Inhibition of telomerase in tumor cells by ribozyme targeting telomerase RNA component

Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. Elimination of template function of telomerase RNA may repress the telomerase activity. A hammer-headed ribozyme (telomerase ribozyme, te-loRZ) directed against the RNA component of human telomerase (hTR) was designed and synthesized. TeloRZ showed a specific cleavage activity against the hTR. The cleavage efficacy reached 60%. A eukaryotic expression plasmid containing teloRZ gene was inducted into HeLa cells by lipofectamine, the telomerase activity in HeLa cells expressing teloRZ decreased to one eighth of that in the control cells. The doubling time increased significantly and the apoptosis ratio was elevated with increasing population doublings (PDS). After 19-20 PDS 95% cells were apoptotic. To further investigate the effect of teloRZ on tumor growth, the eukaryotic expression plasmid containing teloRZ was injected into transplanted tumor of nude mouse. The teloRZ e     

Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells

Although glycogen synthase kinase-3 （GSK-3） might act as a tumor suppressor since its inhibition is expected to mimic the activation of Wnt-signaling pathway, GSK-3β may contribute to NF-κB activation in cancer cells leading to increased cancer cell proliferation and survival. Here we report that GSK-3β activity was involved in the proliferation of human ovarian cancer cell both in vitro and in vivo. Inhibition of GSK-3 activity by pharmacological inhibitors suppressed proliferation of the ovarian cancer cells. Overexpressing constitutively active form of GSK-3β induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. Furthermore, GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovarian cancer cells. Our findings thus suggest that GSK-3β activity is important for the proliferation of ovarian cancer cells, implicating this kinase as a potential therapeutic target in ovarian cancer.     

Roles of sphingosine 1-phosphate on tumorigenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with a variety of biological activities.It is generated from the conversion of ceramide to sphingosine by ceramidase and the subsequent conversion of sphingosine to S1P,which is catalyzed by sphingosine kinases.Through increasing its intracellular levels by sphingolipid metabolism and binding to its cell surface receptors,S1P regulates several physiological and pathological processes,including cell proliferation,migration,angiogenesis and autophagy.These processes are responsible for tumor growth,metastasis and invasion and promote tumor survival.Since ceramide and S1P have distinct functions in regulating in cell fate decision,the balance between the ceramide/sphingosine/S1P rheostat becomes a potent therapeutic target for cancer cells.Herein,we summarize our current understanding of S1P signaling on tumorigenesis and its potential as a target for cancer therapy.     

Human mesenchymal stem cells with adenovirus-mediated TRAIL gene transduction have antitumor effects on esophageal cancer cell line Eca-109

The apoptotic ligand TNF-related apoptosis-inducing ligand （TRAIL） is believed to be a promising candidate for cancer gene therapy, yet gene therapy strategies to tackle this disease systemically are often impaired by inefficient delivery of the vector to the tumor tissue. Mesenchymal stem cells （MSCs） have been shown to home to tumor sites and could potentially act as a shield and vehicle for an anti tumor gene therapy vector. Here, we used an adenoviral vector expressing TRAIL to transduce MSCs and studied the apoptosis-inducing activity of these TRAIL-carrying MSCs on esophageal cancer cell Eca-109. Our results showed that, in vitro, TRAIL-expressing MSCs were able to inhibit proliferation and induce apoptosis in Eca-109 cells by an MTT assay, co-culture experiments and flow cytometry analysis. In vivo, TRAIL-expressing MSCs also displayed an ability to inhibit tumor growth in an Eca-109 xenograft mouse model. Together, our findings indicated that the gene therapy strategy of MSCs-based TRAIL gene delivery has a wide potential value for improving the treatment of esophageal cancer.     

Up-regulation of NKX3.1 expression and inhibition of LNCaP cell proliferation induced by an inhibitory element decoy

NKX3.1 is an androgen-regulated prostate-specific homeobox gene that is thought to play an important role in prostate development and cancerogenesis. NKX3.1 acts as a tumor suppressor gene specifically in the prostate. Up-regulation of NKX3.1 gene offers a promising gene therapy for prostate cancer. The decoy strategy has been developed and is considered a useful tool for regulating gene expression and gene therapy. In our previous studies, we identified a 20 bp inhibitory element upstream of the NKX3.1 promoter.In this study, we focused on using the 20 bp inhibitory element decoy to block negative regulation of the NKX3.1 gene and to up-regulate NKX3.1 expression using synthetic double-stranded oligodeoxynucleotides of the 20 bp inhibitory element. We found in an electrophoretic mobility shift assay experiment that the 20 bp inhibitory decoy presented competitive binding to a specific binding protein of the 20 bp inhibitory element in prostate cancer cell line LNCaP. In luciferase reporter gene assays, we found that the 20 bp inhibitory decoy could enhance NKX3.1 promoter activity, and RT-PCR and Western blot analysis revealed that NKX3.1 expression was up-regulated effectively by the transfection with the 20 bp inhibitory decoy. Furthermore,cell proliferation was inhibited by up-regulated NKX3.1 expression induced by the 20 bp inhibitory decoy.     

Prion Protein Protects Cancer Cells against Endoplasmic Reticulum Stress Induced Apoptosis

Unfolded protein response(UPR) is an adaptive reaction for cells to reduce endoplasmic reticulum(ER) stress. In many types of cancers, such as lung cancer and pancreatic cancer, cancer cells may harness ER stress to facilitate their survival and growth. Prion protein(PrP) is a glycosylated cell surface protein that has been shown to be up-regulated in many cancer cells. Since PrP is a protein prone to misfolding, ER stress can result in under-glycosylated PrP, which in turn may activate ER stress. To assess whether ER stress leads to the production of under-glycosylated PrP and whether underglycosylated PrP may contribute to ER stress thus leading to cancer cell apoptosis, we treated different cancer cells with brefeldin A(BFA), thapsigargin(Thps), and tunicamycin(TM). We found that although BFA, Thps, and TM treatment activated UPR, only ATF4 was consistently activated by these reagents, but not other branches of ER stress. However, the canonical PERK-eIF2α-ATF4 did not account for the observed activation of ATF4 in lung cancer cells. In addition, BFA,but neither Thps nor TM, significantly stimulated the expression of cytosolic PrP. Finally, we found that the levels of PrP contributed to anti-apoptosis activity of BFA-induced cancer cell death. Thus, the pathway of BFA-induced persistent ER stress may be targeted for lung and pancreatic cancer treatment.     

端粒酶RNA和端粒酶活性在人生殖道癌细胞株中的表达

本文采用反转录套式PCR(nested RT-PCR)和PCR端粒重复扩增(TRAP)方法分别检测了5种人生殖道癌细胞株端粒酶RNA(hTR)基因及端粒酶活性的表达。结果表明,宫颈癌HeLa和SiHa细胞株,绒毛膜癌JAR和BeWo细胞株及卵巢癌OCCI细胞株hTR及端粒酶活性均呈强阳性。但正常卵巢、宫颈和胎盘组织hTR和端粒酶呈阴性或弱阳性。提示反转录套式PCR和端粒重复扩增法可以简便而有效地检测端粒酶RNA及端粒酶活性。端粒酶的激活与肿瘤细胞增殖密切相关,并可能成为一项具有临床价值的肿瘤标志物。     

Hammerhead ribozymes to modulate telomerase activity of endometrial carcinoma cells.

Telomerase is an excellent target molecule for cancer therapy, though any effective agents have never been developed in human subjects. We designed a variety of hammerhead ribozymes against human telomerase RNA (hTR) and hTERT mRNA and studied their possibility as a tool for cancer therapy. To search promising target site of hTR, the catalytic actiuity of 3 kinds of hammerhead ribozymes was studied in cell-free system. They showed equivalent catalytic activity, but only 36-ribozyme, which was designed to cleave the template region of hTR, revealed telomerase inhibitory activity in an endometrial carcinoma cell line. Among hTERT-mRNA-targeted ribozymes, the ribozyme to cleave 13 nucleotides downstream from the 5'-end of hTERT mRNA (13-ribozyme) exhibited the strongest telomerase-inhibitory activity, and the ribozyme to cleave 59 nucleotides upstream from the poly(A) tail showed clear activity. Stable transfection studies confirmed that the 36-ribozyme as well as the 13-ribozyme suppressed telomerase. These observations suggest that the template region of hTR and 5'end of hTERT mRNA are promising target sites for ribozymes to reduce telomerase activity.     

端粒酶反义cDNA对乳腺癌细胞损伤修复能力的影响

 利用反义核酸技术将端粒酶RNA的全长cDNA反向导入乳腺癌MCF 7细胞基因组中 .通过单细胞凝胶电泳实验发现 ,其DNA受H2 O2 损伤后的修复能力下降 .但端粒酶活性抑制为何引起其DNA损伤修复能力下降的原因尚待进一步研究 .     

Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80 approximately 90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

Activity of the Human Telomerase Catalytic Subunit (hTERT) Gene Promoter Could Be Increased by the SV40 Enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80～90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

以hTR模板区为靶点的抗肿瘤药物设计策略

端粒酶几乎在所有的人类癌细胞中均异常表达,它的持久活性对肿瘤的增殖是必需的。因此,抑制端粒酶活性代表了一种新的癌症治疗机制。端粒酶全酶复合物有多处可以做为抑制剂的靶点,包括hTR、hTERT、引物锚定位点等。本文对以端粒酶RNA模板区为靶点的抗肿瘤药物设计策略进行了综述,包括对该区域进行点突变、使用反义寡核苷酸封闭模板区、改变端粒酶RNA空间构象等,并探讨了目前抑制端粒酶活性研究中存在的一些问题。     

Adenovirus-mediated suicide SCLC gene therapy using the increased activity of the hTERT promoter by the MMRE and SV40 enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor or stem cells. The aim of this study was to use increased telomerase promoter activity in small-cell lung cancer (SCLC) gene therapy. The hTERT promoter and Myc-Max response elements (MMRE) in pGL3-Control vector containing SV40 enhancer resulted in strong expression of the luciferase gene only in telomerase positive and myc overexpressing SCLC cell line but not in normal human cell line. To investigate the possibility of the utilization of the MMRE, hTERT promoter, and SV40 enhancer in targeted SCLC gene therapy, adenovirus vector expressing HSV-TK controlled by the MMRE, hTERT promoter, and SV40 enhancer for the induction of telomerase positive and myc-overexpressing cancer specific cell death was constructed. SCLC cells infected with Ad-MMRE-hT-TK-enh were significantly suppressed and induced apoptosis more than those of Ad-hT-TK or Ad-hT-TK-enh infected cells. Telomerase and c-myc are activated in 60 approximately 80% of SCLC, so the increased activity of telomerase promoter can be used for targeted SCLC gene therapy. These results show that the MMRE, hTERT promoter, and SV40 enhancer can be used in SCLC targeted cancer gene therapy.