Tumor Cells In Nude Mice Research Articles

SU‐E‐QI‐19: Evaluation of a Clinical 1.5T MRI for Prostate Cancer MRS Imaging Using a In Vivo Tumor Model

Purpose:Magnetic resonance spectroscopic (MRS) imaging may provide important bio‐markers to distinguish normal/cancerous prostate tissue. While MRS imaging requires a high uniform magnetic field, the ability of a clinical 1.5T MRI to achieve a comparable MRS signal is of interest for radiation treatment planning/assessment. This study is to evaluate the MRS imaging of a 1.5T clinical MRI for prostate cancers by comparing with a small animal 7T MRS scanner.Methods:A tumor model was developed by implanting LNCaP tumor cells in nude mice prostates. Tumor was monitored 3 weeks after implantation using MRI, and MRS imaging was performed on the tumor area when the tumor reached around 1cm in diameter. The 1.5T GE clinical MR scanner and the 7T Bruker small animal MR scanner were used for each mouse. MR spectrums acquired with these scanners were analyzed and compared. The signals of Choline and Citrate were considered.Results:The prostate tumor MR spectrum under the 1.5T clinical MRI showed a similar spectrum pattern to that acquired using the 7T animal MRI. The Choline signal (3.2ppm) is clear and there is no clear peak for Citrate (2.6ppm). However, the signal magnitude for Choline is not dominant compared to the background signal under 1.5T MRI. Typical cancerous prostate tissue MR spectrum with an increased Choline signal and a reduced Citrate signal was observed. In addition, signal variation is noticeable between repeated spectrum scans. The average of these scans showed a comparable and consistent spectrum to those under 7T MRI.Conclusion:The clinical 1.5T MRI is able to acquire a MR spectrum for prostate cancer comparable to those acquired using a dedicated 7T MRS scanner. However, to achieve a consistent and reliable spectrum, multiple repeated scans were necessary to get a statistical result and reduce the noise‐induced artifact.This work was supported in part by the National Cancer Institute Grant R21 CA131979 and R01CA172638.

Mechanism of anti-tumor and anti-invasive function of SOCS isoform in colon cancer models

Suppressor of cytokine signaling (SOCS) proteins are the negative regulators in cellular signal transduction system. SOCS family proteins consisting of 8 proteins (CIS and SOCS1-7) inhibit JAK/STAT protein activity to maintain proper cytokine functions. Initially identified for the negative feed-back function of cytokine signal transduction, suppressors of cytokine signaling (SOCS) have emerged as multi-functional protein involved in the regulation of development, apoptosis and oncogenesis. Recent reports showed that SOCS can be also affected by cytokine and other cell signaling factors to regulate cancer development and progression as well as cell death. The present study was aimed to investigate various effects of SOCS1 protein on the tumor suppressive function. We have found that SOCS1 promotes radiation-induced apoptosis of colon cancer cells through cell cycle arrest at G1 involving activation of p53 and p21 induction. SOCS1 also exhibits radiosensitization effects on the growth of implanted colon tumor cells in nude mice. As a mechanism of anti-tumor action of SOCS, we now present the data on the suppressive effects of SOCS1 on tumor cell migration and invasion. Notably SOCS1 over-expression and knock-out inversely regulates the expression of EMT markers such as E-cadherin and vimentin along with modest morphological change of cells from epithelial to fibroblast shape, representing EMT process. Data on the invasion and EMT signaling pathways suggest that SOCS1 potentially regulates tumor cell invasion and metastasis in vitro and in vivo in colon cancer models. SOCS1 may be not only a useful predictive factor for radiation response but also a possible drugable target for human colon cancer.

Targeted delivery of siRNA against hepatitis B virus by preS1 peptide molecular ligand

For chronic hepatitis B virus (HBV) infection, the effects of current therapies are limited. RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. However, a suitable delivery vector is still to be developed. We studied a novel vector transferring siRNA targeting hepatic cells in vivo and in vitro in order to find a new way to cure HBV-related live diseases. The preS1-9Arg ligand was used to deliver siRNA to HepG2 and to HepG2 2.2.15 cells. To validate the antiviral efficacy in vivo, a HBV viremic animal model was established by s.c. inoculation of HepG2 2.2.15 tumor cells in nude mice. The minimal retardation effect on the migration of siRNA was detected by gel electrophoresis to confirm the combination and the optimal ratio. Hepatitis B surface antigen (HBsAg) levels were detected by semiquantitatively enzyme-linked immunosorbent assay RNA levels were quantified with quantitative real-time polymerase chain reaction and protein levels were determined with immunoblots and immunohistochemistry. PreS1-9Arg peptide strongly combined and transferred siRNA into HepG2 cells. PreS1-9Arg-siRNA molecular conjugate effectively reduced the production of HBsAg and HBV DNA without liver toxicity in vitro and in vivo. The results indicated that preS1-9Arg may be a potential novel vector to deliver siRNA targeting liver cells.

Abstract No. 263 - In situ nanocarbon assisted microwave therapy (NAMT) for application of targeting human prostate tumor cells in nude mice causing cytotoxic thermal ablation using extremely short cycle microwave energy as primary therapy

Purpose Evaluate use of spherical nanocarbon (Grafex) injected into known Human Prostatic carcinoma to increase absorption of microwave energy, specifically into tumor cells. This study was selected to evaluate if nanocarbon and microwave can be used as primary treatment and selected to study heat sink associated with microwave treatment. This specific nanocarbon has demonstrated a range of energy absorption capabilities superior to other non-metallic materials. Materials and Methods 10 Nude nu/nu mice were injected with DU145 (ATC#HTB-81) 1x 10^7 Human Prostate carcinoma cells introducted into the dermis and allowed to grow to >1cm. One control mouse received no treatment. One control mouse received only an injection of nanocarbon. Remaining 8 mice received treatment with the microwave and the nanocarbon. Medwave and BSD magnetron generators with temperature probes were used. Nanocarbon and a viscous carrier were injected into the tumors. Short cycle power using 10 watts at 15 seconds used as baseline settings. Target temperature within the tumor was 60 degrees C. Results The control non treated mouse was euthanized due to metastatic prostate cancer 3 weeks after initial injection. Seven of the original treated mice have resolved their prostate tumors with no apparent ill effects from the nanocarbon. One mouse had a non treated skin ulcer from the non cooled microwave probe during the treatment process and was also euthanized ,however, was responding to treatment. The one mouse that received just the nanocarbon treatment, is alive, however the tumor growth has not receded or advanced. All mice were kept in isolation and were observed to have no side effects from the nanocarbon. Conclusion Nanocarbon assisted microwave therapy maximizes energy transfer. Conversion of microwave energy causes thermal ablation to the cancer cells. This reduces heat sink effect and surrounding tissue damage by using much shorter treatment times and lower power output wattage. This suggests Grafex nanocarbon causes enhancement of the dielectric properties of the tumor causing cytotoxic heating. To date, there is no observed toxic side effects in mice from this small study.

Gallic acid inhibits gastric cancer cells metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity

Our previous study demonstrated the therapeutic potential of gallic acid (GA) for controlling tumor metastasis through its inhibitory effect on the motility of AGS cells. A noteworthy finding in our previous experiment was increased RhoB expression in GA-treated cells. The aim of this study was to evaluate the role of RhoB expression on the inhibitory effects of GA on AGS cells. By applying the transfection of RhoB siRNA into AGS cells and an animal model, we tested the effect of GA on inhibition of tumor growth and RhoB expression. The results confirmed that RhoB-siRNA transfection induced GA to inhibit AGS cells’ invasive growth involving blocking the AKT/small GTPase signals pathway and inhibition of NF-κB activity. Finally, we evaluated the effect of GA on AGS cell metastasis by colonization of tumor cells in nude mice. It showed GA inhibited tumor cells growth via the expression of RhoB. These data support the inhibitory effect of GA which was shown to inhibit gastric cancer cell metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity. Thus, GA might be a potential agent in treating gastric cancer.

