The effects of non-invasive radiofrequency treatment and hyperthermia on malignant and nonmalignant cells.

BACKGROUND: Exposure of biological subjects to electromagnetic fields with a high frequency is associated with temperature elevation. In our recent studies, we reported that non-invasive radiofrequency (RF) treatment at 13.56 MHz with the field ranging from 1 KeV to 20 KeV/m2 inhibits tumor progression in animals with abdominal tumor xenografts and enhances the anticancer effect of chemotherapy. The RF treatment was followed by temperature elevation in tumors to approximately 46 °C during 10 min of exposure. In contrast, the temperature of normal tissues remained within a normal range at approximately 37 °C. Whether all biological effects of RF treatment are limited to its hyperthermic property remains unclear. Here, we compared how RF and hyperthermia (HT) treatments change the proliferation rate, oxygen consumption and autophagy in malignant and nonmalignant cells. METHODS: In the current study, cancer and nonmalignant cells of pancreatic origin were exposed to the RF field or to conventional HT at 46 °C, which was chosen based on our previous in vivo studies of the tumor-specific RF-induced hyperthermia. RESULTS: Only RF treatment caused declines in cancer cell viability and proliferation. RF treatment also affected mitochondrial function in cancer cells more than HT treatment did and, unlike HT treatment, was followed by the elevation of autophagosomes in the cytoplasm of cancer cells. Importantly, the effects of RF treatment were negligible in nonmalignant cells. CONCLUSION: The obtained data indicate that the effects of RF treatment are specific to cancer cells and are not limited to its hyperthermic property.

Noninvasive radiofrequency treatment effect on mitochondria in pancreatic cancer cells.

BACKGROUND: The development of novel therapeutic approaches for cancer therapy is important, especially for tumors that have poor response or develop resistance to standard chemotherapy and radiation. We discovered that noninvasive radiofrequency (RF) fields can affect cancer cells but not normal cells, inhibit progression of tumors in mice, and enhance the anticancer effects of chemotherapy. However, it remains unclear what physiological and molecular mechanisms this treatment induces inside cells. Here, we studied the effect of RF treatment on mitochondria in human pancreatic cancer cells. METHODS: The morphology of mitochondria in cells was studied via electron microscopy. The alteration of mitochondrial membrane potential (Δψ) was accessed using a Mitotracker probe. The respiratory activity of mitochondria was evaluated by analyzing changes in oxygen consumption rates determined with a Mito Stress Test Kit. The production of intracellular reactive oxygen species was performed using flow cytometry. The colocalization of mitochondria and autophagosome markers in cells was performed using fluorescence immunostaining and confocal microscopy analysis. RESULTS: RF fields treatment changed the morphology of mitochondria in cancer cells, altered polarization of the mitochondrial membrane, substantially impaired mitochondrial respiration, and increased reactive oxygen species production, indicating RF-induced stress on the mitochondria. We also observed frequent colocalization of the autophagosome marker LC3B with the mitochondrial marker Tom20 inside cancer cells after RF exposure, indicating the presence of mitochondria in the autophagosomes. This suggests that RF-induced stress can damage mitochondria and induce elimination of damaged organelles via autophagy. CONCLUSION: RF treatment impaired the function of mitochondria in cancer cells. Therefore, mitochondria can represent one of the targets of the RF treatment.

Autophagy and enhanced chemosensitivity in experimental pancreatic cancers induced by noninvasive radiofrequency field treatment.

BACKGROUND: Patients with pancreatic ductal adenocarcinoma (PDAC) have limited therapeutic options and poor response to the standard gemcitabine (GCB)-based chemotherapy. In the current study, the authors investigated the feasibility of noninvasive short-wave radiofrequency (RF) electric fields to improve the cytotoxic effect of GCB on PDAC cells and determined its mechanism of action. METHODS: The cytotoxicity of RF alone and in combination with GCB was studied in vitro on normal pancreatic human pancreatic ductal epithelial cells and different PDAC cell lines by flow cytometry, and in vivo on ectopic and orthotopic human PDAC xenograft models in mice. The mechanism of RF activity was studied by Western blot analysis and immunohistochemistry. Toxicity was determined by histopathology. RESULTS: Exposure of different PDAC cells to 13.56-megahertz radio waves resulted in a substantial cytotoxic effect, which was accompanied by the induction of autophagy but not apoptosis. These effects of RF were found to be absent in normal cells. Excessive numbers of autophagosomes in cancer cells persisted 24 to 48 hours after RF exposure and then declined. The addition of a subtoxic dose of GCB to RF treatment inhibited the recovery of cancer cells from the RF-induced autophagy and enhanced the cytotoxic effect of the latter on cancer cells. The treatment of PDAC in situ in mice with the combination of noninvasive RF and GCB was found to have a superior antitumor effect compared with the use of RF or GCB alone, yet there was no evidence of systemic toxicity. CONCLUSIONS: Noninvasive RF treatment induced autophagy but not apoptosis in cancer cells and demonstrated potential as an enhancer of chemotherapy for treating patients with pancreatic cancer without toxicity to normal cells.

