Jinfa Gu

An oncolytic adenoviral vector of Smac increases antitumor activity of TRAIL against HCC in human cells and in mice.

Hepatocellular carcinoma (HCC) displays a high resistance to tumor necrosis factor–related apoptosis–inducing ligand (TRAIL)–mediated cell death. To increase sensitivity of HCC cells to TRAIL, we have constructed an oncolytic adenoviral vector (ZD55) and used this vector to deliver second mitochondria‐derived activator of caspases (Smac) and TRAIL genes (ZD55‐Smac and ZD55‐TRAIL, respectively) into HCC cells. Our data showed that human HCC cells express high levels of inhibitor of apoptosis proteins (IAPs). Transfected HCC cells expressing exogenous X‐linked IAPs (XIAPs) displayed more resistance to TRAIL. The expression of Smac led to rapid and potent activation of apoptosis in HCC cells after infection with ZD55‐Smac. The activation of caspases and induction of apoptosis could be enhanced further through coinfection with ZD55‐TRAIL. The combined treatment of ZD55‐Smac and ZD55‐TRAIL resulted in significant reduction of XIAP expression levels. In addition, our in vivo data in mice showed only a partial response in the established tumor treated either by ZD55‐Smac or ZD55‐TRAIL alone. By contrast, complete tumor regression was observed by combination of ZD55‐Smac and ZD55‐TRAIL in all treated animals. This strong antitumoral activity achieved by this combination was due to a dramatic induction of tumor cell apoptosis in the treated tumors. In conclusion, our data indicate that Smac antagonizes the IAPs in HCC tumor cells and enhances tumor cell death induced by TRAIL in the oncolytic adenoviral vector. The combination of Smac and TRAIL delivered by way of the oncolytic adenoviral vector would provide a useful strategy for therapy of HCC and might also be applied to other IAPs abundant in cancers. (HEPATOLOGY 2004;39:1371–1381.)

Oncolytic adenovirus-mediated shRNA against Apollon inhibits tumor cell growth and enhances antitumor effect of 5-fluorouracil

Apollon, a membrane-associated inhibitor of apoptosis protein, protects cells against apoptosis and is upregulated in certain tumor cells. In this study, the effects of Apollon protein knockdown by RNA interference on the growth of human HeLa, HT-1080 and MCF-7 cells in vitro and in vivo were investigated. An oncolytic adenovirus (ZD55) containing the RNA polymerase III-dependent U6 promoter to express short hairpin RNA (shRNA) directed against Apollon (ZD55-siApollon) was constructed. Our data show that ZD55-siApollon successfully exerts a gene knockdown effect and causes the inhibition of tumor cell growth both in culture and in athymic mice in vivo. Cell cycle analysis, 4′,6-diamidino-2-phenylindole staining and western blot analysis reveal that ZD55-siApollon-mediated suppression of Apollon induces apoptosis. Intratumoral injection of ZD55-siApollon significantly inhibits tumor growth in HT-1080 xenograft mice. Furthermore, ZD55-siApollon enhances the antitumor effect of 5-fluorouracil, a chemotherapeutic agent. In conclusion, these results suggest that the depletion of Apollon by oncolytic adenovirus–shRNA delivery system provides a promising method for cancer therapy.

Adenovirus-mediated interleukin-2 gene therapy of nociception

The effect of adenovirus-mediated interleukin-2 (IL-2) gene on rat basal nociceptive response and chronic neuropathic pain was explored. The paw withdrawal latency induced by radiant heat was used to evaluate the antinociceptive effect of adenovirus type 5 (Ad5) and Ad5-IL-2. The results showed that intrathecal delivery of Ad5-IL-2 exhibited obvious antinociceptive effects on basal nociceptive response and chronic neuropathic pain, which were maintained for 3 and 4 weeks, respectively. This suggested that the antinociceptive effect of Ad5-IL-2 on chronic neuropathic pain was greater than its effect on basal nociceptive response. Human IL-2 mRNA was detected by in situ hybridization in the spinal pia mater and parenchyma of the lumbar, sacral, thoracic and cervical regions, and gray matter had higher level of IL-2 expression than white matter. These data demonstrated that the IL-2 gene was transfected into spinal cord regions relevant to pain modulation. The expressed IL-2 protein profile in spinal cord detected by enzyme-linked immunosorbent assay coincided almost exactly with its antinociceptive effect. This supported the hypothesis that the therapeutic effect of IL-2 gene was related to IL-2 protein expression. The study indicates that intrathecal delivery of adenovirus-mediated IL-2 gene has a relatively long antinociceptive effect.

Cancer targeting Gene-Viro-Therapy of liver carcinoma by dual-regulated oncolytic adenovirus armed with TRAIL gene

Liver cancer is a common and aggressive malignancy, but available treatment approaches remain suboptimal. Cancer targeting Gene-Viro-Therapy (CTGVT) has shown excellent anti-tumor effects in a preclinical study. CTGVT takes advantage of both gene therapy and virotherapy by incorporating an anti-tumor gene into an oncolytic virus vector. Potent anti-tumor activity is achieved by virus replication and exogenous expression of the anti-tumor gene. A dual-regulated oncolytic adenoviral vector designated Ad·AFP·E1A·E1B (Δ55) (Ad·AFP·D55 for short thereafter) was constructed by replacing the native viral E1A promoter with the simian virus 40 enhancer/α-fetoprotein (AFP) composite promoter (AFPep) based on an E1B-55K-deleted construct, ZD55. Ad·AFP·D55 showed specific replication and cytotoxicity in AFP-positive hepatoma cells. It also showed enhanced safety in normal cells when compared with the mono-regulated vector ZD55. To improve the anti-hepatoma activities of the virus, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene was introduced into Ad·AFP·D55. Ad·AFP·D55-TRAIL exhibited remarkable anti-tumor activities in vitro and in vivo. Treatment with Ad·AFP·D55-TRAIL can induce both autophagy owing to the Ad·AFP·D55 vector and caspase-dependent apoptosis owing to the TRAIL protein. Therefore, Ad·AFP·D55-TRAIL could be a potential anti-hepatoma agent with anti-tumor activities due to AFP-specific replication and TRAIL-induced apoptosis.

Interleukin-2 gene has superior antinociceptive effects when delivered intrathecally.

The antinociceptive effect of interleukin-2 gene on rat carrageenan-induced pain was explored using different delivery methods. Intrathecal (i.t.) or plantar s.c. delivery of plasmid harbouring the interleukin-2 gene produced a marked antinociceptive effect, which was maintained up to 6 days; the administration of recombinant human interleukin-2 only had a transitory effect. The antinociceptive effect lasted longer and was more potent when the interleukin-2 gene was administered i.t. than when delivered s.c. The effect of the interleukin-2 gene was related to its protein expression, was dose dependent, and could be potentiated by liposome. The results suggest that the interleukin-2 gene has a good prospect for clinical use.

Adenoviral vector expressing CYLD augments antitumor activity of TRAIL by suppression of NF-kappaB survival signaling in hepatocellular carcinoma.

CYLD is a tumor suppressor gene related to cylindroma and is negative regulator of NF-κB. However, antitumor effect of CYLD has not been reported. The activation of NF-κB induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) renders hepatocellular carcinoma (HCC) resistant to TRAIL-mediated cell apoptosis. Here we described that the adenoviral vector expressing CYLD (Ad/hTERT-CYLD) augmented the cytotoxicity of TRAIL in HCC cells by negatively regulating NF-κB activity since CYLD could reverse the ubiquitination of TNF receptor-associated factor 2 (TRAF2) and interact with the I B kinase? (IKKγ). The combined treatment of Ad/hTERT-CYLD and a conditionally replicating adenovirus carrying TRAIL gene (ZD55-TRAIL) induced rapid and potent apoptosis in HCC cells, characterized by activation of caspase-3, caspase-8, PARP and the reduction of X-linked inhibitor of apoptosis protein (XIAP). In animal study, the combined treatment could eradicate the BEL7404 xenograft tumors. In contrast, treatment with Ad/hTERT-CYLD or ZD55-TRAIL alone achieved less antitumor effect. In conclusion: CYLD inhibits TRAIL-mediated NF-κB activation and enhances the sensitivity of HCC cells to TRAIL-triggered apoptosis. The combined delivery of Ad/hTERT-CYLD and ZD55-TRAIL may be a new useful strategy for HCC or other tumor cells with enhanced NF-κB activity.

A survivin-mediated oncolytic adenovirus induces non-apoptotic cell death in lung cancer cells and shows antitumoral potential in vivo

Conditionally replicating adenoviruses or oncolytic adenoviruses, which can replicate selectively in tumor cells and kill them, represent an innovative class of promising cancer therapeutics. Survivin is the smallest member of the inhibitor of apoptosis (IAP) family, which is transcriptionally upregulated exclusively in most malignant tissues but not in normal tissues. It has been reported that activity of the survivin promoter is tumor‐specific, which makes the survivin promoter a good candidate to construct oncolytic viral vectors.

Combination of Targeting Gene-ViroTherapy with 5-FU Enhances Antitumor Efficacy in Malignant Colorectal Carcinoma

To improve the therapeutic effect of ONYX015, an E1B55kD-deleted replication-competent adenovirus, ZD55 was constructed and armed with the therapeutic gene hTRAIL to form ZD55-hTRAIL, which was used for cancer therapy and which we call Targeting Gene-ViroTherapy. In vitro experiments with SW620, HCT116, and HT29 colorectal carcinoma cell lines demonstrated that they were all sensitive to ZD55-hTRAIL, and especially sensitive to ZD55-hTRAIL plus 5-fluorouracil (5-FU) treatment. In the SW620 xenograft tumor model, various treatment groups showed marked differences at week 11, with the tumor volume for the phosphate-buffered saline (PBS) treatment group >1700 mm3, for 5-FU > 1300 mm3, for ONYX015 1051.3 mm3, for ZD55-hTRAIL 600.05 mm3, and for ZD55-hTRAIL plus 5-FU 230.2 mm3. At the end of week 14, tumor-bearing mice in the other groups almost all died, whereas all the mice in the combined treatment group were alive, with one mouse tumor free. By transmission electron microscopy (TEM) assay, most tumor cells treated with ONYX015 or with ZD55-hTRAIL singly or in combination with 5-FU were lysed due to viral propagation. RT-PCR analysis and immunohistochemistry examination revealed that hTRAIL was expressed in ZD55-hTRAIL-treated SW620 tumor tissue. Furthermore, no detectable hepatoxicity was found by serum enzyme level analysis. These results suggest that ZD55-hTRAIL alone or in combination with 5-FU may have potential clinical implications.

Reexpression of Human Somatostatin Receptor Gene 2 Gene Mediated by Oncolytic Adenovirus Increases Antitumor Activity of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand against Pancreatic Cancer

Purpose: Pancreatic cancer continues to pose an enormous challenge to clinicians and cancer scientists. Clinical studies show that tumor necrosis factor–related apoptosis- inducing ligand (TRAIL) exerts a potent and tumor-specific proapoptotic activity. However, most pancreatic cancer cells are resistant to TRAIL therapy. Human somatostatin receptor gene 2 (hSSTr2) is lost in 90% of pancreatic carcinoma. Oncolytic viruses are able to selectively lyse cancer cells and represent a promising novel anticancer therapy. Here, we investigated whether oncolytic adenovirus–mediated reexpression of hSSTr2 would enhance TRAIL-induced antitumor efficacy against pancreatic cancer. Experimental Design: The antitumor efficacies of combined or single treatment of hSSTr2 and TRAIL mediated by oncolytic adenovirus were compared in pancreatic cancer cell culture and xenografts. The mechanisms involved in hSSTr2-induced sensitization to TRAIL were studied. Results: Oncolytic adenovirus–mediated reexpression of hSSTr2 potentiated TRAIL-induced tumor growth inhibition in vitro and in vivo. Reexpression of hSSTr2 augmented TRAIL-induced apoptosis against pancreatic cancer cells via up-regulation of death receptor 4 and down-regulation of Bcl-2. Conclusions: hSSTr2 restoration mediated by oncolytic adenovirus enhances TRAIL-induced antitumor efficacy against pancreatic cancer. Combined treatment with oncolytic adenovirus–mediated hSSTr2 and TRAIL gene provides the rationale for a clinical trial in pancreatic cancer. (Clin Cancer Res 2009;15(16):5154–60)

Enhanced suicide gene therapy by chimeric tumor‐specific promoter based on HSF1 transcriptional regulation

Two tandem cassettes, one containing the telomerase reverse transcriptase gene (hTERT) promoter upstream of a constitutively activated form of heat shock transcription factor 1 (cHSF1) and followed by the other containing the heat shock protein 70B (hsp70B) promoter (HSE) upstream of the cytosine deaminase (CD) gene, could greatly enhance the efficiency of CD gene therapy while retaining tumor specificity in vitro and in vivo. This hTERT‐cHSF1/HSE promoter could restrict gene expression in tumor cells and was about 1.5–3‐fold more potent than the cytomegalovirus (CMV) promoter. hTERT‐cHSF1/HSE‐CD transfection led to tumor cells more sensitive to 5‐fluorocytosine compared with hTERT‐CD and its toxicity was comparable to that of CMV‐CD. Besides enhancement of promoter activity, cHSF1 overexpression itself could enhance the bystander effect of CD gene therapy that could be reversed by anti‐Fas antibody. This system also led to activation of stress‐related genes such as hsp70 in tumor cells, which in the presence of cell killing by the cytotoxic gene is a highly immunostimulatory event. Furthermore, a more potent anti‐tumor effect of hTERT‐cHSF1/HSE‐CD was observed in nude mice inoculated with Bcap37 cells. No obvious activity of the hTERT‐cHSF1/HSE promoter was observed in normal tissues after intravenous administration. These results indicate that the hTERT‐cHSF1/HSE promoter is highly tumor‐specific and strong with potential application in targeted gene therapy, and therefore may be useful for construction of vectors for systemic therapy.