Zilai Zhang

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.)

A novel oncolytic adenovirus targeting to telomerase activity in tumor cells with potent

Telomerase is a therapeutic target for cancer. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionaly upregulated exclusively in about 90% of cancer cells. Previous studies have demonstrated that hTERT promoter can control the expression of exogenous genes to the telomerase-positive cancer cells, thus hTERT promoter is an excellent candidate for generating cancer-specific oncolytic adenovirus. In this study, we devised a novel oncolytic adenovirus (Ad.TERT) by replacing the normal E1A regulatory elements with hTERT promoter. Ad.TERT displays cancer-specific E1A expression, virus replication and cytolysis in in vitro experiments. In animal experiments, intratumoral administration of Ad.TERT demonstrates potent antitumoral efficacy at least in two xenograft models (Bcap37 and BEL7404). Ad.TERT was targeted by the telomerase activity in cancer cells and has potent antitumoral efficacy in vivo, and since telomerase activity is a wide-ranged tumor marker, Ad.TERT could be a powerful therapeutic agent for a variety of cancers.

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.

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.

Suppression of morphine withdrawal syndrome by interleukin-2 and its gene

The naloxone-precipitated withdrawal syndrome in mice and rats after intrathecal injection of recombinant human interleukin-2 protein (rIL-2) or its gene was studied. The results showed that rIL-2 could significantly decrease the number of jumps in mice. In rats, rIL-2 significantly suppressed irritating, diarrhea, weight loss, abnormal posture and salivation. Tendencies towards reductions in teeth chewing and dog-shaking were also observed. Furthermore, pcDNA3-IL-2 (8 μg DNA) had a similar effect as 1×104 IU rIL-2 protein on inhibition of morphine withdrawal syndrome in mice, and the expression of rIL-2 protein in spinal cord could be detected for 6 days. These findings provided further evidence for the neuroregulatory function of an immunological molecule such as IL-2.

Comprehensive characterization of iron oxide containing mesoporous molecular sieve MCM-41

We have synthesized iron oxide-containing mesoporous silica MCM-41 by a direct route. This material has a significant microporousity, which might be related with the loading of iron oxide in the MCM-41 mesopores, though no crystalline phases of iron oxide were detected by XRD. Intensity deviations and extra bright intensity within the pores, as well as local superstructural phases within a large area, were observed in the TEM images. The position and area of such a crystalline phase could change during the successive electron beam exposure and resulted in a formation of a cycle. These phenomena imply the possible existence of small Fe2O3 nanocrystals loaded in the mesoporous silica materials with regular and oriented arrangement. EELS spectrum of the sample proves the existence of iron, but the chemical environment of iron in the MCM-41 silica should be different from that of bulk amorphous porous iron oxide. The small iron oxide crystals in the MCM-41 sample might be linked with silica wall through oxygen. Another possibility is that iron oxide reacts with silica during synthesis, causing the partial crystallization of the MCM-41 framework. In other words, microcrystals of iron oxide may exist in the silica matrix.

448. Armed Oncolytic Adenovirus with an Antiangiogenic Gene, sflt-1: A Novel Effective Antitumor Approach

Top of pageAbstract Armed oncolytic adenoviruses represent an appealing tumor treatment. In this paper, considing that angiogenesis play an important role in tumor growth, the antiangiogenic gene, sflt-1(1-3) (the first three extracellular domain of hVEGF receptor) was inserted into ZD55 (an E1B55-kD deleted oncolytic adenovirus previously constructed in our lab) to form ZD55-sflt-1. ZD55-sflt-1 could specifically induce cytopathic effect in tumor cells in vitro. The expression of sFLT-1 from ZD55-sflt-1 was much higher than that of replication–deficient Ad-sflt-1, which contributed to its stronger inhibitory effect of VEGF-induced HUVEC proliferation (35.4386% of control). Furthermore, a marked reduction of tumor growth was observed in ZD55-sflt-1-treated nude mice with subcutaneous human SW620 colon tumor. Its efficacy directly corrected with a decrease in microvessel density and an increase in apoptosis of tumor cells. A much more efficient antitumoral effect was obtained when ZD55-sflt-1 was combined with ZD55-trail (another oncolytic adenovirus engineered to express the tumor necrosis factor-related apoptosis-inducing ligand), which resulted in 57.14% complete tumor regression and 71.43% prolonged survival of mice. These results indicate that ZD55-sflt-1, which combines the advantages of oncolytic adenovirus and the antiangiogenic gene system, can result in a striking improvement in treatment efficacy, and is suitable for synergistic therapy with other agents.