Assessment of a combined, adenovirus-mediated oncolytic and immunostimulatory tumor therapy

In this study, we identified murine breast cancer cell lines that support DNA replication of E1-deleted adenovirus vectors and which can be killed by an oncolytic adenovirus expressing adenovirus E1A and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in a replication-dependent manner (Ad.IR-E1A/TRAIL). We showed that systemic or intratumoral (i.t.) injection of adenovirus vectors into mice increases plasma levels of proinflammatory cytokines and chemokines, including TNF-alpha, INF-gamma, and MCP-1, which are potent inducers of dendritic cell maturation. Furthermore, we showed that in vivo expression of Flt3L from an adenovirus vector increases the number of CD11b+ and CD11c+ cells (populations that include dendritic cells) in the blood circulation. Based on these findings, we tested whether Ad.IR-E1A/TRAIL induced killing of tumor cells in combination with dendritic cell mobilization by Ad.Flt3L or, for comparison, Ad.GM-CSF would have an additive antitumor effect. As a model, we used immunocompetent C3H mice with syngeneic s.c. tumors derived from C3L5 cells. We found that vaccination of mice with C3L5 cells that underwent viral oncolysis in combination with Flt3L or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression induces a systemic antitumor immune response. I.t. injection of the oncolytic and Flt3L expressing vectors into established tumors delayed tumor growth but did not cause efficient tumor elimination. This study shows the effectiveness of a combined oncolytic/immunostimulatory tumor therapy approach.     

Transduction of a fiber-mutant adenovirus for the HSVtk gene highly augments the cytopathic effect towards gliomas.

Suicide gene therapy utilizing the herpes simplex thymidine kinase (HSVtk) / ganciclovir (GCV) system has been performed to kill cancer cells. However, the low transduction efficiency of HSVtk gene into cancer cells critically limits its efficacy in cancer treatment in clinical situations. To improve delivery of the HSVtk gene into cancer cells, we transduced U-87MG and U-373MG glioma cells with adenovirus (Adv) vectors with a fiber mutant, F / K20, which has a stretch of 20 lysine residues added at the C-terminus of the fiber, for the HSVtk gene (Adv-TK-F / K20), and compared the cytopathic effect of Adv-TK-F / K20 with that of the Adv for HSVtk with wild-type fiber (Adv-TK). The cytopathic effect of Adv-TK-F / K20 in U-87MG and U-373MG cells was approximately 140 and 40 times, respectively, stronger than that of Adv-TK. At the same multiplicity of infection (MOI) in each cell line, Adv-TK-F / K20 induced a higher degree of apoptosis (U-87MG, 35%; U-373MG, 77%) than Adv-TK (U-87MG, 0.11%; U-373MG, 27%) in U-87MG (MOI 0.03) and U-373MG cells (MOI 0.1). Cleavage of poly(ADP-ribose)polymerase (PARP) was more marked in the cells that were infected with Adv-TK-F / K20 than in cells that were infected with Adv-TK. These results indicate that gene therapy utilizing Adv-TK-F / K20 may be a promising therapeutic modality for the treatment of gliomas.     

Transduction of a Fiber-mutant Adenovirus for the HSVtk Gene Highly Augments the Cytopathic Effect towards Gliomas

Suicide gene therapy utilizing the herpes simplex thymidine kinase (HSVtk)/ganciclovir (GCV) system has been performed to kill cancer cells. However, the low transduction efficiency of HSVtk gene into cancer cells critically limits its efficacy in cancer treatment in clinical situations. To improve delivery of the HSVtk gene into cancer cells, we transduced U-87MG and U-373MG glioma cells with adenovirus (Adv) vectors with a fiber mutant, F/K20, which has a stretch of 20 lysine residues added at the C-terminus of the fiber, for the HSVtk gene (Adv-TK-F/K20), and compared the cytopathic effect of Adv-TK-F/K20 with that of the Adv for HSVtk with wild-type fiber (Adv-TK). The cytopathic effect of Adv-TK-F/K20 in U-87MG and U-373MG cells was approximately 140 and 40 times, respectively, stronger than that of Adv-TK. At the same multiplicity of infection (MOI) in each cell line, Adv-TK-F/K20 induced a higher degree of apoptosis (U-87MG, 35%; U-373MG, 77%) than Adv-TK (U-87MG, 0.11%; U-373MG, 27%) in U-87MG (MOI 0.03) and U-373MG cells (MOI 0.1). Cleavage of poly(ADP-ribose)polymerase (PARP) was more marked in the cells that were infected with Adv-TK-F/K20 than in cells that were infected with Adv-TK. These results indicate that gene therapy utilizing Adv-TK-F/K20 may be a promising therapeutic modality for the treatment of gliomas.     

Introduction of mutant p53 into a wild-type p53-expressing glioma cell line confers sensitivity to Ad-p53-induced apoptosis

Transient expression of the tumor suppressor gene p53 via adenoviral-mediated gene transfer induces apoptosis in glioma cells expressing mutant p53, while causing cell cycle arrest in cells with wild-type p53. To determine whether a change in p53 status of a wild-type p53-expressing cell line such as U-87 MG would alter its apoptotic resistant phenotype in response to Ad-p53 infection, we generated cell lines U-87-175.4 and U-87-175.13 via retroviral-mediated gene transfer of the p53 (175H) mutant into the U-87 MG parental line. Control cell lines U-87-Lux.6 and U-87-Lux.8 were also generated and express the reporter gene luciferase. Both U-87-175.4 and U-87-175.13, but not control cell lines, exhibited morphology characteristic of apoptosis after Ad-p53 infection. Furthermore, expression of other p53 mutants (248W, 273H) in U-87 MG also sensitized cells to Ad-p53-induced apoptosis. Apoptosis was confirmed by TUNEL and cell cycle analysis. Several p53 response genes were examined in cells infected with Ad-p53, and among these, BCL2, p21WAF1/CIP1, CPP32/caspase 3, and PARP showed differences in expression between U87-175 and U87-Lux cell lines. Taken together, our data demonstrate that the introduction of p53 mutants in U-87 MG promotes an apoptotic response in association with adenoviral-mediated wild-type p53 gene transfer. These results underscore the importance of glioma p53 genotype for predicting tumor response to p53-based gene therapy.     

Degree of apoptosis induced by adenovirus-mediated transduction of p53 or p73alpha depends on the p53 status of glioma cells

It has been reported that U-87MG glioma cells with wild-type p53 are resistant to p53 replacement gene therapy. As some gliomas harbor wild-type p53, it would be important to override the resistance mechanism due to wild-type p53 in glioma gene therapy. In this study, we transduced U-87MG cells or U251 glioma cells harboring mutated p53 with the p53 or p73alpha gene (a homologue of p53, that differently induces some p53-responsive genes) via adenovirus vectors (Advs) at same multiplicities of infection (MOIs) into respective cells (U-87MG: MOI 1000, U251: MOI 100), and evaluated the degree of apoptosis. The results demonstrate that the degree of apoptosis induced by Adv-mediated transduction of p53 in U-87MG cells was lower than that in U251 cells, whereas that induced by Adv-mediated transduction of p73alpha in U-87MG cells was higher than that in U251 cells. Bax expression in U-87MG and U251 cells induced by Adv-mediated transduction of p53 was almost the same as that of p73alpha. On the other hand, Adv-mediated transduction of p73alpha induced caspase-9 at higher levels than that of p53 in both cells. The results indicate that Adv-mediated transduction of p73alpha might be beneficial to overcome the resistance mechanism of glioma cells harboring wild-type p53.     

AKIP1 promotes glioblastoma viability,mobility and chemoradiation resistance via regulating CXCL1 and CXCL8 mediated NF-κB and AKT pathways

