腺病毒介导的组织特异性启动子调控活化型Caspase-3用于喉癌的基因治疗研究
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摘要
喉癌是头颈部常见的恶性肿瘤,约占全身恶性肿瘤的3.3-8.1%。据2009年美国国立癌症研究院的流行病学和最终结果监测(Surveillance, Epidemiology and End Results, SEER)数据,仅美国每年新增12250例喉癌病例。在我国喉癌的发病率亦有增高趋势,尽管医疗技术不断进步,喉癌的总体生存率近30年却无明显改善。根治性手术加术后辅助放疗和化疗对晚期喉癌的局部控制有时效果不佳。有些晚期喉癌,特别是治疗后复发的病例,由于头颈部重要血管和颅神经的限制,事实上已经失去了再次根治性手术切除的机会,而辅助放疗和化疗对鳞状细胞癌疗效不佳,因此探讨有效的生物治疗策略作为传统治疗手段的补充具有实际意义。
以腺病毒为基础基因治疗一直是喉癌生物治疗的研究热点。效应基因选择多样,涉及抑癌基因、反义癌基因、免疫调节因子、自杀基因等,虽然这些治疗策略均能在一定程度上抑制喉癌的生长,但凋亡诱导不彻底及缺乏有效的靶向性调控策略影响了喉癌基因治疗的效果及安全性,限制其临床应用。因此,如何高效靶向性诱导肿瘤细胞凋亡是喉癌基因治疗尚需解决的关键问题。目前应用组织和细胞特异性启动子调控目的基因在靶细胞中特异性表达,是实现肿瘤靶向性基因治疗的有效途径。例如：应用分泌性白细胞蛋白酶抑制因子(secretory leukoprotease inhibitor, SLPI)启动子调控的腺病毒对卵巢癌有良好的特异性抗肿瘤作用,而对正常肝脏细胞无明显毒性；应用鳞状细胞癌抗原2(squamous cell carcinoma antigen 2, SCCA2)启动子修饰腺病毒载体,能调控促凋亡基因在SCCA2阳性的肺鳞状细胞癌中的特异性表达,体内外实验均有良好的抑瘤作用。然而,迄今为止尚无喉癌组织特异性启动子靶向性基因治疗的研究报道。由于95%的喉癌属于鳞状细胞癌,而且研究发现SLPI和SCCA2启动子基因在喉癌肿瘤组织中的表达显著高于正常喉粘膜上皮组织。因此,SLPI和SCCA2可作为备选的喉癌组织特异性启动子。
选择合适的效应基因诱导细胞凋亡是喉癌基因治疗最核心的环节。研究提示,天冬氨酸特异性半胱氨酸蛋白酶3(Caspase-3)在凋亡信息传递中居核心地位,因此,以Caspase-3为效应基因的治疗策略可能有助于提高凋亡诱导效果。有一种新合成的活化型Caspase-3分子,与体内的Caspase-3相比,可不需上游分子的切割活化而自发折叠成具有活性的三维结构形式,直接诱导细胞凋亡。如果目的基因产物能像活化型Caspase-3一样直接杀灭肿瘤细胞,则更能提高杀伤肿瘤细胞的效能。
本研究的兴趣是在SLPI与SCCA2启动子中筛选出适合喉鳞状细胞癌细胞株Hep-2的特异性启动子,将筛选出的启动子与活化型Caspase-3构建重组腺病毒。利用腺病毒载体的介导作用和特异性启动子的转录调控作用,将活化型Caspase-3靶向性导入Hep-2细胞,观察重组腺病毒特异性诱导喉癌细胞凋亡、抑制肿瘤生长的作用。
目的
探索一种安全、有效的喉鳞状细胞癌靶向性基因治疗策略,从而特异且高效地诱导喉癌细胞凋亡。
方法
1.PCR扩增SCCA2, SLPI1-3(分别对应SLPI启动子的-849—+22,-650—+22,-531—+22区域)启动子,经测序鉴定后,将SCC A2,SLPI 1-3,Survivin启动子序列定向插入到荧光表达载体pGL4.10的BglII位点上。
2. pGL4.10-SCCA2, pGL4.10-SLPI1-3, pGL4.10-Survivin与pGL4.74载体共转染人喉鳞状细胞癌细胞株Hep-2,人脐静脉内皮细胞HuVEC及人骨肉瘤细胞株MG-63,采用双荧光素酶报告基因检测试剂盒检测各启动子的转录效率(SLPI2启动子转录效率最高)。
3.构建腺病毒穿梭质粒pDC312-SLPI-Caspase-3-TAG-pA, Ad-SLPI-GFP-pA,并与骨架质粒pBGHlox (delta) E1,3Cre共转染HEK293细胞进行腺病毒包装。以PCR, western blot等方法鉴定重组腺病毒Ad-SLPI-Caspase-3, Ad-SLPI-GFP。扩增病毒,并以CsCl密度梯度离心法纯化构建的腺病毒,TCID50法测定病毒滴度。
4.将重组腺病毒Ad-SLPI-Caspase-3及对照病毒Ad-SLPI-GFP分别感染Hep-2和HuVEC细胞株,通过显微镜观察,MTT, DNA片段分析及流式细胞仪细胞凋亡分析检测病毒对喉癌细胞Hep-2的抑制作用及对正常细胞增殖的影响。
5.建立裸鼠喉癌荷瘤模型,观察Ad-SLPI-Caspase-3对喉癌荷瘤裸鼠肿瘤组织生长的抑制作用及毒副作用。
结果
1.启动子的构建及在不同细胞株中启动活性的检测pGL4.10-SCCA2, pGL4.10-SLPI1-3, pGL4.10-Survivin重组质粒构建成功。经双荧光素酶报告基因检测试剂盒检测,Hep-2细胞中,SLPI2启动子启动活性最高,差异经单因素方差分析,有统计学上显著性意义(P<0.05)。而相对于人脐静脉内皮细胞HuVEC,人骨肉瘤细胞MG-63, SLPI2启动子在喉癌细胞内的启动活性明显要高,分别为36.67倍(HuVEC)和17.11倍(MG-63),说明和人脐静脉内皮细胞HuVEC,入骨肉瘤细胞MG-63相比,SLPI2启动子在人喉鳞状细胞癌Hep-2细胞中的转录活性最好。
2.重组腺病毒Ad-SLPI-Caspase-3, Ad-SLPI-GFP的包装,鉴定、扩增、纯化及滴度测定腺病毒穿梭质粒pDC312-SLPI-Caspase-3-TAG-pA、pDC312-SLPI-GFP-pA与骨架质粒pBGHlox (delta) E1,3Cre共转染HEK293细胞,转染第13天左右,倒置显微镜下观察,发现80%以上的细胞都发生细胞病变效应,呈葡萄状,贴壁不牢易脱落。收集感染细胞,反复冻融,病毒上清经蛋白酶K处理后,PCR检测示Ad-SLPI-Caspase-3扩增出330bp片段,Ad-SLPI-GFP扩增出1482bp片段,示重组腺病毒Ad-SLPI-Caspase-3及对照病毒Ad-SLPI-GFP构建成功。予扩增纯化后进行滴度测定。Ad-SLPI-Caspase-3滴度为1×109pfu/ml, Ad-SLPI-GFP滴度为6.3×109pfu/ml,足够进行后期的体内体外实验。
Western blot结果显示,Ad-SLPI-Caspase-3病毒作用72小时的Hep-2细胞,32kd酶原形式的Caspase-3减少,而17kd活化型Caspase-3表达显著增高。而Ad-SLPI-GFP感染72小时后,荧光显微镜下可见大量Hep-2细胞呈现较强的绿色荧光信号,而HuVEC细胞仍无绿色荧光。以上结果表明,SLPI启动子能够在喉癌细胞内有效诱导Caspase-3的活化,并特异性调控绿荧光蛋白在人喉鳞状细胞癌细胞株Hep-2细胞中表达,而在正常人脐静脉内皮细胞HuVEC中无表达,72小时为合适的感染时间。
3.重组腺病毒Ad-SLPI-Caspase-3对喉癌细胞Hep-2的体外抑制作用
3.1重组腺病毒的体外细胞杀伤试验
MTT结果显示,感染72小时后复制缺陷型腺病毒Ad-SLPI-Caspase-3对人喉鳞状细胞癌细胞株Hep-2有较强的杀伤效果,在MOI为50pfu/cell时就能有效地抑制喉癌细胞的生长(细胞存活率为42.8%±0.007),但对正常脐静脉内皮细胞HuVEC却无明显杀伤效果(抑制率为92.2%±0.146)
