Homozygous deletion of glycogen synthase kinase 3beta bypasses senescence allowing Ras transformation of primary murine fibroblasts.

In primary mammalian cells, expression of oncogenes such as activated Ras induces premature senescence rather than transformation. We show that homozygous deletion of glycogen synthase kinase (GSK) 3beta (GSK3beta-/-) bypasses senescence induced by mutant Ras(V12) allowing primary mouse embryo fibroblasts (MEFs) as well as immortalized MEFs to exhibit a transformed phenotype in vitro and in vivo. Both catalytic activity and Axin-binding of GSK3beta are required to optimally suppress Ras transformation. The expression of Ras(V12) in GSK3beta-/-, but not in GSK3beta+/+ MEFs results in translocation of beta-catenin to the nucleus with concomitant up-regulation of cyclin D1. siRNA-mediated knockdown of beta-catenin decreases both cyclin D1 expression and anchorage-independent growth of transformed cells indicating a causal role for beta-catenin. Thus Ras(V12) and the lack of GSK3beta act in concert to activate the beta-catenin pathway, which may underlie the bypass of senescence and tumorigenic transformation by Ras.

Pure ethiodized oil as a capillary embolic agent with and without ethanol-ethiodol mixture in the rabbit kidney: embolic efficacy and temporal histopathologic findings.

PURPOSE: To determine the extent of ablation and the temporal histopathologic findings associated with selective arterial injection of pure Ethiodol in the normal rabbit kidney, with or without arterial occlusion of the main renal artery. MATERIALS AND METHODS: In 19 rabbits, 27 kidneys were embolized by injecting 0.6 mL of pure Ethiodol into the main renal artery to achieve capillary stasis. A 9:1 ethanol-ethiodized oil mixture was then injected into 17 of the 27 kidneys until complete arterial stasis was accomplished. Macro- and microscopic evaluation was performed 10 minutes to 6 weeks and 60 minutes to 1 week, respectively, for kidneys with and without arterial occlusion. RESULTS: Ethiodol followed by ethanol-Ethiodol mixture (mean +/- standard deviation, 0.37 mL +/- 0.03) caused complete and permanent arterial stasis in all 17 kidneys. Thrombosis of the large arteries occurred initially. Ischemic coagulative necrosis of renal tubules and damage to glomeruli were detected 2 hours after embolization. Within 24 hours, the glomeruli and most tubules of the cortex and medulla were necrotic. Without arterial occlusion, the arteriocapillary bed of the kidneys was completely patent, with normal contrast medium excretion. Ethiodol was observed in glomeruli and interstitial capillaries from 60 minutes to 1 week and caused mild acute glomerulitis from day 1. The lesions were confined to the glomeruli, and no significant parenchymal changes were observed. CONCLUSIONS: In the rabbit, selective arterial injection of pure Ethiodol produces complete renal ablation within 24 hours if prompt and permanent occlusion of the arterial compartment guarantees its permanent capillary retention.

The novel triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid inhibits metastatic murine breast tumor growth through inactivation of STAT3 signaling.

We and others have reported that C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) effectively inhibits the growth of multiple cancer cell types. Our previous studies indicated that prolonged CDDO-Me treatment inactivated extracellular signal-regulated kinase signaling in acute myelogenous leukemia cells. Whether treatment with CDDO-Me has an earlier effect on other proteins that are important for either signal transduction or oncogenesis is unknown. Constitutively activated signal transducer and activator of transcription 3 (STAT3) is frequently found in human breast cancer samples. Constitutively activated STAT3 was shown to up-regulate c-Myc in several types of cancer and has a feedback effect on Src and Akt. To examine the effects of CDDO-Me on STAT3 signaling in breast cancer, we used the murine 4T1 breast tumor model, which is largely resistant to chemotherapy. In vitro, after treatment of 4T1 cells with 500 nmol/L CDDO-Me for 2 h, we found (a) inactivation of STAT3, (b) inactivation of Src and Akt, (c) 4-fold reduction of c-Myc mRNA levels, (d) accumulation of cells in G(2)-M cell cycle phase, (e) abrogation of invasive growth of 4T1 cells, and (f) lack of apoptosis induction. In in vivo studies, CDDO-Me completely eliminated 4T1 breast cancer growth and lung metastases induced by 4T1 cells in mice when treatment started 1 day after tumor implantation and significantly inhibited tumor growth when started after 5 days. In vivo studies also indicated that splenic mature dendritic cells were restored after CDDO-Me treatment. In summary, these data suggest that CDDO-Me may have therapeutic potential in breast cancer therapy, in part, through inactivation of STAT3.

Metastasis-associated protein 1 transgenic mice: a new model of spontaneous B-cell lymphomas.

Metastasis-associated protein 1 (MTA1), a component of the nuclear remodeling complex and the founding homologue of the MTA family, has been implicated in metastasis, but definitive causative evidence in an animal model system is currently lacking. Here, we show that MTA1 overexpression in transgenic mice is accompanied by a high incidence of spontaneous B cell lymphomas including diffuse large B cell lymphomas (DLBCL). Lymphocytes and lymphoma cells from MTA1-TG mice are hyperproliferative. Lymphomas were transplantable and of clonal origin and were characterized by down-regulation of p27Kip1 as well as up-regulation of Bcl2 and cyclin D1. The significance of these murine studies was established by evidence showing a widespread up-regulation of MTA1 in DLBCL from humans. These findings reveal a previously unrecognized role for the MTA1 pathway in the development of spontaneous B cell lymphomas, and offer a potential therapeutic target in B cell lymphomas. These observations suggest that MTA1-TG mice represent a new model of spontaneous DLBCL associated with high tumor incidence and could be used for therapeutic intervention studies.

A pilot trial of vascular targeted photodynamic therapy for renal tissue.

PURPOSE: Vascular targeted photodynamic therapy represents the newest generation of photodynamic therapy and a new paradigm for minimally invasive ablative therapy. We report a pilot trial of vascular targeted photodynamic therapy to evaluate the effect on porcine renal tissue. MATERIALS AND METHODS: Pigs underwent continuous infusion of WST-09 (Negma-Lerads, Toussous le Noble, France) and concurrent illumination with interstitial laser at a wavelength of 763 nm to the lower pole of the kidney. Drug doses were 0.5 to 1.0 mg/kg and light doses were 100 to 200 J. Nuclear renography was performed on postoperative day 5. On postoperative day 7 arteriography, pyelography, computerized tomography of the abdomen and necropsy were performed. RESULTS: Four of 7 animals completed therapy and all evaluations. Three animals died, including 1 of surgical complications and 2 of an anaphylactoid reaction to the Cremophor solvent in the compound. All kidneys in surviving animals functioned on nuclear renography. Renal function remained unchanged. No lesions or urine leakage was visible on imaging. On necropsy lesion size was 5 x 4 x 3 to 7 x 7 x 14 mm depending on the drug/light dose. Histology showed a distinct demarcation between the treated zone and the surrounding parenchyma at higher doses. Lesions were well demarcated with necrotic tubules, glomerular fibrinoid necrosis, capillary loop thrombosis, interstitial hemorrhage and lymphocytic infiltrates. CONCLUSIONS: Significant tissue effect with some necrosis was seen at these low drug/light combinations. This study provides the initial proof of principle that justifies further preclinical investigation of vascular targeted photodynamic therapy for renal tumors. A newer, water based formulation should decrease the incidence of reactions in swine. This newer formulation may allow further safe investigation of this novel treatment paradigm.

Development of a macromolecular dual-modality MR-optical imaging for sentinel lymph node mapping.

OBJECTIVE: To evaluate the effectiveness of a dual magnetic resonance-near infrared fluorescence optical imaging agent, poly(l-glutamic acid)-DTPA-Gd-NIR813, for both preoperative and intraoperative visualization and characterization of sentinel lymph nodes (SLN) in mice. MATERIALS AND METHODS: Poly(L-glutamic acid) was conjugated with DTPA-Gd and NIR813 dye to obtain PG-DTPA-Gd-NIR813. To confirm drainage into the SLNs, this agent was injected subcutaneously into the front paw of nude mice followed by isosulfan blue (n = 6). Furthermore, PG-DTPA-Gd-NIR813 was injected subcutaneously at doses of 0.002 mmol Gd/kg (4.8 nmol eq. NIR813) and 0.02 mmol Gd/kg (48 nmol eq. NIR813) (n = 3/dose). To differentiate metastatic from nonmetastatic lymph nodes, nude mice bearing human oral squamous cell carcinoma (DM14) were injected intralingually with 0.02 mmol Gd/kg PG-DTPA-Gd-NIR813 (n = 3). Pre- and postcontrast images were taken using 4.7 T Bruker MRI scanner and Xenogen optical imaging system. The status of lymph nodes resected under the guidance of optical imaging was confirmed by histologic examinations. RESULTS: PG-DTPA-Gd-NIR813 colocalized with isosulfan blue, indicating drainage to the SLN. After subcutaneous injection, axiliary and branchial lymph nodes were clearly visualized with both T1-weighted MR and optical imaging within 3 minutes of contrast injection, even at the lowest dose tested (0.002 mmol Gd/kg). After intralingual injection in tumor-bearing mice, MR imaging identified 4 of the 6 superficial cervical lymph nodes, whereas near-infrared fluorescence (NIRF) optical imaging identified all 6 cervical nodes. The pattern of contrast enhancement of SLN visualized in MR images showed a characteristic ring-shaped appearance with a central filling defect, possibly resulting from nodal infiltration of metastatic lesions. Histopathologic examination of the SLNs resected under NIRF imaging guidance revealed micrometastases in all 6 SLNs identified by NIRF imaging. CONCLUSIONS: The dual modality imaging method demonstrated in this study represents an effective technique for localization and characterization of SLN.

Metastasis tumor antigen family proteins during breast cancer progression and metastasis in a reliable mouse model for human breast cancer.

PURPOSE: Chromatin remodeling pathways are critical in the regulation of cancer-related genes and are currently being explored as potential targets for therapeutic intervention. The metastasis tumor antigen (MTA) family of proteins, MTA1, MTA2, and MTA3, are components of chromatin remodeling pathways with potential roles in breast cancer. Although all three MTA family proteins have been shown to be associated with metastatic progression of breast cancers, the expression characteristic of MTA1-3 proteins in a multistep breast cancer progression model remains unknown. Structural and functional studies have suggested that they are heterogeneous in the Mi-2/NuRD complex, exhibit tissue-specific patterns of expression, and impart unique properties to estrogen receptor-alpha (ERalpha) action. This led us to hypothesize that each member of the MTA family possesses a unique role and interacts with different pathways in the stepwise process of breast cancer development and progression. EXPERIMENTAL DESIGN: MTA family proteins were examined by immunohistochemistry in breast cancer processes ranging from normal duct, to premalignant lesions, to invasive carcinoma, and to metastasized tumors in PyV-mT transgenic mice, which represents a reliable model for multistage tumorigenesis of human breast cancer. We also determined the association of MTA proteins with the status of cell proliferation, ER, E-cadherin and cytoplasmic beta-catenin, and cancer-related coactivators, AIB1 and PELP1. RESULTS: The expression of all three MTA proteins was altered in primary breast tumors. Each MTA protein had a unique expression pattern during the primary breast tumor progression. Altered expression of MTA1 was observed in both premalignant lesion and malignant carcinoma, but an elevated nuclear expression was observed in ER-negative carcinomas. MTA3 was exclusively expressed in a subset of cells of ER-positive premalignant lesions but not in carcinomas. MTA2 expression seems to be unrelated to ER status. Loss of MTA3 expression and more nuclear localization of MTA1 occurred with loss of E-cadherin and decreased cytoplasmic beta-catenin, two molecules essential for epithelial cell adhesion and important tumor cell invasion. At the late stage of tumor formation, MTA1 is usually expressed in the center of tumors. Coincidentally, the distribution of MTA1-positive cells at this stage was complementary to that of AIB1 and PELP1, which were localized to the tumor periphery with relatively active cell proliferation, scattered ER-positive cells and a limited differentiation. In metastasized lung tumors, the expression pattern of MTA-protein expression was distinct from that in primary counterparts. CONCLUSIONS: The findings presented here support the notion that each member of the MTA family might potentially play a stepwise role in a cell type-specific manner during breast cancer progression to metastasis. On the basis of the noted temporal expression patterns of MTA proteins with ER status, cell adhesion-essential regulators (E-cadherin and cytoplasmic beta-catenin), and coactivators, we propose that MTA protein-related chromatin remodeling pathways interact with steroid receptors, growth factor receptors, and other transcriptional signaling pathways to orchestrate the governing of events in breast cancer progression and metastasis.

Mutation of Lkb1 and p53 genes exert a cooperative effect on tumorigenesis.

Peutz-Jeghers syndrome (PJS) is a dominantly inherited disorder characterized by gastrointestinal hamartomatous polyps and mucocutaneous melanin pigmentation. Germ line mutations in LKB1 cause PJS. We have generated mice carrying an Lkb1 exon 2 to 8 deletion by gene targeting in embryonic stem cells. Heterozygotes develop gastric hamartomas that are histologically similar to those found in humans with PJS. LKB1 is also reportedly a mediator of p53-dependent apoptosis. To explore the potential combined effects of p53 and Lkb1 alterations on tumorigenesis, we carried out a series of matings with Lkb1(+/-) and p53 null mice to generate Lkb1(+/-)/p53(+/-) and Lkb1(+/-)/p53(-/-) mice. Similar to the Lkb1(+/-) mice, gastrointestinal hamartomas have also been detected in the mice with these two genotypes. The Lkb1(+/-)/p53(+/-) mice displayed a dramatically reduced life span and increased tumor incidence compared to the mice with either Lkb1 or p53 single gene knockout. The time to onset of polyposis in Lkb1(+/-)/p53(-/-) mice is approximately 2 months earlier than Lkb1(+/-)/p53(+/-) and Lkb1(+/-) mice, whereas the latter two show a similar time to onset which is at approximately 6 months of age. These results strongly suggested that mutations of p53 and Lkb1 gene cooperate in the acceleration of tumorigenesis.

Targeted expression of green fluorescent protein/tumor necrosis factor-related apoptosis-inducing ligand fusion protein from human telomerase reverse transcriptase promoter elicits antitumor activity without toxic effects on primary human hepatocytes.

Liver toxicity is the major concern for use of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) proteins in treatment of cancers. Here we report that normal human primary hepatocytes (NHPHs) are susceptible to the transduction of the wild-type, full-length coding sequence of the human TRAIL gene. To minimize potential toxicity of the TRAIL gene, a bicistronic adenoviral vector that expresses the green fluorescent protein/TRAIL fusion protein from the human telomerase reverse transcriptase promoter (designated Ad/gTRAIL) was constructed. In vitro and in vivo studies have showed that treatment with the adenoviral vector Ad/gTRAIL results in high-level expression of green fluorescent protein/TRAIL in cancer cells but no detectable transgene expression in NHPHs or in normal mouse liver tissues. Furthermore, treatment with Ad/gTRAIL effectively elicited apoptosis in malignant cells but not in NHPHs in vitro and suppressed tumor growth and prolonged duration of survival in vivo. Thus, with the combined advantages of the TRAIL gene and the human telomerase reverse transcriptase target, Ad/gTRAIL can be a potent therapeutic agent for the treatment of cancers.

Long-term effects of neonatal exposure to hydroxylated polychlorinated biphenyls in the BALB/cCrgl mouse.

The neonatal mouse model has been a valuable tool in determining the long-term effects of early exposure to estrogenic agents in mammals. Using this model, we compared the effects of 2',4',6'-trichloro-4-biphenylol (OH-PCB-30) and 2',3',4',5'-tetrachloro-4-biphenylol (OH-PCB-61) as prototype estrogenic hydroxylated PCBs (OH-PCBs) because they are reported to exhibit relatively high estrogenic activity both in vivo and in vitro. The purpose of this study was to examine the relationship between estrogenicity and carcinogenicity of OH-PCB congeners. The OH-PCBs were tested individually and in combination to determine whether effects of combined OH-PCBs differed from those of these OH-PCBs alone. We evaluated the long-term effects of neonatal exposure to OH-PCBs with treatment doses that were based on the reported binding affinity of specific OH-PCB congeners to estrogen receptor alpha. BALB/cCrgl female mice were treated within 16 hr after birth by subcutaneous injections every 24 hr, for 5 days. The mice treated with OH-PCB-30 (200 microg/day) or 17beta-estradiol (5 microg/day) showed similar increased incidences of cervicovaginal (CV) tract carcinomas (43% and 47%, respectively). In addition, when mice were treated with OH-PCBs as a mixture, a change in the type of CV tract tumor was observed, shifting from predominantly squamous cell carcinomas to adenosquamous cell carcinoma. From our results, we conclude that the individual OH-PCBs tested were estrogenic and tumorigenic in mice when exposed during development of the reproductive tract. These data support the hypothesis that mixtures may act differently and unexpectedly than do individual compounds.

Biological and genetic properties of the p53 null preneoplastic mammary epithelium.

The absence of the tumor suppressor gene p53 confers an increased tumorigenic risk for mammary epithelial cells. In this report, we describe the biological and genetic properties of the p53 null preneoplastic mouse mammary epithelium in a p53 wild-type environment. Mammary epithelium from p53 null mice was transplanted serially into the cleared mammary fat pads of p53 wild-type BALB/c female to develop stable outgrowth lines. The outgrowth lines were transplanted for 10 generations. The outgrowths were ductal in morphology and progressed through ductal hyperplasia and ductal carcinoma in situ before invasive cancer. The preneoplastic outgrowth lines were immortal and exhibited activated telomerase activity. They are estrogen and progesterone receptor-positive, and aneuploid, and had various levels of tumorigenic potential. The biological and genetic properties of these lines are distinct from those found in most hyperplastic alveolar outgrowth lines, the form of mammary preneoplasia occurring in most traditional models of murine mammary tumorigenesis. These results indicate that the preneoplastic cell populations found in this genetically engineered model are similar in biological properties to a subset of precurser lesions found in human breast cancer and provide a unique model to identify secondary events critical for tumorigenicity and invasiveness.

The association of p21((WAF-1/CIP1)) with progression to androgen-independent prostate cancer.

The molecular events leading to progression toward androgen-independent prostate cancer (AIPC) are not fully understood. The p21((WAF-1/CIP1)) (p21) gene has been identified as a key factor for the regulation of cell growth. The expression of p21 was examined by immunohistochemical studies in 105 prostate cancer samples: (a) 7 of 30 (23%) androgen-dependent tumors; and (b) 36 of 75 (48%) androgen-independent tumors stained positive for p21 (P < 0.02). No association was found between p21 expression and p53, bcl-2, and the androgen receptor protein expression in bone metastases of patients with AIPC, whereas there was a significant association with a high Ki-67 index (P < 0.05). In 4 of 43 (9%) cases, tumors displayed a p53-negative, bcl-2-negative, and p21-positive phenotype. A xenograft mouse model of prostate cancer using the androgen-responsive MDA PCa 2b prostate cancer cell line was used to study p21 expression after androgen deprivation and at relapse. Androgen deprivation reduced p21 expression to undetectable levels after 14 days. Tumor relapse, defining AIPC, was associated with increased expression of p21 to levels comparable with those found before castration. In this model, p21 expression at relapse was also correlated with a high Ki-67 index. In conclusion, p21 expression is associated with the progression to AIPC. A possible explanation involves a paracrine effect of p21 mediated by the release of mitogenic and antiapoptotic factors. Another explanation involves the regulation of p21 expression by the androgen receptor, which also suggests that p21 may have antiapoptotic function in prostate cancer.

Evidence that transfer of functional p53 protein results in increased apoptosis in prostate cancer.

PURPOSE: INGN 201 (Ad-p53) is a replication-defective adenoviral vector that encodes a wild-type p53 gene driven by the cytomegalovirus promoter. INGN 201 has been shown to have antitumoral activity against human prostate cancer cell lines. This study was undertaken to determine the safety of INGN 201 in patients with locally advanced prostate cancer, to assess transgene expression, and to evaluate antitumoral activity. EXPERIMENTAL DESIGN: Our study included patients with clinical stage T3, T1c-T2a with Gleason score 8-10 disease, or T2a-T2b with Gleason score 7 disease and a prostate-specific antigen level >10 ng/ml. INGN 201 was administered by intraprostatic injection under ultrasonographic guidance. One course of INGN 201 was defined as three separate INGN 201 administrations 2 weeks apart. Biopsies at baseline and 24 h after the first administration were assessed for p53 protein by immunohistochemical staining and for apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. RESULTS: A total of 38 courses of INGN 201 gene therapy were administered to 30 patients, of whom 26 underwent radical prostatectomy. There were no grade 3 or 4 adverse events related to INGN 201 administration. Of the 11 patients with negative baseline immunostaining for p53 protein, 10 had positive p53 immunostaining after the first administration of INGN 201, and 8 had an increase in apoptotic cells by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. All 26 of the patients who underwent radical prostatectomy had significant residual viable prostate cancer, and 12 have experienced biochemical failure (median follow-up, 42 months). CONCLUSION: Intraprostatic INGN 201 gene therapy is safe and can reliably result in p53 protein production and apoptosis.

Cell proliferation and apoptotic indices predict adenoma regression in a placebo-controlled trial of celecoxib in familial adenomatous polyposis patients.

BACKGROUND: Celecoxib was shown to regress colorectal adenomas in familial adenomatous polyposis (FAP) patients relative to placebo. To address the mechanism of polyp regression, we determined whether celecoxib can modulate cell proliferation, apoptosis, and prostaglandin E(2) (PGE(2)) levels in colorectal epithelia from FAP trial participants and whether such alterations correlate with observed reductions in polyp number. MATERIALS AND METHODS: Colorectal mucosal biopsies were obtained at baseline and on last day of celecoxib (100 or 400 mg twice daily) or placebo administration (6 months). Residual paraffin-embedded adenomas and normal mucosa from the same patients (n = 17) or normal tissue alone (n = 15) were analyzed. Immunoperoxidase staining for Ki-67 was performed and apoptotic cells were identified by their morphology. Ki-67 and apoptotic labeling indices and their ratios were calculated in superficials (s) and nonsuperficial (ns) regions of adenomas and normal mucosa, and baseline to 6-month differences were calculated. PGE(2) levels were analyzed by mass spectroscopy (normal, n = 64; adenoma, n = 56). Biomarkers were analyzed by treatment arm and correlated with previously determined mean percentage reductions in colorectal polyp number. RESULTS: In adenomas, a reduction in the superficial proliferative activity i.e., Ki-67(s) labeling index, accompanied polyp regression (r = -0.76, P = 0.006). An increase in the apoptotic ratio [i.e., superficial apoptotic index (AI(s))/nonsuperficial apoptotic index (AI(ns))] was found to correlate with reduced polyp counts in that higher apoptotic ratios correlated with better response to celecoxib (r = 0.71, P = 0.004). Furthermore, the AI(s)/Ki-67(s) ratio (r = 0.58, P = 0.026) accompanied polyp regression. In normal mucosa, a trend toward increased AI(s) (r = 0.33, P = 0.053) and polyp regression was found. PGE(2) levels did not significantly correlate with polyp regression. Changes in biomarker levels (baseline to 6 months) were correlated in adenomas and normal mucosa (AI(s), r = 0.29, P = 0.024; AI(ns), r = 0.34, P = 0.009; PGE(2), r = 0.50, P = 0.059) within individual patients. CONCLUSION: Suppression of cell proliferation and an increased apoptotic ratio, as well as the ratio of apoptosis to cell proliferation, accompany polyp regression in a chemoprevention trial in FAP patients. These findings suggest potential mechanisms for the efficacy of celecoxib and warrant further study of these biomarkers as intermediate endpoints in FAP patients.

Liposomal vector mediated delivery of the 3p FUS1 gene demonstrates potent antitumor activity against human lung cancer in vivo.

Lung cancer is one of the leading causes of death in the world. The underlying cause for lung cancer has been attributed to various factors that include alteration and mutation in the tumor suppressor genes. Restoration of normal function of the tumor suppressor gene is a potential therapeutic strategy. Recent studies have identified a group of candidate tumor suppressor genes on human chromosome 3p21.3 that are frequently deleted in human lung and breast cancers. Among the various genes identified in the 3p21.3 region, we tested the antitumor activity of the FUS1 gene in two human non-small-cell lung cancer (NSCLC) xenografts in vivo. Intratumoral administration of FUS1 gene complexed to DOTAP:cholesterol (DOTAP:Chol) liposome into subcutaneous H1299 and A549 lung tumor xenograft resulted in significant (P = .02) inhibition of tumor growth. Furthermore, intravenous injections of DOTAP:Chol-FUS1 complex into mice bearing experimental A549 lung metastasis demonstrated significant (P = .001) decrease in the number of metastatic tumor nodules. Finally, lung tumor-bearing animals when treated with DOTAP:Chol-FUS1 complex demonstrate prolonged survival (median survival time: 80 days, P = .01) compared to control animals. This result demonstrates the potent tumor suppressive activity of the FUS1 gene and is a promising therapeutic agent for treatment of primary and disseminated human lung cancer.

The natural progression of microvasculature in primary tumor and lymph node metastases in a breast carcinoma model: relationship between microvessel density, vascular endothelial growth factor expression, and metastatic invasion.

The natural course of tumor microvascularity in rat MTLn3 mammary adenocarcinomas was studied. The relationship between microvessel density (MVD), vascular endothelial growth factor (VEGF) expression, and histopathology was compared in primary and metastatic axillary (ALN) and inguinal lymph node (ILN) tumors over 5-6 tumor doublings. Excised tumors were examined for (i) MVD assessed by immunostaining with anti-CD31 antibody, (ii) VEGF expression assessed by immunostaining with anti-VEGF antibody, and (iii) histopathologic extent of metastatic lymph node invasion. MVD and VEGF scores rose asymptotically with increasing tumor weight in both primary and metastatic tumors. The MVD saturation level was significantly greater for primary tumors (MVD = 22) than for ALNs or ILNs (MVD = 14). Maximal VEGF score was not statistically different between the three kinds of tumors, however the rate of rise in VEGF expression was different. Near-maximal VEGF expression occurred early in tumor growth, preceding microvessel development. Both MVD and VEGF expression in lymph nodes were proportional to the pathology score characterizing increasing metastatic invasion. LNMs limited to the subcapsular sinus had the lowest MVD, indicating an ability to survive without significant vasculature. These findings underscore the differences in angiogenesis between primary tumors and LNMs and have implications for therapy of metastatic cancer.

Pure ethiodized oil-based transcatheter ablative therapy in normal rabbit kidneys and kidneys inoculated with VX-2 carcinoma.

PURPOSE: To evaluate the efficacy of ablation with selective arterial injection of pure ethiodized oil followed by arterial occlusion with 9:1 ethanol-Ethiodol mixture (EEM) and coil placement in normal rabbit kidneys and kidneys inoculated with VX-2 carcinoma. MATERIALS AND METHODS: All experiments were conducted with Animal Care and Use Committee approval. In six rabbits (group 1), one kidney was embolized with pure Ethiodol until capillary stasis, followed by injection of 9:1 EEM until arterial stasis and then coil placement into the main renal artery. In 12 other rabbits, one kidney was inoculated with VX-2 tumor. Ethiodol and EEM embolization and coil placement followed 7 days later (group 2, n = 6) or 11-14 days later (group 3, n = 6). Kidneys were evaluated (angiography, computed tomography, macro- and microscopy) 7 days after treatment. RESULTS: Capillary stasis was achieved in groups 1, 2, and 3 with (mean ± standard deviation) 0.47 ± 0.03, 0.53 ± 0.02, and 0.56 ± 0.04 ml of pure Ethiodol, followed by 0.47 ± 0.05, 0.42 ± 0.03, and 0.38 ± 0.04 ml of EEM, respectively, which caused complete arterial occlusion in 17 of 18 kidneys. In group 1, all but one kidney showed at least 95% generalized coagulative necrosis. In group 2, all six kidneys exhibited 100% coagulative necrosis, with no viable tumor present. In group 3, 100% coagulative necrosis was present in all kidneys, with a small viable tumor in one. CONCLUSION: In the rabbit, selective arterial injection of pure Ethiodol can cause complete renal parenchyma and tumor ablation when it is followed by prompt, contiguous, and permanent occlusion of the arterial compartment.

Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases.

Lysophosphatidic acid (LPA) acts through high-affinity G protein-coupled receptors to mediate a plethora of physiological and pathological activities associated with tumorigenesis. LPA receptors and autotaxin (ATX/LysoPLD), the primary enzyme producing LPA, are aberrantly expressed in multiple cancer lineages. However, the role of ATX and LPA receptors in the initiation and progression of breast cancer has not been evaluated. We demonstrate that expression of ATX or each edg family LPA receptor in mammary epithelium of transgenic mice is sufficient to induce a high frequency of late-onset, estrogen receptor (ER)-positive, invasive, and metastatic mammary cancer. Thus, ATX and LPA receptors can contribute to the initiation and progression of breast cancer.

Evaluation of cerebral microemboli during radiofrequency ablation of lung tumors in a canine model with use of impedance-controlled devices.

PURPOSE: To evaluate the incidence of cerebral microemboli during radiofrequency (RF) ablation of lung tumors in a canine model and evaluate the adverse effects of these microemboli on the brain parenchyma with use of magnetic resonance (MR) imaging and histopathologic examination. MATERIALS AND METHODS: Percutaneous RF ablation of 12 lung tumors in 12 dogs was performed under computed tomography (CT) guidance with use of impedance-controlled devices. The common carotid artery was continuously monitored in each animal during RF ablation with duplex Doppler ultrasonography. All animals underwent brain MR imaging shortly after RF ablation. Delayed brain MR imaging (5-8 days after RF ablation) was performed in eight animals. The MR examinations included diffusion-weighted echo-planar imaging. The animals were euthanized 3-11 days after RF ablation. The brain was harvested from each animal and examined by an experienced veterinary pathologist for evidence of ischemia. RESULTS: RF ablation was technically successful in all animals. Microbubbles were detected in the carotid artery in two animals (17%). Acute and delayed MR studies demonstrated no evidence of ischemic brain injury in any of the animals. Gross and histopathologic assessment of brain tissue also demonstrated no ischemic changes. CONCLUSIONS: During RF ablation of lung tumors, microbubbles are detected in the carotid arteries in a small number of cases. These microbubbles are too few and too small to be detected by CT imaging of the brain and do not cause ischemic brain injury.

mda-7 In combination with bevacizumab treatment produces a synergistic and complete inhibitory effect on lung tumor xenograft.

Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), has shown antitumor activity by inhibiting tumor angiogenesis in preclinical and clinical studies. However, bevacizumab monotherapy does not induce complete tumor regression. Therefore, additional treatments must be combined with bevacizumab to promote tumor regression. We previously showed that melanoma differentiation associated gene-7 (mda-7) protein exerts potent antitumor and antiangiogenic activity. Thus, in this study, we investigated the therapeutic effects of mda-7 in combination with bevacizumab using lung cancer as a model. In vitro, treatment of human umbilical vein endothelial cells with conditioned medium from Ad-mda7 plus bevacizumab-treated lung tumor cells showed reduced VEGF ligand-receptor binding, and decreased cell survival, resulting in growth arrest and apoptosis. In vivo, treatment of subcutaneous lung tumor xenografts with bevacizumab plus Ad-mda7 resulted in significant tumor growth inhibition and improved survival compared to tumor growth in control mice. Furthermore, tumors in all the Ad-mda7 plus bevacizumab-treated mice completely regressed, and these were tumor free through the study's end. Molecular analysis showed enhanced tumor cell apoptosis and reduced VEGF and CD31 expression in Ad-mda7 plus bevacizumab-treated tumors. Thus, Ad-mda7 and bevacizumab treatment produces a synergistic and complete therapeutic effect against human lung cancer.