This study aimed to investigate the interaction of A-kinase-interacting protein 1 (AKIP1) with C-X-C motif chemokine ligand (CXCL)1, CXCL2, CXCL8, and their effects on regulating glioblastoma multiforme (GBM) malignant behaviors. AKIP1 expression was modified by pcDNA and pGPH1 vectors in U-87 MG and U-251 MG cells. Subsequently, multiple compensative experiments were conducted via adding CXCL1, CXCL2 and CXCL8 in the pGPH1-AKIP1 (AKIP1 knockdown) transfected U-87 MG and U-251 MG cells, respectively. Furthermore, AKIP1, CXCL1/2/8 expressions in 10 GBM and 10 low-grade glioma (LGG) tumor samples were detected. AKIP1 was elevated in various GBM cell lines compared to normal human astrocytes. AKIP1 overexpression promoted U-87 MG and U-251 MG cell proliferation and invasion while inhibited apoptosis; and it enhanced chemoresistance to temozolomide (but not cisplatin) and radiation resistance; then AKIP1 knockdown showed the opposite effects. Meanwhile, AKIP1 positively regulated CXCL1/2/8, NF-κB pathway, AKT pathway and PD-L1 expression. Further multiple compensative experiments uncovered that CXCL1 and CXCL8 promoted proliferation, invasion, chemoradiation resistance, NF-κB pathway, AKT pathway and PD-L1 expression in U-87 MG and U-251 MG cells, also in pGPH1-AKIP1 (AKIP1 knockdown) transfected U-87 MG and U-251 MG cells; although CXCL2 exhibited similar treads, but its effect was much weaker. Besides, NF-κB pathway inhibitor and AKT pathway inhibitor attenuated the effect of CXCL1&CXCL8 on promoting GBM cell malignant behaviors. Clinically AKIP1 and CXCL1/8 were elevated in GBM compared to LGG tumor samples, and they were inter-correlated. AKIP1 promotes GBM viability, mobility and chemoradiation resistance via regulating CXCL1 and CXCL8 mediated NF-κB and AKT pathways.     

Function of aberrant EGFR in malignant gliomas

The most common alteration of the epidermal growth factor receptor (EGFR) gene in human malignant gliomas is an in-frame deletion of exon 2–7 from the extracellular domain. To study the relationship between the expression of this aberrant EGFR and cell proliferation, as well as apoptosis in malignant gliomas, we have developed U-87MG cell transfectants that express the aberrant (mutant-type) or normal (wild-type) EGFR. We analyzed cell number, tumor volume, and MIB-1 positive rate as proliferation markers, and found that in tissue culture, tumors derived from U-87 MG cells (mutant-type) have the same proliferative activity as those derived from U-87 MG cells (wild-type). However, when cells expressing mutant EGFR were implanted into nude mice subcutaneously, the tumorigenic capacity was much enhanced. We also found that the apototic index of tumors derived from U-87 MG cells (mutant-type) was less than 0.1%, whereas that of wild-type tumor was 1%. These results suggest that aberrant EGFR affects the malignancy of glioma by stimulating proliferation and inhibiting apoptosis.     

Tissue inhibitor of metalloproteinase-3 expression from an oncolytic adenovirus inhibits matrix metalloproteinase activity in vivo without affecting antitumor efficacy in malignant glioma

Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, AdDelta24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus AdDelta24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by AdDelta24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2-activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the AdDelta24TIMP-3-treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87deltaEGFR) tumors with AdDelta24TIMP-3 and AdDelta24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of AdDelta24 did not significantly affect the antitumor efficacy of this oncolytic agent.     

Long-term tumor-free survival from treatment with the GFP-TRAIL fusion gene expressed from the hTERT promoter in breast cancer cells

We evaluated anti-tumor activity and toxic effect of an adenoviral vector expressing the GFP/TRAIL fusion gene from the hTERT promoter (designated Ad/gTRAIL) on human breast cancer cell lines and on normal human breast cells. Treatment with Ad/gTRAIL elicited high levels of transgene expression and apoptosis in a variety of breast cancer cell lines. Furthermore, treatment with Ad/gTRAIL was effective in killing breast cancer lines resistant to doxorubicin or soluble TRAIL protein. In contrast, only minimal transgene expression and toxicity was detected in normal human primary mammary epithelial cells after treatment with this vector. An in vivo study further showed that the intralesional administration of Ad/gTRAIL effectively suppressed the growth of human tumor xenografts derived from both doxorubicin-sensitive and doxorubicin-resistant breast cancer lines. Specifically, about 50% of animals bearing doxorubicin-sensitive and doxorubicin-resistant breast cancer xenografts showed complete tumor regression and remained tumor-free for over 5 months. These results suggest that the adenovirus encoding the GFP/TRAIL gene driven by the hTERT promoter has potential application in cancer therapy.     

In vivo studies of adenovirus-mediated p53 gene therapy for cis-platinum-resistant human ovarian tumor xenografts.