3.2细胞被重组腺病毒Ad-SLPI-Caspase-3作用后形态学变化
重组腺病毒Ad-SLPI-Caspase-3作用72小时后的喉癌细胞在形态学上发生了显著的变化：细胞变圆,皱缩,部分呈串珠状、浮起。而人正常脐静脉内皮细胞HuVEC在病毒作用前后细胞形态差异不明显。
3.3 DNA fragmentation定性分析细胞凋亡
重组腺病毒Ad-SLPI-Caspase-3感染喉癌细胞72小时后可见明显的DNA阶梯状条带,有效地诱导了喉癌细胞Hep-2的凋亡,但不能诱导正常脐静脉内皮细胞HuVEC发生凋亡。Ad-SLPI-GFP对Hep-2和HuVEC均不能有效诱导细胞凋亡,说明Caspase-3作为效应基因成功诱导了细胞凋亡,而腺病毒载体本身不对细胞的生长产生明显影响。
3.4流式细胞仪定量分析细胞凋亡
流式细胞仪检测显示Ad-SLPI-Caspase-3作用72小时,Hep-2细胞中晚期凋亡细胞及坏死细胞(即Annexin V-R-PE和7-AAD均染色的细胞)占80.6%,明显高于Ad-SLPI-GFP作用72小时后15.3%的比例,两者差异显著,从定量角度说明Caspase-3能高效地诱导喉癌细胞凋亡。而在正常脐静脉内皮细胞中,Ad-SLPI-Caspase-3, Ad-SLPI-GFP及PBS对照组的晚期凋亡及坏死率分别为29.4%,44.3%,15.2%,三者差异不显著,证实所构建的病毒不能在正常细胞中有效诱导凋亡。
4.重组腺病毒Ad-SLPI-Caspase-3对喉癌荷瘤模型肿瘤组织生长的抑制作用
4.1重组腺病毒Ad-SLPI-Caspase-3的体内抗瘤活性
重组腺病毒Ad-SLPI-Caspase-3在裸鼠体内具有一定的抗肿瘤活性,与对照组Ad-SLPI-GFP和PBS组相比,Ad-SLPI-Caspase-3治疗组的瘤体生长在第16、19天得到了显著地抑制(P<0.05)。而在观察结束时(首次给药后第25天)治疗组瘤体体积及瘤重均数虽较对照组小,但无统计学差异(P>0.05),这可能与治疗组中肿瘤细胞未被完全抑制,残留的肿瘤细胞继续增殖导致了肿瘤进展及实验观察后期个体差异增大有关。在末次病毒注射后12天,在每组中随机选取一只裸鼠,取出瘤体,固定包埋,做石蜡切片,并HE染色。结果显示,大量的坏死细胞存在于Ad-SLPI-Caspase-3治疗组的镜下视野内,而对照组未见明显的大片坏死细胞灶。
4.2重组腺病毒Ad-SLPI-Caspase-3的体内毒副作用观察
在整个观察期内,三组裸鼠均未出现个体死亡,生存情况良好。重组腺病毒Ad-SLPI-Caspase-3与对照组Ad-SLPI-GFP和PBS组相比,裸鼠的活动及饮食情况均无明显差异。在观察结束时,治疗组裸鼠体重及肝脏重量较对照组无显著性差异(P>0.05)。肝脏HE染色结果显示,Ad-SLPI-Caspase-3、对照病毒Ad-SLPI-GFP及PBS治疗组的镜下视野内未见肝细胞明显的变性坏死。
结论
1.与人脐静脉内皮细胞HuVEC,人骨肉瘤细胞MG-63相比,SLPI2启动子在人喉鳞状细胞癌Hep-2细胞中的转录效率最高,能有效地调控和启动下游基因的表达,具有高效性和特异性。
2.复制缺陷型腺病毒Ad-SLPI-Caspase-3和Ad-SLPI-GFP均能有效感染Hep-2细胞,并表达相应的基因,具有良好的喉癌组织特异性。重组腺病毒Ad-SLPI-Caspase-3在喉癌细胞中成功表达活化型Caspase-3的同时,可在喉癌细胞中促进内源性Caspase-3的活化。
3.复制缺陷型腺病毒Ad-SLPI-Caspase-3,能够有效的诱导喉癌细胞凋亡,而对正常细胞的生长无明显的影响,具有良好的特异性。Ad-SLPI-Caspase-3在体内具有一定的抗瘤活性,能有效抑制喉癌细胞株Hep-2的皮下移植瘤生长,促进肿瘤组织坏死,而无明显的肝脏毒性。
Laryngeal carcinoma is one of the most common cancers in head and neck, with 12,250 new cases reported every year in the United States alone according to the data from Surveillance, Epidemiology and End Results (SEER) in 2009. The incidence is also gradually increasing in our country. Despite advances in conventional therapy including surgery, chemotherapy and radiation, the 5-year survival rate has not been significantly improved during the past three decades. The regional control for advanced laryngeal carcinoma is sometimes unsatisfactory, even under the combined therapy of radical surgery, adjuvant radiotherapy and chemotherapy. Big hurdles also lie in front of the therapeutic regimen for terminal relapsed cases:the tumor was unresectable due to invasion to the important blood vessels in head and neck or to the important cranial nerves, while the sensitivity to the administration of adjuvant radiotherapy and chemotherapy is rather low in squamous cell carcinoma. Therefore, local and regional disease control is paramount, underscoring an urgent need for more effective, minimally invasive and lower toxic biotherapy in laryngeal carcinoma.