Tumor selective hyperthermia induced by short-wave capacitively-coupled RF electric-fields.

There is a renewed interest in developing high-intensity short wave capacitively-coupled radiofrequency (RF) electric-fields for nanoparticle-mediated tumor-targeted hyperthermia. However, the direct thermal effects of such high-intensity electric-fields (13.56 MHZ, 600 W) on normal and tumor tissues are not completely understood. In this study, we investigate the heating behavior and dielectric properties of normal mouse tissues and orthotopically-implanted human hepatocellular and pancreatic carcinoma xenografts. We note tumor-selective hyperthermia (relative to normal mouse tissues) in implanted xenografts that can be explained on the basis of differential dielectric properties. Furthermore, we demonstrate that repeated RF exposure of tumor-bearing mice can result in significant anti-tumor effects compared to control groups without detectable harm to normal mouse tissues.

The histone deacetylase inhibitor, MS-275 (entinostat), downregulates c-FLIP, sensitizes osteosarcoma cells to FasL, and induces the regression of osteosarcoma lung metastases.

The purpose of this study was to determine the effects of the histone deacetylase inhibitor, MS-275, on the Fas signaling pathway and susceptibility of osteosarcoma (OS) to Fas ligand (FasL)-induced cell death. OS metastasizes almost exclusively to the lungs. We have shown that Fas expression in OS cells is inversely correlated with their metastatic potential. Fas(+) cells are rapidly eliminated when they enter the lungs via interaction with FasL, which is constitutively expressed in the lungs. Fas(-) OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Moreover, upregulation of Fas in established OS lung metastases results in tumor regression. Therefore, agents that upregulate Fas expression or activate the Fas signaling pathway may have therapeutic potential. Treatment of Fas(-) metastatic OS cell lines with 2 µM MS-275 sensitized cells to FasL-induced cell death in vitro. We found that MS-275 did not alter the expression of Fas on the cell surface; rather it resulted in the downregulation of the anti-apoptotic protein, c-FLIP (cellular FLICE-inhibitory protein), by inhibiting c-FLIP mRNA. Downregulation of c- FLIP correlated with caspase activation and apoptosis induction. Treatment of nu/nu-mice with established OS lung metastases with oral MS-275 resulted in tumor regression, increased apoptosis and a significant inhibition of c-FLIP expression in tumors. Histopathological examination of mice showed no evidence of significant toxicity. Overall, these results suggest that the mechanism by which MS-275 sensitizes OS cells and lung metastases to FasL-induced cell death may be by a direct reduction in the expression of c-FLIP.

Inhibition of Cdc42-interacting protein 4 (CIP4) impairs osteosarcoma tumor progression.

BACKGROUND: In patients with Osteosarcoma (OS), larger primary tumor size correlates with higher metastasis incidence and lower overall survival. Identifying the mechanisms that control primary tumor growth can lead to the new therapeutic approaches and improve prognosis. Cytoskeleton regulatory molecules play an important role in tumor cells proliferation and motility. In the current study, we investigated the role of scaffolding protein Cdc42-interacting protein 4 (CIP4) in OS. METHODS: Murine OS cells, DLM8 were stably transfected with shCIP4 plasmid and their tumorigenic activity was studied in vitro and in vivo. The effect of CIP4 downregulation on cytoskeleton was studied by immunohistochemical analysis. RESULTS: In vivo studies revealed that downregulation of CIP4 in OS tumor cells can inhibit primary subcutaneous tumor growth and delay spontaneous metastases growth in the animal lungs. In vitro studies confirmed that inhibiting CIP4 expression significantly reduced tumor cells invasiveness and migration. Changes in CIP4 expression altered its cellular localization pattern and tumor cell morphology, which could explain changes in OS cell behavior in vivo and in vitro. Cells with downregulated CIP4 showed lower levels of actin in cells along with fewer perinuclear actin stress filaments and actin bridges to the lipid bilayer of the cell membrane than control cells. CONCLUSIONS: This study presents compelling evidence that inhibition of CIP4 expression in OS cells impairs metastatic behavior of cells in vitro and prolongs survival of animals by inhibiting primary tumor growth. Although the lack of CIP4 in tumor cells did not prevent formation of metastasis, it did substantially delay their formation in the animals' lungs.

Effect of the histone deacetylase inhibitor SNDX-275 on Fas signaling in osteosarcoma cells and the feasibility of its topical application for the treatment of osteosarcoma lung metastases.

BACKGROUND: Patients with lung metastases from osteosarcoma (OS) have poor response to salvage therapy. Understanding the mechanisms involved in the metastatic process of OS may lead to new effective therapeutic approaches. The authors reported previously that up-regulation of the Fas receptor by transfecting OS cells with Fas plasmid inhibited the in vivo growth of metastases in the lungs. METHODS: In the current study, the authors treated OS cells with the histone deacetylase inhibitor SNDX-275 and studied its cytotoxicity and its effect on Fas signaling in vitro and in vivo. RESULTS: Subtoxic doses of SNDX-275 were able to activate the Fas pathway in OS cells by increasing the expression of Fas messenger RNA; however, the increased expression was not always followed by increased levels of Fas receptor expression on the cell surface. The treatment of cells with a combination of SNDX-275 and Fas ligand (FasL) had a stronger cytotoxic effect on tested OS cells than either agent alone. Inhibition of the Fas pathway in cells by inhibition of the Fas-associated death domain (FADD) molecule eliminated this combination effect, indicating that activity of FADD is important for the efficacy of this agent in the FasL-expressing environment of the lungs. Intranasal administration of SNDX-275 in mice with OS lung metastases revealed that SNDX-275 may inhibit metastatic growth at a dose of 0.13 mg/kg, which is approximately 200-fold lower than the therapeutically effective oral dose reported previously. CONCLUSIONS: The current findings indicated that SNDX-275 can activate Fas signaling in OS cells in vitro and in vivo and that the administration of SDNX-275 by inhalation is feasible as a treatment for OS metastases and warrants its further investigation.

Fas expression in metastatic osteosarcoma cells is not regulated by CpG island methylation.

Fas expression in osteosarcoma (OS) cells is inversely correlated with the metastatic potential of OS to the lung. The purpose of this study was to determine whether loss of Fas expression in metastatic OS cells is secondary to DNA methylation of CpG islands in the Fas gene. SAOS-2 cells have high levels of Fas expression and do not form lung metastases when injected intravenously, whereas LM7 cells have low levels of Fas expression and do produce lung metastases. Using the endonucleases HpaII and MspI and a polymerase chain reaction-based methylation assay, we found that all four CpG sites in the CCGG sequence in the Fas promoter region were unmethylated in both SAOS-2 and LM7 cells. We performed detailed analysis of the 28 and 46 CpG sites in the Fas promoter and first intron region, respectively, using bisulfite-modified genomic DNA sequencing. More than 99.8% of the examined CpG sites were unmethylated and there was no difference of CpG methylation in SAOS-2 and LM7 cells as well as LM7 metastatic lung tumor tissue samples. Treatment of LM7 cells and another OS cell line, DLM8 with low levels of Fas expression, with demethylation agent, 5-azadeoxycitidine (AzadC), did not change the Fas expression and did not increase sensitivity of AzadC-treated cells to Fas ligand (FasL) treatment. In conclusion, our data indicate that decreased Fas expression in OS cells is not secondary to DNA methylation of CpG islands in the Fas gene and that Fas expression cannot be increased by using demethylation agents.

COX-2 expression correlates with survival in patients with osteosarcoma lung metastases.

SUMMARY: The purpose of this study was to determine whether a correlation exists between tumor cyclooxygenase (COX)-2 expression and disease-specific survival in patients with osteosarcoma lung metastases. Thirty-six patients diagnosed with osteosarcoma lung metastases between the years 1990 and 2001 were included in this retrospective study. The majority of the patients (72%) presented newly -diagnosed osteosarcoma lung metastases whereas the remaining patients (28%) presented recurrent disease. Clinicopathologic parameters were obtained from patients' clinical records. Tissue samples were obtained at the time of resection of the lung metastases and stained for COX-2 using immunohistochemistry. Samples were graded according to the intensity of COX-2 staining (grade 0: negative, grade 1: very weak, grade 2: weak, grade 3: moderate, and grade 4: strong). COX-2 staining was correlated with disease-specific survival and clinicopathologic parameters using the Jonckheere-Terpstra and the Kruskal-Wallis tests. All patients with grade 3 or 4 COX-2 expression died of osteosarcoma lung metastases. Ten percent of patients with grade 2 COX-2 expression and 29% of patients with grade 1 expression were alive and free of disease at the last follow-up. By contrast, 60% of the patients with grade 0 COX-2 expression were alive and free of disease at the last follow-up. No association between COX-2 expression and clinicopathologic parameters was found. However, COX-2 expression correlated inversely with disease-specific survival in patients with osteosarcoma lung metastases. Our data indicate that COX-2 expression in metastatic osteosarcoma may have prognostic significance.

Intranasal granulocyte-macrophage colony-stimulating factor reduces the Aspergillus burden in an immunosuppressed murine model of pulmonary aspergillosis.

We demonstrated that intranasal granulocyte-macrophage colony-stimulating factor given to immunosuppressed mice infected with pulmonary aspergillosis resulted in a sixfold reduction in the lung fungal burden compared to the result for saline-treated mice (P = 0.045). These data suggest that lung-targeted immunotherapy may be complementary to antifungal agents and may improve patient responses.

Exploratory analysis of Fas gene polymorphisms in pediatric osteosarcoma patients.

Defective apoptosis signaling by the Fas pathway has carcinogenic implications. We analyzed 123 pediatric patients with osteosarcoma for Fas single nucleotide polymorphisms: 2 of the promoter region (-1377 G>A and -670 A>G) and 2 of the coding region (exon 3 18272 A>G and exon 7 22628 C>T). As a comparison group, we used 510 adults without a history of cancer. We found an increased risk of osteosarcoma associated with the heterozygous genotype Fas exon 3 AG (genotype frequency 19.5% in cases vs. 12.0% in controls, P=0.028; adjusted odds ratio=1.6, 95% confidence interval=0.9-2.7], and this association was more pronounced in non-Hispanic whites (20.6% in cases vs. 10.1% in controls, P=0.014; adjusted odds ratio=2.3, 95% confidence interval=1.2-4.6). Additionally, the frequency of the variant allele (exon 3 G) was significantly higher in cases than in controls for both the entire group and non-Hispanic whites (P=0.046 and P=0.030, respectively). We found no significant association between osteosarcoma risk and the other Fas polymorphisms. This study suggests an association between the Fas exon 3 A>G polymorphism and osteosarcoma risk; however, further study is needed with pediatric controls and a larger sample size.

Fas-negative osteosarcoma tumor cells are selected during metastasis to the lungs: the role of the Fas pathway in the metastatic process of osteosarcoma.

Low expression of Fas by different tumors including osteosarcoma, correlates with poor prognosis. We found that osteosarcoma lung metastases from patients expressed negligible amounts of Fas, but primary tumors often expressed high Fas levels. The reason for this discrepancy is unknown. We hypothesized that because FasL is constitutively expressed in the lungs, Fas-positive (Fas(+)) tumor cells entering the lungs would bind with FasL and die from Fas-induced apoptosis, resulting in the "selection" of Fas-negative (Fas(-)) cells, which would eventually form metastases. To test this hypothesis, we injected K7 osteosarcoma cells, which express functional Fas in vitro, into mice and confirmed that its bone tumors were Fas(+), but lung metastases were Fas(-). Next, to inhibit Fas signaling without affecting Fas expression, we transfected these cells with a FADD-dominant negative (FDN) plasmid and developed K7/FDN cells. Metastases formed by K7/FDN cells contained Fas(+) tumor cells. Moreover, K7/FDN cells were retained in the lungs longer and formed more lung metastases than K7 cells. In addition, the incidence of lung metastases in FasL-deficient mice injected with K7 cells was higher than that in wild-type mice. Metastases from FasL-deficient mice but not from wild-type mice contained Fas(+) tumor cells. Based on that, we conclude that Fas(-) osteosarcoma cells are selected during lung metastases formation and that inhibition of Fas signaling in tumors or lack of FasL in the host environment allows the proliferation of Fas(+) osteosarcoma cells in the lungs and promotes metastases growth. Therefore, Fas may be considered as a new therapeutic target for osteosarcoma treatment.

