Pancreatic cancer–Outlook: gene therapy

Gene therapy offers an elegant alternative to toxic chemotherapy regimens, mostly without severe side effects. Cancer gene therapy was among the first applications. Following the enthusiasm in the early nineties, a more rationale view is the recent way to look at it. This tutorial review looks upon the tools of gene therapy and the principte elements （vector, promoter, targeting, therapeutic gene）. The principles of gene therapy such as gene directed enzyme prodrug therapy （GDEPT） and gene directed tumor vaccination are explained. Further, published protocols and clinical studies for pancreatic carcinoma gene therapy are reviewed. Finally, an outlook is given on the latest developments, some of them beyond conventional gene therapy.

Non-transmissible Sendai virus vector encoding c-myc suppressor FBP-interacting repressor for cancer therapy

AIM: To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR).

An Oncolytic Adenovirus Expressing Herpes Simplex Virus-Thymidine Kinase for Targeting Cancer Therapy:An in vitro Evaluation

Objective： Oncolytic adenovirus, also called conditionally replicating adenovirus （CRAD）, has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage. Methods： A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase （hTERT）, the coding sequence of E1A gene, an internal ribosome entry site sequence （IRES） and the coding sequence of herpes simplex virus-thymidine kinase （HSV-TK） was constructed. Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK. The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured. The infection efficiency was determined by flow cytometry. Results： Ad-ETK deliverys E1A and HSV-TK gene, which selectively replicates in hTERT-positive tumor cells, and the progeny virus can reach up to 150 IU/cell. Our in vitro study showed that Ad-ETK plus ganciclovir （GCV） induced an obvious cell death. Conclusion： An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.

Melanoma differentiation-associated gene-7, MDA-7/IL-24, selectively induces growth suppression, apoptosis in human hepatocellular carcinoma cell line HepG2 by replication-incompetent adenovirus vector

AIM： To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatocellular carcinoma （HCC） cell line HepG2 and normal liver cell line L02. METHODS： We constructed the recombinant replication-incornpetent Ad.rnda-7 virus vector and infected it into the human HCC cell line HepG2 and normal liver cell line L02. RT-PCR was performed to detect the rnRNA expressing in cells, by ELISA was used to detect MDA-7/IL-24 protein expression in the culture supernatant. The effect of apoptosis induced by Ad.rnda-7 was confirmed by Hoechst staining and flow cytometry assay with Annexin-V and PI staining. MTT assay was used to determine growth inhibition of HepG2 cells, and cell-cycle and hypodiploidy analyses were performed by flow cytometry. RESULTS： Recombinant replication-defective virus expressing MDA-7/IL-24 was constructed successfully. RTPCR showed that the Ad.rnda-7 could mediate the expression of the exogenous gene MDA-7/IL-24 into HepG2 and L02. The concentration of MDA-7/IL-24 protein in supernatant was 130 pg/mL and 110 pg/mL in Ad.rnda-7-infected L02 and HepG2 ceils, respectively. Ad.mda-7 infection obviously induced apoptosis （from 2.604±0.72% to 33.64±13.2%, P=0.00012） and growth suppression in HepG2 （inhibition ratio IR=68%） and an increase in the percentage of specific cancer cell types at the G2/M phase of the cell cycle （from 6.44% to 32.29%, P〈 0.01）, but not in L02 cells.CONCLUSION： These results confirm selectively induction of apoptosis and growth suppression by the mda-7/ IL-24 gene with replication-incompetent adenovirus vector in human hepatocellular carcinoma cell line HepG2.

Increased Activity of the Human Telomerase Catalytic Subunit Promoter by the VEGF Enhancer in Human Cancer Cells

We attempted to improve the activity of hTERT promoter by fusing the vascular endothelial growth factor （VEGF） enhancer. To determine the potential as cancer specific promoters, we measured the reporter gene transfection assay driven by the hTERT promoter and the VEGF enhancer in human cancer cells. We found that the hTERT promoter containing VEGF enhancer conferred strong expression of the reporter gene only in different cancer cell lines but not in normal human cells. Retrovirus vector expressing HSV-TK controlled by the hTERT promoter and the VEGF enhancer was constructed. A549 cells infected with LN-enh-hT-TK was significantly suppressed and induced to apoptosis more than those infected with LN-hT-TK. The apoptosis ratio ofA549 cell infected with two kinds of retrovirus cell with GCV in lower concentration is 20.94% and 50.7%. It suggested that there is significant differentiation between the assay groups. Our results demonstrated the possible application of hTERT promoter and the VEGF enhancer in targeted cancer gene therapy.