STAT3 Knockdown Reduces Pancreatic Cancer Cell Invasiveness and Matrix Metalloproteinase-7 Expression in Nude Mice

AimsTransducer and activator of transcription-3 (STAT3) plays an important role in tumor cell invasion and metastasis. The aim of the present study was to investigate the effects of STAT3 knockdown in nude mouse xenografts of pancreatic cancer cells and underlying gene expression.MethodsA STAT3 shRNA lentiviral vector was constructed and infected into SW1990 cells. qRT-PCR and western immunoblot were performed to detect gene expression. Nude mouse xenograft assays were used to assess changes in phenotypes of these stable cells in vivo. HE staining was utilized to evaluate tumor cell invasion and immunohistochemistry was performed to analyze gene expression.ResultsSTAT3 shRNA successfully silenced expression of STAT3 mRNA and protein in SW1990 cells compared to control cells. Growth rate of the STAT3-silenced tumor cells in nude mice was significantly reduced compared to in the control vector tumors and parental cells-generated tumors. Tumor invasion into the vessel and muscle were also suppressed in the STAT3-silenced tumors compared to controls. Collagen IV expression was complete and continuous surrounding the tumors of STAT3-silenced SW1990 cells, whereas collagen IV expression was incomplete and discontinuous surrounding the control tumors. Moreover, microvessel density was significantly lower in STAT3-silenced tumors than parental or control tumors of SW1990 cells. In addition, MMP-7 expression was reduced in STAT3-silenced tumors compared to parental SW1990 xenografts and controls. In contrast, expression of IL-1β and IgT7α was not altered.ConclusionThese data clearly demonstrate that STAT3 plays an important role in regulation of tumor growth, invasion, and angiogenesis, which could be act by reducing MMP-7 expression in pancreatic cancer cells.

WE-G-220-07: Treatment Assessment for Pulsed Focused Ultrasound Therapy of Prostate Tumor Using a Clinical 1.5T MRI

Purpose: Recent in vivo experiments have showed non‐thermal effects of pulsed focused ultrasound (pFUS) on tumor growth delay. This study investigates the application of a clinical 1.5T MR scanner for treatment assessment of prostate tumors after pFUS treatment, which provides a non‐ invasive and accurate way to measure tumor volume for treatment efficacy evaluation, and also an alternative way for small animaltumor model study that generally requires high‐magnetic‐field MRI facility (e.g., the Bruker NMR system). Methods: A mouse tumor model was developed by implanting LNCaP tumor cells in nude mice prostates. When tumor volume reached ∼130 mm3 on MRI, mice were randomly assigned into two groups (n=8): (1) pFUS treatment; (2) control. MRI‐guided pFUS treatment was performed using InSightec ExAblate 2000 with a 1.5T GE MR scanner. Non‐thermal pFUS was delivered for 60 sec per sonication (1MHz; 25W acoustic power; 10% duty cycle, on/off=0.1/0.9 sec). The body temperature was kept 40% reduction in tumor volume was shown in mice treated with pFUS compared to that for the control mice Conclusion: Clinical 1.5T MRI is able to assess the tumor volume change after pFUS treatment for a small animal model (tumor size: 3–10mm). It may provide an alternative way for treatment assessment for small animal studies. This work is supported by Focused Ultrasound Surgery Foundation, Varian Medical Systems, DOD PC073127 and DOD BC102806.

Mechanism of apoptosis of esophageal cancer EC9706 in nude mice induced by all-trans retinoic acid

To investigate the mechanism of apoptosis of EC9706 tumor-bearing nude mice induced by all-trans retinoic acid (ATRA). Human esophageal carcinoma cell line EC9706 cells were inoculated into nude mice to establish the solid tumor model. The tumor models were divided into the following groups: ATRA group, fluorouracil group, the two-drugs combination group, and with an equal volume fraction of solvent as the control group. The nude mice were sacrificed after 10 days of medication. TUNEL staining was used to detect cell apoptosis. RT-PCR was used to detect the expression level of mRNA and immunohistochemistry was used to detect the expression level of protein of caspase-3 and survivin, the apoptosis-related genes in the tumor tissue. The apoptosis rates of the ATRA group, 5-Fu group and ATRA + 5-Fu group were 44.3%, 39.7% and 91.0%, respectively. There was a significant difference in comparison with the control group (0.7%), and the ATRA group had no significant difference compared with that of the fluorouracil group (P > 0.05), but the apoptosis rate of the two-drugs combination group was significantly higher than that in the two single-drug groups (P < 0.05). The average gray value of caspase-3 protein expressed in the control group was 46.12 ± 0.33 and the relative expression of caspase-3 mRNA was 0.14 ± 0.03, both were significantly lower than that in the ATRA group, 5-Fu group and the two-drugs combination group (P < 0.05). The average gray value of survivin protein expressed in the control group was 96.07 ± 0.13 and the relative expression of survivin mRNA was 0.84 ± 0.04, both were significantly higher than those of other groups (P < 0.05). The ATRA group had no significant difference compared with the fluorouracil group (P > 0.05), but the two-drugs combination group was significantly different compared with the single-drug groups (P < 0.05). Apoptosis in the EC9706 tumor cells in nude mice can be induced by ATRA. The mechanism may be related with down-regulation of the level of survivin gene expression and up-regulation of the level of caspase-3 gene expression.

HVEGF siRNA inhibits initial growth of A549 tumor cells in nude mice and angiogenesis on CAM assay

To investigate the effect of small interfering RNA (siRNA) targeting human vascular endothelial growth factor (hVEGF) on A549 cell growth in nude mice and angiogenesis on chorioallantoic membrane (CAM) assay. Three pairs of hVEGF siRNA-plasmid and non-silencing-plasmid were constructed, and transfected into A549 cells through lipofectamine 2000, respectively. The most effective pair of hVEGF siRNA-plasmid was selected by ELISA and real-time RT-PCR. A549 cells transfected with selected hVEGF siRNA- plasmid, A549 cells transfected with non-silencing-plasmid and A549 cells without transfection were inoculated into nude mice, respectively. Chick embryos were randomly divided into four groups and CAM was treated by different solutions for 48 h: culture media DMEM as negative control group,un-transfected A549 cell culture supernatants as positive control group, hVEGF siRNA A549 cell culture supernatants as hVEGF siRNA group and nonsilencing siRNA A549 cell culture supernatants as non-silencing siRNA group. The CAMs were harvested on d12 for microscopic assays. Compared with control group, hVEGF siRNA-plasmid induced 48% reduction in hVEGF secretion by A549 cells accompanied by 70% reduction in hVEGF mRNA. Compared with non-silencing siRNA group, the mean tumor volume of murine xenograft was reduced by 58% in hVEGF siRNA group; time for xenografts growing to 50 mm(3)was delayed by 5.4 d. hVEGF contents in xenograft were reduced by 54%; but mean doubling time of tumors and the growth rate of tumors were not significantly reduced. In CAM assays, hVEGF content was zero in negative group, and in hVEGF siRNA group that was 40%-44% of non-silencing siRNA group or positive group; vessels branch points of CAM in hVEGF siRNA group or non-silencing siRNA group or positive group were increased by 45%-55% compared with negative group; total vessel length of CAM in hVEGF siRNA group was increased by 53% compared with negative group, while in non-silencing siRNA group or positive group that was increased by 97% or 99%. Compared with negative control group, the proliferation of microvessels was increased when cell culture supernatant with hVEGF was added in hVEGF siRNA group, significant proliferated vessels were observed in non-silencing siRNA group or positive group. A plasmid-mediated hVEGF siRNA has been constructed and verified, which can effectively downregulate the expression of hVEGF in human A549 cells, resulting in the inhibition of angiogenesis. hVEGF siRNA can delay initial growth of A549 tumor xenograft but not reduce the growth rate.

In vivo gamma imaging of the secondary tumors of transplanted human fetal striatum neural stem cells-derived primary tumor cells

This study describes gamma-imaging of the secondary tumors from the transplanted human fetal striatum neural stem cells-derived primary tumor cells in nude mice. The subcutaneous primary tumors were detected to express integrin alphavbeta3, and the corresponding cells were isolated and enriched in vitro, then transplanted to the nude mice. The technetium-99m-labeled Arg-Gly-Asp peptide, with high affinity to integrin alphavbeta3, was prepared for biodistribution and gamma-imaging. The secondary tumors were readily visualized at 1-h postinjection, and the tumor uptake of radiotracer was similar to that of positive control animals transplanted with U87MG human glioma cells. The tumor specificity of radiotracer was demonstrated by blocking experiment. We concluded that gamma-imaging is a promising approach in imaging the tumorigenesis of transplanted stem cells in vivo.

Inhibitory effect of interferon-α-2b on expression of cyclooxygenase-2 and vascular endothelial growth factor in human hepatocellular carcinoma inoculated in nude mice

To evaluate the effects of interferon-alpha-2b (IFN-alpha-2b) on expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in human hepatocellular carcinoma (HCC) inoculated in nude mice and to study the underlying mechanism of IFN-alpha-2b against HCC growth. Thirty-two nude mice bearing human HCC were randomly divided into four groups (n = 8). On the 10th day after implantation of HCC cells, the mice in test groups (groups A, B and C) received IFN-alpha-2b at a serial dose (10000 IU for group A, 20000 IU for group B, 40000 IU for group C sc daily) for 35 d. The mice in control group received normal saline (NS). The growth conditions of transplanted tumors were observed. Both genes and proteins of COX-2 and VEGF were detected by RT-PCR and Western blot. Apoptosis of tumor cells in nude mice was detected by TUNEL assay after treatment with IFN-alpha-2b. Tumors were significantly smaller and had a lower weight in the IFN-alpha-2b treatment groups than those in the control group (P < 0.01), and the tumor growth inhibition rate in groups A, B and C was 27.78%, 65.22% and 49.64%, respectively. The expression levels of both genes and proteins of COX-2 and VEGF were much lower in the IFN-alpha-2b treatment groups than in the control group (P < 0.01). The apoptosis index (AI) of tumor cells in the IFN-alpha-2b treatment groups was markedly higher than that in the control group (P < 0.01). Group B had a higher inhibition rate of tumor growth, a lower expression level of COX-2 and VEGF and a higher AI than groups A and C (P < 0.05), but there was no significant difference between groups A and C. The inhibitory effects of IFN-alpha-2b on implanted tumor growth and apoptosis may be associated with the down-regulation of COX-2 and VEGF expression. There is a dose-effect relationship. The medium dose of IFN-alpha-2b for inhibiting tumor growth is 20000 IU/d.

Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and β4 integrin function in MDA-MB-231 breast cancer cells

Hepatocyte growth factor (HGF) and its receptor, Met, known to control invasive growth program have recently been shown to play crucial roles in the survival of breast cancer patients. The diet-derived flavonoids have been reported to possess anti-invasion properties; however, knowledge on the pharmacological and molecular mechanisms in suppressing HGF/Met-mediated tumor invasion and metastasis is poorly understood. In our preliminary study, we use HGF as an invasive inducer to investigate the effect of flavonoids including apigenin, naringenin, genistein and kaempferol on HGF-dependent invasive growth of MDA-MB-231 human breast cancer cells. Results show that apigenin presents the most potent anti-migration and anti-invasion properties by Boyden chamber assay. Furthermore, apigenin represses the HGF-induced cell motility and scattering and inhibits the HGF-promoted cell migration and invasion in a dose-dependent manner. The effect of apigenin on HGF-induced signaling activation involving invasive growth was evaluated by immunoblotting analysis, it shows that apigenin blocks the HGF-induced Akt phosphorylation but not Met, ERK, and JNK phosphorylation. In addition to MDA-MB-231 cells, apigenin exhibits inhibitory effect on HGF-induced Akt phosphorylation in hepatoma SK-Hep1 cells and lung carcinoma A549 cells. By indirect immunofluorescence microscopy assay, apigenin inhibits the HGF-induced clustering of β4 integrin at actin-rich adhesive site and lamellipodia through PI3K-dependent manner. Treatment of apigenin inhibited HGF-stimulated integrin β4 function including cell–matrix adhesion and cell–endothelial cells adhesion in MDA-MB-231 cells. By Akt-siRNA transfection analysis, it confirmed that apigenin inhibited HGF-promoted invasive growth involving blocking PI3K/Akt pathway. Finally, we evaluated the effect of apigenin on HGF-promoted metastasis by lung colonization of tumor cells in nude mice and organ metastasis of tumor cells in chick embryo. By histological and gross examination of mouse lung and real-time PCR analysis of human alu in host tissues, it showed that apigenin, wortmannin, as well as anti-β4 antibody all inhibit HGF-promoted metastasis. These data support the inhibitory effect of apigenin on HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and integrin β4 function.

Evaluating Drug Efficacy and Toxicology in Three Dimensions: Using Synthetic Extracellular Matrices in Drug Discovery

The acceptance of the new paradigm of 3-D cell culture is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. I describe the development of a covalently cross-linked mimic of the extracellular matrix (sECM), now commercially available, for 3-D culture of cells in vitro and for translational use in vivo. These bio-inspired, biomimetic materials can be used "as is" in drug discovery, toxicology, cell banking, and, ultimately, medicine. For cell therapy and the development of clinical combination products, the sECM biomaterials must be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end users, physicians and patients, must dictate the key design criteria. In chemical terms, the sECM consists of chemically-modified hyaluronan (HA), other glycosaminoglycans (GAGs), and ECM polypeptides containing thiol residues that are cross-linked using biocompatible polyvalent electrophiles. For example, co-cross-linking the semisynthetic thiol-modified HA-like GAG with thiol-modified gelatin produces Extracel as a hydrogel. This hydrogel may be formed in situ in the presence of cells or tissues to provide an injectable cell-delivery vehicle. Alternately, an Extracel hyrogel can be lyophilized to create a macroporous scaffold, which can then be employed for 3-D cell culture. In this Account, we describe four applications of sECMs that are relevant to the evaluation of drug efficacy and drug toxicity. First, the uses of sECMs to promote both in vitro and in vivo growth of healthy cellularized 3-D tissues are summarized. Primary or cell-line-derived cells, including fibroblasts, chondrocytes, hepatocytes, adult and embryonic stem cells, and endothelial and epithelial cells have been used. Second, primary hepatocytes retain their biochemical phenotypes and achieve greater longevity in 3-D culture in Extracel. This constitutes a new 3-D method for rapid evaluation of hepatotoxicity in vitro. Third, cancer cell lines are readily grown in 3-D culture in Extracel, offering a method for rapid evaluation of new anticancer agents in a more physiological ex vivo tumor model. This system has been used to evaluate signal transduction modifiers obtained from our research on lipid signaling. Fourth, a new "tumor engineering" xenograft model uses orthotopic injection of Extracel-containing tumor cells in nude mice. This approach allows production of patient-specific mice using primary human tumor samples and offers a superior metastatic cancer model. Future applications of the injectable cell delivery and 3-D cell culture methods include chemoattractant and angiogenesis assays, high-content automated screening of chemical libraries, pharmacogenomic and toxicogenomic studies with cultured organoids, and personalized treatment models. In summary, the sECM technology offers a versatile "translational bridge" from in vitro to in vivo to facilitate drug discovery in both academic and pharmaceutical laboratories.

Inhibitory effects of transfected Bcl-XL antisense oligodeoxynucleotide on proliferation of esophageal cancer cells and growth of human esophageal carcinoma in nude mice

To investigate the biologic effects of Bcl-XL antisense oligodeoxynucleotide (ASODN) transfected into cultured esophageal carcinoma cells and human esophageal carcinoma xenograft in nude mice. Cationic liposome-mediated ASODN was used to transfect esophageal carcinoma cells. RT-PCR, Western blot, MTT assay, flow cytometry and in-situ apoptosis cells detection (TUNEL detection) were used to systematically study the biological effects of the transfected cells in vitro and in vivo. MTT assay showed that the proliferation of esophageal carcinoma cells in the ASODN group decreased significantly as compared with control (P < 0.05), along with a 57.3% inhibitory rate of Bcl-XL mRNA, a significant decrease of Bcl-XL protein and the apoptosis rates of (31.1 +/- 5.8)% and 35.0% by flow cytometry and TUNEL assay, respectively (P < 0.01, as compared with controls). The growth of human esophageal carcinoma in nude mice was also significantly inhibited in the ASODN group (P < 0.05), along with a significant decrease of Bcl-XL mRNA and protein expression, and also an enhanced apoptosis of the tumor cells in nude mice. Bcl-XL ASODN can effectively inhibit the proliferation of esophageal carcinoma cells in vitro and the growth of the tumor in vivo. The suppression of Bcl-XL expression by ASODN may offer both a therapeutic approach and an important theoretic foundation for gene therapy against esophageal carcinoma.

