Wenhui Jiang

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)

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 at and replicate in hypoxia and necrosis 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 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 HSP70 expression sensitizes NSCLC cell lines to TRAIL-induced apoptosis by upregulating DR4 and DR5 and downregulating c-FLIP-L expressions

Many cancer cell types are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Here, we examined whether HSP70 suppression by small interfering RNA (siRNA) sensitized non-small cell lung cancer (NSCLC) cells to TRAIL-induced apoptosis and the underlying mechanisms. We demonstrated that HSP70 suppression by siRNA sensitized NSCLC cells to TRAIL-induced apoptosis by upregulating the expressions of death receptor 4 (DR4) and death receptor 5 (DR5) through activating NF-κB, JNK, and, subsequently, p53, consequently significantly amplifying TRAIL-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Consistently, the pro-apoptotic proteins Bad and Bax were upregulated, while the anti-apoptotic protein Bcl-2 was downregulated. The luciferase activity of the DR4 promoter was blocked by a NF-κB pathway inhibitor BAY11-7082, suggesting that NF-κB activation plays an important role in the transcriptional upregulation of DR4. Additionally, HSP70 suppression inhibited the phosphorylation of ERK, AKT, and PKC, thereby downregulating c-FLIP-L. A549 xenografts in mice receiving HSP70 siRNA showed TRAIL-induced cell death and increased DR4/DR5 levels and reduced tumor growth. The combination of psiHSP70 gene therapy with TRAIL also significantly increased the survival benefits induced by TRAIL therapy alone. Interestingly, HSP27 siRNA and TRAIL together could not suppress tumor growth or prolong the survival of tumor-bearing mice significantly, although the combination could efficiently induce the apoptosis of A549 cells in vitro. Our findings suggest that HSP70 suppression or downregulation might be promising to overcome TRAIL resistance in cancer.

Proteomic Screening of Anaerobically Regulated Promoters from Salmonella and Its Antitumor Applications

Solid tumors often contain hypoxic and necrotic areas that can be targeted by attenuated Salmonella typhimurium VNP20009 (VNP). We sought to develop a hypoxia- inducible promoter system based on the tumor-specific delivered strain VNP to confine expression of therapeutic gene specifically or selectively within the tumor microenvironment. A hypoxia-inducible promoter - adhE promoter was screened from the hypoxia-regulated endogenous proteins of Salmonella through two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight MS-based proteomics approaches. The efficiency and specificity of the selected adhE promoter were validated first in both bacteria and animal tumor models. The adhE promoter could specifically drive GFP gene expression under hypoxia, but not under normoxia. Furthermore, luciferase reporter expression controlled by the system was also confined to the tumors. Finally, we investigated the anticancer efficacy of VNP delivering human endostatin controlled by our adhE promoter system in both murine melanoma and Lewis lung carcinoma models. Our results demonstrated that by the dual effects of tumoricidal and anti-angiogenic activities, the recombinant Salmonella strain could generate enhanced antitumor effects compared with those of unarmed VNP treatment or untreated control. The recombinant VNP could retard tumor growth significantly and extend survival of tumor-bearing mice by inducing more apoptosis and more severe necrosis as well as inhibiting blood vessel density within tumors. Therefore, VNP carrying the endostatin gene under our tumor-targeted expression system holds promise for the treatment of solid tumors.

Salmonella-mediated tumor-targeting TRAIL gene therapy significantly suppresses melanoma growth in mouse model.

Attenuated Salmonella typhimurium (S. typhimurium) strains can selectively grow and express exogenous genes in tumors for targeted therapy. We engineered S. typhimurium strain VNP20009 to secrete tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) under the control of a hypoxia‐induced nirB promoter and examined the efficacy of Salmonella‐mediated targeted expression of TRAIL in mice bearing melanoma tumor and in TRAIL‐resistant RM‐1 tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. Compared with recombinant TRAIL protein and VNP20009 combination therapy, VNP20009 expressing TRAIL significantly suppressed melanoma growth but failed to suppress RM‐1 tumor growth. Furthermore, we confirmed that VNP20009 expressing TRAIL yielded its antitumor effect by inducing melanoma apoptosis. Our findings indicate that Salmonella‐mediated tumor‐targeted therapy with TRAIL could reduce tumor growth and extend host survival. (Cancer Sci 2012; 103: 325–333)

Requirement of PEA3 for Transcriptional Activation of FAK Gene in Tumor Metastasis

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase critically involved in cancer metastasis. We found an elevation of FAK expression in highly metastatic melanoma B16F10 cells compared with its less metastatic partner B16F1 cells. Down-regulation of the FAK expression by either small interfering RNA or dominant negative FAK (FAK Related Non-Kinase, FRNK) inhibited the B16F10 cell migration in vitro and invasiveness in vivo. The mechanism by which FAK activity is up-regulated in highly metastatic cells remains unclear. In this study, we reported for the first time that one of the Est family proteins, PEA3, is able to transactivate FAK expression through binding to the promoter region of FAK. We identified a PEA3-binding site between nucleotides −170 and +43 in the FAK promoter that was critical for the responsiveness to PEA3. A stronger affinity of PEA3 to this region contributed to the elevation of FAK expression in B16F10 cells. Both in vitro and in vivo knockdown of PEA3 gene successfully mimicked the cell migration and invasiveness as that induced by FAK down-regulation. The activation of the well-known upstream of PEA3, such as epidermal growth factor, JNK, and ERK can also induce FAK expression. Furthermore, in the metastatic human clinic tumor specimens from the patients with human primary oral squamous cell carcinoma, we observed a strong positive correlation among PEA3, FAK, and carcinoma metastasis. Taking together, we hypothesized that PEA3 might play an essential role in the activation of the FAK gene during tumor metastasis.

Keratocystoma of the parotid gland: a clinicopathological study and literature review §

The authors investigated the clinicopathological characteristics of keratocystoma of the parotid gland. Two cases of parotid gland keratocystoma in the files of Nanjing Stomatological Hospital were analysed. These slowly growing parotid gland tumours occurred in two women aged 29 and 49 years. The cut surface showed multilocular cystic lesions filled with keratin materials. Histologically, there were multi-cystic spaces and solid epithelium islands, containing keratinized lamellae. Without cytological atypia, the lining stratified squamous epithelium showed apparent keratinization through an orthokeratotic or parakeratotic pathway. No skin appendage formation was observed. Both cases immunoreactively stained positively for AE1/AE3 and CK5/6 but negatively for CK8/18, S-100 and Calponin. There was no evidence of recurrence 3 or 4 years after superficial parotidectomy. The data from these two cases and cases previously published suggest that keratocystoma of the parotid gland is a benign cystic neoplasm. Surgical resection is apparently sufficient for cure.

FADD is a key regulator of lipid metabolism

FADD, a classical apoptotic signaling adaptor, was recently reported to have non‐apoptotic functions. Here, we report the discovery that FADD regulates lipid metabolism. PPAR‐α is a dietary lipid sensor, whose activation results in hypolipidemic effects. We show that FADD interacts with RIP140, which is a corepressor for PPAR‐α, and FADD phosphorylation‐mimic mutation (FADD‐D) or FADD deficiency abolishes RIP140‐mediated transcriptional repression, leading to the activation of PPAR‐α. FADD‐D‐mutant mice exhibit significantly decreased adipose tissue mass and triglyceride accumulation. Also, they exhibit increased energy expenditure with enhanced fatty acid oxidation in adipocytes due to the activation of PPAR‐α. Similar metabolic phenotypes, such as reduced fat formation, insulin resistance, and resistance to HFD‐induced obesity, are shown in adipose‐specific FADD knockout mice. Additionally, FADD‐D mutation can reverse the severe genetic obesity phenotype of ob/ob mice, with elevated fatty acid oxidation and oxygen consumption in adipose tissue, improved insulin resistance, and decreased triglyceride storage. We conclude that FADD is a master regulator of glucose and fat metabolism with potential applications for treatment of insulin resistance and obesity.

Modulation of Salmonella Tumor-Colonization and Intratumoral Anti-angiogenesis by Triptolide and Its Mechanism

The weakened tumour colonization of attenuated Salmonella has severely hampered its clinical development. In this study, we investigated whether an anti-inflammation and antiangiogenesis compound triptolide could improve the efficacy of VNP20009, a highly attenuated Salmonella strain, against mice melanoma. By comparing the effects of conventional VNP20009 monotherapy and a combination therapy that uses both triptolide and VNP20009, we found that triptolide significantly improved the tumour colonization of VNP20009 by reducing the number of infiltrated neutrophils in the melanoma, which led to a larger necrotic area in the melanoma. Moreover, the combination therapy suppressed tumour angiogenesis by reducing the expression of VEGF in a synergistic manner, retarding the growth of the melanoma. Our study revealed that triptolide could significantly enhance the antitumour effect of VNP20009 by modulating tumour angiogenesis and the host immune response, providing a new understanding of the strategy to improve Salmonella-mediated tumour therapy.

Absence of PTHrP nuclear localization and C-terminus sequences leads to abnormal development of T cells

Parathyroid hormone-related protein (PTHrP), a ubiquitously expressed protein, is composed of four functional domains including N-terminus, mid region, nuclear localization signal (NLS) and C-terminus. Under the direction of NLS, PTHrP can enter cell nucleus from cytoplasm and stimulate mitogenesis. Although PTHrP is considered to have important developmental roles, the role of PTHrP NLS and C-terminus in developmental process remains unknown, especially in T-cell development. Here, we used a knock-in mouse model, which expresses a truncated form of PTHrP missing the NLS (87-107) and C-terminus (108-139) of the protein, to examine the role of PTHrP NLS and C-terminus in T-cell development. Our results showed that the truncated PTHrP (1-84) led to abnormal subpopulations, impaired proliferation and increased apoptosis in the thymus, indicating that PTHrP is involved in the development of T cells, and the NLS and C-terminus part is necessary for the normal role of PTHrP in T-cell development.