pIL-12 delivered by polymer based nanovector for anti-tumor genetherapy

Finding more effective and safe non-viral vectors to transfer genes into cancer cells has become the key of immune gene therapy for cancer.Herein a triblock compound MPEG_(2000)-PDLLA_(4000)-MPEG_(2000) modified by cationic liposome DOTAP was used as a non-viral vector DOTAP/MPEG_(2000)-PDLLA_(4000)-MPEG_(2000)(DMPM)to effectively transfer interleukin(IL)-12 plasmid(pIL-12)into tumor tissue.IL-12 produced by transfected tumor cells successfully inducing lymphocyte proliferation and promoting interferon-γ(IFN-γ)secretion,which resulted in tumor cells death.The ability of DMPM to transfer pIL-12 and the immune effect induced by IL-12 in cells had been explored.The anti-tumor effect,mechanism and safety of pIL-12/DMPM in mice cancer model were investigated in this study.Our results showed that the pIL-12 transferred by DMPM was highly expressed both in CT26 cells and B16-F10 cells.IL-12 expressed in the culture supernatant of transfected tumor cells stimulated lymphocyte proliferation and promoted IFN-γsecretion.The experimental result confirmed that pIL-12/DMPM therapy significantly reduced tumor growth in mice model.We designed the nanocomposite DMPM to deliver pIL-12 for cancer treatment and explored its therapeutic efficacy and the underlying anti-tumor mechanism.Our study suggested pIL-12 loaded by DMPM complex would be an effective strategy for cancer treatment.

Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines

AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), enzymelinked immunosorbent assay (ELISA), and Western blotting, respectively. Apoptosis of HCC cells and normal liver cells was detected by cytometric assay with Hoechst33258 staining. 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used to investigate proliferation of HCC cells and normal liver cells, and cell cycle was assayed by flow cytometry. RESULTS: RT-PCR, ELISA and Western blotting showed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24. MTT indicated that SG600-IL24 could suppress the growth of HepG2, Hep3B, MHCC97L, with an inhibition rate of 75% ± 2.5%, 85% ± 2.0%, 72% ± 1.8%, respectively (P < 0.01), promote the apoptosis of HepG2, Hep3B, MHCC97L, with an apoptosis rate of 56.59% ± 4.0%, 78.36% ± 3.5%, 43.39% ± 2.5%, respectively (P < 0.01), and block the HCC cell lines in the G2/M phase with a blocking rate of 35.4% ± 4.2%, 47.3% ± 6.2%, 42% ± 5.0%, respectively (P < 0.01) but not the normal liver cell line in a p53-independent manner. CONCLUSION: SG600-IL24 can selectively suppress the proliferation and apoptosis of HCC cell lines in vitro but not normal liver cell line L02 in a p53-independent manner. Compared with Ad.IL-24, SG600-IL24 can significantly enhance the antitumor activity in HCC cell lines.

Replication-incompetent Adenovirus Vector-mediated MDA-7/IL-24 Selectively Induces Growth Suppression and Apoptosis of Hepatoma Cell Line SMMC-7721

In order to investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis of human hepatocellular carcinoma （HCC） cell line SMMC-7721 and normal liver cell line L02, the recombinant replication-incompetent Ad.mda-7 virus vector was constructed and infected into the HCC cell line SMMC-7721 and normal liver cell line L02. RT-PCR was performed to examine the expression of MDA-7 mRNA. The concentrations of MDA-7/IL-4 in culture superuatants were determined by using ELISA. MTT and Hoechst staining assay were applied to observe the inhibitory and killing effects of MDA-7 on the HCC cells. By using flow cytometry, the apoptosis, cell cycle and proliferation of SMMC-7721 and L02 cells were measured. The results showed recombinant replication-incompetent virus expressing MDA-7/IL-24 was constructed successfully, and RT-PCR revealed that it could mediate the high expression of the exogenous gene MDA-7/IL-24 in SMMC-7721 and L02 cells. The expression of MDA-7/IL-24 proteins in the culture superuatant was detectable by ELISA. Ad.mda-7 infection induced apoptosis and growth suppression in SMMC-7721 cells and an increased percentage of HCC cells in the GyM phase of the cell cycle, but not in L02 cells. It was concluded that mda-7/IL-24 gene, mediated with replication-incompetent adenovirus vector, could selectively induce growth suppression and apoptosis in HCC cell line SMMC-7721 but without any toxic side-effect on normal liver line L02.

Construction and in vitro Study of an E1B-Defective Adenovirus

An E1B-defective adenovirus named r1/Ad was constructed by homologous recombination. The construction, selection and propagation of recombinant virus was done in the human embryonic kidney 293 cells (HEK293). Thein vitro study demonstrated that the recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as the human glioblastoma tumor cells (U251) and human bladder tumer cells (EJ) but not in the normal cells with functional p53 such as the human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated that the U251 cells were more sensitive to the infection of r1/Ad than that of EJ cells under identical conditions. In this paper, it was found that r1/Ad could be very useful in studying thein vitro selective replication of E1B-defective adenovirus. This may help to determine the safety of using any E1B-defective adenoviruses in cancer gene therapy. Key words E1B-defective adenovirus - cytopathic effect - cancer gene therapy CLC number Q78 Foundation item: Supported by the National Natural Science Foundation of China (3988003)Biography: Xue Feng (1978-), male, Master, research direction: cancer gene therapy by recombinant virus.

Characteristics of ovarian cancer cells transduced by the bicistronic retroviral vector containing GM-CSF and HSV-TK genes

Objectives To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and to observe the characteristics of the transduced cells.Methods Retroviral vector pLGM-I-TK was constructed by linking the HSV-TK gene and GM-CSF gene with the IRES sequence. By using the “ping-pong' technique, pLGM-I-TK was transfected into the packaging cell line, PA317, to produce a PA317/TK-GM cell line. Using the resulting viral supernatant to infect the ovarian cancer cell line SKOV3, PCR and RT-PCR were used to explore the integration and transcription of HSV-TK and GM-CSF genes. The cytotoxicity of GCV (gancyclovir) on SKOV3/TK-GM was determined by MTT assay and the bystander effect of the HSV-TK/GCV system was also assessed. ELISA was used to measure the expression of GM-CSF by the transgene cells.Results The bicistronic retroviral vector constructed could be successfully transduced into PA317 and the titer of the retroviral vector was about 8.6×105?cfu/ml. PCR and RT-PCR demonstrated the successful integration and transcription of HSV-TK and GM-CSF genes transduced into the SKOV3 cell. SKOV3/TK-GM cells could be killed by GCV, and the IC50 was 0.7?μg/ml. The bystander effect was demonstrated. The expression level of GM-CSF in SKOV3/TK-GM was 60.4?ng*ml-1*106 cells-1*2 days-1.Conclusion The IRES sequence can be used to construct retroviral vectors to facilitate co-transfection of two genes. SKOV3/TK-GM cells can simultaneously express the HSV-TK and GM-CSF genes with biological activities which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.

Effects of anti-HPV16E6-ribozyme on phenotype and gene expression of a cervical cancer cell line

To investigate the effects of anti HPV16E6 ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line Methods HRz was designed by computer programs HRz’s activity was identified by cleavage experiments in vitro HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi R and CaSKi P, respectively The expression of ribozyme in transfected cells was observed by RNA dot blot The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot Cell growth curves and soft agar forming ability were studied The ability of each cell line to form tumors was assessed in nude mice Apoptosis rates and expression of c myc, bcl 2, p53 and Fas were detected by flow cytometry (FCM) Antigens of tumor cells, HLA 1, HLA 2, B7 1 and B7 2 were also detected NK, LAK, and CD 3AK cells were induced Their cytotoxicities were detected in CaSKi R, CaSKi P, and CaSKi cells Results In vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site specific manner HRz could be expressed stably in transfected CaSKi cells Northern blot analysis showed that E6 mRNA levels were lower in CaSKi R than in CaSKi The growth rate of CaSKi R was slower than those of CaSKi and CaSKi P The soft agar forming rate of CaSKi R was lower compared with those of CaSKi and CaSKi P cells The ability of CaSKi R to form tumors in nude mice was also poor The apoptosis rate of CaSKi R cells was much higher than those of CaSKi and CaSKi P HRz could reduce the expression of E6, c myc and bcl 2 proteins, and increase the expression of p53 as well HRz could increase the expression of HLA 2, B7 1 and B7 2 antigens The cytotoxicity of NK, LAK and CD 3AK cells was much higher in CaSKi R than in CaSKi P and CaSKi cells Conclusion HRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells

Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo

OBJECTIVE： Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine（TCM） drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus（r AAV） vectors has not been attempted. METHODS： We synthesized the c DNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged c DNAs were subcloned into a r AAV plasmid vector. The protein expression was confi rmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into r AAV vectors, under the control of a liver cancer-specifi c promoter. The liver tumor growth was then monitored following intratumor administration of the r AAV vectors.RESULTS： The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin（TCS） gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of r AAV vectors containing the TCS gene signifi cantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model.CONCLUSION： Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

Sensitization of prostate cancer cell lines to 5-fluorocytosine induced by adenoviral vector carrying a CD transcription unit

Objective To investigate the efficiency of the cytosine deaminase adenoviral/5-fluorocytosine system on prostate cancer cell lines.Methods We used cell culture, infectivity and sensitivity tests, to observe bystander effect by animal tests.Results Established prostate cancer cell lines are eventually infectible by adenoviral vector. The ratio of vector/cell at which infection occurs depends on the specific cell line. The peak of expression of the transferred cytosine deaminase gene occurred in cells at different time, but persisted beyond 11 days. These prostate cell lines are sensitized to 5-fluorocytosine by infection with adenoviral vector carrying the cytosine deaminase gene. Only 5% of the LNCap and 10% of the RM-1 cells were infected and produced 100% cell death. In the animal test, there was significant inhibition of tumor growth at a ratio of 400 vector particles/cell with the systematic treatment of 5-fluorocytosine.Conclusions Adenoviral vector carrying a cytosine deaminase transcription unit can sensitize prostate cancer cell lines to 5-fluorocytosine. The system can significantly inhibit the growth of prostatic tumors in mice.