Human antibody SC-1 reduces disseminated tumor cells in nude mice with human gastric cancer

Advanced gastric cancer is a systemic disease that requires adjuvant therapy targeted at eliminating disseminated tumor cells (DTCs). We investigated whether the apoptosis-inducing human monoclonal IgM antibody SC-1 was able to reduce the number of disseminated gastric cancer cells in blood and bone marrow. Human gastric tumor specimens with positive expression of the SC-1 receptor were transplanted in nude mice with metastasizing gastric cancer. After tumor growth (4-6 weeks) animals were randomly allocated to intraperitoneal 100 microg SC-1 (n=23) or 100 microg human IgM (n=23). One week later, animals were sacrificed and blood and bone marrow specimens were obtained. A nested RT-PCR for cytokeratin 20 (CK-20) from blood and bone marrow of mice was performed for detection of disseminated tumor cells. Animals receiving SC-1 had significantly fewer DTCs than did control animals (p=0.0011). None of the SC-1 mice had DTCs simultaneously in both blood and bone marrow versus four of the control animals (p=0.0363). The reduction of DTCs in SC-1 animals was due to reduction in bone marrow (p=0.032 compared to controls), but not in blood (p=0.1158). Treatment with SC-1 significantly reduced the number of DTCs in bone marrow in this animal model.

In vivo selective and distant killing of cancer cells using adenovirus-mediated decorin gene transfer.

Decorin is a well-known, ubiquitous proteoglycan that is a normal component of the ECM. Upon transgenic expression of decorin, tumor cells with diverse histogenetic background overexpress p21WAF1, a potent inhibitor of cyclin-dependent kinase activity, become arrested in G1, and fail to generate tumors in immunocompromised animals. Because decorin is a secreted protein, it has been recently suggested that decorin could act as an autocrine and paracrine regulator of tumor growth. Here, we demonstrate that adenovirus (Ad)-mediated transfer and expression of human decorin cDNA induced in vivo apoptosis of xenograft tumor cells in nude mice. This oncolytic activity was observed when the Ad vector encoding the decorin cDNA was injected intratumorally (i.t.) or i.v. Importantly, i.t. injection of the decorin Ad vector led to growth inhibition of the injected tumor associated with similar growth inhibition of a distant contralateral tumor, demonstrating a distant decorin antitumoral effect. Immunochemistry against human decorin and decorin quantitation in tumors confirmed that decorin migrated to the tumor distant site. Furthermore, decorin effect was specific to tumor cells, because neither apoptosis nor growth inhibition were observed in nontumoral human cells such as hepatocytes, endothelial cells, and fibroblasts, despite p21 overexpression.

Detection of disseminated tumor cells in nude mice with human gastric cancer.

Disseminated tumor cells (DTC) are a potential contributor to relapse of cancer. In the present study we developed a model for induction of disseminated tumor cells in nude mice, which can aid in the search for therapeutic approaches as well as improve our understanding of metastasizing gastric cancer. To detect DTC in blood and bone marrow we established a modified animal model of orthotopic transplantation. Two groups of nude mice were used for xenotransplantation of gastric cancer specimens. In group I tumor specimens originating from a gastric adenocarcinoma cell line were transplanted onto the stomach; in group II they were transplanted subcutaneously into both axillaries. Tumor growth, metastases and presence of DTC were compared in both groups. For detection of DTC a nested reverse transcriptase polymerase chain reaction (RT-PCR) for human cytokeratin (CK)-20 was performed on blood and bone marrow of all mice. Tumor growth occurred in both groups (9/10 animals in group I, 10/10 in group II) within 14 weeks. Only animals in group I developed local invasive tumor growth, stenosis of the stomach and distant metastases. Tumors in animals of group II grew with local displacement only and developed no metastases. There were no signals of CK-20 detected in the blood in both groups. In group I, 5 of 9 animals had positive signals of human CK-20 in their bone marrow as a sign of DTC. In group II no DTC were detected in bone marrow. We conclude that orthotopic transplantation is a prerequisite for the development of DTC and metastasizing tumor growth in this modified gastric cancer model.

