An armed oncolytic adenovirus system, ZD55-gene, demonstrating potent antitumoral efficacy

ONYX-015 is an attractive therapeutic adenovirus for cancer because it can selectively replicate in tumor cells and kill them. To date, clinical trials of this adenovirus have demonstrated marked safety but not potent enough when it was used alone. In this paper, we put forward a novel concept of Gene-ViroTherapy strategy and in this way, we constructed an armed therapeutic oncolytic adenovirus system, ZD55-gene, which is not only deleted of E1B 55-kD gene similar to ONYX-015, but also armed with foreign antitumor gene. ZD55-gene exhibited similar cytopathic effects and replication kinetics to that of ONYX-015 in vitro. Importantly, the carried gene is expressed and the expression level can increase with the replication of virus. Consequently, a significant antitumoral efficacy was observed when ZD55-CD/5-FU was used as an example in nude mice with subcutaneous human SW620 colon cancer. Our data demonstrated that ZD55-gene, which utilizing the Gene-Viro Therapy strategy, is more efficacious than each individual component in vivo.     

癌症靶向基因-病毒ZD55-XAF1抗肝癌移植瘤的生长及其安全性研究

目的:探讨溶瘤腺病毒(ZD55-gene)作为载体携带外源抗癌基因(XAF1)抗肝癌移植瘤的生长及其安全性。方法:抽提溶瘤腺病毒ZD55-XAF1的基因组DNA,PCR扩增鉴定病毒;细菌平板培养和支原体检测试剂盒检测细胞有无细菌、支原体污染;通过荷瘤小鼠动物实验,观察溶瘤腺病毒ZD55-XAF1对肝癌移植瘤生长的抑制、小鼠的临床反应指标、血清肝毒性指标、各脏器组织中的病毒残留分布及病理切片观察。结果:细胞培养过程无细菌和支原体污染;较对照组,受试小鼠血清肝酶AST活性上升(P0.05),而ALT和ALP活性基本无变化(P0.05);PCR检测各脏器均有病毒基因组DNA存在;HE染色显示受试小鼠各脏器具有不同程度的损伤,病毒处理对肿瘤细胞具有明显的杀伤效果,而受试小鼠的临床反应并无明显异常。结论:溶瘤腺病毒ZD55-XAF1能够抑制肿瘤生长,杀死肿瘤细胞,对小鼠血清肝酶活性影响较小而对各脏器有不同程度的轻微损伤,作为癌症基因治疗载体有潜在的应用价值但其安全性还有待提高。     

Synergistic antitumor effect of TRAIL and IL-24 with complete eradication of hepatoma in the CTGVT-DG strategy

The ZD55-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and ZD55-interleukin (IL)-24 were constructed by inserting TRAIL or IL-24 gene separately into the oncolytic adenovirus named ZD55 (with adenovirus E1B-55kD deletion). The resulting ZD55-TRAIL and ZD55-IL-24 were used in combination to treat xenograft tumors in nude mice model. The results showed that it can not only completely eliminate BEL7404 hepatoma xenograft but also have excellent antitumor effect against gaster, lung, prostate, and breast carcinomas. It was also found that ZD55-TRAIL could not only suppress the tumor growth promoting effect by ZD55-IL-24 at lower dosage, but also substantially reduce the cancer cell viability in their combined use. This is because ZD55-IL-24 and ZD55-TRAIL could mutually enhance each other's antitumor effect greatly. All these findings conspicuously showed the synergistic antitumor effect of TRAIL and IL-24, which is also the reason for the antitumor effect by the combined use of TRAIL and IL-24 in vitro and also in vivo.     