Currently adenovirus-based therapy is a useful approach for effective gene delivery and expression in laryngeal carcinoma, by targeting various kinds of genes, like tumor suppressor genes, antisense oncogenes, immune regulatory factors, suicide genes. Although these strategies could be effective in inhibiting the growth of laryngeal cancer to some extend, no ideal strategy has yet been developed to achieve specific and efficient anti-tumor effect and avoid any adverse reactions in normal cells. Therefore, effective targeting strategies are necessary to increase target transduction and reduce the risk of unspecific side effects to nontarget tissues. So far, modifying adenovirus by tissue specific promoters (TSPs) is one of the most effective ways for targeting gene therapy for adenovirus vectors. However, no effective tissue specific promoter has been reported in laryngeal carcinoma. As 95% of the laryngeal carcinoma is composed by squamous cell carcinoma, the TSPs for some squamous cell carcinomas could also be a suitable candidate for laryngeal carcinoma. In recent years, some researches have reported the successful appliance for secretory leukocyte protease inhibitor (SLPI) promoter and squamous cell carcinoma antigen 2 (SCCA2) promoter in the targeting gene therapy in ovarian cancer, lung cancer, and other squamous cell carcinomas. Meanwhile, SLPI, SCCA2 were confirmed as potential biomarkers for laryngeal carcinoma, with significant over-expression in tumor tissues and extremely low expression in normal hepatic tissues. These characteristics have provided strong evidences for the usage of the two above promoters for targeting gene therapy in laryngeal carcinoma.
In addition, the selection of suitable effect genes to induce efficient apoptosis is an essential part in gene therapy. The latest research results indicate that Caspase-3, a member of aspartate-specific cysteine protease, has taken a central stage in the induction of apoptosis, which make it a potential candidate as an apoptosis-induced factor with wide range of applicability in almost all kinds of malignant tumors. Recently, a kind of activated Caspase-3 was addressed to spontaneously fold into three-dimensional structure, inducing cell apoptosis directly without activation of the upstream elements. It would be more convenient and efficient by using activated Caspase-3 to kill tumor cells. By taking all these evidence into consideration, activated Caspase-3 could also be a powerful effect gene in the practice for gene therapy in laryngeal carcinoma.
Objective
To specifically and efficiently induce apoptosis in laryngeal carcinomas by a novel strategy for adenovirus-based targeting gene therapy
Methods
1. The region of SCCA2, SLPI1-3 promoters (corresponding to the region of SLPI promoter -849-+22,-650-+22,-531-+22) was amplified by PCR. After sequence analysis, they were inserted separately into the multiple cloning sites (MCS) in luciferase reporter gene vector of pGL4.10.
2. The recombinant plasmids of pGL4.10-SCCA2, pGL4.10-SLPI1-3, and pGL4.10-Survivin were separately cotransfected with pGL4.74 into Hep-2, HuVEC and MG-63. The efficiency of each promoter was detected by using Dual-Luciferase Reporter Gene Assay Kit (SLPI2 is the most efficient promoter in our study).
3. The shuttle plasmids of adenovirus pDC312-SLPI-Caspase-3-TAG-pA and Ad-SLPI-GFP-pA were constructed and then separately cotransfected into HEK293 with the backbone plasmid of pBGHlox (delta) E1,3Cre for adenovirus packaging. Recombinant adenovirus of Ad-SLPI-Caspase-3 and Ad-SLPI-GFP were identified by PCR and western blot. Recombinant adenovirus were amplified and purified by CsCl density gradient centrifugation. The titers for recombinant adenovirus were assessed by TCID50.
4. The inhibition of the growth for Hep-2 and HuVEC by recombinant adenoviruses in vitro was evaluated by the microscope, MTT, DNA fragmentation analysis and flow cytometry analysis.
5. The antitumor activity and side effects in vivo by recombinant adenoviruses were observed in human laryngeal carcinoma xenograft in Nude Mice.
Results
1. Construction of SCCA2, SLPI1-3 promoters and detection for the activity of different promoters in each cell line
Recombinant plasmids of pGL4.10-SCCA2, pGL4.10-SLPI1-3, and pGL4.10-Survivin were successfully constructed. Among all the promoters for Hep-2, promoter for SLPI2 showed the highest activity, the difference was statistically significant (P<0.05) by one-way ANOVA test. By comparing with the activities in the cell lines for HuVEC and MG-63, promoter for SLPI2 also showed the highest activity in Hep-2, with approximately 36.67 fold and 17.11 fold respectively.
2. Construction for the recombinant adenoviruses for Ad-SLPI-Caspase-3, Ad-SLPI-GFP
An obvious cytopathic effect (CPE) was observed in HEK293 after about 13 days from cotransfection. Recombinant adenoviruses were then harvested and identified by PCR. The expected fragments of 330bp and 1482bp could be found in the amplified products of the virus supernatants from Ad-SLPI-Caspase-3 and Ad-SLPI-GFP respectively. The recombinant adenoviruses were successfully constructed. After amplification, purification and concentration, the titers for Ad-SLPI-Caspase-3 and Ad-SLPI-GFP reached 1×109pfu/ml,6.3×109pfu/ml respectively, which was sufficient for future studies both in vivo and in vitro.
Western blot showed that the 17kd activated Caspase-3 was significantly increased after 72 hours by the infection of Ad-SLPI-Caspase-3 in Hep-2. Meanwhile, strong signals of green fluorescence could be seen in a large number of Hep-2 cells after 72 hours by the infection of Ad-SLPI-GFP. However, it showed absent expression in HuVEC. These results indicated SLPI promoter could effectively induce the expression of activated Caspase-3 in laryngeal carcinoma cells. The expression of green fluorescent protein was specifically restricted in Hep-2 cells and absent in normal human umbilical vein endothelial cells of HuVEC. And 72 hours is the right time for infection.
3. In vitro Study on cytotoxicity of Ad-SLPI-Caspase-3 to human laryngeal carcinoma cells Hep-2
3.1 MTT Assay
The proliferation of Hep-2 cells was noticeable inhibited by Ad-SLPI-Caspase-3 after transfecting 72 hours, while no significant inhibition was observed in HuVEC cells. The cell survival rate in Hep-2(42.8%±0.007) was much lower than that in HuVEC (92.2%±0.146).
3.2 Morphological changes by infecting with Ad-SLPI-Caspase-3
After 72 hours, the infected Hep-2 cells showed an obvious change in morphology:cells became round and shrieked, some even floated in cluster. However, no significant morphological change was observed in the infected Hove cells.
3.3 DNA fragmentation assay
Noticeable DNA ladder was detected in the infected Hep-2 cells by Ad-SLPI-Caspase-3, which clearly indicated effective induction of apoptosis in laryngeal carcinoma cells, but none was detected in HuVEC cells, as well as the Hep-2 cells and HuVEC cells infected by Ad-SLPI-GFP. This revealed that Caspase-3 was an effective gene to induce apoptosis, while the adenovirus vector itself did not produce any effect on cell apoptosis.
3.4 Flow cytometry for quantitative analysis of apoptosis
The rate of late apoptosis in the infected Hep-2 cells by Ad-SLPI-Caspase-3 was 80.6%, which was significantly higher than that by Ad-SLPI-GFP and PBS. No significant difference was observed in the infected HuVEC cells by Ad-SLPI-Caspase-3, Ad-SLPI-GFP and the control PBS group. All these evidence confirmed that Caspase-3 could efficiently induce apoptosis in laryngeal cancer cells. And the apoptosis-inducing effect was specific in Hep-2 cells under the control of SLPI promoter.
4. The antitumor activity by Ad-SLPI-Caspase-3 in human laryngeal carcinoma xenograft in Nude Mice
4.1 The antitumor activity by Ad-SLPI-Caspase-3 in human laryngeal carcinoma in vivo
Statistical analysis showed that Ad-SLPI-Caspase-3 achieved significant antitumor efficacy at the thirteenth and sixteenth day after first injection (P< 0.05). However, by the end of the whole observation period (twenty-five days after first injection), the difference of the average tumor sizes and tumor weights among the three groups was not statistically significant(P> 0.05), which might probably be related with the incomplete suppression of tumor cells, the residual tumor continued to proliferate and lead to tumor progression. Large area of typical necrotic appearance was observed in the paraffin sections from tumor tissues of the Ad-SLPI-Caspase-3 group by HE staining. However, there was no necrosis in large area in the other two control group.
4.2 Observation on toxicity in vivo by recombinant adenovirus
During the whole period of study, all of the three groups of nude mice survived in good condition. There was no significant difference in the activities and diets among them. No significant difference in body weight and liver weight was seen in the adenovirus treated groups, compared with the control group. And no obvious degeneration and necrosis of liver cells was found by HE-staining for the hepatic tissues in the three groups.
Conclusion
1. SLPI2 promoter is an efficient and tissue-specific promoter for human laryngeal squamous cell carcinoma cell line Hep-2.
2. Replication-defective adenovirus of Ad-SLPI-Caspase-3 and Ad-SLPI-GFP could infect Hep-2 cells specifically and effectively, inducing the expression of target genes. The activated Caspase-3 could be successfully expressed, meanwhile the endogenous Caspase-3 could also be activated by Ad-SLPI-Caspase-3 in Hep-2 cells.
3. Replication-defective adenovirus of Ad-SLPI-Caspase-3 could inhibit growth of laryngeal carcinoma xenografts and induce necrosis efficiently in tumor tissues, without any obvious toxicity to the liver.
以腺病毒为基础基因治疗一直是喉癌生物治疗的研究热点。效应基因选择多样,涉及抑癌基因、反义癌基因、免疫调节因子、自杀基因等,虽然这些治疗策略均能在一定程度上抑制喉癌的生长,但凋亡诱导不彻底及缺乏有效的靶向性调控策略影响了喉癌基因治疗的效果及安全性,限制其临床应用。因此,如何高效靶向性诱导肿瘤细胞凋亡是喉癌基因治疗尚需解决的关键问题。目前应用组织和细胞特异性启动子调控目的基因在靶细胞中特异性表达,是实现肿瘤靶向性基因治疗的有效途径。例如：应用分泌性白细胞蛋白酶抑制因子(secretory leukoprotease inhibitor, SLPI)启动子调控的腺病毒对卵巢癌有良好的特异性抗肿瘤作用,而对正常肝脏细胞无明显毒性；应用鳞状细胞癌抗原2(squamous cell carcinoma antigen 2, SCCA2)启动子修饰腺病毒载体,能调控促凋亡基因在SCCA2阳性的肺鳞状细胞癌中的特异性表达,体内外实验均有良好的抑瘤作用。然而,迄今为止尚无喉癌组织特异性启动子靶向性基因治疗的研究报道。由于95%的喉癌属于鳞状细胞癌,而且研究发现SLPI和SCCA2启动子基因在喉癌肿瘤组织中的表达显著高于正常喉粘膜上皮组织。因此,SLPI和SCCA2可作为备选的喉癌组织特异性启动子。
选择合适的效应基因诱导细胞凋亡是喉癌基因治疗最核心的环节。研究提示,天冬氨酸特异性半胱氨酸蛋白酶3(Caspase-3)在凋亡信息传递中居核心地位,因此,以Caspase-3为效应基因的治疗策略可能有助于提高凋亡诱导效果。有一种新合成的活化型Caspase-3分子,与体内的Caspase-3相比,可不需上游分子的切割活化而自发折叠成具有活性的三维结构形式,直接诱导细胞凋亡。如果目的基因产物能像活化型Caspase-3一样直接杀灭肿瘤细胞,则更能提高杀伤肿瘤细胞的效能。
本研究的兴趣是在SLPI与SCCA2启动子中筛选出适合喉鳞状细胞癌细胞株Hep-2的特异性启动子,将筛选出的启动子与活化型Caspase-3构建重组腺病毒。利用腺病毒载体的介导作用和特异性启动子的转录调控作用,将活化型Caspase-3靶向性导入Hep-2细胞,观察重组腺病毒特异性诱导喉癌细胞凋亡、抑制肿瘤生长的作用。
目的
探索一种安全、有效的喉鳞状细胞癌靶向性基因治疗策略,从而特异且高效地诱导喉癌细胞凋亡。
方法
1.PCR扩增SCCA2, SLPI1-3(分别对应SLPI启动子的-849—+22,-650—+22,-531—+22区域)启动子,经测序鉴定后,将SCC A2,SLPI 1-3,Survivin启动子序列定向插入到荧光表达载体pGL4.10的BglII位点上。
2. pGL4.10-SCCA2, pGL4.10-SLPI1-3, pGL4.10-Survivin与pGL4.74载体共转染人喉鳞状细胞癌细胞株Hep-2,人脐静脉内皮细胞HuVEC及人骨肉瘤细胞株MG-63,采用双荧光素酶报告基因检测试剂盒检测各启动子的转录效率(SLPI2启动子转录效率最高)。
3.构建腺病毒穿梭质粒pDC312-SLPI-Caspase-3-TAG-pA, Ad-SLPI-GFP-pA,并与骨架质粒pBGHlox (delta) E1,3Cre共转染HEK293细胞进行腺病毒包装。以PCR, western blot等方法鉴定重组腺病毒Ad-SLPI-Caspase-3, Ad-SLPI-GFP。扩增病毒,并以CsCl密度梯度离心法纯化构建的腺病毒,TCID50法测定病毒滴度。
4.将重组腺病毒Ad-SLPI-Caspase-3及对照病毒Ad-SLPI-GFP分别感染Hep-2和HuVEC细胞株,通过显微镜观察,MTT, DNA片段分析及流式细胞仪细胞凋亡分析检测病毒对喉癌细胞Hep-2的抑制作用及对正常细胞增殖的影响。
5.建立裸鼠喉癌荷瘤模型,观察Ad-SLPI-Caspase-3对喉癌荷瘤裸鼠肿瘤组织生长的抑制作用及毒副作用。
结果
1.启动子的构建及在不同细胞株中启动活性的检测pGL4.10-SCCA2, pGL4.10-SLPI1-3, pGL4.10-Survivin重组质粒构建成功。经双荧光素酶报告基因检测试剂盒检测,Hep-2细胞中,SLPI2启动子启动活性最高,差异经单因素方差分析,有统计学上显著性意义(P<0.05)。而相对于人脐静脉内皮细胞HuVEC,人骨肉瘤细胞MG-63, SLPI2启动子在喉癌细胞内的启动活性明显要高,分别为36.67倍(HuVEC)和17.11倍(MG-63),说明和人脐静脉内皮细胞HuVEC,入骨肉瘤细胞MG-63相比,SLPI2启动子在人喉鳞状细胞癌Hep-2细胞中的转录活性最好。
2.重组腺病毒Ad-SLPI-Caspase-3, Ad-SLPI-GFP的包装,鉴定、扩增、纯化及滴度测定腺病毒穿梭质粒pDC312-SLPI-Caspase-3-TAG-pA、pDC312-SLPI-GFP-pA与骨架质粒pBGHlox (delta) E1,3Cre共转染HEK293细胞,转染第13天左右,倒置显微镜下观察,发现80%以上的细胞都发生细胞病变效应,呈葡萄状,贴壁不牢易脱落。收集感染细胞,反复冻融,病毒上清经蛋白酶K处理后,PCR检测示Ad-SLPI-Caspase-3扩增出330bp片段,Ad-SLPI-GFP扩增出1482bp片段,示重组腺病毒Ad-SLPI-Caspase-3及对照病毒Ad-SLPI-GFP构建成功。予扩增纯化后进行滴度测定。Ad-SLPI-Caspase-3滴度为1×109pfu/ml, Ad-SLPI-GFP滴度为6.3×109pfu/ml,足够进行后期的体内体外实验。
Western blot结果显示,Ad-SLPI-Caspase-3病毒作用72小时的Hep-2细胞,32kd酶原形式的Caspase-3减少,而17kd活化型Caspase-3表达显著增高。而Ad-SLPI-GFP感染72小时后,荧光显微镜下可见大量Hep-2细胞呈现较强的绿色荧光信号,而HuVEC细胞仍无绿色荧光。以上结果表明,SLPI启动子能够在喉癌细胞内有效诱导Caspase-3的活化,并特异性调控绿荧光蛋白在人喉鳞状细胞癌细胞株Hep-2细胞中表达,而在正常人脐静脉内皮细胞HuVEC中无表达,72小时为合适的感染时间。
3.重组腺病毒Ad-SLPI-Caspase-3对喉癌细胞Hep-2的体外抑制作用
3.1重组腺病毒的体外细胞杀伤试验
MTT结果显示,感染72小时后复制缺陷型腺病毒Ad-SLPI-Caspase-3对人喉鳞状细胞癌细胞株Hep-2有较强的杀伤效果,在MOI为50pfu/cell时就能有效地抑制喉癌细胞的生长(细胞存活率为42.8%±0.007),但对正常脐静脉内皮细胞HuVEC却无明显杀伤效果(抑制率为92.2%±0.146)
3.2细胞被重组腺病毒Ad-SLPI-Caspase-3作用后形态学变化
重组腺病毒Ad-SLPI-Caspase-3作用72小时后的喉癌细胞在形态学上发生了显著的变化：细胞变圆,皱缩,部分呈串珠状、浮起。而人正常脐静脉内皮细胞HuVEC在病毒作用前后细胞形态差异不明显。
3.3 DNA fragmentation定性分析细胞凋亡
重组腺病毒Ad-SLPI-Caspase-3感染喉癌细胞72小时后可见明显的DNA阶梯状条带,有效地诱导了喉癌细胞Hep-2的凋亡,但不能诱导正常脐静脉内皮细胞HuVEC发生凋亡。Ad-SLPI-GFP对Hep-2和HuVEC均不能有效诱导细胞凋亡,说明Caspase-3作为效应基因成功诱导了细胞凋亡,而腺病毒载体本身不对细胞的生长产生明显影响。
3.4流式细胞仪定量分析细胞凋亡
流式细胞仪检测显示Ad-SLPI-Caspase-3作用72小时,Hep-2细胞中晚期凋亡细胞及坏死细胞(即Annexin V-R-PE和7-AAD均染色的细胞)占80.6%,明显高于Ad-SLPI-GFP作用72小时后15.3%的比例,两者差异显著,从定量角度说明Caspase-3能高效地诱导喉癌细胞凋亡。而在正常脐静脉内皮细胞中,Ad-SLPI-Caspase-3, Ad-SLPI-GFP及PBS对照组的晚期凋亡及坏死率分别为29.4%,44.3%,15.2%,三者差异不显著,证实所构建的病毒不能在正常细胞中有效诱导凋亡。
4.重组腺病毒Ad-SLPI-Caspase-3对喉癌荷瘤模型肿瘤组织生长的抑制作用
4.1重组腺病毒Ad-SLPI-Caspase-3的体内抗瘤活性
重组腺病毒Ad-SLPI-Caspase-3在裸鼠体内具有一定的抗肿瘤活性,与对照组Ad-SLPI-GFP和PBS组相比,Ad-SLPI-Caspase-3治疗组的瘤体生长在第16、19天得到了显著地抑制(P<0.05)。而在观察结束时(首次给药后第25天)治疗组瘤体体积及瘤重均数虽较对照组小,但无统计学差异(P>0.05),这可能与治疗组中肿瘤细胞未被完全抑制,残留的肿瘤细胞继续增殖导致了肿瘤进展及实验观察后期个体差异增大有关。在末次病毒注射后12天,在每组中随机选取一只裸鼠,取出瘤体,固定包埋,做石蜡切片,并HE染色。结果显示,大量的坏死细胞存在于Ad-SLPI-Caspase-3治疗组的镜下视野内,而对照组未见明显的大片坏死细胞灶。
4.2重组腺病毒Ad-SLPI-Caspase-3的体内毒副作用观察
在整个观察期内,三组裸鼠均未出现个体死亡,生存情况良好。重组腺病毒Ad-SLPI-Caspase-3与对照组Ad-SLPI-GFP和PBS组相比,裸鼠的活动及饮食情况均无明显差异。在观察结束时,治疗组裸鼠体重及肝脏重量较对照组无显著性差异(P>0.05)。肝脏HE染色结果显示,Ad-SLPI-Caspase-3、对照病毒Ad-SLPI-GFP及PBS治疗组的镜下视野内未见肝细胞明显的变性坏死。
结论
1.与人脐静脉内皮细胞HuVEC,人骨肉瘤细胞MG-63相比,SLPI2启动子在人喉鳞状细胞癌Hep-2细胞中的转录效率最高,能有效地调控和启动下游基因的表达,具有高效性和特异性。
2.复制缺陷型腺病毒Ad-SLPI-Caspase-3和Ad-SLPI-GFP均能有效感染Hep-2细胞,并表达相应的基因,具有良好的喉癌组织特异性。重组腺病毒Ad-SLPI-Caspase-3在喉癌细胞中成功表达活化型Caspase-3的同时,可在喉癌细胞中促进内源性Caspase-3的活化。
3.复制缺陷型腺病毒Ad-SLPI-Caspase-3,能够有效的诱导喉癌细胞凋亡,而对正常细胞的生长无明显的影响,具有良好的特异性。Ad-SLPI-Caspase-3在体内具有一定的抗瘤活性,能有效抑制喉癌细胞株Hep-2的皮下移植瘤生长,促进肿瘤组织坏死,而无明显的肝脏毒性。
Laryngeal carcinoma is one of the most common cancers in head and neck, with 12,250 new cases reported every year in the United States alone according to the data from Surveillance, Epidemiology and End Results (SEER) in 2009. The incidence is also gradually increasing in our country. Despite advances in conventional therapy including surgery, chemotherapy and radiation, the 5-year survival rate has not been significantly improved during the past three decades. The regional control for advanced laryngeal carcinoma is sometimes unsatisfactory, even under the combined therapy of radical surgery, adjuvant radiotherapy and chemotherapy. Big hurdles also lie in front of the therapeutic regimen for terminal relapsed cases:the tumor was unresectable due to invasion to the important blood vessels in head and neck or to the important cranial nerves, while the sensitivity to the administration of adjuvant radiotherapy and chemotherapy is rather low in squamous cell carcinoma. Therefore, local and regional disease control is paramount, underscoring an urgent need for more effective, minimally invasive and lower toxic biotherapy in laryngeal carcinoma.
Currently adenovirus-based therapy is a useful approach for effective gene delivery and expression in laryngeal carcinoma, by targeting various kinds of genes, like tumor suppressor genes, antisense oncogenes, immune regulatory factors, suicide genes. Although these strategies could be effective in inhibiting the growth of laryngeal cancer to some extend, no ideal strategy has yet been developed to achieve specific and efficient anti-tumor effect and avoid any adverse reactions in normal cells. Therefore, effective targeting strategies are necessary to increase target transduction and reduce the risk of unspecific side effects to nontarget tissues. So far, modifying adenovirus by tissue specific promoters (TSPs) is one of the most effective ways for targeting gene therapy for adenovirus vectors. However, no effective tissue specific promoter has been reported in laryngeal carcinoma. As 95% of the laryngeal carcinoma is composed by squamous cell carcinoma, the TSPs for some squamous cell carcinomas could also be a suitable candidate for laryngeal carcinoma. In recent years, some researches have reported the successful appliance for secretory leukocyte protease inhibitor (SLPI) promoter and squamous cell carcinoma antigen 2 (SCCA2) promoter in the targeting gene therapy in ovarian cancer, lung cancer, and other squamous cell carcinomas. Meanwhile, SLPI, SCCA2 were confirmed as potential biomarkers for laryngeal carcinoma, with significant over-expression in tumor tissues and extremely low expression in normal hepatic tissues. These characteristics have provided strong evidences for the usage of the two above promoters for targeting gene therapy in laryngeal carcinoma.
In addition, the selection of suitable effect genes to induce efficient apoptosis is an essential part in gene therapy. The latest research results indicate that Caspase-3, a member of aspartate-specific cysteine protease, has taken a central stage in the induction of apoptosis, which make it a potential candidate as an apoptosis-induced factor with wide range of applicability in almost all kinds of malignant tumors. Recently, a kind of activated Caspase-3 was addressed to spontaneously fold into three-dimensional structure, inducing cell apoptosis directly without activation of the upstream elements. It would be more convenient and efficient by using activated Caspase-3 to kill tumor cells. By taking all these evidence into consideration, activated Caspase-3 could also be a powerful effect gene in the practice for gene therapy in laryngeal carcinoma.
Objective
To specifically and efficiently induce apoptosis in laryngeal carcinomas by a novel strategy for adenovirus-based targeting gene therapy
Methods
1. The region of SCCA2, SLPI1-3 promoters (corresponding to the region of SLPI promoter -849-+22,-650-+22,-531-+22) was amplified by PCR. After sequence analysis, they were inserted separately into the multiple cloning sites (MCS) in luciferase reporter gene vector of pGL4.10.
2. The recombinant plasmids of pGL4.10-SCCA2, pGL4.10-SLPI1-3, and pGL4.10-Survivin were separately cotransfected with pGL4.74 into Hep-2, HuVEC and MG-63. The efficiency of each promoter was detected by using Dual-Luciferase Reporter Gene Assay Kit (SLPI2 is the most efficient promoter in our study).
3. The shuttle plasmids of adenovirus pDC312-SLPI-Caspase-3-TAG-pA and Ad-SLPI-GFP-pA were constructed and then separately cotransfected into HEK293 with the backbone plasmid of pBGHlox (delta) E1,3Cre for adenovirus packaging. Recombinant adenovirus of Ad-SLPI-Caspase-3 and Ad-SLPI-GFP were identified by PCR and western blot. Recombinant adenovirus were amplified and purified by CsCl density gradient centrifugation. The titers for recombinant adenovirus were assessed by TCID50.
4. The inhibition of the growth for Hep-2 and HuVEC by recombinant adenoviruses in vitro was evaluated by the microscope, MTT, DNA fragmentation analysis and flow cytometry analysis.
5. The antitumor activity and side effects in vivo by recombinant adenoviruses were observed in human laryngeal carcinoma xenograft in Nude Mice.
Results
1. Construction of SCCA2, SLPI1-3 promoters and detection for the activity of different promoters in each cell line
Recombinant plasmids of pGL4.10-SCCA2, pGL4.10-SLPI1-3, and pGL4.10-Survivin were successfully constructed. Among all the promoters for Hep-2, promoter for SLPI2 showed the highest activity, the difference was statistically significant (P<0.05) by one-way ANOVA test. By comparing with the activities in the cell lines for HuVEC and MG-63, promoter for SLPI2 also showed the highest activity in Hep-2, with approximately 36.67 fold and 17.11 fold respectively.
2. Construction for the recombinant adenoviruses for Ad-SLPI-Caspase-3, Ad-SLPI-GFP
An obvious cytopathic effect (CPE) was observed in HEK293 after about 13 days from cotransfection. Recombinant adenoviruses were then harvested and identified by PCR. The expected fragments of 330bp and 1482bp could be found in the amplified products of the virus supernatants from Ad-SLPI-Caspase-3 and Ad-SLPI-GFP respectively. The recombinant adenoviruses were successfully constructed. After amplification, purification and concentration, the titers for Ad-SLPI-Caspase-3 and Ad-SLPI-GFP reached 1×109pfu/ml,6.3×109pfu/ml respectively, which was sufficient for future studies both in vivo and in vitro.
Western blot showed that the 17kd activated Caspase-3 was significantly increased after 72 hours by the infection of Ad-SLPI-Caspase-3 in Hep-2. Meanwhile, strong signals of green fluorescence could be seen in a large number of Hep-2 cells after 72 hours by the infection of Ad-SLPI-GFP. However, it showed absent expression in HuVEC. These results indicated SLPI promoter could effectively induce the expression of activated Caspase-3 in laryngeal carcinoma cells. The expression of green fluorescent protein was specifically restricted in Hep-2 cells and absent in normal human umbilical vein endothelial cells of HuVEC. And 72 hours is the right time for infection.
3. In vitro Study on cytotoxicity of Ad-SLPI-Caspase-3 to human laryngeal carcinoma cells Hep-2
3.1 MTT Assay
The proliferation of Hep-2 cells was noticeable inhibited by Ad-SLPI-Caspase-3 after transfecting 72 hours, while no significant inhibition was observed in HuVEC cells. The cell survival rate in Hep-2(42.8%±0.007) was much lower than that in HuVEC (92.2%±0.146).
3.2 Morphological changes by infecting with Ad-SLPI-Caspase-3
After 72 hours, the infected Hep-2 cells showed an obvious change in morphology:cells became round and shrieked, some even floated in cluster. However, no significant morphological change was observed in the infected Hove cells.
3.3 DNA fragmentation assay
Noticeable DNA ladder was detected in the infected Hep-2 cells by Ad-SLPI-Caspase-3, which clearly indicated effective induction of apoptosis in laryngeal carcinoma cells, but none was detected in HuVEC cells, as well as the Hep-2 cells and HuVEC cells infected by Ad-SLPI-GFP. This revealed that Caspase-3 was an effective gene to induce apoptosis, while the adenovirus vector itself did not produce any effect on cell apoptosis.
3.4 Flow cytometry for quantitative analysis of apoptosis
The rate of late apoptosis in the infected Hep-2 cells by Ad-SLPI-Caspase-3 was 80.6%, which was significantly higher than that by Ad-SLPI-GFP and PBS. No significant difference was observed in the infected HuVEC cells by Ad-SLPI-Caspase-3, Ad-SLPI-GFP and the control PBS group. All these evidence confirmed that Caspase-3 could efficiently induce apoptosis in laryngeal cancer cells. And the apoptosis-inducing effect was specific in Hep-2 cells under the control of SLPI promoter.
4. The antitumor activity by Ad-SLPI-Caspase-3 in human laryngeal carcinoma xenograft in Nude Mice
4.1 The antitumor activity by Ad-SLPI-Caspase-3 in human laryngeal carcinoma in vivo
Statistical analysis showed that Ad-SLPI-Caspase-3 achieved significant antitumor efficacy at the thirteenth and sixteenth day after first injection (P< 0.05). However, by the end of the whole observation period (twenty-five days after first injection), the difference of the average tumor sizes and tumor weights among the three groups was not statistically significant(P> 0.05), which might probably be related with the incomplete suppression of tumor cells, the residual tumor continued to proliferate and lead to tumor progression. Large area of typical necrotic appearance was observed in the paraffin sections from tumor tissues of the Ad-SLPI-Caspase-3 group by HE staining. However, there was no necrosis in large area in the other two control group.
4.2 Observation on toxicity in vivo by recombinant adenovirus
During the whole period of study, all of the three groups of nude mice survived in good condition. There was no significant difference in the activities and diets among them. No significant difference in body weight and liver weight was seen in the adenovirus treated groups, compared with the control group. And no obvious degeneration and necrosis of liver cells was found by HE-staining for the hepatic tissues in the three groups.
Conclusion
1. SLPI2 promoter is an efficient and tissue-specific promoter for human laryngeal squamous cell carcinoma cell line Hep-2.
2. Replication-defective adenovirus of Ad-SLPI-Caspase-3 and Ad-SLPI-GFP could infect Hep-2 cells specifically and effectively, inducing the expression of target genes. The activated Caspase-3 could be successfully expressed, meanwhile the endogenous Caspase-3 could also be activated by Ad-SLPI-Caspase-3 in Hep-2 cells.
3. Replication-defective adenovirus of Ad-SLPI-Caspase-3 could inhibit growth of laryngeal carcinoma xenografts and induce necrosis efficiently in tumor tissues, without any obvious toxicity to the liver.
引文
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