Suppressor of Fused Negatively Regulates β-Catenin Signaling

Suppressor of fused (Su(fu)) is a negative regulator of the Hedgehog signaling pathway that controls the nuclear-cytoplasmic distribution of Gli/Ci transcription factors through direct protein-protein interactions. We show here that Su(fu) is present in a complex with the oncogenic transcriptional activator beta-catenin and functions as a negative regulator of T-cell factor (Tcf)-dependent transcription. Overexpression of Su(fu) in SW480 (APC(mut)) colon cancer cells in which beta-catenin protein is stabilized leads to a reduction in nuclear beta-catenin levels and in Tcf-dependent transcription. This effect of Su(fu) overexpression can be blocked by treatment of these cells with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export. Overexpression of Su(fu) suppresses growth of SW480 (APC(mut)) tumor cells in nude mice. These observations indicate that Su(fu) negatively regulates beta-catenin signaling and that CRM-1-mediated nuclear export plays a role in this regulation. Our results also suggest that Su(fu) acts as a tumor suppressor.

A Transplantable Human Carcinoid as Model for Somatostatin Receptor-Mediated and Amine Transporter-Mediated Radionuclide Uptake

A human midgut carcinoid tumor was successfully transplanted into nude mice and propagated for five consecutive generations (30 months) with well-preserved phenotype. Tumor cells in nude mice expressed identical neuroendocrine markers as the original tumor, including somatostatin receptors (somatostatin receptors 1 to 5) and vesicular monoamine transporters (VMAT1 and VMAT2). Because of the expression of somatostatin receptors and VMAT1 and VMAT2 the grafted tumors could be visualized scintigraphically using the somatostatin analogue 111In-octreotide and the catecholamine analogue 123I-metaiodobenzylguanidine. The biokinetics of the somatostatin analogue 111In-octreotide in the tumors was studied and showed a high retention 7 days after administration. Cell cultures were re-established from transplanted tumors. Immunocytochemical and ultrastructural studies confirmed the neuroendocrine differentiation. The human origin of transplanted tumor cells was confirmed by cytogenetic and fluorescence it situ hybridization analyses. Spontaneous secretion of serotonin and its metabolite, 5-hydroxyindole acetic acid, from tumor cells was demonstrated. The tumor cells increased their [Ca2+]i in response to beta-adrenoceptor stimulation (isoproterenol) and K+-depolarization. All somatostatin receptor subtypes could be demonstrated in cultured cells. This human transplantable carcinoid tumor, designated GOT1, grafted to nude mice, will give unique possibilities for studies of somatostatin receptor- and VMAT-mediated radionuclide uptake as well as for studies of secretory mechanisms.

Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development.

Ras proteins are guanine nucleotide-binding proteins that play pivotal roles in the control of normal and transformed cell growth and are among the most intensively studied proteins of the past decade. After stimulation by various growth factors and cytokines, Ras activates several downstream effectors, including the Raf-1/mitogen-activated protein kinase pathway and the Rac/Rho pathway. In approximately 30% of human cancers, including a substantial proportion of pancreatic and colon adenocarcinomas, mutated ras genes produce mutated proteins that remain locked in an active state, thereby relaying uncontrolled proliferative signals. Ras undergoes several posttranslational modifications that facilitate its attachment to the inner surface of the plasma membrane. The first-and most critical-modification is the addition of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein farnesyltransferase (FTase). It follows that inhibiting FTase would prevent Ras from maturing into its biologically active form, and FTase is of considerable interest as a potential therapeutic target. Different classes of FTase inhibitors have been identified that block farnesylation of Ras, reverse Ras-mediated cell transformation in human cell lines, and inhibit the growth of human tumor cells in nude mice. In transgenic mice with established tumors, FTase inhibitors cause regression in some tumors, which appears to be mediated through both apoptosis and cell cycle regulation. FTase inhibitors have been well tolerated in animal studies and do not produce the generalized cytotoxic effects in normal tissues that are a major limitation of most conventional anticancer agents. There are ongoing clinical evaluations of FTase inhibitors to determine the feasibility of administering them on dose schedules like those that portend optimal therapeutic indices in preclinical studies. Because of the unique biologic aspects of FTase, designing disease-directed phase II and III evaluations of their effectiveness presents formidable challenges.