We have recently reported that mutations of the tumor suppressor p53 gene are associated with the development of resistance to cis-platinum in human ovarian cancer cells, and that adenovirus-mediated reintroduction of the wild-type p53 (wtp53) gene in ovarian tumor cells resulted in the sensitization of tumor cells to cis-diamminedichloroplatinum (II) (CDDP). The purpose of this study was to evaluate whether i.p. treatment of CDDP-resistant tumor cells expressing mutant p53 (mutp53) with a recombinant adenovirus expressing wtp53 (Adwtp53) would result in the sensitization of resistant cells to CDDP. In order to determine whether i.p. injection of a recombinant adenovirus would result in expression of the transgene in tumor cells growing intraperitoneally, we first injected A2780/CP cells in nude mice and 10 days later the mice were injected i.p. with a recombinant adenovirus expressing beta-galactosidase (Ad beta-gal). Twenty-four hours following i.p. injection of Ad beta-gal, tumors were removed and stained for beta-gal. While tumors showed extensive staining for beta-gal, indicating internalization of adenovirus and the expression of the transgene in tumors, no expression of beta-gal protein was detected in liver. I.p. treatment of A2780/CP tumor xenografts with Adwtp53 caused extensive tumor cell death, which was further enhanced by CDDP. Treatment with Adwtp53 (5 x 10(7) pfu/day, 3-5 treatments) resulted in a significant decrease in tumor volume and increase in animal survival compared to either no treatment or treatment with vector alone without p53 gene. Additional therapy with CDDP (1 mg/kg/day x 3-4) further reduced tumor volume and increased survival (30-40%), suggesting that combination therapy of Adwtp53 and CDDP was better than single agents alone. Our results indicate that i.p. dosing with adenovirus-mediated wtp53 gene therapy could be beneficial in combination with CDDP for the treatment of ovarian tumors expressing mutp53.     

Concomitant use of Ad5/35 chimeric oncolytic adenovirus with TRAIL gene and taxol produces synergistic cytotoxicity in gastric cancer cells

Chimeric adenoviral vectors possessing fiber derived from human adenovirus subgroup B (Ad35) have been developed for their high infection efficiency in cell types which are refractory to adenovirus serotype 5 (Subgroup C). The present study constructed an E1B-deleted chimeric oncolytic adenovirus, SG235-TRAIL, which carries a human TRAIL gene expression cassette and whose fiber shaft and knob domains are from serotype Ad35. It was found that SG235-TRAIL preferentially replicated in gastric cancer cell lines, SGC-7901 and BGC-823 compared to in normal human fibroblast BJ cells. Also, when compared with a replication-deficient chimeric vector Ad5/35-TRAIL, SG235-TRAIL mediated a higher level of the transgene expression via viral replication in the cancer cells. Further, because of the more efficient cell-entry and infection, SG235-TRAIL induced stronger cell apoptosis than the Ad5 CRAD vector, ZD55-TRAIL. In addition, SG235-TRAIL in combination with the chemotherapeutic drug, taxol, produced a synergistic cytotoxic effect in cancer cells in vitro without causing significant toxicity to normal cells. In the gastric tumor xenograft mouse model, intratumoral SG235-TRAIL injection produced a significant antitumor effect 14 days after treatment. Pathological examination demonstrated TRAIL expression and associated apoptosis in majority of SG235-TRAIL-treated tumor cells. These results suggest that SG235-TRAIL is a potential novel, efficient anti-cancer agent, and in combination with taxol, it would be even more useful with considerably low toxic side effects.     

慢病毒载体介导RNAi稳定抑制VASH1表达对人脑胶质瘤细胞增殖、凋亡影响的体外研究

目的 探讨运用慢病毒载体介导的RNA干扰技术抑制血管生成抑制因子1（VASH1）的表达对人脑胶质瘤U-87MG细胞增殖和凋亡的影响。方法 应用pGCL-GFP质粒构建针对VASH1的慢病毒shRNA载体,转染入工具细胞293T,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞;通过RT-PCR和Western blot评价其对U-87MG细胞内VASH1表达的影响;用四甲基偶氮唑蓝（MTT）法观察U-87MG细胞增殖活性的改变;用流式细胞仪（FCM）检测U-87MG细胞凋亡的变化。结果 与转染阴性对照组和空白组相比,转染pGCL-GFP-VASH1慢病毒载体的U-87MG细胞VASH1的mRNA 蛋白表达水平明显降低（P<0.01）;细胞的增殖活性显著上调（P<0.01）,细胞凋亡明显减少（P<0.01）。结论 VASH1 shRNA慢病毒载体可抑制VASH1在人脑胶质瘤U-87MG细胞中的表达,并增加肿瘤细胞的增殖活性,抑制细胞凋亡。     

Selective effects of a fiber chimeric conditionally replicative adenovirus armed with hep27 gene on renal cancer cell

Adenoviruses mediated cancer gene therapies are widely investigated and show a promising effect on cancer treatment. However, efficient gene transfer varies among different cancer cell lines based on the expression of coxsakie adenovirus receptor (CAR). Hep27, a member of dehydrogenase/reductase (SDR) family, can bind to Mdm2, resulting in the attenuation of Mdm2-mediated p53 degradation. Here we constructed a fiber chimeric adenovirus carrying hep27 gene (F5/35-ZD55-Hep27), in which the fiber protein of 5-serotype adenovirus (Ad5) was substituted by that of 35-serotype adenovirus (Ad35), aiming to facilitate the infection for renal cancer cells and develop the role of hep27 in cancer therapy. We evaluated the CAR and CD46 (a membrane cofactor protein for Ad35) expression in four kinds of renal cancer cells and assessed the relationship between receptors and infection efficiency. 5/35 fiber-modified adenovirus had a much promising infectivity compared with Ad5-based vector in renal cancer cells. F5/35-ZD55-Hep27 had enhanced antitumor activity against human renal cancer cells compared to the other groups. Further, hep27 mediated p53 and cleaved-PARP upregulation and mdm2 downregulation was involved and caused increased apoptosis. Moreover, F5/35-ZD55-Hep27 significantly suppressed tumor growth in subcutaneous renal cancer cell xenograft models. Our data demonstrated that 5/35 fiber-modified adenovirus F5/35-ZD55-Hep27 transferred into renal cancers efficiently and increased p53 to induce cancer cell apoptosis. Thus 5/35 fiber-modified adenoviral vector F5/35-ZD55-Hep27 might a promising vector and antitumor reagent for renal cancer gene therapy.     

A tumor-specific conditionally replicative adenovirus vector expressing TRAIL for gene therapy of hepatocellular carcinoma

We constructed a novel hepatocellular carcinoma-specific conditionally replicative adenovirus (CRAd). This adenovirus, designated Ad.HS4.AFP.E1A/TRAIL, expresses E1A to mediate viral replication and TRAIL to enhance HCC-killing efficacy under the control of a modified AFP promoter. An insulator HS-4 was placed in front of the AFP promoter to enhance the fidelity of the heterologous promoter. This virus was shown to have specific cytolytic activity in AFP-expressing HCC cells in vitro. Furthermore, the replication efficiency of Ad.HS4.AFP.E1A/TRAIL correlated well with AFP expression of the host cells, showing a 100-fold and 1 000 000-fold decrease in the low-and non-AFP-expressing HCC cells, respectively, compared to the high AFP-expressing HCC cells. An increase in mRNA of TRAIL and the elevated Caspase-3 activity were also observed in Ad.HS4.AFP.E1A/TRAIL-infected HCC cells. These results indicated that TRAIL expression from the viral vector activated the Caspase-3 enzymatic capacity and the HCC cells were sensitive to TRAIL. In vivo, Ad.HS4.AFP.E1A/TRAIL effectively prevented the growth of low AFP-expressing BEL-7404 xenografts. These results indicate that Ad.HS4.AFP.E1A/TRAIL could provide a new strategy of gene therapy for HCC.     

Introduction of wild-type p53 enhances thrombospondin-1 expression in human glioma cells

Malignant gliomas are distinguished from low-grade gliomas by their intense angiogenesis. In gliomas, p53 is the most frequently altered gene and is involved in the early phase of glioma development. In contrast, homozygous p16 gene deletion is more common in high-grade gliomas. In order to understand the mechanism by which gliomas become more angiogenic during the malignant transformation, we examined the relationship between thrombospondin-1, a negative regulator in angiogenesis, and these tumor suppressor genes in malignant gliomas. Human glioma cell line U-251 MG, which has mutated p53 and deleted p16, was transduced with recombinant replication-defective adenovirus vectors containing the cDNA of wild-type p53, p16, and p21. Only the induction of wild-type p53 enhanced expression of thrombospondin-1 mRNA and the protein in U-251 MG cells. Furthermore, thrombospondin-1 that was secreted in the culture medium was significantly increased (3.8-fold) as compared with that of the viral control 36 h after infection with Ad5CMV-p53. In the presence of wild-type p53 plasmid DNA, the promoter activity was increased 7.4-fold as compared with an empty expression vector control. These studies may suggest that mutation of p53 gene endows gliomas with an angiogenic phenotype by reducing thrombospondin-1 production as well as enhancing the angiogenesis inducers in the early phase of malignant progression.     

Adenovirus-mediated IKKbetaKA expression sensitizes prostate carcinoma cells to TRAIL-induced apoptosis

Despite the fact that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in cancer cells, TRAIL resistance in cancer cells has challenged the use of TRAIL as a therapeutic agent. First, prostate carcinoma cell lines (DU145, LNCaP and PC3) were screened for sensitivity to adenovirus delivery of TRAIL (Ad5hTRAIL). As amplified Ikappa B kinase (IKK) activity is responsible for the constitutive nuclear factor-kappaB (NF-kappaB) activation leading to uncontrolled cell growth and metastasis, a dual vector approach using both an adenovirus vector (Ad) expressing the dominant-negative mutant of IKKbeta (AdIKKbetaKA) and Ad5hTRAIL was employed to determine if prostate cancer cells were sensitized to TRAIL in the setting of IKK inhibition. Inhibition of the NF-kappaB pathway through IKK blockade sensitized all three prostate cancer cell lines to TRAIL, regardless of NF-kappaB activation or decoy receptor gene expression. Moreover, a novel quantitative real-time RT-PCR assay and conventional flow cytometry analysis indicated that TRAIL-resistant DU145 and LNCaP cells, but not TRAIL-sensitive PC3 cells, expressed substantial amounts of TRAIL Decoy Receptor 4. In conclusion, TRAIL decoy receptor expression appeared to be the chief determinant of TRAIL resistance encountered in prostate carcinoma cell lines.     

Effect of adenovirus-mediated heat shock protein expression and oncolysis in combination with low-dose cyclophosphamide treatment on antitumor immune responses

Heat shock proteins such as gp96 have the ability to chaperone peptides and activate antigen-presenting cells. In this study, we tested whether adenovirus-mediated overexpression of secreted or membrane-associated forms of gp96 in tumor cells would stimulate an antitumor immune response. Studies were carried out in C57Bl/6 mice bearing aggressively growing s.c. tumors derived from syngeneic TC-1 cells, a cell line that expresses HPV16 E6 and E7 proteins. We found that secreted gp96 can induce protective and therapeutic antitumor immune responses. Our data also indicate that the antitumor effect of sgp96 expression seems to be limited by the induction of suppressive regulatory T cells (Treg). TC-1 tumor transplantation increased the number of splenic and tumor-infiltrating Tregs. Importantly, treatment of mice with low-dose cyclophosphamide decreased the number of Tregs and enhanced the immunostimulatory effect of sgp96 expression. We also tested whether an oncolytic vector (Ad.IR-E1A/TRAIL), that is able to induce tumor cell apoptosis and, potentially, release cryptic tumor epitopes in immunogenic form, could stimulate antitumor immune responses. Although tumor cells infected ex vivo with Ad.IR-E1A/TRAIL had no antitumor effect when used as a vaccine alone, the additional treatment with low-dose cyclophosphamide resulted in the elimination of pre-established tumors. This study gives a rationale for testing approaches that suppress Tregs in combination with oncolytic or immunostimulatory vectors.     

Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IkappaBalpha and HSV thymidine kinase

To improve the effectiveness of herpes simplex virus (HSV) thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication-defective HSV vector TOIkappaB expressing both HSV-TK and a mutant form of the NF-kappaB inhibitor IkappaBalpha (IkappaBalphaM) was developed. TOIkappaB was constructed by recombining the IkappaBalphaM gene into the U(L)41 locus of a replication-defective lacZ expression vector, TOZ.1. Expression of IkappaBalphaM was confirmed by Western blotting, and the ability of the mutant protein to inhibit NF-kappaB nuclear translocation was examined by electrophoretic mobility shift assay. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-kappaB was already translocated to the nucleus without receptor-dependent signaling by TNF-alpha. Following infection with TOIkappaB, nuclear translocation of NF-kappaB in U-87MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIkappaB for U-87MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIkappaB infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95-100% for TOIkappaB and 80-85% for TOZ.1. TOIkappaB neurotoxicity measured on cultured murine neurons was relatively low and similar to that of TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOIkappaB injection and GCV administration. Survival of TOIkappaB+GCV group was significantly longer (P<.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIkappaB only, PBS or PBS+GCV). These results suggest that IkappaBalphaM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo.