Tumor-specifically hypoxia-induced therapy of SPRY1/2 displayed differential therapeutic efficacy for melanoma

Activation of receptor tyrosine kinase (RTK) signalling pathways is frequently correlated to cancer cell proliferation, angiogenesis and cell survival. Sprouty (SPRY) proteins function as a physiological endogenous inhibitor of RTK signalling pathways, have been shown to be deregulated in most cancer cells. Here, we demonstrated that over-expression of SPRY1 and SPRY2 inhibited B16F10 cell proliferation through G1 phase arrest in vitro, and SPRY2 showed more potent inhibitory effects than SPRY1. In order to tumor-specific delivery of SPRY1/2 in vivo, two strains of attenuated Salmonella typhimurium VNP20009 (VNP-PQE-SPRY1 and VNP-PQE-SPRY2) were constructed to specifically express SPRY1 or SPRY2 under the control of a hypoxia-induced nirB promoter. The efficiency and specificity of the recombinant strains were validated in both bacteria and animal tumor models. SPRY1 and SPRY2 gene could be specifically driven by the nirB promoter under hypoxia, but not normoxia conditions. In addition, the tumor-targeting ability of VNP-PQE-SPRY1 or VNP-PQE-SPRY2 was similar with VNP. VNP-PQE-SPRY2 significantly suppressed melanoma growth in vivo, suggesting that SPRY2 is a more efficient agent for melanoma therapy. Moreover, the antitumor effect of VNP-SPRY2 is mainly mediated through the inhibition of ERK1/2 phosphorylation, which leads to the inhibition of proliferation in melanoma. Taken together, our results indicated that SPRY2 displayed more potent melanoma suppression than SPRY1 both in vitro and in vivo, and the hypoxia-induced tumor-specific gene therapy of SPRY2 delivered by VNP20009 is a promising strategy for melanoma therapy.     

Antitumor effect of VNP20009, an attenuated Salmonella, in murine tumor models

VNP20009, a genetically modified strain of Salmonella typhimurium with deletions in the msbB and purI loci, exhibited antitumor activities when given systemically to tumor-bearing mice. VNP20009 inhibited the growth of subcutaneously implanted B16F10 murine melanoma, and the human tumor xenografts Lox, DLD-1, A549, WiDr, HTB177, and MDA-MB-231. A single intravenous injection of VNP20009, at doses ranging from 1 x 10(4) to 3 x 10(6) cfu/mouse, produced tumor growth inhibitions of 57-95%. Tumor volume doubling time, another indicator for tumor growth inhibition, also significantly increased in mice treated with VNP20009. Using mice with immune system deficiencies, we also demonstrated that the antitumor effects of VNP20009 did not depend on the presence of T and B cells. In addition, VNP20009, given intravenously, inhibited the growth of lung metastases in mice. Only live bacteria showed the antitumor effect.     

Tumor-targeting Salmonella typhimurium improves cyclophosphamide chemotherapy at maximum tolerated dose and low-dose metronomic regimens in a murine melanoma model

Chemotherapy for cancer is partly limited by the inability of drugs to act on poorly vascularized or avascularized areas of tumors. Tumor-targeting bacteria are capable of preferentially replicating in these poorly perfused regions. Some strains have been combined with chemotherapeutic agents and the results have been promising. However, no systematic work has been carried out to test the effect of bacteria on clinical modes of chemotherapy, such as standard maximum tolerated dose (MTD) and novel low-dose metronomic (LDM) chemotherapy. Here Salmonella typhimurium VNP20009 was combined with cyclophosphamide (CTX) at both MTD and LDM schedules in a murine melanoma model. The results showed that VNP20009 significantly improved the effects of all forms of CTX treatments. The combination of VNP20009 and CTX led to a more significant decrease in tumor microvessel density and serum vascular endothelial growth factor (VEGF) level, compared with either treatment alone. Furthermore, combination therapy remarkably increased the number of bacteria within tumors when compared with bacteria treatment alone. These findings suggest that tumor-targeting bacteria, in conjunction with CTX at standard MTD and LDM regimens, might be of clinical value for the treatment of melanoma.     

Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma.

PURPOSE: A strain of Salmonella typhimurium (VNP20009), attenuated by chromosomal deletion of the purI and msbB genes, was found to target to tumor and inhibit tumor growth in mice. These findings led to the present phase I study of the intravenous infusion of VNP20009 to patients with metastatic cancer. PATIENTS AND METHODS: In cohorts consisting of three to six patients, 24 patients with metastatic melanoma and one patient with metastatic renal cell carcinoma received 30-minute intravenous bolus infusions containing 10(6) to 10(9) cfu/m(2) of VNP20009. Patients were evaluated for dose-related toxicities, selective replication within tumors, and antitumor effects. RESULTS: The maximum-tolerated dose was 3 x 10(8) cfu/m(2). Dose-limiting toxicity was observed in patients receiving 1 x 10(9) cfu/m(2), which included thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia. VNP20009 induced a dose-related increase in the circulation of proinflammatory cytokines, such as interleukin (IL)-1beta, tumor necrosis factor alpha, IL-6, and IL-12. Focal tumor colonization was observed in two patients receiving 1 x 10(9) cfu/m(2) and in one patient receiving 3 x 10(8) cfu/m(2). None of the patients experienced objective tumor regression, including those patients with colonized tumors. CONCLUSION: The VNP20009 strain of Salmonella typhimurium can be safely administered to patients, and at the highest tolerated dose, some tumor colonization was observed. No antitumor effects were seen, and additional studies are required to reduce dose-related toxicity and improve tumor localization.     

Oral delivery of tumor-targeting Salmonella for cancer therapy in murine tumor models

Tumor-targeting bacteria have been investigated intensively in recent years as anticancer agents. To ensure the reliability of infection, bacteria have conventionally been injected intravenously or intraperitoneally into animals or humans. However, systemic infection of bacteria is rather inconvenient and carries the risk of obvious toxicity. Here we tested whether Salmonella typhimurium VNP20009, a tumor-targeting strain, could be administrated orally for tumor therapy. Tumor-targeting potential, antitumor effects, as well as toxicity of orally administrated VNP20009 were investigated in this study. Oral delivery of VNP20009 not only exhibited high tumor-targeting potential, but also led to a significant anticancer effect by delaying tumor growth and prolonging survival in murine tumor models. As part of combination therapy, orally administrated bacteria notably improved the antitumor effect of cyclophosphamide. In vitro and in vivo studies showed that VNP20009 significantly induced tumor cell apoptosis. No obvious toxicity was observed during the treatments with oral inoculation of VNP20009. Comparative analysis of toxicity in tumor-bearing and tumor-free mice further revealed that orally administrated Salmonella had high safety compared to conventional systemic infection of bacteria. The findings indicated that oral administration of tumor-targeting bacteria is effective and safe. This approach provides a novel avenue in the application of bacteria as a potential antitumor agent.     

Tumor amplified protein expression therapy: Salmonella as a tumor-selective protein delivery vector

Attenuated strains of Salmonella typhimurium, VNP20009 and YS7212, when injected systemically to tumor-bearing mice, accumulated preferentially in tumors at levels at least 200-fold and, more commonly, 1000-fold greater than in other normal tissues. This selectivity occurred in subcutaneously implanted murine tumors, including B16F10 melanoma, M27 lung carcinoma, and colon 38 carcinoma. The preferential accumulation was also manifested in animals bearing human tumor xenografts, including Lox, C8186, DLD1, SW620, HCT116, HTB177, DU145, MDA-MB-231, and Caki. Four to five days after a single IV injection of 1 x 10(6) colony-forming unit (cfu)/mouse, we routinely detected VNP20009 proliferation and accumulation at levels ranging from 1 x 10(8) to 2 x 10(9) cfu/g tumor. The amount of VNP20009 accumulated in the liver ranged from 3 x 10(4) to 2 x 10(6) cfu/g. The distribution of Salmonella in tumors was homogenous; YS7212 could be detected from the periphery to the interior portion of the tumors. Using mice with various immunodeficiencies, we also discovered the same preferential accumulation of Salmonella in tumors implanted in these mice. The use of Salmonella as a protein delivery vector was shown by IV administration of the bacteria expressing either green fluorescent protein (GFP) or cytosine deaminase (CD) into tumor-bearing mice. GFP and CD were detected in tumors, but not in livers, taken from mice inoculated with Salmonella carrying these genes. Bacteria accumulation and CD expression persisted in the tumors for up to 14 days after a single bolus IV administration of bacteria to tumor-bearing mice.     

Enhanced therapeutic effect by combination of tumor-targeting Salmonella and endostatin in murine melanoma model

The growth of tumor is angiogenesis-dependent and it often contains hypoxia and necrotic areas. Salmonella VNP20009 could target and replicate in hypoxia and necrotic areas within tumor and induce antitumor effect. Angiogenesis inhibitor endostatin could reduce tumor angiogenesis and inhibit its growth. However, in the phase I trials of VNP20009 and endostatin at the maximum-tolerated dose, no antitumor effects for bacteria therapy and minor therapeutic effects for endostatin treatment were seen. The ineffectiveness of these agents in clinical trials suggests that the combination of these agents with synergic modalities might be necessary. Here we described antitumor effects mediated by the combination of VNP20009 with recombinant human endostatin in B16F10 murine melanoma model with the aim to exploit tumor-targeting of bacteria and anti-angiogenesis strategy to enhance therapeutic efficacy. Combination therapy of these agents significantly enhanced antitumor effects by inducing greater tumor growth inhibition, more severe tumor tissue necrosis as well as less blood vessel density than those induced by either of treatments. The findings suggest that the combination of tumor-targeting bacteria with angiogenesis inhibitor might be of value for the treatment of solid tumors.     

Suppression of pancreatic ductal adenocarcinoma growth by intratumoral delivery of attenuated Salmonella typhimurium using a dual fluorescent live tracking system

Pancreatic ductal adenocarcinoma (PDAC) has the poorest prognosis among all malignancies and is resistant to almost all current therapies. Attenuated Salmonella typhimurium strain VNP20009 has been deployed as powerful anticancer agent in a variety of animal cancer models, and previous phase 1 clinical trials have proven its safety profiles. However, thus far, little is known about its effect on PDAC. Here, we established CFPAC-1 cell lines expressing an mKate2 protein and thus emitting far-red fluorescence in the subsequent xenograft implant. VNP20009 strain was further engineered to carry a luciferase cDNA, which catalyzes the light-emitting reaction to allow the observation of salmonella distribution and accumulation within tumor with live imaging. Using such VNP20009 strain and intratumoral delivery, we could reduce the growth of pancreatic cancer by inducing apoptosis and severe necrosis in a dosage dependent manner. Consistent with this finding, intratumoral delivery of VNP20009 also increase caspase-3 activity and the expression of Bax protein. In summary, we revealed that VNP20009 is a promising bacterial agent for the treatment of PDAC, and that we have established a dual fluorescent imaging system as a valuable tool for noninvasive live imaging of solid tumor and engineered bacterial drug.     

Comparison of the selective targeting efficacy of Salmonella typhimurium A1-R and VNP20009 on the Lewis lung carcinoma in nude mice

Salmonella typhimurium A1-R is auxotrophic for arg and leu, which attenuates growth in normal tissue but allows high tumor targeting and virulence. A1-R is effective against metastatic human prostate, breast, and pancreatic cancer as well as osteosarcoma, fibrosarcoma, and glioma in clinically-relevant mouse models. VNP20009 is also a genetically-modified strain of Salmonella typhimurium that has been tested in Phase I clinical trials, but is more attenuated than S. typhimurium A1-R and in addition of multiple amino-acid auxotrophs, is purine auxotropic with the purI mutation. In the present study, mouse Lewis lung carcinoma-bearing nude mouse models were treated with S. typhimurium A1-R or VNP20009. S. typhimurium A1-R and VNP20009 were both eliminated from the liver and spleen approximately 3-5 days after administration via the tail vein. However, A1-R showed higher tumor targeting and inhibited the Lewis lung carcinoma to a greater extent than VNP20009, with less body weight loss. The mice tolerated S. typhimurium A1-R to at a least 2-fold higher dose than VNP20009 when the bacteria were administered iv. The results of the present study suggest that S. typhimurium A1-R has greater clinical potential than VNP20009.     

Systemic administration of an attenuated, tumor-targeting Salmonella typhimurium to dogs with spontaneous neoplasia: phase I evaluation.

PURPOSE: Genetically modified bacteria are a potentially powerful anticancer therapy due to their tumor targeting capacity, inherent antitumor activity, and ability to serve as efficient vectors for gene delivery. This study sought to characterize the acute and short-term toxicities and tumor colonization rates of a genetically modified Salmonella typhimurium (VNP20009) in dogs with spontaneous tumors, in the context of a phase I dose escalation trial. EXPERIMENTAL DESIGN: Forty-one pet dogs with a variety of malignant tumors received weekly or biweekly i.v. infusions of VNP20009, at doses ranging from 1.5 x 10(5) to 1 x 10(8) cfu/kg. Vital signs and clinicopathologic variables were monitored regularly. Incisional biopsies were obtained before and 1 week following the first infusion for histopathology and bacterial culture. RESULTS: The nominal maximum tolerated dose was 3 x 10(7) cfu/kg, with refractory fever and vomiting being the dose-limiting toxicities. One treatment-related acute death occurred. Bacteria were cultured from tumor tissue in 42% of cases. Thirty-five patients were evaluable for antitumor response. Major antitumor responses were seen in 15% (4 complete response and 2 partial response), and disease stabilization for at least 6 weeks in 10%. CONCLUSIONS: Administration of VNP20009 at doses with acceptable toxicity results in detectable bacterial colonization of tumor tissue and significant antitumor activity in tumor-bearing dogs.     

A Salmonella Typhimurium mutant strain capable of RNAi delivery: Higher tumor-targeting and lower toxicity

Bacteria are highly versatile and useful tools that could deliver short interfering RNA. In this study, a phoP/phoQ double-deleted Salmonella Typhimurium named VNP(PhoP/Q⁻) based on the genetic background of VNP20009. The biological safety and function of VNP(PhoP/Q⁻) were also analyzed. Our study revealed the following results: (1) VNP(PhoP/Q⁻) exhibited lower titers in tumor-free livers and spleens than VNP20009, (2) The survival of VNP(PhoP/Q⁻) in macrophages and 4T1 tumor cells was significantly reduced compared with that of VNP20009, (3) The tumor-targeting ability of VNP(PhoP/Q⁻) was significantly enhanced compared with that of VNP20009, and the anticancer effects of VNP(pPhoP/Q⁻) and VNP20009 on tumor-bearing mice were similar, (4) VNP(PhoP/Q⁻) could release an shRNA-expressing plasmid and express the EGFP reporter gene in tumor tissue. Therefore, VNP(PhoP/Q⁻) exhibited a better safety level in tumor-free mice and elicited an anti-tumor effect on tumor-bearing mice. Moreover, VNP(PhoP/Q⁻) could release an shRNA-expressing plasmid into the cytoplasm of host cells to silence targeted genes.     

Oral delivery of tumor-targeting Salmonella exhibits promising therapeutic efficacy and low toxicity

Tumor‐targeting bacteria have been developed as powerful anticancer agents. Salmonella typhimurium VNP20009, a representative tumor‐targeting strain, has been systemically administered as a single‐agent therapy at doses of 1 × 10⁶ to 3 × 10⁶ colony‐forming unit (cfu)/mouse, or in combination with other antitumor agents at doses of 1 × 10⁴ to 2 × 10⁵ cfu/mouse. Recently, we reported that oral delivery of VNP20009 at the dose of 1 × 10⁹ cfu/mouse induced significant anticancer effects comparable to that induced by systemic administration of this strain at 1 × 10⁴ cfu/mouse. To further address the efficacy and safety of oral administration of bacteria, here we performed a systemically comparative analysis of anticancer efficacy and toxicity of VNP20009 administered: (i) orally at a dose of 1 × 10⁹ cfu/mouse (VNP9‐oral); (ii) intraperitoneally at a dose of 1 × 10⁴ cfu/mouse (VNP4‐i.p.); or (iii) intraperitoneally at a dose of 1 × 10⁶ cfu/mouse in tumor‐free and tumor‐bearing murine models. The results showed that VNP9‐oral, similar to VNP4‐i.p., induced significant tumor growth inhibition whereas VNP6‐i.p. induced better anticancer effect in the B16F10 melanoma model. Among three treatments, VNP9‐oral induced the mildest and reversible toxicity whereas VNP6‐i.p. resulted in the most serious and irreversible toxicities when compared to other two treatments. Moreover, the combination of VNP9‐oral with a low dose of chemotherapeutics produced comparable antitumor effects but displayed significantly reduced toxicity when compared to VNP6‐i.p. The findings demonstrated that oral administration, as a novel avenue in the application of bacteria, is highly safe and effective. Moreover, the present preclinical study should facilitate the optimization of bacterial therapies with improved anticancer efficacy and reduced adverse effects in future clinical trials. (Cancer Sci 2009; 100: 2437–2443)     

Synergistic effects of arsenic trioxide combined with Salmonella typhimurium in treating the advanced hepatocellular carcinoma in rat models

BackgroundTo evaluate the safety and efficacy of arsenic trioxide (ATO) combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 in treating the advanced hepatocellular carcinoma (HCC).MethodsThe proliferation assay, migration assay and real-time PCR analyses were performed to assess the impact of ATO combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 on the McA-RH7777 cells. Forty Buffalo rats were orthotopically implanted with HCC in the livers and randomly divided into four groups: (A) ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009; (B) ATO; (C) {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009; and (D) control. ATO (2 mg/kg) was administered by peritoneal injection once a day and continued for five days. {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 (about 1×10⁷ CFU) was directly injected into the tail vein. MRI examinations were performed to access the tumor responses one and 2 weeks later, respectively. Micro CT scans of chest were performed to assess the lung metastases. Hematoxylin-eosin (HE) staining and immunohistochemical analyses were performed to analyze the tumor tissues.ResultsIn the in vitro experiments, {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 suppressed the proliferation of McA-RH7777 cells, attenuated their migration ability, and weakened the potential of metastases. MRI examinations showed that the mean residual tumor volumes of ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group on the 7th day and 14th day after the administration of ATO combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 were significantly smaller than those of other groups. Micro CT scans revealed that the lung metastases rates of ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group and {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group were significantly lower than those of other groups. Immunohistochemical analyses displayed that the levels of VEGF and Vimentin in the tumors of ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group were obviously lower than those of other groups. The median survival of rats in the ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group was longer than those of other groups.ConclusionsThe strategy of ATO combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 was safe and had a potential to inhibit tumor growth, decrease the lung metastases, and prolong the overall survival in treating the advanced HCC. The two complementary interventions may have synergistic effects.     

重组沙门氏菌VNP20009-M对骨肉瘤的治疗作用

目的 研究携带甲硫氨酸酶基因的减毒沙门氏菌VNP20009-M对骨肉瘤的治疗作用及其机制。方法 将重组沙门氏菌VNP20009-M与骨肉瘤细胞MNNG-HOS共培养;骨肉瘤细胞MNNGHOS、U2OS及SaoS-2均高表达甲硫氨酸酶基因后探索细胞增殖、迁移及凋亡能力的变化;构建MNNG-HOS裸鼠皮下荷瘤模型,评价不同剂量的VNP20009-M在动物模型中的治疗效果。结果 通过质粒PCR验证甲硫氨酸酶基因只存在于目的菌株VNP20009-M中,且具有较高的甲硫氨酸酶活性,重组沙门氏菌构建成功。VNP20009-M显著诱导骨肉瘤细胞MNNG-HOS的凋亡。相比对照组,甲硫氨酸酶基因过表达的骨肉瘤细胞增殖、迁移能力被显著抑制。VNP20009-M治疗后小鼠皮下肿瘤生长被显著抑制。结论 VNP20009-M可诱导骨肉瘤细胞凋亡并抑制癌细胞增殖和迁移,可以作为一种新型高效的药物为临床提供新的治疗方式。     

Mcl-1 targeting strategies unlock the proapoptotic potential of TRAIL in melanoma cells

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis selectively in cancer cells. For melanoma, the targeting of TRAIL signaling appears highly attractive, due to pronounced TRAIL receptor expression in tumor tissue. However, mechanisms of TRAIL resistance observed in melanoma cells may limit its clinical use. The Bcl-2 family members are critical regulators of cell-intrinsic apoptotic pathways. Thus, the antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1) is overexpressed in many tumor types and was linked to chemotherapy resistance in melanoma. In this study, we evaluated the involvement of antiapoptotic Bcl-2 proteins (Bcl-2, Bcl-x_L, Bcl-w, Mcl-1, Bcl-A1, and Bcl-B) in TRAIL resistance. They were targeted by small interfering RNA-mediated silencing in TRAIL-sensitive (A-375, Mel-HO) and in TRAIL-resistant melanoma cell lines (Mel-2a, MeWo). This highlighted Mcl-1 as the most efficient target to overcome TRAIL resistance. In this context, we investigated the effects of Mcl-1-targeting microRNAs as well as the Mcl-1-selective inhibitor S63845. Both miR-193b and S63845 resulted in significant enhancement of TRAIL-induced apoptosis, associated with decreased cell viability. Apoptosis induction was mediated by caspase-3 processing as well as by Bax and Bak activation, indicating the critical involvement of intrinsic apoptosis pathways. These data may indicate a high relevance of Mcl-1 targeting also in melanoma therapy. Furthermore, the data may suggest to consider the use of the tumor suppressor miR-193b as a strategy for countering TRAIL resistance in melanoma.     

Restoration of XAF1 expression induces apoptosis and inhibits tumor growth in gastric cancer

XAF1 (XIAP‐associated factor 1) is a novel XIAP binding protein that can antagonize XIAP and sensitize cells to other cell death triggers. Our previous results have shown that aberrant hypermethylation of the CpG sites in XAF1 promoter is strongly associated with lower expression of XAF1 in gastric cancers. In our study, we investigated the effect of restoration of XAF1 expression on growth of gastric cancers. We found that the restoration of XAF1 expression suppressed anchorage‐dependent and ‐independent growth and increased sensitivity to TRAIL and drug‐induced apoptosis. Stable cell clones expressing XAF1 exhibited delayed tumor initiation in nude mice. Restoration of XAF1 expression mediated by adenovirus vector greatly increased apoptosis in gastric cancer cell lines in a time‐ and dose‐dependent manner and sensitized cancer cells to TRAIL and drugs‐induced apoptosis. Adeno‐XAF1 transduction induced cell cycle G2/M arrest and upregulated the expression of p21 and downregulated the expression of cyclin B1 and cdc2. Notably, adeno‐XAF1 treatment significantly inhibited tumor growth, strongly enhanced the antitumor activity of TRAIL in a gastric cancer xenograft model in vivo, and significantly prolonged the survival time of animals bearing tumor xenografts. Complete eradication of established tumors was achieved on combined treatment with adeno‐XAF1 and TRAIL. Our results document that the restoration of XAF1 inhibits gastric tumorigenesis and tumor growth and that XAF1 is a promising candidate for cancer gene therapy. © 2009 UICC     

Prediction of proapoptotic anticancer therapeutic response in vivo based on cell death visualization and TRAIL death ligand-receptor interaction

Tumor growth is often associated with insufficient apoptosis. The Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) and its proapoptotic receptors death receptor 4 (DR4) and DR5 agonistic monoclonal antibodies are being developed as targeted therapeutics because they kill cancer cells while sparing normal cells. A challenge to targeted therapeutics is the selection of patients who are most likely to benefit from targeted drugs because of the heterogeneity of cancer. Molecular imaging may be useful in targeted drug development by assessing the target expression and drug-target interaction, and for predicting therapeutic response. We hypothesized that the cell surface expression level of DR4/5 may predict the proapoptotic targeted therapeutic response if the signaling pathway downstream is intact. The goal of this proof-of-concept study was to develop a molecular imaging strategy to predict proapoptotic anti-cancer therapy response at an early stage of treatment. TRAIL and the DR5 agonistic monoclonal antibody HGS-ETR2 (Lexatumumab, TRM-2) were labeled with a near-infrared dye and these were used to image the TRAIL receptors on cultured TRAIL sensitive and TRAIL resistant human tumor cells as well as tumor xenografts. Imaging of cells and tumor-bearing animals was conducted with near infrared fluorescence imagers and apoptosis in cells was assessed by western blots of PARP-cleavage and flow cytometry of sub-G1 content. Apoptosis in tumors was evaluated by imaging near-infrared dye-labeled Annexin V and tumor tissue activated caspase-3 staining. Both in vitro and in vivo studies showed that imaging of death inducing ligand-receptor interaction was consistent with the apoptosis readout. Thus TRAIL sensitive tumors that express TRAIL receptors underwent cell death following treatment whereas tumors lacking TRAIL receptor expression were shown to be TRAIL resistant. In vivo molecular imaging of TRAIL receptor expression correlated with response to TRAIL therapy and an apoptotic response in vivo.     

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.