ODN转入卵巢癌细胞的转染效率。
2.探讨STAT3 decoy ODN对体外培养卵巢癌细胞系生长、凋亡及细胞周期的影响。
3.观察STAT3 decoy ODN对体外培养卵巢癌细胞系紫杉醇敏感性的影响。
研究方法：
1.应用阳离子脂质体LipofectamineTM 2000介导STAT3 decoy ODN的转染,流式细胞术及荧光显微镜检测FITC标记STAT3 decoy ODN在卵巢癌细胞中的转染效率。
2.采用四甲基偶氮唑盐(Dimethylthiazol-2-yl-diphenyl tetrazalium bromide, MTT)比色法,检测不同浓度STAT3 decoy ODN(0,12.5,25,50nmol/L)转染卵巢癌细胞SKOV3和OVCAR3后在不同时间点(24h,48h,72h)的生长抑制率(growth inhibition rate, GIR); MTT比色法检测STAT3 decoy ODN对SKOV3、OVCAR3细胞紫杉醇敏感性的影响。
3. Annexin V-FITC双染法检测：STAT3 decoy ODN对SKOV3、OVCAR3细胞凋亡的影响；流式细胞术(FCM)检测：STAT3 decoy ODN对SKOV3、OVCAR3细胞周期的影响。
4. Western blot法检测：STAT3 decoy ODN对卵巢癌细胞SKOV3、OVCAR3中相关蛋白P-gp、pAkt、caspase-3表达的影响。
结果：
1. STAT3 decoy ODN在卵巢癌细胞中的转染效率：应用阳离子脂质体LipofectamineTM 2000介导的DNA转染技术,能够将STAT3 decoy ODN高效转导入人卵巢癌细胞中；当转染浓度为25nmol／L时,SKOV3和OVCAR3细胞的转染效率分别为81.6±3.66%和79.5±3.15%。
2. STAT3 decoy ODN对卵巢癌细胞生长抑制作用：STAT3 decoy ODN 25nmol/L转染24h、48h、72h,SKOV3细胞的生长抑制率分别为25.87±3.02%、35.78±2.98%、44.65±3.21%,OVCAR3细胞的生长抑制率分别为20.17±3.01%、34.78±3.35%、40.67±4.23%；在两种细胞系中,与空白对照组及阴性对照组相比,decoy组生长抑制率均显著增高,差异具有统计学意义(P<0.05)。
3. STAT3 decoy ODN对卵巢癌细胞凋亡的影响：STAT3 decoy ODN 25 nmol/L转染卵巢癌细胞24h,转染组与空白对照组SKOV3细胞凋亡比例分别为27.76±5.06%、5.32±0.98%,差异具有显著性(P<0.05)；转染组与空白对照组OVCAR3细胞凋亡比例分别为27.04±4.89%、6.34±1.20%,差异具有显著性(P＜0.05); STAT3 decoy ODN转染卵巢癌细胞后可使细胞凋亡比例显著增加,差异具有统计学意义(P<0.05)。
4. STAT3 decoy ODN对卵巢癌细胞周期的影响：STAT3 decoy ODN 25 nmol/L转染细胞后,转染组与空白对照组SKOV3细胞中G0／G1期细胞所占比例分别为64.38±3.10%、50.71±1.32%,差异具有统计学意义(P<0.05)；转染组与空白对照组SKOV3细胞中S期细胞所占比例分别为26.30±1.50%、37.85±2.31%,差异具有统计学意义(P<0.05)；转染组与空白对照组OVCAR3细胞中G0／G1期细胞所占比例分别为63.84±3.31%、47.23±1.32%,差异具有统计学意义(P<0.05)；转染组与空白对照组OVCAR3细胞中S期细胞所占比例分别为27.72±1.92%、39.62±1.62%,差异具有统计学意义(P<0.05)。统计学分析结果显示,STAT3 decoy ODN转染SKOV3、OVCAR3细胞后,G0／G1期细胞所占比例显著增加,S期细胞所占比例显著减少,差异具有统计学意义(P<0.05)。
5. STAT3 decoy ODN对卵巢癌细胞紫杉醇敏感性的影响：STAT3 decoy ODN25nmol／L和紫杉醇2μmol/L单独或联合作用于卵巢癌细胞24h。在SKOV3细胞中,STAT3 decoy ODN、紫杉醇单独作用时细胞生长抑制率分别为24.86±2.87%、37.80±3.64%,两者联合作用时细胞生长抑制率为63.45±3.82%。在OVCAR3细胞中,STAT3 decoy ODN、紫杉醇单独作用时细胞生长抑制率分别为34.67±3.12%、48.67±3.78%,两者联合作用时细胞生长抑制率为65.23±3.90%。统计学分析显示,STAT3 decoy ODN和紫杉醇联合作用产生的生长抑制率显著高于单独作用产生的抑制率(P<0.05)。
6. STAT3 decoy ODN对卵巢癌细胞中相关蛋白表达的影响：与空白对照组和阴性对照组相比,STAT3 decoy ODN转染组SKOV3和OVCAR3细胞中P-gp、磷酸化Akt(pAkt)的表达水平明显下调,凋亡相关蛋白caspase-3表达显著增加。
结论：
1.阳离子脂质体LipofectamineTM 2000介导的STAT3 decoy ODN能够高效转导入人卵巢癌细胞中,STAT3 decoy ODN能够有效抑制人卵巢癌细胞的生长,诱导卵巢癌细胞凋亡,抑制细胞周期从G0／G1期向S转化,并增加卵巢癌细胞对紫杉醇的敏感性。
2. STAT3 decoy ODN发挥作用的机制与降调卵巢癌细胞中P-gp、pAkt的表达,以及增加细胞中凋亡相关caspase-3活性裂解片段的表达有关。
第二部分诱骗寡核苷酸靶向阻断STAT3对人卵巢癌细胞侵袭力影响的研究
研究目的：探讨STAT3 decoy ODN对卵巢癌细胞侵袭力的影响及其机制。
研究方法：
1.应用肿瘤细胞侵袭实验(Transwell侵袭实验)检测转染STAT3 decoy ODN对卵巢癌细胞SKOV3、OVCAR3体外侵袭力的影响。
2.应用Western blot法检测STAT3 decoy ODN对卵巢癌细胞SKOV3、OVCAR3
侵袭相关蛋白EMMPRIN、MMP-2、MMP-9表达水平的影响。
结果：
1. STAT3 decoy ODN对卵巢癌细胞侵袭力的影响：STAT3 decoy ODN 25nmol/L转染卵巢癌细胞SKOV3和OVCAR3 24h后,Transwell侵袭实验结果显示,在SKOV3细胞中,空白对照组、阴性对照组、STAT3 decoy ODN转染组细胞每高倍视野穿过人工基底膜的细胞数目分别为68±7.30个、70.2±5.0个、36±4.33个；在OVCAR3细胞中,空白对照组、阴性对照组、STAT3 decoy ODN转染组细胞每高倍视野穿过人工基底膜的细胞数目分别为73.2+7.4个、69.4±5.6个、39±4.2个。统计学分析显示,与空白对照组和阴性对照组相比,STAT3 decoy ODN转染组卵巢癌细胞穿过人工基底膜的数目显著减少,差异具有统计学意义(P<0.05)。
2. STAT3 decoy ODN对卵巢癌细胞侵袭相关蛋白表达水平的影响：Western blot检测结果显示,STAT3 decoy ODN转染卵巢癌细胞SKOV3、OVCAR3后,细胞中EMMPRIN、MMP-2、MMP-9蛋白的表达水平受抑制。
结论：
1. STAT3 decoy ODN能够明显抑制卵巢癌细胞的体外侵袭力。
2. STAT3 decoy ODN发挥作用的机制与降调卵巢癌细胞中侵袭相关蛋白EMMPRIN、MMP-2、MMP-9的表达有关。
第三部分诱骗寡核苷酸靶向阻断STAT3对人卵巢癌裸鼠种植瘤治疗作用的研究
研究目的：
1.建立人卵巢癌裸鼠皮下种植瘤模型,探讨STAT3 decoy ODN对荷瘤裸鼠的体内抗瘤效应。
2.探讨STAT3 decoy ODN体内抗瘤效应的机制及对裸鼠正常器官的影响。
研究方法：
1.皮下注射法将常规培养的SKOV3细胞(浓度：2×107／ml)接种到裸鼠右侧胸部皮下,裸鼠在无菌SPF级的动物培养设备中饲养。
2.接种后约12天,待皮下肿瘤结节明显触到,将裸鼠随机分成三组,分别给予STAT3 decoy ODN、STAT3 decoy ODN、PBS隔日瘤周注射,连续处理30天；通过监测肿瘤体积及治疗结束后瘤体重量,评价其对荷瘤裸鼠的抗瘤效应。
3.治疗结束后,取肿瘤组织及裸鼠心、肝、肾组织,制作石蜡切片,HE染色镜检。
4. TUNEL法检测：STAT3 decoy ODN在体内对人卵巢癌细胞的凋亡诱导作用。
5.应用Western blot法检测STAT3 decoy ODN对裸鼠肿瘤组织中凋亡相关蛋白及肿瘤转移相关蛋白表达水平的影响。
结果：
1. STAT3 decoy ODN对裸鼠体内肿瘤生长抑制作用：人卵巢癌细胞SKOV3可以在裸鼠体内成瘤,成瘤时间为10-12天。成瘤后将裸鼠随机分成三组,隔日进行瘤周注射,连续处理30天。治疗结束后,PBS组、scramble组、decoy组三组裸鼠肿瘤平均体积分别为620±68mm3、540±55mm3、226±35mm3。数据统计分析显示,decoy组的肿瘤体积与scramble、PBS两个对照组肿瘤体积有显著性差异(P<0.05),scramble组和PBS组之间的差异无统计学意义。治疗结束后,摘除并称量三组裸鼠肿瘤组织,计算每组裸鼠肿瘤组织的平均重量,结果显示,STAT3 decoy ODN治疗组肿瘤组织的重量比两个对照组肿瘤组织的重量明显降低(P<0.05)。
2. STAT3 decoy ODN能够诱导裸鼠体内肿瘤细胞凋亡：TUNEL法检测结果显示,decoy组每高倍视野中凋亡细胞数为43±7个,scramble对照组每高倍视野中凋亡细胞数为11±3个,PBS对照组每高倍视野中凋亡细胞数为13±4个。Decoy治疗组与另外两个对照组相比,凋亡细胞数目显著增多,差异具有统计学意义(P<0.05)。
3. STAT3 decoy ODN对肿瘤组织及裸鼠心、肝、肾组织的影响：裸鼠肿瘤组织切片HE染色显示,肿瘤细胞核大、深染,可见核分裂相；肿瘤组织内部有坏死灶。裸鼠心、肾组织HE染色未见异常；但在STAT3 decoy ODN治疗组的裸鼠中有1只裸鼠肝脏标本中出现部分肝小叶结构异常,存在炎症和坏死。
4. STAT3 decoy ODN在体内对肿瘤侵袭相关蛋白及凋亡相关蛋白表达水平的影响：Western blot检测结果显示,与PBS治疗组和STAT3 scramble decoy治疗组相比,STAT3 decoy ODN治疗组卵巢癌组织中侵袭转移相关蛋白MMP-2、MMP-9、抗凋亡蛋白Bcl-2的表达受抑制,而凋亡相关蛋白caspase-3活性片段的表达水平增加。
结论：
1.人卵巢癌细胞SKOV3可以在裸鼠体内成瘤,成瘤时间为10.12天。STAT3 decoy ODN可以显著抑制裸鼠体内种植瘤的生长,并对肿瘤细胞具有凋亡诱导作用。
2. STAT3 decoy ODN在体内发挥作用的机制与调控肿瘤侵袭转移相关蛋白及凋亡相关蛋白的表达有关。
Background
JAK/STAT (Janus kinases/signal transducer and activator of transcription) signaling pathway is an important pathway by which cytokines can transfer information from surface of the cell into nucleus. The signal transducers and activators of transcription 3 (STAT3) is a crucial member in the JAK/STAT signaling pathway. The activation of STAT3 is quickly and transient during the normal signal transduction. However, the constitutive activation of STAT3 is closely related to malignant transformation. Previous studies have found that STAT3 is constitutively activated in many different tumor cell lines and primary tumors, including breast, ovarian, head and neck cancer, prostate cancer, melanoma, leukemia, and so on. The functions of activated STAT3 proteins vary, including cell growth, differentiation, development, apoptosis, and angiogenesis. STAT3 has been recognized as an oncogene, and researchers have paid close attention to the effects of STAT3 on malignant tumor. Therefore, STAT3 may be a potential molecular target for cancer treatment.
Epithelial ovarian cancer (EOC) is the most common malignant tumors in women. EOC has been described as a silent killer because when it is first diagnosed the disease always has aggravation, local invasion or distant metastasis. As a result, it is a disease with high mortality, and the survival rate within 5 years is only about 15%-30%. The development of ovarian cancer is multi-steps, multi-factors, and complicated biological progress, which involves many oncogenes activation and tumor suppressor genes inactivation. It is essential to make further study on mechanisms of carcinogenesis, development, and metastasis of ovarian cancer.
The advantages of targeting STAT3 for ovarian cancer treatment are as follows: (1) STAT3 plays important roles in carcinogenesis, development, and metastasis of ovarian cancer. Previous studies have found that STAT3 is constitutively activated in ovarian carcinoma cell lines and clinical specimens, and there is evidence indicating that STAT3 phosphorylation is associated with aggressive epithelial ovarian carcinomas. Recent studies reported that STAT3 activation in tumors may be associated with metastasis and poor prognosis. (2) STAT3 is the focus of multiple cancerigenic pathways, so there will be strong therapeutic effects by targeting STAT3. (3) Constitutively activated STAT3 inhibits the expression of mediators necessary for immune activation against tumour cells. Furthermore, STAT3 activity promotes the production of immunosuppressive factors that activate STAT3 in diverse immune-cell subsets, altering gene-expression programmes and, thereby, restraining anti-tumour immune responses. Consequently, STAT3 has emerged as a promising target for cancer immunotherapy. (4) The decoy oligodeoxynucleotides (decoy ODN) technology, which has high affinity for a target transcription factor (TF) and is a kind of "one-drug, multiple-target" strategy. This technology has advantages of specificity, simplicity, and effectiveness. (5) Intraperitoneal injection or peritoneal lavage is reasonable administration route of STAT3 decoy ODN.
The decoy ODN technology is a novel tool, which has advantages of low cost, specificity, simplicity, and effectiveness, moreover, the sequence of decoy ODN is relatively stable. This technology has been successfully used to inhibit STAT3 pathway activation in squamous cell carcinoma of the head and neck (SCCHN) pancreatic cancer, and in human lung cancer. However, the study of STAT3 decoy ODN on ovarian cancer is limited, especially the biological effects of STAT3 decoy ODN in vivo is limited, too. This research aims to study the inhibiting potency and mechanisms of STAT3 decoy ODN on ovarian cancer cells proliferation, growth, and invasive power; to evaluate the anti-tumor effects and study the mechanisms of STAT3 decoy ODN on xenografted nude mice.
Part I Growth Inhibition of Human Ovarian Cancer Cells by Blocking Activated STAT3 with Decoy ODN in Vitro
Objective:
1. To assay the transfection efficiency of STAT3 decoy ODN with LipofectamineTM 2000 in ovarian cancer cells.
2. To study the influence of STAT3 decoy ODN on cell proliferation, apoptosis and cell cycle of ovarian cancer cells in vitro.
3. To study the influence of STAT3 decoy ODN on chemotherapy sensitivity of ovarian cancer cells in vitro.
Methods:
1. STAT3 decoy ODN transfection was carried out with Lipofectamine TM 2000. Flow cytometry and fluorescence microscope were used to assay the transfection efficiency.
2. MTT assay were used to evaluate the cell growth inhibition rate (GIR) induced by different doses of STAT3 decoy ODN at different time in SKOV3 and OVCAR3 cells. MTT assay were used to evaluate chemotherapy sensitivity to paclitaxel in SKOV3/decoy and OVCAR3/decoy cells.
3. Annexin V/PI dual staining assay was used to examine cell apoptosis after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells. Flow cytometry (FCM) was used to investigate the cell cycle distribution after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.
4. Western blot analysis was used to examine the protein expression level of P-gp、pAkt、caspase-3 after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.
Results:
1. The transfection efficiency of STAT3 decoy ODN:STAT3 decoy ODN could be efficiently transfected into ovarian cancer cells LipofectamineTM 2000. Flow cytometry assay showed the ODN was efficiently introduced into SKOV3 and OVCAR3 cells in a dose-dependent manner. The transfection rate was 81.6±3.66% for SKOV3 and 79.5±3.15% for OVCAR3 at the dosage of 25nmol/L.
2. The growth inhibition of STAT3 decoy ODN on ovarian cancer cells:With transfection of 25nM STAT3 decoy ODN for 24h,48h, and 72h, the growth inhibition rate (GIR) was respectively 25.87±3.02%,35.78±2.98%, and 44.65±3.21% in SKOV3 cells; and GIR was respectively 20.17±3.01%, 34.78±3.35%, and 40.67±4.23% in OVCAR3 cells. GIR induced by STAT3 decoy ODN was significantly increased, and the difference had statistic significance (P＜0.05).
3. STAT3 decoy ODN could induce apoptosis of SKOV3 and OVCAR3 cells:With transfection of 25nM STAT3 decoy ODN, the percentages of early apoptotic cells and later apoptotic cells were both increased. The percentage of apoptotic cells was significantly increased from 5.32%±0.98% to 27.76%±5.06% (P＜0.05) in SKOV3 cells. The percentage of apoptotic cells was also significantly increased from 6.34%±1.20% to 27.04%±4.89%(P＜0.05) in SKOV3 cells. However, the difference of apoptotic percentage induced by STAT3 scramble ODN had no statistic significance.
4. STAT3 decoy ODN could induce cell cycle arrest in SKOV3 and OVCAR3 cells: With transfection of 25nM STAT3 decoy ODN, the percentage of GO/G1 phase in SKOV3 cells was increased from 50.71±1.32% to 64.38±3.10%, and the percentage of S phase in SKOV3 cells was decreased from 37.85±2.31% to 26.30±1.50%. With transfection of 25nM STAT3 decoy ODN, the percentage of GO/G1 phase in OVCAR3 cells was increased from 47.23±0.95% to 63.84±3.31%, and the percentage of S phase in OVCAR3 cells was decreased from 39.62±1.62% to 27.72±1.92%. Results showed that with transfection of STAT3 decoy ODN, the percentages of G0/G1 phase were significantly increased and the percentages of S phase were significantly decreased, the difference had statistic significance (P＜0.05).
5. STAT3 decoy ODN could enhance the chemotherapy sensitivity of ovarian cancer cells:In SKOV3 cells, the growth inhibit rate (GIR) was respectively 24.86±2.87% and 37.80±3.64% with single treatment of STAT3 decoy ODN (25nmol/L) or paclitaxel (2μmol/L), while the GIR was 63.45±3.82% with combined treatment of STAT3 decoy ODN and paclitaxel. In 0VCAR3 cells, the growth inhibit rate (GIR) was respectively 34.67±3.12% and 48.67±3.78% with single treatment of STAT3 decoy ODN (25nmol/L) or paclitaxel (2μmol/L), while the GIR was 65.23±3.90% with combined treatment of STAT3 decoy ODN and paclitaxel. Results showed that GIR caused by combined treatment of STAT3 decoy ODN and paclitaxel was significantly higher than single treatment, the difference had statistic significance (P＜O.05).
6. STAT3 decoy ODN could regulate the expression of proteins:In contrast to vacant and scramble groups, the expression of protein P-gp and pAkt in decoy groups of cells were decreased, and the expression of protein caspase-3 was up-regulated.
Conclusions:
1. STAT3 decoy ODN can be efficiently transfected into ovarian cancer cells by using LipofectamineTM 2000. STAT3 decoy ODN can inhibit cell growth, induce cell apoptosis and cell cycle arrest, and enhance the sensitivity to paclitaxel for ovarian cancer cells.
2. The mechanism of STAT3 decoy ODN is to down-regulate the protein expression of P-gp, pAkt, and up-regulate the expression of caspase-3 in ovarian cancer cells.
PartⅡSTAT3 decoy ODN Inhibits the Invasive Power of Ovarian Cancer Cells in vitro
Objective:
1. To study the influence of STAT3 decoy ODN on invasive power of ovarian cancer cells in vitro. To study the mechanisms of STAT3 decoy ODN influencing the invasive power of ovarian cancer cells.
Methods:
1. The invasive power was tested by transwell matrigel invasion assays after SKOV3 and OVCAR3 were transfected with STAT3 decoy ODN.
2. Western blot analysis was used to examine the protein expression level of EMMPRIN、MMP-2、MMP-9 after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.
Results:
1. The influence of STAT3 decoy ODN on invasive power of ovarian cancer cells: With transfection of 25nM STAT3 decoy ODN, the percentages of SKOV3 cells invading the membrane were reduced from 68±7.30 to 36±4.33 (P＜0.05), and the percentages of OVCAR3 cells invading the membrane were reduced from 69.4±5.6 to 39±4.2 (P＜0.05). The difference between vacant and scramble control groups had no statistic significance.
2. Western blot analysis showed the protein expression levels of EMMPRIN、MMP-2、MMP-9 were decreased with transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.
Conclusions:
1. STAT3 decoy ODN can significantly inhibit the invasive power of ovarian cancer cells.
2. The mechanisms of STAT3 decoy ODN on cancer cell invasion involve the down-regulation of protein EMMPRIN、MMP-2、MMP-9 in ovarian cancer cells.
Part III Therapeutic Effects of STAT3 decoy ODN on Human Ovarian Cancer Cells in vivo
Objective:
1. Establish subcutaneous xenografts of human ovarian cancer cells SKOV3 in nude mice, and evaluate the anti-tumor effects of STAT3 decoy ODN on xenografted nude mice.
2. To study the mechanisms of STAT3 decoy ODN on anti-tumor effects in vivo.
Methods:
1. SKOV3 cells (2×107/ml) were subcutaneously injected into the right chest of nude mice. The nude mice were cultured according to the regulation of SPF grade.
2. STAT3 decoy ODN was peritumorally injected after the tumor formation (about 13 days after SKOV3 cells injection). Evaluate the anti-tumor effects of STAT3 decoy ODN by monitoring the tumor volume and measuring the tumor weight in the end.
3. After the treatment was finished, make paraffin sections for tissues of tumor, heart, liver and kidney.
4. The effects on apoptotic induction of STAT3 decoy ODN in vivo was detected by TUNEL.
5. Western blot analysis was used to examine the expression of associated proteins in the tumor tissues of nude mice.
Results:
1. SKOV3 cells could form xenografts in the nude mice, and the average time was about 10-12 days. The xenografted nude mice were divided into 3 groups randomly. STAT3 decoy ODN was peritumorally injected into the xenografts every other day. In the end of the treatment, the average volume of xenografts was as follows:620±68mm3 in PBS group,540±55mm3 in scramble group, 226±35mm3 in decoy group. The average volume of xenografts in decoy group was decreased significantly contrast to PBS and scramble groups (P＜0.05). However, the difference between PBS and scramble groups had no statistically significance. The average weight of tumor tissues in decoy group was significantly lower than that in the other control groups (P<0.05).
2. STAT3 decoy ODN could induce ovarian cancer cells apoptosis in vivo. TUNEL analysis showed that there were 43±7 positive cells per high power field in the group treated with STAT3 decoy ODN, while 11±3 positive cells in scramble group and 13±4 positive cells in PBS group. The difference between decoy group and the other two groups had statistically significance (P＜0.05).
3. The biological effects of STAT3 decoy ODN on tissues of xenograft, heart, liver and kidney in nude mice. The sections of xenograft tissues (HE staining) showed karyomegaly, anachromasis, and karyokinesis in cancer cells. There was necrosis partly in the xenograft tissues. The sections of heart and liver tissues had no significant abnormality. However, one in five nude mice treated with STAT3 decoy ODN had abnormality, inflammation and necrosis in part of hepatic lobule.
4. The expression of MMP2, MMP9, Bcl-2, and caspase-3 in the xenograft tissues of nude mice:Compared with PBS and scramble treatment group, the protein expression level of MMP2, MMP9, Bcl-2 in STAT3 decoy ODN treatment group decreased significantly, while the protein expression level of caspase-3 increased significantly.
Conclusions:
1. SKOV3 cells can form xenografts in the nude mice. STAT3 decoy ODN can inhibit the growth of ovarian cancer cells and induce apoptosis of ovarian cancer cells in vivo.
2. STAT3 decoy ODN can down-regulate the protein expression level of MMP2, MMP9, Bcl-2, and up-regulate the protein expression level of caspase-3 in vivo.
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