Targeting gene-virotherapy of cancer

Our purpose is to completely elimination of xenograft tumor in animal tumor model in order to work out a protocal for the cure of patient. Gene therapy and viral therapy for cancer have got some therapeutic effects, but both have no great breakthrough. Therefore, we worked out a new strategy called Targeting Gene-Virotherapy of Cancer which is a combination of the advantage of gene therapy and virotherapy. This new strategy has stronger antitumor effect than either of them alone. A tumor specific replicative adenovirus vector ZD55 (E1B 55KD deleted Adv.) which is similar to ONYX-015 in targeting fuction but significant different in construction was produced and various single therapeutic gene was inserted into ZD55. Now such a conception as Targeting Gene-Virotherapy of Cancer was raised and systemically studied before, although there are some works on ONYX-015-tk, -cd or cd/-tk etc. separately. The antitumor effect of ZD55-Gene (for example IL-24 gene) is much better than ZD55 (virotherapy) alone and hundred fold high than that of Ad-IL-24 (gene therapy) alone. ZD55-IL-24 was in preclinal studying in the ZD55-IL-24 therapy, completely elimination of tumor mass was occurred in some mice but not in all mice, that means one gene was not effictive enough to eliminate all the tumor mass in all mice. Therefore two genes with compensative or synergetic effect were inserted into ZD55 separately and used in combination. This strategy was called Targeting Dual Gene-Virotherapy of Cancer (with PCT patent). Then much better results were obtained and all the xenograft tumor masses were completely eliminated in all mice, if two suitable genes were chosen. On the basis of the initiation of two gene results, it was thought about that using two tumors promoter to control the virus vector will be better for the targeting effect and the safty of the drugs. Then double tumor controlled virus vector harboring two genes for cancer therapy was worked out. Better results have been obtained and another patent has been applied. This antitumor strategy could be used to kill all the tumor cells completely in all mice with minimum damage to normal cells.     

Synergistic induction of tumor cell death by combining cisplatin with an oncolytic adenovirus carrying TRAIL

Chemoresistance and side effects are considered as the major obstacles in cisplatin-based chemotherapy of various human malignant tumors. Conjugation with cancer-specific apoptotic stimuli TRAIL or typical viro-agent ONYX-015 has been extensively investigated to enhance the antitumor activity of cisplatin. In this study, we presented a novel chemo-gene-virotherapeutic strategy to further improve the toxic effects in cancer cells and reduce the damage in normal cells. Here, an oncolytic adenoviral vector (ZD55), with a deletion of E1B 55-kDa gene, was employed to express the therapeutic TRAIL gene by constructing a recombinant virus ZD55-TRAIL. Exogenous gene delivery efficacy was determined by both in vitro and in vivo experiments and enhanced cytotoxicity of combined treatment of ZD55-TRAIL with cisplatin was evaluated in several cancer cell lines. Moreover, negative effects on normal cells have been tested in both L-02 and MRC-5 cell lines by MTT assay and apoptotic cell staining. According to our observation, combination of ZD55-TRAIL with cisplatin exhibited an apparent synergistic cytotoxicity in cancer cells, yet significantly abolished the negative toxicity in normal cells by reducing the dosage. Thus, a novel chemo-gene-virotherapeutic strategy for cancer therapy was proposed.     

Targeting gene-virotherapy for cancer

Gene therapy and viral therapy for cancer have therapeutic effects, but there has been no significant breakthrough in these two forms of therapy. Therefore, a new strategy called “targeting gene-virotherapy”, which combines the advantages of gene therapy and viral therapy, has been formulated. This new therapy has stronger antitumor effects than either gene therapy or viral therapy. A tumor-specific replicative adenovirus vector ZD55 （E1B55KD deleted Adv.） was constructed and various single therapeutic genes were inserted into ZD55 to form ZD55-gene. These are the targeting gene-virotherapy genes. But experiments showed that a single gene was not effective in eliminating the tumor mass, and therefore two genes were separately inserted into ZD55. This strategy is called “targeting dual gene-virotherapy” （with PCT patent）. Better results were obtained with this strategy, and all the xenograft tumor masses were completely eliminated in all mice when two suitable genes producing a synergetic or compensative effect were chosen. Twenty-six papers on these strategies have been published by researchers in our laboratory. Furthermore, an adenoviral vector with two targeting promoters harboring two antitumor genes has been constructed for cancer therapy. Promising results have been obtained with this adenoviral vector and another patent has been applied for. This antitumor strategy can be used to kill tumor cells completely with minimum damage to normal cells.     

Cancer targeting gene-viro-therapy for pancreatic cancer using oncolytic adenovirus ZD55-IL-24 in immune-competent mice

Cancer targeting gene-viro-therapy (CTGVT) may prove to be an effective treatment for pancreatic cancer (PC). This study was intended to explore the anti-tumor effect of ZD55-IL-24 (oncolytic adenovirus ZD55 harboring IL-24) on PC in immune-competent mice. The expression of gene harbored by oncolytic adenovirus ZD55 in PC cells was detected by reporter-gene assays. The in vitro anti PC ability of ZD55-IL-24 was tested by MTT, crystal violet staining and apoptosis assays. The in vivo anti PC effect of ZD55-IL-24 was further observed in an immune-competent mice model by detecting anti-tumor immunity and induction of apoptosis. The expression of gene harbored by ZD55 in PC cells was significantly higher than that harbored by the replicated-deficient adenovirus, and the amount of gene expression was time-dependent and dose-dependent. Both ZD55-IL-24 and ZD55 inhibited PC cells growth, but the anti-tumor effect of ZD55-IL-24 was significantly stronger than that of ZD55, and the ability of ZD55-IL-24 in inducing PC apoptosis was significantly stronger than that of ZD55. The tumor-forming rate of group ZD55-IL-24 was the lowest, and the tumor-growing rate was also significantly lower than that of group ZD55 in immune-competent PC models. Moreover, ZD55-IL-24 mediated more anti-cancer immunity effects by induction of stronger T-lymphocytes response to PC cells, higher levels of γ-IFN and IL-6 cytokines. ZD55-IL-24-mediated CTGVT could inhibit PC growth not only by inducing oncolysis and apoptosis but enhancing the anti-cancer immune effects by inducing T cell response to PC and up-regulating γ-IFN and IL-6 cytokine in immune-competent mice. This may serve as a candidate therapeutic approach for the treatment of PC.     

Anti-tumor activity of the combination of cetuximab, an anti-EGFR blocking monoclonal antibody and ZD6474, an inhibitor of VEGFR and EGFR tyrosine kinases

PURPOSE: The epidermal growth factor receptor (EGFR) autocrine pathway plays an important role in cancer cell growth. Vascular endothelial growth factor A (VEGF-A) is a key regulator of tumor-induced endothelial cell proliferation and vascular permeability. ZD6474 is an orally available, small molecule inhibitor of VEGF receptor-2 (VEGFR-2), EGFR and RET tyrosine kinase activity. We investigated the activity of ZD6474 in combination with cetuximab, an anti-EGFR blocking monoclonal antibody, to determine the anti-tumor activity of EGFR blockade through the combined use of two agents targeting the receptor at different molecular sites in cancer cells and of VEGFR-2 blockade in endothelial cells. EXPERIMENTAL DESIGN: The anti-tumor activity in vitro and in vivo of ZD6474 and/or cetuximab was tested in human cancer cell lines with a functional EGFR autocrine pathway. RESULTS: The combination of ZD6474 and cetuximab determined synergistic growth inhibition in all cancer cell lines tested as assessed by the Chou and Talalay method. In nude mice bearing established human colon carcinoma (GEO) or lung adenocarcinoma (A549) xenografts and treated with ZD6474 and/or cetuximab for 4 weeks, a reversible tumor growth inhibition was caused by each drug. In contrast, a more significant tumor growth delay resulted from the combination of the two agents with an approximately 100-110 days increase in mice median overall survival as compared to single agent treatment. CONCLUSIONS: This study provides a rationale for evaluating in a clinical setting the double blockade of EGFR in combination with inhibition of VEGFR-2 signaling as cancer therapy.     

Vascular disrupting agents

A clear definition for vascular targeting agents (VTAs) and vascular disrupting agents (VDAs) has separated the two as distinct methods of cancer treatment. VDAs differ from VTAs (antiangiogenesis drugs) in their mechanism of action. VTAs attempt to keep new blood vessels from forming and do not act on blood vessels that already feed existing tumors. In contrast, VDAs cause the vascular structure inside a solid tumor to collapse, depriving the tumor of blood and oxygen it needs to survive. Therefore, VDAs are an attractive way to approach the cancer problem by combating developed tumors. The following review discusses six small molecule VDAs, namely DMXAA, ZD6126, TZT1027, CA4P, AVE8062, and Oxi4503, their synthesis, biological mechanism of action, and current clinical status.     

A double-regulated oncolytic adenovirus with improved safety for adenocarcinoma therapy

Safety and efficiency are equally important to be considered in developing oncolytic adenovirus. Previously, we have reported that ZD55, an oncolytic adenovirus with the deletion of E1B-55K gene, exhibited potent antitumor activity. In this study, to improve the safety of ZD55, we utilized MUC1 promoter to replace the native promoter of E1A on the basis of ZD55, and generated a double-regulated adenovirus, named MUD55. Our data demonstrated that the expression of early and late genes of MUD55 was both reduced in MUC1-negative cells, resulting in its stricter glandular-tumor selective progeny production. The cytopathic effect of MUD55 was about 10-fold lower than mono-regulated adenovirus ZD55 or Ad.MUC1 in normal cells and not obviously attenuated in glandular tumor cells. Moreover, MUD55 showed the least liver toxicity when administrated by intravenous injection in nude mice. These results indicate that MUD55 could be a promising candidate for the treatment of adenocarcinoma.     

Targeting gene-virotherapy of cancer and its prosperity

Gene and viral therapies for cancer have shown some therapeutic effects, but there has been a lack of real breakthrough. To achieve the goal of complete elimination of tumor xenograft in animal models, we have developed a new strategy called Targeting Gene-Virotherapy of Cancer, which aims to combine the advantages of both gene therapy and virotherapy. This new strategy has produced stronger anti-tumor effects than either gene or viral therapy alone. A tumorspecific replicative adenovirus vector, designated as ZD55, was constructed by deletion of the 55kDa E1B region of adenovirus. The resulting viral construct not only retains a similar function to ONYX-015 by specifically targeting p53 negative tumors, but also allows for the insertion of various therapeutic genes to form appropriate ZD55 derivatives due to the newly introduced cloning site, a task not feasible with the original ONYX-015 virus. We showed that the anti-tumor effect of one such derivative, ZD55-IL-24, is at least 100 times more potent than that of either ZD55 virotherapy or Ad-IL-24 gene therapy. Nevertheless, complete elimination of tumor mass by the use of ZD55-1L-24 was only observed in some but not all mice, indicating that one therapeutic gene was not sufficient to ＂cure＂ these mice. When genes with complementary or synergetic effects were separately cloned into the ZD55 vector and used in combination （designated as the Dual Gene Therapy strategy）, much better results were obtained; and it was possible to achieve complete elimination of all the xenograft tumor masses in all mice if two suitable genes were chosen. More comprehensive studies based on this new strategy will likely lead to a protocol for clinical trial. Finally, the concept of Double Controlled Targeting Virus-Dual Gene Therapy for cancer treatment, and the implication of the recent progress in cancer stem cells are also discussed.     

Dichloroacetate (DCA) enhances tumor cell death in combination with oncolytic adenovirus armed with MDA-7/IL-24

Dichloroacetate (DCA) is a metabolic modulator for the treatment of lactic acidosis and inherited mitochondrial diseases. A recent study showed that DCA treatment could induce apoptosis in many kinds of tumor cell lines via mitochondrial apoptotic pathway while sparing normal cells. ONYX-015 (dl 1520) is one of the oncolytic adenoviruses developed by the deletion of E1B-55kD gene of type 5 adenoviral DNA, and it replicates efficiently and selectively in tumor cells. ZD55-IL-24, an E1B-55kD deleted oncolytic adenovirus carrying interleukin-24 (IL-24, also called melanoma differentiation associated gene-7), had showed potent antitumor efficacy in a variety of tumor cells and exerted no apparent toxicity on normal cells. Given both the good therapeutic effect and low toxicity of these agents, here we investigated whether DCA in combination with ZD55-IL-24 or ONYX-015 could have more efficient antitumor activity in vitro experiments. Therefore, we tested the cytotoxicity of combination therapy in normal hepatic cells L-02 and QSG-7701 using the MTT assay. Our results showed that DCA combined with ONYX-015 or ZD55-IL-24 exhibited more potent antitumor activity than DCA or virus alone, and the combination treatment did not have superimposed toxicities in normal cells. Thus, a novel combination therapy associating oncolytic adenoviruses with relatively low toxic drug without severe side effects was proposed.     

Inhibition of proliferation and induction of apoptosis in breast cancer cells by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 ('Iressa') is independent of EGFR expression level

High expression of the epidermal growth factor receptor (EGFR) in breast carcinoma confers a growth advantage to the tumor cells. The EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 ('Iressa') has clinical activity in a wide range of tumor types, although the mechanism(s) by which it exerts its antitumor activity effects remain unclear. We analyzed the ability of ZD1839 to induce apoptosis and/or inhibition of proliferation in breast carcinoma cell lines, as well any association between this ability and the downregulation activity of MAPK and Akt, two recently proposed markers of ZD1839 activity. Proliferation, survival, and activation of Akt and MAPK were evaluated in six human breast cancer cell lines expressing various levels of EGFR and HER2 and exposed to ZD1839. EGFR and HER2 expression levels were determined using specific monoclonal antibodies and FACS analysis. The effects of ZD1839 were independent of EGFR expression levels, but were influenced by high HER2 expression. ZD1839 significantly reduced the rate of [3H]-thymidine incorporation in the four sensitive cell lines, while apoptosis was also induced in two of these cell lines. No correlation was found between the cytostatic or cytotoxic effects of ZD1839 and its ability to downregulate MAPK and Akt activity in the tumor cell lines. Our data suggest that the antitumor activity of ZD1839 is due to a cytostatic effect, and involves apoptosis induction in a subset of sensitive cells only, and that neither MAPK nor Akt is a reliable marker of ZD1839 activity.     

Interferon-β-armed oncolytic adenovirus induces both apoptosis and necroptosis in cancer cells

Interferon-β (IFN-β) has been widely used in cancer therapy, but the clinical trial results are generally disappointing. Our previous studies have shown that an oncolytic adenovirus carrying IFN-β (ZD55-IFN-β) exhibits significant anti-tumor activities. However, the underlying mechanisms are not clear. Here we showed that ZD55-IFN-β infection-induced S-phase cell cycle arrest in a p53-dependent manner by activating the ataxia telangiectasia mutated-dependent DNA damage pathway. In addition, ZD55-IFN-β infection could initiate both caspase-dependent apoptosis and necroptosis in cancer cells. More importantly, ZD55-IFN-β showed a synergistic effect on cancer cells when combined with doxorubicin. These results suggest that the combination of ZD55-IFN-β with doxorubicin may represent a promising clinical strategy in cancer therapy.     

Vascular remodeling marks tumors that recur during chronic suppression of angiogenesis

The potential for avoiding acquired resistance to therapy has been proposed as one compelling theoretical advantage of antiangiogenic therapy based on the normal genetic status of the target vasculature. However, previous work has demonstrated that tumors may resume growth after initial inhibition if antiangiogenic blockade is continued for an extended period. The mechanisms of this recurrent growth are unclear. In these studies, we characterized molecular changes in vasculature during apparent resumption of xenograft growth after initial inhibition by vascular endothelial growth factor blockade, "metronome" topotecan chemotherapy, and combined agents in a xenograft murine model of human Wilms' tumor. Tumors that grew during antiangiogenic blockade developed as viable clusters surrounding strikingly remodeled vessels. These vessels displayed significant increases in diameter and active proliferation of vascular mural cells and expressed platelet-derived growth factor-B, a factor that functions to enhance vascular integrity via stromal cell recruitment. In addition, remodeled vessels were marked by expression of ephrinB2, required for proper assembly of stromal cells into vasculature. Thus, enhanced vascular stability appears to characterize tumor vessel response to chronic antiangiogenesis, features that potentially support increased perfusion and recurrent tumor growth.     

基质衍生的血管生成抑制剂研究进展

血管的生成与肿瘤密切相关,抑制肿瘤血管生成可以调节肿瘤的生长。体内存在着内源性的促血管生成因子和抑制因子的平衡,当促血管形成因子增强就会产生新生血管供肿瘤生长,而当抑制因子增强则会抑制肿瘤的生长。本文即对细胞外基质衍生的内源性血管生成抑制因子TSP、内皮他丁、Arresten;Canstatin、Endorepellin、Fibulin、Tumstatin等的特性、应用和作用机制等作一总结。     

Targeting lung cancer stem‐like cells with TRAIL gene armed oncolytic adenovirus

Lung cancer stem cell (LCSC) is critical in cancer initiation, progression, drug resistance and relapse. Disadvantages showed in conventional lung cancer therapy probably because of its existence. In this study, lung cancer cell line A549 cells propagated as spheroid bodies (named as A549 sphere cells) in growth factors‐defined serum‐free medium. A549 sphere cells displayed CSC properties, including chemo‐resistance, increased proportion of G0/G1 cells, slower proliferation rate, ability of differentiation and enhanced tumour formation ability in vivo. Oncolytic adenovirus ZD55 carrying EGFP gene, ZD55‐EGFP, infected A549 sphere cells and inhibited cell growth. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) armed oncolytic adenovirus, ZD55‐TRAIL, exhibited enhanced cytotoxicity and induced A549 sphere cells apoptosis through mitochondrial pathway. Moreover, small molecules embelin, LY294002 and resveratrol improved the cytotoxicity of ZD55‐TRAIL. In the A549 sphere cells xenograft models, ZD55‐TRAIL significantly inhibited tumour growth and improved survival status of mice. These results suggested that gene armed oncolytic adenovirus is a potential approach for lung cancer therapy through targeting LCSCs.     

雷帕霉素联合ZD55-TRAIL病毒对肿瘤抑制及其机制探讨

溶瘤腺病毒的肿瘤靶向性研究一直是一个热点。目前已有商业化的ONYX-015、H101溶瘤腺病毒。在此基础上,科学家又进一步发展形成基因-病毒治疗方案,如文中应用的ZD55-TRAIL病毒。本研究利用刘新垣实验室提供的携带TRAIL(TNF-related apoptosis-inducing ligand,TNF相关的凋亡诱导配体)的溶瘤腺病毒ZD55-TRAIL联合雷帕霉素杀伤肿瘤     

A new oncolytic adenoviral vector carrying dual tumour suppressor genes shows potent anti-tumour effect

Cancer Targeting Gene-Viro-Therapy (CTGVT) is a promising cancer therapeutical strategy that strengthens the anti-tumour effect of oncolytic virus by expressing inserted foreign anti-tumour genes. In this work, we constructed a novel adenoviral vector controlled by the tumour-specific survivin promoter on the basis of the ZD55 vector, which is an E1B55KD gene deleted vector we previously constructed. Compared with the original ZD55 vector, this new adenoviral vector (ZD55SP/E1A) showed much better ability of replication and reporter gene expression. We then combined anti-tumour gene interleukine-24 (IL-24) with an RNA polymerase III-dependent U6 promoter driving short hairpin RNA (shRNA) that targets M-phase phosphoprotein 1 (MPHOSPH1, a newly identified oncogene) by inserting the IL-24 and the shRNA of MPHOSPH1 (shMPP1) expression cassettes into the new ZD55SP/E1A vector. Our results demonstrated excellent anti-tumour effect of ZD55SP/E1A-IL-24-shMPP1 in vitro on multiple cancer cell lines such as lung cancer, liver cancer and ovarian caner. At high multiplicity-of-infection (MOI), ZD55SP/E1A-IL-24-shMPP1 triggered post-mitotic apoptosis in cancer cells by inducing prolonged mitotic arrest; while at low MOI, senescence was induced. More importantly, ZD55SP/E1A-IL-24-shMPP1 also showed excellent anti-tumour effects in vivo on SW620 xenograft nude mice. In conclusion, our strategy of constructing an IL-24 and shMPP1 dual gene expressing oncolytic adenoviral vector, which is regulated by the survivin promoter and E1B55KD deletion, could be a promising method of cancer gene therapy.     

Characterization of mouse tissue kallikrein 5

Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (Klk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling.