Long-term tumor-free survival from treatment with the GFP-TRAIL fusion gene expressed from the hTERT promoter in breast cancer cells

We evaluated anti-tumor activity and toxic effect of an adenoviral vector expressing the GFP/TRAIL fusion gene from the hTERT promoter (designated Ad/gTRAIL) on human breast cancer cell lines and on normal human breast cells. Treatment with Ad/gTRAIL elicited high levels of transgene expression and apoptosis in a variety of breast cancer cell lines. Furthermore, treatment with Ad/gTRAIL was effective in killing breast cancer lines resistant to doxorubicin or soluble TRAIL protein. In contrast, only minimal transgene expression and toxicity was detected in normal human primary mammary epithelial cells after treatment with this vector. An in vivo study further showed that the intralesional administration of Ad/gTRAIL effectively suppressed the growth of human tumor xenografts derived from both doxorubicin-sensitive and doxorubicin-resistant breast cancer lines. Specifically, about 50% of animals bearing doxorubicin-sensitive and doxorubicin-resistant breast cancer xenografts showed complete tumor regression and remained tumor-free for over 5 months. These results suggest that the adenovirus encoding the GFP/TRAIL gene driven by the hTERT promoter has potential application in cancer therapy.     

Combination effect of oncolytic adenovirotherapy and TRAIL gene therapy in syngeneic murine breast cancer models

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and oncolytic adenovirotherapy have been investigated extensively in xenografic human tumor models established in immunocompromised nude mice. However, the effects of these therapies on syngeneic murine tumors in immunocompetent settings were not well documented. We hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. In this study, we evaluated the antitumor effects of an oncolytic adenovirus, Delta24, in both human and murine breast cancer cell lines. We also analyzed the effects of TRAIL gene therapy combined with oncolytic virotherapy in these cancer cells. Our results showed that Delta24 can replicate and help the E1-deleted adenovector replicate in murine cancer cells. We also found that these two therapies combined had greater antitumor activity than either one alone in both human and murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. Moreover, Delta24 virotherapy alone and combined with TRAIL gene therapy dramatically reduced the spontaneous liver metastasis that originated in the subcutaneous 4T1 tumor established in Balb/c mice. These findings provide important considerations in the development and preclinical assessments of oncolytic virotherapy.     

腺相关病毒携带hTERT启动子调控的TRAIL基因特异性杀伤肿瘤细胞

背景与目的:腺相关病毒(adeno-associated virus)作为载体已被广泛用于肿瘤的基因治疗研究.肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factorrelated apoptosis.inducing ligand,TRAIL)基因可迅速诱导多种肿瘤细胞的凋亡.是一个安全有效的肿瘤杀伤基因.本研究旨在构建能在肿瘤细胞内特异性表达TRAIL基因的靶向腺相关病毒,并探讨其体外抗肿瘤效应的可能机制.方法:利用肿瘤特异性启动子端粒酶逆转录酶(human telomerase reverse transcfiptase,hTERT)构建特异性杀伤肿瘤细胞的腺相关病毒载体pAAV-hTERT-TRAIL.通过与pAAV-Rc、pHelper共转染HEK293细胞包装出病毒AAV-hTERT-TRAIL.将该病毒体外转染人结肠癌SW620细胞、人肝癌HepG2细胞、人肺癌A549细胞和正常细胞NHLF、MRC5后,检测TRAIL基因的肿瘤特异性表达.MTT法检测其对细胞增殖的影响,ELISA、Western blot法以及流式细胞仪检测细胞的凋亡,并分析其体外抗肿瘤效应的可能机制.结果:成功包装出病毒AAVhTERT-TRAIL.RT-PCR、Western blot和免疫组化法均证实AAV-hTERT-TRAIL能介导TRAIL基因在肿瘤细胞内特异性表达,但在正常细胞内不表达.以100 MOI AAV-IlTERTTRAIL感染细胞96 h后,SW620、A549和HepG2细胞的增殖率分别是41.55%、44.29%、49.95%,NHLF和MRC5细胞的增殖率分别是84.59%和87.22%.Western blot检测发现AAV-hTERT-TRAIL可激活Caspase通路.流式细胞仪和ELISA方法检测证实AAV-hTERT-TRAIL可诱导细胞凋亡.结论:hTERT的存在增强了腺相关病毒所携带TRAIL基因表达的肿瘤靶向性和对正常细胞的安全性.由它调控的杀伤基因可介导肿瘤细胞特异性的细胞毒效应.     

Death receptor 4 (DR4) efficiently kills breast cancer cells irrespective of their sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Breast cancer cells are generally resistant to induction of apoptosis by treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we demonstrate that both TRAIL-sensitive and TRAIL-resistant breast cancer cell lines can be efficiently killed by overexpression of the TRAIL receptor, death receptor 4 (DR4). The extent of cell death depended on the strength of the promoter driving DR4 expression. When driven by the strong CMV promoter, expression of DR4 killed over 90% of cells in five out of six cell lines tested in the absence of exogenous TRAIL. When driven by the relatively weak tumor-specific hTERT promoter, DR4 was less effective alone, but sensitized cells to killing by TRAIL. The extent of TRAIL sensitization depended on the magnitude of hTERT promoter activity. MCF-7 cells were relatively resistant to the action of DR4. We compared expression of the genes involved in transduction and execution of the death receptor-initiated apoptotic stimuli between MCF-7 and DR4-sensitive cell lines. We confirmed that in the panel of cell lines, MCF-7 was the only line deficient in expression of caspase 3. Bcl-2 and FLIP proteins, implicated in suppression of TRAIL-induced apoptosis, were expressed at a higher level.     

Recombinant adenoviruses expressing TRAIL demonstrate antitumor effects on non-small cell lung cancer (NSCLC)

INTRODUCTION: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of malignant cells, but not in normal cells. This preferential toxicity to the abnormal cells renders TRAIL potentially a very powerful therapeutic weapon against cancer. However, a requirement for large quantities of TRAIL to suppress tumor growth in vivo is one of the major factors that has hindered it from being widely applied clinically. To overcome this, we constructed a replication-deficient adenovirus that carries a human full-length TRAIL gene (Ad-TRAIL) and tested its efficacy against a lung cancer model system in comparison to that of the recombinant soluble TRAIL protein. METHODS: To investigate the antitumor activity and therapeutic value of the Ad-TRAIL on the non-small cell lung cancer (NSCLC), four NSCLC cell lines, namely, YTMLC, GLC, A549, and H460 cells, were used. TRAIL protein expression was determined by Western blotting and flow cytometry. Cell viability was analyzed by proliferation assay, and DNA ladder and cell-cycle analysis were used to identify apoptosis. To further evaluate the effect of Ad-TRAIL in vivo, YTMLC cells were inoculated to the subcutis of nude mice. The Ad-TRAIL was subsequently administered into the established tumors. Tumor growth and the TRAIL toxicity were evaluated after treatment. RESULTS: YTMLC cells infected with Ad-TRAIL showed decreased cell viability and a higher percentage of apoptosis. Similar, Ad-TRAIL treatment also significantly suppressed tumor growth in vivo. CONCLUSIONS: TRAIL gene therapy provides a promising therapy for the treatment of NSCLC.     

The sequential treatment with ionizing radiation followed by TRAIL/Apo-2L reduces tumor growth and induces apoptosis of breast tumor xenografts in nude mice

TRAIL primarily induces apoptosis in cancer cells but not in normal cells. However, some TRAIL-resistant cancer cell lines have recently been discovered. Ionizing radiation may enhance the apoptosis inducing potential of TRAIL in sensitive cells, and sensitize TRAIL-resistant cancer cells. We assessed the influence of sequential treatment of irradiation followed by TRAIL on intracellular mechanisms of apoptosis of breast tumor cells in vitro and on tumor regression in xenografted athymic nude mice. Irradiation augmented TRAIL-induced apoptosis in breast cancer cells through up-regulation of DR5, and subsequent activation of caspases-3, -8 and -9. Inhibition of p53 by siRNA abrogated irradiation-induced DR5 expression, suggesting the requirement of p53 for DR5 induction. The pretreatment of cells with irradiation followed by TRAIL significantly induced more apoptosis than single agent alone or concurrent treatment with irradiation and TRAIL. The sequential treatment of xenografted mice with irradiation followed by TRAIL-induced apoptosis through caspase-3 activation, completely eradicated the established breast tumors, and enhanced survival of mice without detectable toxicity to normal tissues. The sequential treatment with irradiation followed by TRAIL provides an approach to enhance therapeutic potential of TRAIL. Thus, irradiation can be combined with TRAIL in breast cancer therapy.     

TRAIL作用机制及其在肿瘤治疗中的研究

肿瘤坏死因子相关凋亡诱导配体(TRAIL)具有特异性抗肿瘤活性,可通过死亡受体途径及转录因子途径发挥诱导肿瘤细胞凋亡作用.而正常细胞则通过诱骗受体、FLIP及凋亡抑制蛋白来逃逸TRAIL诱导的凋亡.TRAIL与化疗药物、基因治疗联合应用能明显提高肿瘤治疗的靶向性,同时还可以逆转肿瘤细胞对TRAIL的抵抗现象.因此TRAIL作为新型抗肿瘤药物有望应用于临床治疗.     

重组人可溶性凋亡素-2在人喉癌株中表达及其体外诱导凋亡作用的实验研究

目的:研究凋亡因子TRAIL结合端粒酶启动子对肿瘤的特异性杀伤作用。方法:刺激人外周血淋巴细胞增殖,提取总RNA。采用RTPCR扩增IL2信号肽基因和TRAIL基因的融合基因,并克隆入真核表达载体pGL3181hTERT肿瘤特异性端粒酶启动子的下游,构建TRAIL基因的重组真核表达载体pGL3181hTERT/TRAIL。用Westernblot鉴定喉癌细胞株Hepa2中表达产物。将该重组载体经阳离子脂质体转染入人喉癌细胞株Hepa2中,用电镜观察细胞的形态变化,用流式细胞仪技术(FCM)分析转染后细胞凋亡率及细胞周期的变化。结果:PCR扩增得到了613bp的cDNA片断。与GenBank中报道的IL2信号肽和TRAIL凋亡诱导功能区cDNA序列完全一致,成功构建了重组真核表达载体pGL3181hTERT/TRAIL。Westernblot结果显示,获得的瞬时转染TRAIL在喉癌细胞Hepa2中特异表达,且能诱导其凋亡。结论:重组真核表达载体pGL3181hTERT/TRAIL的成功构建,为肿瘤基因治疗的靶向性提供了可行的理想工具。     

转染TRAIL的内皮祖细胞对人卵巢癌裸鼠皮下移植瘤的抑制作用

背景与目的:肿瘤坏死因子相关凋亡诱导配体(tumornecrosisfactor-relatedapoptosisinducingligand,TRAIL)有广谱的抗瘤作用,且对正常组织细胞无毒性,因此有望应用于肿瘤基因治疗。内皮祖细胞(endothelialprogenitorcells,EPCs)在体内能定向归巢于肿瘤,参与肿瘤新生血管的建立。本研究以EPC为载体,观察TRAIL转染EPCs对人卵巢上皮癌裸鼠皮下移植瘤的治疗作用。方法:用磁珠分离法从脐血中分离EPCs,并进行体外培养扩增。用脂质体将带有GFP-TRAIL基因的质粒转入EPCs(TRAIL-EPCs)。将转染后的EPC经尾静脉注入3AO卵巢癌裸鼠皮下移植瘤模型。流式细胞仪检测各组中绿色荧光蛋白(Green-Fluoroprotein,GFP)表达情况,观察各组移植瘤体积的变化,计算抑瘤率。结果:静脉注射转染TRAIL后的EPC,对卵巢上皮癌裸鼠皮下移植瘤生长具有明显抑制作用,对照组裸鼠的瘤重(0.226±0.209)g,而TRAIL细胞因子治疗组、GFP-TRAIL转染组裸小鼠的瘤重分别为(0.118±0.164)g、(0.075±0.084)g;TRAIL细胞因子组的抑瘤率为48.1%,TRAIL转染组抑瘤率为66.9%。肿瘤组织石蜡切片HE染色检查显示TRAIL细胞因子组,TRAIL转染组转对照组有更多的出血坏死区。TRAIL细胞因子组,TRAIL转染组均无明显毒副作用的表现。结论:TRAIL细胞因子和TRAIL-EPCs对人卵巢癌裸鼠皮下移植瘤均有明显的抑制作用,EPC在裸鼠皮下移植瘤模型体内有一定的导向作用,有希望成为基因治疗的载体。     

Antitumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene and transferred them into malignant cells in vitro and tumors in vivo. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of activation of pro-caspase-8 and cleavage of poly(ADP-ribose) polymerase. The intratumoral delivery elicited tumor cell apoptosis and suppressed tumor growth. In comparison with Bax gene treatment, which is toxic to normal cells, TRAIL gene treatment caused no detectable toxicity in cultured normal fibroblasts nor in mouse hepatocytes after systemic gene delivery. Furthermore, coculture of cancer cells expressing TRAIL with those expressing green fluorescent protein (GFP) resulted in apoptosis of both cells, whereas coculture of Bax-expressing cells with GFP-expressing cells resulted in the cell death of the Bax-expressing cells only, which suggested that the transfer of the TRAIL gene resulted in bystander effects. Moreover, culture of cells with medium from TRAIL-expressing cells showed the proapoptotic activity and bystander effect of the TRAIL gene to be not transferable with medium. To further demonstrate the bystander effect of the TRAIL gene, we constructed plasmid vectors encoding GFP-TRAIL or GFP-Bik chimeric proteins. Transfection of the GFP-TRAIL gene into cancer cells resulted in the death of GFP-positive cells and their neighbors, whereas transfection of the GFP-Bik gene killed GFP-positive cells only. Finally, GFP-TRAIL genes, transfected into normal human fibroblasts or bronchial epithelial cells, did not kill such cells, whereas transfected GFP-Bik genes did. Thus, the direct transfer of the TRAIL gene led to selective killing of malignant cells with bystander effect, which suggests that the TRAIL gene could be valuable for treatment for cancers. Together, these results suggest that delivering the TRAIL gene to cancerous cells may be an alternative approach to cancer treatment.     

Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo

Tumor necrosis factor-related apoptosis-inducing-ligand (TRAIL/Apo-2 ligand) induces apoptosis in the majority of cancer cells without appreciable effect in normal cells. Here, we report the effects of TRAIL on apoptosis in several human breast cancer cell lines, primary memory epithelial cells, and immortalized nontransformed cell lines, and we examine whether chemotherapeutic agents augment TRAIL-induced cytotoxicity in breast cancer cells in vitro and in vivo. TRAIL induced apoptosis with different sensitivities, and the majority of cancer cell lines were resistant to TRAIL. The chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, camptothecin, and Adriamycin) induced death receptors (DRs) TRAIL receptor 1/DR4 and TRAIL receptor 2/DR5, and successive treatment with TRAIL resulted in apoptosis of both TRAIL-sensitive and -resistant cells. Actinomycin D sensitized TRAIL-resistant cells through up-regulation of caspases (caspase-3, -9, and -8). TRAIL induces apoptosis in Adriamycin-resistant MCF7 cells already expressing high levels of death receptors DR4 and DR5. The pretreatment of breast cancer cells with chemotherapeutic drugs followed by TRAIL reversed their resistance by triggering caspase-3, -9, and -8 activation. The sequential treatment of nude mice with chemotherapeutic drugs followed by TRAIL induced caspase-3 activity and apoptosis in xenografted tumors. Complete eradication of established tumors and survival of mice were achieved without detectable toxicity. Thus, the sequential administration of chemotherapeutic drugs followed by TRAIL may be used as a new therapeutic approach for cancer therapy.