Expression of granulocyte-colony-stimulating factor and its receptor in human Ewing sarcoma cells and patient tumor specimens: potential consequences of granulocyte-colony-stimulating factor administration.

BACKGROUND: Ewing sarcoma (ES) is a highly vascular malignancy. It has been demonstrated that both angiogenesis and vasculogenesis contribute to the growth of ES tumors. Granulocyte-colony-stimulating factor (G-CSF), a cytokine known to stimulate bone marrow (BM) stem cell production and angiogenesis, is routinely administered to ES patients after chemotherapy. Whether ES cells and patient tumor samples express G-CSF and its receptor (G-CSFR) and whether treatment with this factor enhances tumor growth was examined. METHODS: Human ES cell lines were analyzed for expression of G-CSF and G-CSFR in vitro and in vivo. Sixty-eight paraffin-embedded and 15 frozen tumor specimens from patients with ES were also evaluated for the presence of G-CSF and G-CSFR. The in vivo effect of G-CSF on angiogenesis and BM cell migration was determined. Using a TC/7-1 human ES mouse model, the effect of G-CSF administration on ES tumors was investigated. RESULTS: G-CSF and G-CSFR protein and RNA expression was identified in all ES cell lines and patient samples analyzed. In addition, G-CSF was found to stimulate angiogenesis and BM cell migration in vivo. Tumor growth was found to be significantly increased in mice treated with G-CSF. The average tumor volume for the group treated with G-CSF was 1218 mm(3) compared with 577 mm(3) for the control group (P = .006). CONCLUSIONS: The findings that ES cells and patient tumors expressed both G-CSF and its receptor in vitro and in vivo and that the administration of G-CSF promoted tumor growth in vivo suggest that the potential consequences of G-CSF administration should be investigated further.

Corruption of the Fas pathway delays the pulmonary clearance of murine osteosarcoma cells, enhances their metastatic potential, and reduces the effect of aerosol gemcitabine.

PURPOSE: Pulmonary metastases continue to be a significant problem in osteosarcoma. Apoptosis dysfunction is known to influence tumor development. Fas (CD95, APO-1)/FasL is one of the most extensively studied apoptotic pathways. Because FasL is constitutively expressed in the lung, cells that express Fas should be eliminated by lung endothelium. Cells with low or no cell surface Fas expression may be able to evade this innate defense mechanism. The purpose of these studies was to evaluate Fas expression in osteosarcoma lung metastases and the effect of gemcitabine on Fas expression and tumor growth. EXPERIMENTAL DESIGN AND RESULTS: Using the K7M2 murine osteosarcoma model, Fas expression was quantified using immunohistochemistry. High levels of Fas were present in primary tumors, but no Fas expression was present in actively growing lung metastases. Blocking the Fas pathway using Fas-associated death domain dominant-negative delayed tumor cell clearance from the lung and increased metastatic potential. Treatment of mice with aerosol gemcitabine resulted in increased Fas expression and subsequent tumor regression. CONCLUSIONS: We conclude that corruption of the Fas pathway is critical to the ability of osteosarcoma cells to grow in the lung. Agents such as gemcitabine that up-regulate cell surface Fas expression may therefore be effective in treating osteosarcoma lung metastases. These data also suggest that an additional mechanism by which gemcitabine induces regression of osteosarcoma lung metastases is mediated by enhancing the sensitivity of the tumor cells to the constitutive FasL in the lung.

Interleukin-12 up-regulates Fas expression in human osteosarcoma and Ewing's sarcoma cells by enhancing its promoter activity.

Interleukin-12 (IL-12) has shown significant antitumor activity in several preclinical animal tumor models. Our previous studies showed that IL-12 inhibited tumor growth in human osteosarcoma and Ewing's sarcoma animal model. Decreased Fas expression in osteosarcoma increased the lung metastatic potential. In this study, we further examined the mechanism of IL-12 antitumor activity and showed that IL-12 significantly increased Fas expression in both human osteosarcoma cells LM7 and Ewing's sarcoma cells TC71. Up-regulation of Fas expression increased their sensitivity to Fas-induced cell apoptosis. Constructs of the Fas promoter linked to a luciferase reporter gene were used to determine the promoter activity. IL-12 increased Fas promoter activity 4.2- and 4.9-fold in TC71 and LM7 cells, respectively. Time course studies have shown that recombinant IL-12 stimulated Fas promoter activity at 2 hours, reached the peak level at 4 hours, and then declined at 24 hours. To investigate whether IL-12 specifically enhanced Fas promoter activity, we determined whether another gene (E1A) was able to stimulate Fas promoter activity. We also evaluated effect of IL-12 on the topoisomerase IIalpha promoter. The results indicated that E1A but not IL-12 stimulated topoisomerase IIalpha promoter activity. E1A failed to increase Fas promoter activity. We also found that kappaB-Sp1 element at position -295 to -286 in Fas promoter was essential for IL-12-induced activation, and nuclear factor-kappaB transcription factor was activated after IL-12 treatment in TC71 cells. These results indicate that IL-12 up-regulates Fas expression in human osteosarcoma and Ewing's sarcoma by enhancing Fas promoter activity. Understanding this mechanism may lead to new therapeutic approaches for the treatment of sarcoma involving the use of IL-12.

Fas expression in lung metastasis from osteosarcoma patients.

The authors' animal studies have shown that the metastatic potential of osteosarcoma (OS) cells correlates inversely with Fas expression-that is, Fas-negative cells metastasize but Fas-positive cells do not. One reason for this in the context of OS lung metastases may be that Fas-positive cells are eliminated by engagement with the Fas ligand (FasL) constitutively expressed on the surface of pneumocytes, whereas Fas-negative tumor cells are not. The purpose of this study was to determine the status of Fas expression in OS lung metastases from patients. Specifically, archived paraffin-embedded specimens of lung metastases from 38 patients with OS were analyzed by immunohistochemistry. Lung nodules from 23 of the 38 patients (60%) were Fas negative, those from 12 patients (32%) were weakly positive, and that from only 1 patient (3%) was strongly positive. Findings in the samples from the remaining two patients (5%) could not be interpreted because of extensive necrosis. Most patients with the weakly positive tumors and the single patient with the strongly positive tumor received chemotherapy prior to lung resection. There was a significant correlation between Fas expression and the administration of preoperative salvage chemotherapy (P = 0.0013). These data indicate that loss of Fas may be one mechanism by which OS cells evade host resistance in the lung. Chemotherapy may induce regression by upregulating Fas.

Aerosol gemcitabine inhibits the growth of primary osteosarcoma and osteosarcoma lung metastases.

Osteosacarcoma (OS) lung metastases are often resistant to chemotherapy. Most anticancer drugs are administered systemically. In many cases this is followed by dose-dependent toxicity, which may not allow the achievement of therapeutic levels in lungs to eradicate metastases. We determined the efficacy of gemcitabine (GCB) by administering it directly to the lungs via aerosol and studied the role of the Fas pathway in response to the therapy. We used 2 osteosarcoma lung metastases animal models: human LM7 cells that form lung metastases in mice following intravenous injection and murine LM8 cells, which grows subcutaneously in mice and spontaneously metastasize to the lung. Treatment was initiated when the presence of lung metastases had been established. Aerosol GCB inhibited the growth of lung metastases in mice. Intraperitoneal GCB administration at similar dosage had no effect on lung metastases. Besides its direct effect on lung metastases, aerosol GCB suppressed the growth of subcutaneous LM8 tumor. Histopathological examination of mice receiving aerosol GCB showed no evidence of toxicity. Lungs are distinguished from other tissues by the constitutive expression of FasL. Since exposure of tumor cells to GCB upregulated Fas expression, we hypothesized that the susceptibility of the tumor cells to ligand-induced cell death by resident lung cells may be increased. Therefore, the Fas pathway may contribute to the therapeutic effect of aerosol GCB.

Increased Fas expression reduces the metastatic potential of human osteosarcoma cells.

PURPOSE: The process of metastasis requires the single tumor cell that seeds the metastatic clone to complete a complex series of steps. Identifying factors responsible for these steps is essential in developing and improving targeted therapy for metastasis. Resistance to receptor-mediated cell death, such as the Fas/Fas ligand pathway, is one mechanism commonly exploited by metastatic cell populations. EXPERIMENTAL DESIGN AND RESULTS: LM7, a subline of the SAOS human osteosarcoma cell line with low Fas expression, was selected for its high metastatic potential in an experimental nude mouse model. When transfected with the full-length Fas gene (LM7-Fas), these cells expressed higher levels of Fas than the parental LM7 cells or LM7-neo control-transfected cells. These cells were also more sensitive to Fas-induced cell death than controls. When injected intravenously into nude mice, the LM7-Fas cell line produced a significantly lower incidence of tumor nodules than control cell lines. Lung weight and tumor nodule size were also decreased in those mice injected with LM7-Fas. Levels of Fas were quantified in osteosarcoma lung nodules from 17 patients. Eight samples were Fas negative, whereas the remaining 9 were only weakly positive compared with normal human liver (positive control). CONCLUSIONS: Our results demonstrate that altering Fas expression can impact the metastatic potential of osteosarcoma cells. We conclude that the increase of Fas on the surface of the LM7 osteosarcoma cells increased their sensitivity to Fas-induced cell death in the microenvironment of the lung, where Fas ligand is constitutively expressed. Thus, loss of Fas expression is one mechanism by which osteosarcoma cells may evade host resistance mechanisms in the lung, increasing metastatic potential. Fas may therefore be a new therapeutic target for osteosarcoma.

Cyclosporin A aerosol improves the anticancer effect of paclitaxel aerosol in mice.

The objective of this study was to assess the effect of cyclosporin A liposome aerosol on the anticancer activity of paclitaxel (PTX) liposome aerosol against renal cell carcinoma (Renca) pulmonary metastases in mice. Cyclosporin A (CsA) was administered as a liposome aerosol for one-half hour before starting one-half hour treatment with PTX liposome aerosol (CsA/PTX), and in a second groups of animals cyclosporin A liposome aerosol was given before PTX for one-half hour and also later by mixing a second dose of cyclosporin A aerosol with PTX aerosol and extending the treatment period to one hour (CsA/PTX + CsA). In one experiment, PTX and CsA/PTX aerosols were significantly more effective compared to untreated controls against renal cell cancer as measured by lung weights and tumor surface areas. CsA/PTX was significantly better that PTX alone as measured by lung weights and tumor area. In a second experiment, tumor areas of PTX and CsA/PTX treated mice were significantly reduced compared to untreated controls and CsA/PTX treated mice had significantly smaller tumor areas than PTX treated mice. In contrast, tumor numbers were not significantly fewer than controls in either therapeutic group. In a third experiment, tumor numbers and tumor areas were significantly fewer in mice treated with CsA/PTX and CsA/PTX + CsA compared to untreated controls. Mice treated with CsA/PTX + CsA had significantly fewer tumors and less tumor area than mice receiving CsA/PTX. While PTX treated mice were not different than untreated controls with respect to tumor numbers or tumor volumes, PTX treated mice had significantly greater tumor numbers and tumor areas than CsA/PTX and CsA/PTX + CsA treated mice. Co-administration of CsA with PTX demonstrated significant dose dependent anticancer effects against renal cell pulmonary metastases in mice. Toxicity manifested by weight loss was associated with the highest dose of CsA.

Biodistribution and pharmacokinetics of aerosol and intravenously administered DNA-polyethyleneimine complexes: optimization of pulmonary delivery and retention.

This report describes the time-dependent biodistribution of human p53 plasmid delivered in aerosol with polyethyleneimine in mice compared to the distribution of this material following intravenous injection. Area-under-the-curve values for p53 plasmid after inhalation were 2.8-fold greater than values after intravenous administration, despite the fact that the delivered aerosol dose was one-fifth the intravenous dose. After aerosol administration, pulmonary concentrations of p53 plasmid were high and other organs showed amounts not distinguishable from untreated control. High concentrations of p53 plasmid in the lungs remained with negligible reduction for at least 24 h. Shortly after intravenous injection, organs exhibited the following relative levels of exogenously administered p53: liver > spleen > blood > or = lungs > heart > kidney. These results demonstrate effective pulmonary delivery of DNA in complex with PEI by aerosol, without significant systemic dissemination. In contrast, intravenous administration caused a prompt systemic distribution of DNA with a shorter half-life of the administered gene in the lungs.