AIM: To explore the antitumor bioactivity of adenovirus- mediated mutant type p27kip1 gene in a colorectal cancer cell line SW480. METHODS: We constructed recombinant adenovirus vector expressing a mutant type p27kip1...AIM: To explore the antitumor bioactivity of adenovirus- mediated mutant type p27kip1 gene in a colorectal cancer cell line SW480. METHODS: We constructed recombinant adenovirus vector expressing a mutant type p27kip1 gene (ad- p27mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), and transduced into SW480 cells. Then we detected expression of p27, Bcl-2 and Bax protein in the transductants by Western blotting, cell cycle of transductants by a digital flow cytometric system, migrating potential with Boyden Chamber and SW480 tumor cell growth inhibition in vitro and in vivo. RESULTS: We found that a recombinant adenovirus vector of expressing ad-p27mt, with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) has potent inhibition of SW480 tumor cell growth in vitro and in vivo. Furthermore, ad-p27mt induced cell apoptosis via regulating bax and bcl-2 expressions, and G1/S arrest in SW480 cells and inhibited cell migration. CONCLUSION: ad-p27mt has a strong anti-tumor bioactivity and has the potential to develop into new therapeutic agents for colorectal cancer.展开更多
AIM: To construct a tumor-selective replication-competent adenovirus (RCAd), SG300, using a modified promoter of human telomerase reverse transcriptase (hTERT). METHODS: The antitumor efficacy of SG300 in hepatocellul...AIM: To construct a tumor-selective replication-competent adenovirus (RCAd), SG300, using a modified promoter of human telomerase reverse transcriptase (hTERT). METHODS: The antitumor efficacy of SG300 in hepatocellular carcinoma was assessed in vitro and in vivo. In vitro cell viability by MTT assay was used to assess the tumor-selective oncolysis and safety features of SG300, and in vivo antitumor activity of SG300 was assessed in established hepatocellular carcinoma models in nude mice. RESULTS: SG300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection (MOI), but could not affect growth of normal cells even at a high MOI. Both in Hep3B and SMMC-7721 xenograft models of hepatocellular carcinoma, SG300 had an obvious antitumor effect, resulting in a decrease in tumor volume. Its selective oncolysis to tumor cells and safety to normal cells was also superior to that of ONYX-015. Pathological examination of tumor specimens showed that SG300 replicated selectively in cancer cells and resulted in apoptosis and necrosis of cancer cells. CONCLUSION: hTERT promoter-regulated replicativeadenovirus SG300 has a better cancer-selective replication-competent ability, and can specifically kill a wide range of cancer cells with positive telomerase activity, and thus has better potential for targeting therapy of hepatocellular carcinoma.展开更多
Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bac...Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or aftermalignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper wedemonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression inairway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targetinghuman IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-α, IL-1β orheat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reducedby shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels ofIκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for thetreatment of inflammatory diseases.展开更多
OBJECTIVE Chemotherapy is an important therapy for hepatocellular carcinoma （HCC）. However, it is not effective in many cases due to recurrence and metastasis even if the initial treatment produces a response. Multi...OBJECTIVE Chemotherapy is an important therapy for hepatocellular carcinoma （HCC）. However, it is not effective in many cases due to recurrence and metastasis even if the initial treatment produces a response. Multidrug resistance （MDR） is considered to be one of the considerable causes. The aim of this study was to reverse MDR of HepG2/ADM cells by blocking mdr1 with an adenovirus vector carrying antisense mdr1 in a tumor transplantated in athymic mice. METHODS PCMV IE was removed from the pshuttle vector. A 0.3 kb AFP promoter was inserted into the pshuttle vector and pCMV changed into pAFP. The pAFP and asmdr1 PCR products were doubly digested with Kpnl and Apal, the digested products were ligated by T4 ligase, the asmdr1 gene was inserted into pAFP and a newly plasmid pAFP-asmdr1 was constructed. Following digestion with PI-SceI/I-Ceu I, pAFP-asmdr1 was ligated with Adeno-X genome DNA and amplified in E.coli XL1-Blue. The HEK293 cells were transfected and virus collected. The HepG2 MDR cells （HepG2/ADM） were induced by graded resistance to ADM and were inoculated into athymic mice. After adeno-asmdr1 was injected, the expression of mdr1-mRNA and the volume of the transplantated tumor and its cells were observed. RESULTS Following injection with Adeno-asmdr1, the tumor volume in the ADM＋Adeno-asmdr1 group did not increase. However the tumor volume in the PBS plus ADM group did significantly increase （P〈0.05）. In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and was found to be reduced at 1 week and 4 weeks in the ADM＋asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM＋asmdr1 group compared to the ADM group at the 4th week （P〈0.05）. Evidence of apoptosis was observed in the tumor xenograft cells treated with Adeno-asmdr1, but there was rare or no apoptosis in the group treated with ADM and PBS. CONCLUSION Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.展开更多
