A novel MCT1 and MCT4 dual inhibitor reduces mitochondrial metabolism and inhibits tumour growth of feline oral squamous cell carcinoma.

Monocarboxylate transporters (MCTs) support tumour growth by regulating the transport of metabolites in the tumour microenvironment. High MCT1 or MCT4 expression is correlated with poor outcomes in human patients with head and neck squamous cell carcinoma (HNSCC). Recently, drugs targeting these transporters have been developed and may prove to be an effective treatment strategy for HNSCC. Feline oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy resembling advanced or recurrent HNSCC. The goals of this study were to investigate the effects of a previously characterized dual MCT1 and MCT4 inhibitor, MD-1, in OSCC as a novel treatment approach for feline oral cancer. We also sought to determine the potential of feline OSCC as a large animal model for the further development of MCT inhibitors to treat human HNSCC. In vitro, MD-1 reduced the viability of feline OSCC and human HNSCC cell lines, altered glycolytic and mitochondrial metabolism and synergized with platinum-based chemotherapies. While MD-1 treatment increased lactate concentrations in an HNSCC cell line, the inhibitor failed to alter lactate levels in feline OSCC cells, suggesting an MCT-independent activity. In vivo, MD-1 significantly inhibited tumour growth in a subcutaneous xenograft model and prolonged overall survival in an orthotopic model of feline OSCC. Our results show that MD-1 may be an effective therapy for the treatment of feline oral cancer. Our findings also support the further investigation of feline OSCC as a large animal model to inform the development of MCT inhibitors and future clinical studies in human HNSCC.

Cap-dependent translational control of oncolytic measles virus infection in malignant mesothelioma.

Malignant mesothelioma has a poor prognosis for which there remains an urgent need for successful treatment approaches. Infection with the Edmonston vaccine strain (MV-Edm) derivative of measles virus results in lysis of cancer cells and has been tested in clinical trials for numerous tumor types including mesothelioma. Many factors play a role in MV-Edm tumor cell selectivity and cytopathic activity while also sparing non-cancerous cells. The MV-Edm receptor CD46 (cluster of differentiation 46) was demonstrated to be significantly higher in mesothelioma cells than in control cells. In contrast, mesothelioma cells are not reliant upon the alternative MV-Edm receptor nectin-4 for entry. MV-Edm treatment of mesothelioma reduced cell viability and also invoked apoptotic cell death. Forced expression of eIF4E or translation stimulation following IGF-I (insulin-like growth factor 1) exposure strengthened the potency of measles virus oncolytic activity. It was also shown that repression of cap-dependent translation by treatment with agents [4EASO, 4EGI-1] that suppress host cell translation or by forcing cells to produce an activated repressor protein diminishes the strength of oncolytic viral efficacy.

Involvement of calprotectin (S100A8/A9) in molecular pathways associated with HNSCC.

Calprotectin (S100A8/A9), a heterodimeric protein complex of calcium-binding proteins S100A8 and S100A9, plays key roles in cell cycle regulation and inflammation, with potential functions in squamous cell differentiation. While upregulated in many cancers, S100A8/A9 is downregulated in squamous cell carcinomas of the cervix, esophagus, and the head and neck (HNSCC). We previously reported that ectopic S100A8/A9 expression inhibits cell cycle progression in carcinoma cells. Here, we show that declining expression of S100A8/A9 in patients with HNSCC is associated with increased DNA methylation, less differentiated tumors, and reduced overall survival. Upon ectopic over-expression of S100A8/A9, the cancer phenotype of S100A8/A9-negative carcinoma cells was suppressed in vitro and tumor growth in vivo was significantly decreased. MMP1, INHBA, FST, LAMC2, CCL3, SULF1, and SLC16A1 were significantly upregulated in HNSCC but were downregulated by S100A8/A9 expression. Our findings strongly suggest that downregulation of S100A8/A9 through epigenetic mechanisms may contribute to increased proliferation, malignant transformation, and disease progression in HNSCC.

Augmentation of epithelial resistance to invading bacteria by using mRNA transfections.

To protect against invading bacteria, oral epithelial cells appear to use two effector antimicrobial peptides (AMPs): calprotectin (S100A8-S100A9 heterodimer [S100A8/A9]) in the cytosol and cathelicidin antimicrobial protein (CAMP) in endosomes. We sought to learn whether innate immunity might be augmented benignly to increase resistance against invasive bacteria. Epithelial cells were transiently transfected with mRNA constructs containing either the CAMP, S100A8, and S100A9 open reading frames, A8-IRES-A9 (fusion sequence), or A8-nIRES-A9 (fusion with native internal ribosome entry site [IRES] sequence). CAMP, S100A8, and S100A9 protein levels generally peaked between 16 and 44 h after mRNA transfection, depending on the construct; CAMP was processed to LL-37 over time. Following transfection with the respective mRNAs, CAMP and S100A8/A9 each independently increased resistance of epithelial cells to invasion by Listeria and Salmonella for up to 48 h; tandem S100A8/A9 constructs were also effective. Cotransfection to express S100A8/A9 and CAMP together augmented resistance, but synergy was not seen. Independent of the new proteins produced, transfection reduced cell viability after 48 h by 20%, with only 2% attributable to apoptosis. Taken together, these results suggest that epithelial cell resistance to invasive pathogens can be augmented by transient transfection of antimicrobial mRNAs into epithelial cells.

S100A8/A9 (calprotectin) negatively regulates G2/M cell cycle progression and growth of squamous cell carcinoma.

Malignant transformation results in abnormal cell cycle regulation and uncontrolled growth in head and neck squamous cell carcinoma (HNSCC) and other cancers. S100A8/A9 (calprotectin) is a calcium-binding heterodimeric protein complex implicated in cell cycle regulation, but the specific mechanism and role in cell cycle control and carcinoma growth are not well understood. In HNSCC, S100A8/A9 is downregulated at both mRNA and protein levels. We now report that downregulation of S100A8/A9 correlates strongly with a loss of cell cycle control and increased growth of carcinoma cells. To show its role in carcinogenesis in an in vitro model, S100A8/A9 was stably expressed in an S100A8/A9-negative human carcinoma cell line (KB cells, HeLa-like). S100A8/A9 expression increases PP2A phosphatase activity and p-Chk1 (Ser345) phosphorylation, which appears to signal inhibitory phosphorylation of mitotic p-Cdc25C (Ser216) and p-Cdc2 (Thr14/Tyr15) to inactivate the G2/M Cdc2/cyclin B1 complex. Cyclin B1 expression then downregulates and the cell cycle arrests at the G2/M checkpoint, reducing cell division. As expected, S100A8/A9-expressing cells show both decreased anchorage-dependent and -independent growth and mitotic progression. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increases growth and survival and reduces Cdc2 inhibitory phosphorylation at Thr14/Tyr15. The level of S100A8/A9 endogenous expression correlates strongly with the reduced p-Cdc2 (Thr14/Tyr14) level in HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B. S100A8/A9-mediated control of the G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may suggest new therapeutic approaches.

PR_b functionalized stealth liposomes for targeted delivery to metastatic colon cancer.

A gene delivery system was designed to carry a payload to integrin overexpressing cells. Branched-polyethyleneimine (bPEI) condensed plasmid DNA was encapsulated into targeted stealth liposomes, thereby combining the condensing and transfection properties of bPEI with the stealth and targeting properties of the liposomal carrier system. PR_b was used as a targeting ligand - a peptide we designed to bind specifically to the cancer cell surface marker α5ß1 integrin - and such a robust receptor-ligand interaction achieved higher specificity than what has been previously reported for targeted delivery systems. In the process of formulating the PR_b functionalized gene delivery vehicle, we developed a protocol to fully encapsulate condensed DNA in liposomes and accurately quantify the total DNA in the system. We demonstrate that compared to non-targeted stealth liposomes and non-encapsulated condensed DNA, the PR_b functionalized stealth liposomes mediated improved in vitro transfection specifically to colon cancer cells overexpressing the α5ß1 integrin. Furthermore, when administered in vivo to metastatic tumor bearing mice, PR_b functionalized stealth liposomes outperformed non-targeted liposomes and delivered genes specifically to the tumor site.

Antioxidant oils and Salmonella enterica Typhimurium reduce tumor in an experimental model of hepatic metastasis.

Fruit seeds high in antioxidants have been shown to have anticancer properties and enhance host protection against microbial infection. Recently we showed that a single oral dose of Salmonella enterica serovar Typhimurium expressing a truncated human interleukin-2 gene (SalpIL2) is avirulent, immunogenic, and reduces hepatic metastases through increased natural killer cell populations in mice. To determine whether antioxidant compounds enhance the antitumor effect seen in SalpIL2-treated animals, we assayed black cumin (BC), black raspberry (BR), and milk thistle (MT) seed oils for the ability to reduce experimental hepatic metastases in mice. In animals without tumor, BC and BR oil diets altered the kinetics of the splenic lymphocyte response to SalpIL2. Consistent with previous reports, BR and BC seed oils demonstrated independent antitumor properties and moderate adjuvant potential with SalpIL2. MT oil, however, inhibited the efficacy of SalpIL2 in our model. Based on these data, we conclude that a diet high in antioxidant oils promoted a more robust immune response to SalpIL2, thus enhancing its antitumor efficacy.

Inhibition of angiogenesis and suppression of colorectal cancer metastatic to the liver using the Sleeping Beauty Transposon System.

BACKGROUND: Metastatic colon cancer is one of the leading causes of cancer-related death worldwide, with disease progression and metastatic spread being closely associated with angiogenesis. We investigated whether an antiangiogenic gene transfer approach using the Sleeping Beauty (SB) transposon system could be used to inhibit growth of colorectal tumors metastatic to the liver. RESULTS: Liver CT26 tumor-bearing mice were hydrodynamically injected with different doses of a plasmid containing a transposon encoding an angiostatin-endostatin fusion gene (Statin AE) along with varying amounts of SB transposase-encoding plasmid. Animals that were injected with a low dose (10 µg) of Statin AE transposon plasmid showed a significant decrease in tumor formation only when co-injected with SB transposase-encoding plasmid, while for animals injected with a higher dose (25 µg) of Statin AE transposon, co-injection of SB transposase-encoding plasmid did not significantly affect tumor load. For animals injected with 10 µg Statin AE transposon plasmid, the number of tumor nodules was inversely proportional to the amount of co-injected SB plasmid. Suppression of metastases was further evident in histological analyses, in which untreated animals showed higher levels of tumor cell proliferation and tumor vascularization than animals treated with low dose transposon plasmid. CONCLUSION: These results demonstrate that hepatic colorectal metastases can be reduced using antiangiogenic transposons, and provide evidence for the importance of the transposition process in mediating suppression of these tumors.

Triptolide therapy for neuroblastoma decreases cell viability in vitro and inhibits tumor growth in vivo.

BACKGROUND: Heat shock protein (Hsp)-70 is overexpressed in several human malignancies, and its inhibition has been shown to kill cancer cells. Our objectives were to assess the effectiveness of triptolide, an Hsp-70 inhibitor, in treating neuroblastoma in vitro and in vivo, and to measure the associated effects on Hsp-70 levels and apoptosis markers. METHODS: After exposing N2a and SKNSH cell lines to triptolide, cell viability was assessed. Caspase-3 and -9 activities were measured and annexin staining performed to determine if cell death occurred via apoptosis. Hsp-70 protein and mRNA levels were determined using Western blot and real-time polymerase chain reaction. In an orthotopic tumor model, mice received daily triptolide injections and were humanely killed at study completion with tumor measurement. RESULTS: Triptolide treatment resulted in dose- and time-dependent N2a cell death and dose-dependent SKNSH killing. Triptolide exposure was associated with dose-dependent increases in caspase activity and annexin staining. Triptolide decreased Hsp-70 protein and mRNA levels in a dose-dependent fashion. Mice receiving triptolide therapy had significantly smaller tumors than controls. CONCLUSION: Triptolide therapy decreased neuroblastoma cell viability in vitro and inhibited tumor growth in vivo. Our studies suggest that triptolide killed cells via apoptosis and in association with inhibition of Hsp-70 expression. Triptolide may provide a novel therapy for neuroblastoma.

ANTI-INFECTIVE PROTECTIVE PROPERTIES OF S100 CALGRANULINS.

The calgranulins are a subgroup of proteins in the S100 family (calgranulin A, S100A8; calgranulin B, S100A9 and calgranulin C, S100A12) that provide protective anti-infective and anti-inflammatory functions for the mammalian host. In this review, we discuss the structure-function relationships whereby S100A8 and S100A9, and for comparison, S100A12, provide intra- and extracellular protection during the complex interplay between infection and inflammation and how the calgranulins are regulated to optimally protect the host. Ideally located to support epithelial barrier function, calprotectin, a complex of S100A8/S100A9, is expressed in squamous mucosal keratinocytes and innate immune cells present at mucosal surfaces. The calgranulins are also abundantly produced in neutrophils and monocytes, whereas expression is induced in epidermal keratinocytes, gastrointestinal epithelial cells and fibroblasts during inflammation. The calgranulins show species-specific expression and function. For example, S100A8 is chemotactic in rodents but not in humans. In humans, S100A12 appears to serve as a functional chemotactic homolog to murine S100A8. Transition metal-binding and oxidation sites within calgranulins are able to create structural changes that may orchestrate new protective functions or binding targets. The calgranulins thus appear to adopt a variety of roles to protect the host. In addition to serving as a leukocyte chemoattractant, protective functions include oxidant scavenging, antimicrobial activity, and chemokine-like activities. Each function may reflect the concentration of the calgranulin, post-transcriptional modifications, oligomeric forms, and the proximal intracellular or extracellular environments. Calprotectin and the calgranulins are remarkable as multifunctional proteins dedicated to protecting the intra- and extracellular environments during infection and inflammation.

Attenuated Salmonella typhimurium with interleukin 2 gene prevents the establishment of pulmonary metastases in a model of osteosarcoma.

PURPOSE: The current management of osteosarcoma (OS) entails an aggressive preoperative and postoperative chemotherapeutic regimen with limb salvage surgery. Despite these efforts, relapse-free survival is less than 60% in patients with classic OS, whereas most patients relapse with pulmonary metastases. In these studies, we sought to prevent the establishment of pulmonary metastases from OS with a single oral dose of SalpIL2. METHODS: Mice were administered attenuated Salmonella typhimurium with (SalpIL2) and without a gene for human interleukin 2 (Sal-NG) 7 days before challenge with 2 x 10(5) OS cells via tail vein. Three weeks after injection, mice were harvested for splenic lymphocytes and tumor enumeration. RESULTS: Prophylaxis with attenuated SalpIL2 significantly reduces pulmonary metastases in number and volume (P < .0001 and P < .0001) with respect to saline controls. Furthermore, splenic natural killer cell populations were increased 396% with SalpIL2 (P < .0007) and 426% with Sal-NG (P < .0003) compared to nontreated groups. CONCLUSIONS: Host natural killer response is greatly amplified and maybe partially responsible for the effective immune response against the formation of pulmonary metastases. A single oral dose of SalpIL2 may be a novel form of adjuvant therapy for patients after early detection of primary OS.

Attenuated Salmonella typhimurium with IL-2 gene reduces pulmonary metastases in murine osteosarcoma.

Historically, osteosarcoma has been a problematic metastatic disease, with 40-80% of patients developing pulmonary metastasis after primary tumor resection. Recent treatment advancements have reduced the occurrence of metastatic lesions to less than 30%. Using attenuated Salmonella typhimurium, we previously demonstrated regression in tumor burden in murine solid tumor and metastatic models. We established a murine model for metastatic osteosarcoma to determine the effect of treatment with a single oral dose of attenuated S. typhimurium with (SalpIL2) and without (Sal-NG) a gene for a truncated human interleukin-2. Female balb/c mice were administered 2 x 10(5) (ATCC K7M2) osteosarcoma cells via tail vein injection from culture and treated by oral gavage of Salmonella species 3 days later. Mice were harvested for splenic lymphocytes and tumor enumeration by intratracheal injection with India ink 21 days after injection. Treatment with attenuated SalpIL2 reduced pulmonary metastases in number and volume compared to saline controls. Furthermore, splenic natural killer cell populations were increased 93% with SalpIL2 and 114% with Sal-NG compared to nontreated groups. This pulmonary metastasis model demonstrates attenuated Salmonella typhimurium with human interleukin-2 reduced metastatic osteosarcoma in mice and confirm the need for further investigation into the immunologic properties of SalpIL2 as a possible treatment for metastatic osteosarcoma.

Attenuated Salmonella typhimurium invades and decreases tumor burden in neuroblastoma.

INTRODUCTION: We have previously shown that Salmonella elicits an antitumor response against hepatic adenocarcinomatous metastases. In vitro studies have demonstrated both intracellular invasion and proliferation of Salmonella within cultured neuroblastoma cells. We sought to demonstrate in vivo invasion, proliferation, and a potential antitumor response. METHODS: A murine model for retroperitoneal neuroblastoma was established with viable neuroblastoma cells. A green fluorescent protein (GFP--Clontech, Palo Alto, CA) gene was inserted into our attenuated Salmonella species via electroporation. Fourteen days after retroperitoneal injection, the Salmonella typhimurium-pGFP construct was administered and studied. In separate experiments, the antitumor effect against neuroblastoma was studied in controls, Salmonella lacking an interleukin 2 (IL-2) gene (Sal-NG), and Salmonella containing an IL-2 gene (Salmonella-pIL2 ). RESULTS: Consistent with previous reports, 74% of mice injected were found to have recognizable tumors. Salmonella was present within tumor cells. Average tumor volumes for control, Sal-NG, and Salmonella-pIL2 mice were 2024.3, 749.5, and 332.4 mm3 , respectively (P < .0001). Tumor weights for control, Sal-NG, and Salmonella-pIL2 mice were 2.218, 0.880, and 0.377 g, respectively (P < .0001). CONCLUSIONS: Attenuated Salmonella species can now be tracked via fluorescent microscopy within tumor cells. Furthermore, an 84% reduction in tumor burden was observed in those animals gavage fed Salmonella with the IL-2 gene.

Preferential proliferation of attenuated Salmonella typhimurium within neuroblastoma.

BACKGROUND/PURPOSE: Attenuated Salmonella typhimurium, a facultative intracellular parasite that colonizes the liver, has been shown to accumulate within extrahepatic malignancies. The authors sought to define a mechanism for attenuated Salmonella accumulation within cancer cells compared with hepatocytes. METHODS: Invasion and intracellular proliferation of attenuated Salmonella was assessed through an in vitro assay performed on neuroblasoma, osteosarcoma, hepatoma, and colon adenocarcinoma cell lines and compared with freshly isolated mouse hepatocytes. RESULTS: The efficiency of attenuated S typhimurium invasion into hepatocytes was greater than any malignant cell line (3.8 v 0.46; P <.04). However, the intracellular proliferation of the bacteria was most abundant within neuroblastoma, exceeding the proliferation within hepatocytes (14.3 v 6.2; P <.01). CONCLUSIONS: Attenuated S typhimurium may prove to be an effective in vivo immunotherapy for the local delivery of therapeutic proteins to neuroblastoma.

Attenuated Salmonella typhimurium prevents the establishment of unresectable hepatic metastases and improves survival in a murine model.

PURPOSE: The authors investigated the utility of attenuated Salmonella typhimurium for preventing the establishment of hepatic metastases in a murine model. METHODS: A single, oral 10(8) cfu dose of attenuated S typhimurium was given 8 days before the establishment of a model of unresectable hepatic metastases. Animals were assessed for hepatic tumor number and volume, hepatic lymphocyte population analysis, and survival. RESULTS: Pretreatment with Salmonella provided a 10-fold reduction in hepatic tumor burden compared with saline-treated controls. The antitumor effect is associated with markedly elevated natural killer (NK), CD8+ and CD4+ hepatic lymphocytes. Pretreatment with Salmonella provided a 90-day survival rate of 30%, whereas control animals were dead by 30 days. All long-term survivors were devoid of hepatic tumor. CONCLUSIONS: Attenuated S typhimurium effectively prevents the establishment of hepatic metastases in a murine model, providing a clear survival benefit. Thus, it may represent a novel form of in vivo immunotherapy for the prevention of hepatic metastases for patients with locally advanced colorectal cancer.