Objective： To prepare and identity monoclonal antibodies （McAbs） against the capsid proteins of adenovirus vector. Methods： BALB/c mice were immunized with a mixture of the purified adenovirus vector （Adv） and A...Objective： To prepare and identity monoclonal antibodies （McAbs） against the capsid proteins of adenovirus vector. Methods： BALB/c mice were immunized with a mixture of the purified adenovirus vector （Adv） and AI（OH）3. McAbs were produced using cell fusion technique in a conventional way. The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay （ELISA）, immunocytochemical staining and Western blotting. Results： Six strains of hybridoma cells （A4H11, A8C7, F1H5, G1D2, G4E3 and H2G8） that can stably secrete the IgG1 McAb against Adv were obtained. After 3 months subculture and low concentration of serum adapting culture, six strains retained their stability to secrete McAb. The ascites titers were between 1：10^6 and 1：10^8. Western blot analysis demonstrated that all the McAbs reacted with one protein （about 114 kDa） which is present in wild type 3 adenovirus （wtAd3）, wild type 5 adenovirus （wtAd5）, wild type 7 adenovirus （wtAd7） and adenovirus vector. Conclusion： Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector, and provided the substantial foundation of preclinical research of adenovirus vectors.展开更多
Breast cancer （BC） is diagnosed in 〉 65 year old women in about half of cases. Experts currently recommend that systemic therapy is offered to elderly patients with BC, if, based on their overall conditions and lif...Breast cancer （BC） is diagnosed in 〉 65 year old women in about half of cases. Experts currently recommend that systemic therapy is offered to elderly patients with BC, if, based on their overall conditions and life expectancy, it can be reasonably anticipated that the benefits will outweigh the risks of treatment. Like for young subjects, the monoclonal antibody against human epidermal growth factor receptor-2 （HER-2）, trastuzumab, represents a valid therapeutic option when BC over-expresses this receptor. Unforttmately, administration of trastu- zumab is associated with the occurrence of left ventricular dysfunction and chronic heart failure （CHF）, possibly because of interference with the homeostatic functions of HER-2 in the heart. Registry-based, retrospective analyses have reported an incidence of CHF around 25% in elderly women receiving trastuzumab compared with 10%-15% in those not given any therapy for BC, and the risk of CHF has been estimated to be two-fold higher in 〉 60455 year old trastuzumab users vs. non-users. Extremely advanced age and preexisting cardiac disease have been shown to predispose to trastuzumab cardiotoxicity. Therefore, selection of older patients for treatment with trastuzumab should be primarily based on their general status and the presence of comorbidities; previous chemotherapy, especially with anthracyclines, should be also taken into account. Once therapy has started, efforts should be made to ensure regular cardiac surveillance. The role of selected biomarkers, such as cardiac troponin, or new imaging techniques （three-dimension, tissue Doppler echocardiography, magnetic resonance imaging） is promising, but must be further investigated especially in the elderly. Moreover, additional studies are needed in order to better understand the mechanisms by which trastuzumab affects the old heart.展开更多
基金Grant from the Natural Science Foundation of Hubei Province, NO.2003ABA193
文摘AIM: To explore the antitumor bioactivity of adenovirus- mediated mutant type p27kip1 gene in a colorectal cancer cell line SW480. METHODS: We constructed recombinant adenovirus vector expressing a mutant type p27kip1 gene (ad- p27mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), and transduced into SW480 cells. Then we detected expression of p27, Bcl-2 and Bax protein in the transductants by Western blotting, cell cycle of transductants by a digital flow cytometric system, migrating potential with Boyden Chamber and SW480 tumor cell growth inhibition in vitro and in vivo. RESULTS: We found that a recombinant adenovirus vector of expressing ad-p27mt, with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) has potent inhibition of SW480 tumor cell growth in vitro and in vivo. Furthermore, ad-p27mt induced cell apoptosis via regulating bax and bcl-2 expressions, and G1/S arrest in SW480 cells and inhibited cell migration. CONCLUSION: ad-p27mt has a strong anti-tumor bioactivity and has the potential to develop into new therapeutic agents for colorectal cancer.
基金Supported by the Natural Science Foundation of China, No. 30572149 the National 863 High Technology R&D Project of China, No. 2003AA216030
文摘AIM: To construct a tumor-selective replication-competent adenovirus (RCAd), SG300, using a modified promoter of human telomerase reverse transcriptase (hTERT). METHODS: The antitumor efficacy of SG300 in hepatocellular carcinoma was assessed in vitro and in vivo. In vitro cell viability by MTT assay was used to assess the tumor-selective oncolysis and safety features of SG300, and in vivo antitumor activity of SG300 was assessed in established hepatocellular carcinoma models in nude mice. RESULTS: SG300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection (MOI), but could not affect growth of normal cells even at a high MOI. Both in Hep3B and SMMC-7721 xenograft models of hepatocellular carcinoma, SG300 had an obvious antitumor effect, resulting in a decrease in tumor volume. Its selective oncolysis to tumor cells and safety to normal cells was also superior to that of ONYX-015. Pathological examination of tumor specimens showed that SG300 replicated selectively in cancer cells and resulted in apoptosis and necrosis of cancer cells. CONCLUSION: hTERT promoter-regulated replicativeadenovirus SG300 has a better cancer-selective replication-competent ability, and can specifically kill a wide range of cancer cells with positive telomerase activity, and thus has better potential for targeting therapy of hepatocellular carcinoma.
文摘Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or aftermalignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper wedemonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression inairway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targetinghuman IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-α, IL-1β orheat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reducedby shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels ofIκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for thetreatment of inflammatory diseases.
基金This work was supported by the Key Program of Medical Science Foundation of Chongqing Public Health Bureau (No. [2001]01-1-018).
文摘OBJECTIVE Chemotherapy is an important therapy for hepatocellular carcinoma （HCC）. However, it is not effective in many cases due to recurrence and metastasis even if the initial treatment produces a response. Multidrug resistance （MDR） is considered to be one of the considerable causes. The aim of this study was to reverse MDR of HepG2/ADM cells by blocking mdr1 with an adenovirus vector carrying antisense mdr1 in a tumor transplantated in athymic mice. METHODS PCMV IE was removed from the pshuttle vector. A 0.3 kb AFP promoter was inserted into the pshuttle vector and pCMV changed into pAFP. The pAFP and asmdr1 PCR products were doubly digested with Kpnl and Apal, the digested products were ligated by T4 ligase, the asmdr1 gene was inserted into pAFP and a newly plasmid pAFP-asmdr1 was constructed. Following digestion with PI-SceI/I-Ceu I, pAFP-asmdr1 was ligated with Adeno-X genome DNA and amplified in E.coli XL1-Blue. The HEK293 cells were transfected and virus collected. The HepG2 MDR cells （HepG2/ADM） were induced by graded resistance to ADM and were inoculated into athymic mice. After adeno-asmdr1 was injected, the expression of mdr1-mRNA and the volume of the transplantated tumor and its cells were observed. RESULTS Following injection with Adeno-asmdr1, the tumor volume in the ADM＋Adeno-asmdr1 group did not increase. However the tumor volume in the PBS plus ADM group did significantly increase （P〈0.05）. In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and was found to be reduced at 1 week and 4 weeks in the ADM＋asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM＋asmdr1 group compared to the ADM group at the 4th week （P〈0.05）. Evidence of apoptosis was observed in the tumor xenograft cells treated with Adeno-asmdr1, but there was rare or no apoptosis in the group treated with ADM and PBS. CONCLUSION Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.
文摘Objective： To prepare and identity monoclonal antibodies （McAbs） against the capsid proteins of adenovirus vector. Methods： BALB/c mice were immunized with a mixture of the purified adenovirus vector （Adv） and AI（OH）3. McAbs were produced using cell fusion technique in a conventional way. The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay （ELISA）, immunocytochemical staining and Western blotting. Results： Six strains of hybridoma cells （A4H11, A8C7, F1H5, G1D2, G4E3 and H2G8） that can stably secrete the IgG1 McAb against Adv were obtained. After 3 months subculture and low concentration of serum adapting culture, six strains retained their stability to secrete McAb. The ascites titers were between 1：10^6 and 1：10^8. Western blot analysis demonstrated that all the McAbs reacted with one protein （about 114 kDa） which is present in wild type 3 adenovirus （wtAd3）, wild type 5 adenovirus （wtAd5）, wild type 7 adenovirus （wtAd7） and adenovirus vector. Conclusion： Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector, and provided the substantial foundation of preclinical research of adenovirus vectors.
文摘Breast cancer （BC） is diagnosed in 〉 65 year old women in about half of cases. Experts currently recommend that systemic therapy is offered to elderly patients with BC, if, based on their overall conditions and life expectancy, it can be reasonably anticipated that the benefits will outweigh the risks of treatment. Like for young subjects, the monoclonal antibody against human epidermal growth factor receptor-2 （HER-2）, trastuzumab, represents a valid therapeutic option when BC over-expresses this receptor. Unforttmately, administration of trastu- zumab is associated with the occurrence of left ventricular dysfunction and chronic heart failure （CHF）, possibly because of interference with the homeostatic functions of HER-2 in the heart. Registry-based, retrospective analyses have reported an incidence of CHF around 25% in elderly women receiving trastuzumab compared with 10%-15% in those not given any therapy for BC, and the risk of CHF has been estimated to be two-fold higher in 〉 60455 year old trastuzumab users vs. non-users. Extremely advanced age and preexisting cardiac disease have been shown to predispose to trastuzumab cardiotoxicity. Therefore, selection of older patients for treatment with trastuzumab should be primarily based on their general status and the presence of comorbidities; previous chemotherapy, especially with anthracyclines, should be also taken into account. Once therapy has started, efforts should be made to ensure regular cardiac surveillance. The role of selected biomarkers, such as cardiac troponin, or new imaging techniques （three-dimension, tissue Doppler echocardiography, magnetic resonance imaging） is promising, but must be further investigated especially in the elderly. Moreover, additional studies are needed in order to better understand the mechanisms by which trastuzumab affects the old heart.
