Mean Tumor Weight Research Articles

Vascular endothelial growth factor-A and changes in a tumor-bearing mouse model with Lewis lung cancer

Vascular endothelial growth factor-A (VEGF-A) affects tumor growth and metastasis through stimulation of angiogenesis. The purpose of this study was to describe features of Lewis lung cancer (LLC) in mice and compare the serum VEGF-A levels with those of normal control mice. Two groups of mice were compared: one was subcutaneously injected with LLC cells (n=16) and the other served as the normal control (n=6). The serum VEGF-A levels were measured by ELISA prior to inoculation, and at 7, 21 and 35 days post-inoculation. The tumor weight and the metastatic condition were evaluated on day 35. Changes in body weight and serum VEGF-A concentration over a period of time were compared between the groups using generalized estimating equations. The relationship between the primary tumor and the metastatic condition was analyzed using the Spearman's rank correlation test. The survival rate was 56.3% on day 35 post-tumor inoculation. No difference was found between the groups with regard to gastrocnemius muscle weight on day 35 post-inoculation [0.1315±0.0066 g vs. 0.1308±0.0069 g (normal control)]. In tumor-bearing mice, the weight gain at sacrifice was less (0.24±0.45 vs. 1.93±0.47 g, P=0.01), the final mean tumor volume and weight were 4264.69±1038.32 mm(3) and 3.70±0.83 g, the number of nodules in the lungs and livers was 6.33 (range 0-20) and 2.22 (range 0-11), respectively, and the serum VEGF-A levels were significantly higher than those of control mice. In conclusion, lower body weight gain, metastasis in the liver and lungs, and elevated VEGF-A levels are features of LLC in mice.

Apoptosis Induction in Primary Human Colorectal Cancer Cell Lines and Retarded Tumor Growth in SCID Mice by Sulforaphane

We have investigated the anticancer effects of the dietary isothiocyanate sulforaphane (SFN) on colorectal cancer (CRC), using primary cancer cells lines isolated from five Taiwanese colorectal cancer patients as the model for colorectal cancer. SFN-treated cells accumulated in metaphase (SFN 6.25 μM) and subG1 (SFN 12.5 and 25 μM) as determined by flow cytometry. In addition, treated cells showed nuclear apoptotic morphology that coincided with an activation of caspase-3, and loss of mitochondrial membrane potential (ΔΨm). Incubations at higher SFN doses (12.5 and 25 μM) resulted in cleavage of procaspase-3 and elevated caspase-2, -3, -8, and -9 activity, suggesting that the induction of apoptosis and the sulforaphane-induced mitosis delay at the lower dose are independently regulated. Daily SFN s.c. injections (400 micromol/kg/d for 3 weeks) in severe combined immunodeficient mice with primary human CRC (CP1 to CP5) s.c. tumors resulted in a decrease of mean tumor weight by 70% compared with vehicle-treated controls. Our findings suggest that, in addition to the known effects on cancer prevention, sulforaphane may have antitumor activity in established colorectal cancer.

Abstract LB-458: Additive effects of caloric restriction and IGF-I receptor blockade on the progression of 22RV1 prostate cancer xenografts

Abstract INTRODUCTION: IGF-I receptor (IGF-1R) blockade has been shown to slow prostate cancer xenograft growth. Caloric restriction inhibits xenograft growth through modulation of the insulin-IGF axis. We hypothesized that caloric restriction combined with IGF-I receptor blockade would result in additive suppression of prostate cancer growth. METHODS: Sixty SCID mice were subcutaneously injected with 22RV1 cells (suppressible with IGF-1R blockade in vitro) and randomized to four groups: 1) ad libitum feeding with intraperitoneal saline (ad lib), 2) ad libitum with 20 mg/kg twice weekly intraperitoneal AMG479 IGF-I receptor blocking antibody (Amgen), (ad lib/Ab), 3) 40% calorie restriction with intraperitoneal saline (CR), 4) 40% calorie restriction with intraperitoneal AMG479, (CR/Ab). Diet and treatment with either antibody or saline were initiated one week after tumor injection. The animals were euthanized after 19 days of treatment and tumors were weighed. Fasting plasma levels of insulin, IGF-I, IGFBP-3, and IGFBP-1 were measured and tumors were stained for Ki67, TUNEL, and CD31. Cleaved caspase 3 and AKT activation in tumors were assessed by western blot. RESULTS: Mean tumor weight at sacrifice in the CR/Ab group was significantly lower (p<0.05) than the other three groups (166 mg ± 23 vs. Ad lib: 467 mg ± 58, Ad lib/Ab: 502 mg ± 52 and CR: 295 mg ± 56). Mean tumor weight was also lower in the CR group as compared to the ad lib groups (p< 0.05). Cleaved Caspase 3/Total Caspase 3 ratios were increased by 2 to 3 fold in the CR groups compared to the ad lib groups irrespectively of antibody therapy. Ki67 expression was significantly decreased in the CR/Ab group compared to the ad lib group (77 ± 1.2 vs. 81± 1.4 p<0.05). Plasma IGF-1 and IGFBP-3 levels were significantly reduced in the CR groups compared to the Ad lib groups irrespectively of treatment. AKT activation correlated to plasma IGF-1 levels. Mice in the Ad lib/Ab group had increased plasma insulin levels compared to the Ad lib group. The CR and CR/AB groups had significantly lower plasma insulin (80 and 60% reduction respectively) relative to the ad lib group. Angiogenesis was not affected by these interventions. CONCLUSION: Combining caloric restriction with antibody therapy targeting the IGF-1 receptor had additive anticancer effects on 22RV1 tumor growth. This additive anticancer effect may be due to suppression of the insulin elevation observed with AMG479 resulting in increased apoptosis and decreased proliferation in the CR/Ab combination arm. Further pre-clinical and clinical trials are warranted to evaluate the role of dietary interventions in combination with anti IGF1R antibody therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-458. doi:10.1158/1538-7445.AM2011-LB-458

Abstract LB-230: Enhancing anti-angiogenic therapy by blocking focal adhesion kinase

Abstract Focal adhesion kinase (FAK) represents a central point of convergence for many signaling pathways implicated in cancer progression and metastasis. Pazopanib is a pan-VEGFR and PDGFR inhibitor. In the present study, we examined whether pazopanib treatment would result in greater anti-tumor activity in combination with the novel FAK-inhibitor, GSK2256098. The in vitro effects of GSK2256098 on invasion and migration were examined using the HeyA8 and SKOV3-IP1 human ovarian cancer cell lines. In vivo effects of pazopanib with and without GSK2256098 were then assessed using an orthotopic mouse model of human ovarian cancer. GSK2256098 resulted in reduced levels of FAK phosphorylation at Y397 (pFAKY397) at 1μM concentration in SKOV3-IP1 cells. GSK2256098 resulted in a reduced invasion (p<0.001) and decreased migration (p<0.001) in SKOV3-ip1 cells. Dose-finding studies performed in vivo demonstrated that a 75 mg/kg dose resulted in a significant reduction in pFAKY397. Monotherapy with GSK2256098 resulted in a 58% decrease in mean tumor weight compared to control (p = 0.038). The combination of GSK2256098 with pazopanib resulted in a 71% decrease in mean tumor weight compared to pazopanib monotherapy (p = 0.04). We also tested treatment combinations including traditional cytotoxic chemotherapy. The combination of GSK2256098 with docetaxel resulted in a 44% decrease in mean tumor weight compared to docetaxel monotherapy (p = 0.17). The triplet combination of GSK2256098 with pazopanib and docetaxel resulted in the greatest overall decrease mean tumor weight, 99% compared to control and 92% compared to the doublet alone (p = 0.001). Similar trends were noted with mean number of tumor nodules and ascites volume. On the basis of our recent findings that FAK is highly phosphorylated in platelets in cancer-bearing mice, we also examined FAK phosphorylation in the platelets of treated and untreated mice. Phosphorylation of platelet FAK (pFAKY397) was inhibited by 68% at 4 hours after treatment compared to control (p = 0.07). Treatment with pazopanib decreased MVD by 49% (p < 0.01), which was further enhanced in combination with GSK2256098 (p < 0.01). In summary, FAK inhibition results in substantial anti-angiogenic and anti-tumor effects in combination with pazopanib and represents a viable strategy for further development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-230. doi:10.1158/1538-7445.AM2011-LB-230

Solid Tumor-Targeted Infiltrating Cytotoxic T Lymphocytes Retained by a Superantigen Fusion Protein

Successful immune-mediated regression of solid tumors is difficult because of the small number of cytotoxic T lymphocytes (CTLs) that were traffic to the tumor site. Here, the targeting of tumor-specific infiltrating CTLs was dependent on a fusion protein consisting of human epidermal growth factor (EGF) and staphylococcal enterotoxin A (SEA) with the D227A mutation. EGF-SEA strongly restrained the growth of murine solid sarcoma 180 (S180) tumors (control versus EGF-SEA, mean tumor weight: 1.013 versus 0.197 g, difference = 0.816 g). In mice treated with EGF-SEA, CD4+, CD8+ and SEA-reactive T lymphocytes were enriched around the EGFR expressing tumor cells. The EGF receptors were potentially phosphorylated by EGF-SEA stimulation and the fusion protein promoted T cells to release the tumoricidal cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Intratumoral CTLs secreted cytolytic pore-forming perforins and granzyme B proteins near the surface of carcinomas, causing the death of many tumor cells. We additionally show that labeled EGF-SEA was directly targeted to the tumor tissue after intravenous (i.v.) injection. The findings demonstrate that antibody-like EGF-SEA plays an important role in arresting CTLs in the solid tumor site and has therapeutic potential as a tumor-targeting agent.

Selective inhibition of cyclooxygenase-2 suppresses metastatic disease without affecting primary tumor growth in a murine model of Ewing sarcoma

Background/Purpose Mammalian target of rapamycin suppression by rapamycin inhibits tumor growth and neovascularization via cyclooxygenase-2 (COX-2) downregulation with no effect on lung metastases. We hypothesize that combining a selective COX-2 antagonist (celecoxib) with rapamycin would decrease lung metastases. Methods Ewing sarcoma cells (SK-NEP-1) were surgically implanted into the left kidney of athymic mice (n = 40). The mice were divided into 4 treatment groups (control, rapamycin only, celecoxib only, and combination) and then killed at 6 weeks. Primary tumors were weighed. Vasculature was examined using lectin angiography and immunohistochemistry, and lung metastases were examined using H&E and CD99 immunostaining. Tumor weight and lung metastases were analyzed. Results Mean primary tumor weights were significantly reduced in the rapamycin-treated groups but not in the celecoxib-only group. Lectin angiography and endothelial markers immunostaining showed markedly decreased vascularity in the rapamycin-treated groups but not in the celecoxib-only group. Celecoxib-treated groups showed significantly fewer mice with lung metastases than non–celecoxib-treated groups. Conclusion Celecoxib prevents lung metastasis in a murine model of Ewing sarcoma with no effect on tumor size or neovascularization. Cyclooxygenase-2 may represent a future potential target for metastatic disease prevention.

Emodin enhances antitumor effect of gemcitabine in model of SW1990 cell xenograft on athymic mouse

To evaluate the enhanced effect of gemcitabine by emodin and the possible mechanisms of the enhancement. Based on the model of SW1990 cell xenograft on athymic mouse, the mice were randomized to four groups with intraperitoneal (IP) injections of different drugs: group N (injecting 0.9% sodium chloride), group E (emodin, 40 mg x kg(-1)), group G (gemcitabine, 125 mg x kg(-1)), and group E + G (emodin 40 mg x kg(-1) and gemcitabine 80 mg x kg(-1) in combination). The tumor volume, tumor weight and body weight of mice were measured during the drug therapy. The mice were sacrificed one week after last injection of drug. Tunel assay were used used to detect the apoptosis of tumor cells. And immunohistochemistry (IHC) and Western blot (WB) were used to detect the variance of the apoptosis relative protein expression of Bax, Bcl-2, and Cytochrome C . One week after the last administration, the mean tumor volume and tumor weight in group E + G were significantly decreased compared to the other groups. Tunel assay showed group E + G presented apparently more apoptosis than the other groups. Immunohistochemistry (IHC) and Western blot (WB) analysis showed the expression of Cytochrome C in cytoplasmin and Bax in group E + G was apparently upregulated while the expression of Bcl-2 was apparently downregulated compared to the other groups. As a result, Bcl-2/Bax ratio was significantly decreased in group E + G. Emodin can significantly improve the antitumor effect of gemcitabine on transplanted tumor of SW1990 cell line through apparently enhancing the tumor cell apoptosis by gemcitabine. Downregulation of Bcl-2/Bax ratio and promoting release of Cytochrome C from mitochondria is possibly one of the mechanisms of the augmented apoptosis.

Wnt/β-Catenin Pathway Activation in Adrenocortical Adenomas Is Frequently due to Somatic CTNNB1-Activating Mutations, Which Are Associated with Larger and Nonsecreting Tumors: A Study in Cortisol-Secreting and -Nonsecreting Tumors

Abstract Background: Abnormal β-catenin immunohistochemistry and mutations of the β-catenin gene (CTNNB1) have been reported in adrenocortical adenomas (ACAs), but the frequencies of these defects and the phenotype of such tumors have not been clearly determined. Objective: The objective of the study was to describe the Wnt/β-catenin pathway alterations in 100 ACAs and their association with clinicopathological characteristics. Patients and methods: One hundred consecutive ACAs (excluding Conn’s adenomas) were studied clinically by β-catenin immunohistochemistry and direct sequencing of CTNNB1. Results: Thirty-five ACAs were nonsecreting adenomas (NSAs), 19 were subclinical cortisol secreting adenomas (SCSAs), and 46 were cortisol secreting adenomas (CSAs). Fifty-one tumors had abnormal cytoplasmic and/or nuclear β-catenin immunohistochemical staining, indicating Wnt/β-catenin pathway alteration. Thirty-six tumors showed CTNNB1 mutations, which all showed abnormal immunohistochemical β-catenin accumulation. Among the 64 nonmutated tumors, only 15 (23%) showed cytoplasmic and/or nuclear β-catenin staining (P < 0.0001). Tumors with CTNNB1 mutations were predominantly nonsecreting (61% NSAs, 22% SCSAs, 16% CSAs) whereas nonmutated tumors were predominantly secreting (20% NSAs, 17% SCSAs, 62% CSAs) (P < 0.0001). Mean tumor size and weight were, respectively, 4.2 cm (±1.2) and 28.4 g (±21.1) for tumors with CTNNB1 mutations vs. 3.4 cm (±0.9) and 18.2 g (±8.1) for nonmutated tumors (P < 0.01). Conclusions: Abnormal cytoplasmic and/or nuclear β-catenin immunohistochemical staining occurs in about half of ACAs. This suggests the activation of the Wnt/β-catenin pathway, which could be explained by activating mutations of CTNNB1 in 70% of the cases. CTNNB1 mutations are mainly observed in larger and nonsecreting ACAs, suggesting that the Wnt/β-catenin pathway activation is associated with the development of less differentiated ACAs.

Wnt/β-Catenin Pathway Activation in Adrenocortical Adenomas Is Frequently due to Somatic CTNNB1-Activating Mutations, Which Are Associated with Larger and Nonsecreting Tumors: A Study in Cortisol-Secreting and -Nonsecreting Tumors

Background Abnormal β-catenin immunohistochemistry and mutations of the β-catenin gene (CTNNB1) have been reported in adrenocortical adenomas (ACAs), but the frequencies of these defects and the phenotype of such tumors have not been clearly determined. Objective The objective of the study was to describe the Wnt/β-catenin pathway alterations in 100 ACAs and their association with clinicopathological characteristics. Patients and methods One hundred consecutive ACAs (excluding Conn’s adenomas) were studied clinically by β-catenin immunohistochemistry and direct sequencing of CTNNB1. Results Thirty-five ACAs were nonsecreting adenomas (NSAs), 19 were subclinical cortisol secreting adenomas (SCSAs), and 46 were cortisol secreting adenomas (CSAs). Fifty-one tumors had abnormal cytoplasmic and/or nuclear β-catenin immunohistochemical staining, indicating Wnt/β-catenin pathway alteration. Thirty-six tumors showed CTNNB1 mutations, which all showed abnormal immunohistochemical β-catenin accumulation. Among the 64 nonmutated tumors, only 15 (23%) showed cytoplasmic and/or nuclear β-catenin staining (P < 0.0001). Tumors with CTNNB1 mutations were predominantly nonsecreting (61% NSAs, 22% SCSAs, 16% CSAs) whereas nonmutated tumors were predominantly secreting (20% NSAs, 17% SCSAs, 62% CSAs) (P < 0.0001). Mean tumor size and weight were, respectively, 4.2 cm (±1.2) and 28.4 g (±21.1) for tumors with CTNNB1 mutations vs. 3.4 cm (±0.9) and 18.2 g (±8.1) for nonmutated tumors (P < 0.01). Conclusions Abnormal cytoplasmic and/or nuclear β-catenin immunohistochemical staining occurs in about half of ACAs. This suggests the activation of the Wnt/β-catenin pathway, which could be explained by activating mutations of CTNNB1 in 70% of the cases. CTNNB1 mutations are mainly observed in larger and nonsecreting ACAs, suggesting that the Wnt/β-catenin pathway activation is associated with the development of less differentiated ACAs.

Wnt/β-Catenin Pathway Activation in Adrenocortical Adenomas Is Frequently due to Somatic CTNNB1-Activating Mutations, Which Are Associated with Larger and Nonsecreting Tumors: A Study in Cortisol-Secreting and -Nonsecreting Tumors

Abnormal β-catenin immunohistochemistry and mutations of the β-catenin gene (CTNNB1) have been reported in adrenocortical adenomas (ACAs), but the frequencies of these defects and the phenotype of such tumors have not been clearly determined. The objective of the study was to describe the Wnt/β-catenin pathway alterations in 100 ACAs and their association with clinicopathological characteristics. One hundred consecutive ACAs (excluding Conn's adenomas) were studied clinically by β-catenin immunohistochemistry and direct sequencing of CTNNB1. Thirty-five ACAs were nonsecreting adenomas (NSAs), 19 were subclinical cortisol secreting adenomas (SCSAs), and 46 were cortisol secreting adenomas (CSAs). Fifty-one tumors had abnormal cytoplasmic and/or nuclear β-catenin immunohistochemical staining, indicating Wnt/β-catenin pathway alteration. Thirty-six tumors showed CTNNB1 mutations, which all showed abnormal immunohistochemical β-catenin accumulation. Among the 64 nonmutated tumors, only 15 (23%) showed cytoplasmic and/or nuclear β-catenin staining (P < 0.0001). Tumors with CTNNB1 mutations were predominantly nonsecreting (61% NSAs, 22% SCSAs, 16% CSAs) whereas nonmutated tumors were predominantly secreting (20% NSAs, 17% SCSAs, 62% CSAs) (P < 0.0001). Mean tumor size and weight were, respectively, 4.2 cm (± 1.3) and 28.4 g (± 21.4) for tumors with CTNNB1 mutations vs. 3.4 cm (± 0.9) and 18.2 g (± 8.2) for nonmutated tumors (P < 0.01). Abnormal cytoplasmic and/or nuclear β-catenin immunohistochemical staining occurs in about half of ACAs. This suggests the activation of the Wnt/β-catenin pathway, which could be explained by activating mutations of CTNNB1 in 70% of the cases. CTNNB1 mutations are mainly observed in larger and nonsecreting ACAs, suggesting that the Wnt/β-catenin pathway activation is associated with the development of less differentiated ACAs.

Salirasib inhibits the growth of hepatocarcinoma cell lines in vitro and tumor growth in vivo through ras and mTOR inhibition

BackgroundDysregulation of epidermal growth factor and insulin-like growth factor signaling play important roles in human hepatocellular carcinoma (HCC), leading to frequent activation of their downstream targets, the ras/raf/extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) pathways. Salirasib is an S-prenyl-cysteine analog that has been shown to block ras and/or mTOR activation in several non hepatic tumor cell lines. We investigated in vitro the effect of salirasib on cell growth as well as its mechanism of action in human hepatoma cell lines (HepG2, Huh7, and Hep3B) and its in vivo effect in a subcutaneous xenograft model with HepG2 cells.ResultsSalirasib induced a time and dose dependent growth inhibition in hepatocarcinoma cells through inhibition of proliferation and partially through induction of apoptosis. A 50 percent reduction in cell growth was obtained in all three cell lines at a dose of 150 μM when they were cultured with serum. By contrast, salirasib was more potent at reducing cell growth after stimulation with EGF or IGF2 under serum-free conditions, with an IC50 ranging from 60 μM to 85 μM. The drug-induced anti-proliferative effect was associated with downregulation of cyclin A and to a lesser extent of cyclin D1, and upregulation of p21 and p27. Apoptosis induction was related to a global pro-apoptotic balance with caspase 3 activation, cytochrome c release, death receptor upregulation, and a reduced mRNA expression of the apoptosis inhibitors cFLIP and survivin. These effects were associated with ras downregulation and mTOR inhibition, without reduction of ERK and Akt activation. In vivo, salirasib reduced tumour growth from day 5 onwards. After 12 days of treatment, mean tumor weight was diminished by 56 percent in the treated animals.ConclusionsOur results show for the first time that salirasib inhibits the growth of human hepatoma cell lines through inhibition of proliferation and induction of apoptosis, which is associated with ras and mTOR inhibition. The therapeutic potential of salirasib in human HCC was further confirmed in a subcutaneous xenograft model.

Efficient Inhibition of C-26 Colon Carcinoma by VSVMP Gene Delivered by Biodegradable Cationic Nanogel Derived from Polyethyleneimine

Biodegradable cationic nanoparticles have promising application as a gene delivery system. In this article, heparin-polyethyleneimine (HPEI) nanogels were prepared, and these nanogels were developed as a nonviral gene vector. The transfection efficiency of HPEI nanogels was comparable with that of PEI25K, while the cytotoxicity was lower than that of PEI2K and much lower than that of PEI25K in vitro. These HPEI nanogels also had better blood compatibility than PEI25K. After intravenous administration, HPEI nanogels degraded, and the degradation products were excreted through urine. The plasmid expressing vesicular stomatitis virus matrix protein (pVSVMP) could be efficiently transfected into C-26 colon carcinoma cells by HPEI nanogels in vitro, inhibiting the cell proliferation through apoptosis induction. Intraperitoneal injection of pVSVMP/HPEI complexes efficiently inhibited the abdominal metastases of C-26 colon carcinoma through apoptosis induction (mean tumor weight in mice treated with pVSVMP/HPEI complex = 0.93 g and in control mice = 3.28 g, difference = 2.35 g, 95% confidence interval [CI] = 1.75-2.95 g, P < 0.001) and prolonged the survival of treated mice. Moreover, intravenous application of pVSVMP/HPEI complexes also inhibited the growth of pulmonary metastases of C-26 colon carcinoma through apoptosis induction. The HPEI nanogels delivering pVSVMP have promising application in treating colon carcinoma.

Efficient inhibition of lung cancer in murine model by plasmid-encoding VEGF short hairpin RNA in combination with low-dose DDP

BackgroundVEGF is a well-validated target for antiangiogenic intervention in cancer. To date, RNAi technology has been proven to be a promising approach for targeted therapy. DDP is frequently used as a first-line drug in chemotherapy for lung cancer but usually causes severe toxicity. In this study, we investigated a novel strategy of administering and combining RNAi mediated VEGF-targeted therapy with DDP for treatment of lung cancer, with the aim of increasing efficacy and decreasing toxicity.MethodsIn this study, a plasmid encoding VEGF shRNA was constructed to knockdown VEGF both in vitro and in vivo. In vitro, specificity and potency of the targeting sequence were validated in A549 lung adenocarcinoma cells by RT-PCR and ELISA assays. In vivo, therapy experiments were conducted on nude mice bearing A549 xenograft tumors. The VEGF shRNA expressing plasmids were administered systemically in combination with low-dose DDP on a frequent basis. The tumor volume and weight were measured. MVD, the number of apoptotic cells and proliferation index in tumor tissues were assessed by CD31, TUNEL and PCNA immunostaining.ResultsThe VEGF shRNA was highly effective in attenuating VEGF expression both in vitro and in vivo. The treatment with the VEGF shRNA alone reduced the mean tumor weight by 49.40% compared with the blank control (P < 0.05). The treatment with the VEGF shRNA plus DDP yielded maximal benefits by reducing the mean tumor weight by 83.13% compared with the blank control (P < 0.01). The enhanced antitumor efficacy was associated with decreased angiogenesis and increased induction of apoptosis.ConclusionsOur study demonstrated synergistic antitumor activity of combined VEGF shRNA expressing plasmids and low-dose DDP with no overt toxicity, suggesting potential applications of the combined approach in the treatment of lung cancer.

Abstract 1793: Platelet-derived growth factor receptor alpha blockade significantly enhances sensitization to docetaxel in ovarian carcinoma

Abstract Objective: Platelet-derived growth factor receptor alpha (PDGFRα) is a member of class III receptor tyrosine kinase and is associated with cell survival. Here, we examined whether PDGFRα blockade enhances the anti-tumor activity of taxanes in ovarian carcinoma. Methods: PDGFRα expression was evaluated with RT-PCR and Western blot in multiple ovarian cancer cell lines. Human-specific monoclonal antibody to PDGFRα (IMC-3G3, ImClone Systems, NJ, USA) was used for in-vitro (cell viability assay, apoptosis assay) and in-vivo experiments with or without docetaxel. Results: All eleven tested ovarian cancer cell lines had variable expression of PDGFRα. OVCA433 cell line had the highest level of expression of PDGFRα. IMC-3G3 blocked PDGFRα phosphorylation in response to PDGF-AA stimulation. IMC-3G3 alone had no affect on cell viability in all tested cell lines. However, IMC-3G3 significantly enhanced sensitization to docetaxel: IC50 reduction rate, SKOV3-ip1, 25.5%; HeyA8-parental, 31.7%; OVCA433, 35.3%; and HeyA8-MDR, 44.2% (all, p&amp;lt;0.05). While treatment with IMC-3G3 alone did not increase apoptosis, concurrent use with docetaxel significantly enhanced docetaxel-mediated apoptosis (mean percentage of total apoptotic cells in SKOV3-ip1, untreated vs IMC-3G3 alone vs docetaxel alone vs docetaxel with IMC-3G3, 7.3% vs 7.1% vs 15.4% vs 22.2%, p&amp;lt;0.001). In the in-vivo experiment with SKOV3-ip1 bearing mice, administration of IMC-3G3 monotherapy had no significant anti-tumor effects compared to the control group (mean tumor weight, 2.5 ± 0.32 vs 2.9 ± 0.51 grams, p&amp;gt;0.05). Concurrent use of IMC-3G3 with docetaxel significantly reduced tumor weight compared to docetaxel alone (mean tumor weight, 0.22 ± 0.07 vs 0.63 ± 0.14 grams, tumor reduction rate 65.1%, p&amp;lt;0.05). Conclusion: IMC-3G3 significantly enhances the anti-tumor effects of docetaxel. Therefore, targeting PDGFRα may be an attractive approach for increasing the cytotoxic effects of chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1793.

Abstract LB-429: Synergistic effects of low-fat diet combined with IGF-I receptor blockade on 22RV1 prostate cancer xenografts

Abstract Introduction: IGF-I receptor (IGF-1R) blockade has previously been shown to slow prostate cancer xenograft growth. Dietary fat reduction also inhibits xenograft growth, possibly by modulation of the IGF axis. We hypothesized that a low-fat (LF) diet combined with IGF-I receptor blockade would cause additive retardation of prostate cancer growth and offset the possible untoward metabolic effects of IGF-1R antibody therapy. Methods: Fifty severe combined immunodeficient mice were injected subcutaneously with 22RV1 cells (suppressible with IGF-1R blockade in vitro) and were randomized to four groups: 1) high-fat (HF) diet with intraperitoneal saline, 2) high-fat diet with intraperitoneal IGF-I receptor blocking antibody from Amgen: AMG479, (HFAb), 3) low-fat diet with intraperitoneal saline (LF), 4) low-fat diet with intraperitoneal AMG479, (LFAb). Treatment with either antibody or saline was initiated 10 days after tumor injection and given twice weekly, tumors were measured serially. The animals were euthanized at 19 days of treatment. Tumors were then weighed and stained for Ki67, TUNEL, and CD31. Serum levels of insulin, resistin, murine IGF-I, murine IGFBP-3, and TNF-alpha were measured. Results: Mean tumor weights were significantly lower (by ∼ 40%) in the HFAb and LFAb group relative to the HF group. There was no difference in mean tumor weight between the HFAb and LFAb groups. Ki67 expression (% positive cells) was significantly decreased in the LFAb group compared to the HFAb group (62 + 29 vs. 94 + 4.3, p&amp;lt;0.001). IGF-1R antibody caused a significant increase in serum insulin levels compared to the LF and HF group, but combining the LF diet with the IGF-1R antibody significantly reduced serum insulin levels by 40% relative to the HFAb group. Likewise, combining a LF diet with the IGF-1R antibody also reduced serum TNF-alpha levels relative to the HF group. Conclusion: IGF-1R antibody therapy inhibited xenograft growth in mice on HF and LF diets. Whereas combining a LF diet with IGF-1R antibody did not have additive growth inhibitory effects on tumor size, there was evidence of decreased tumor cell proliferation, reduced serum insulin levels and reduced pro-inflammatory cytokine levels. Further pre-clinical and clinical trials are warranted to evaluate the role of dietary interventions to enhance anti-tumor effects and offset the metabolic consequences of IGF-1R antibody therapy. Aknowledgements: This work was supported by the NCI grant # P50CA92131 and “The Ruby Family Foundation”. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-429.

Abstract 4524: Targeting Bcl-2 family proteins in adult T-cell leukemia/lymphoma: in vitro and in vivo effects of a novel Bcl-2 family inhibitor, ABT-737

Abstract Adult T-cell lymphoma/leukemia (ATLL) is a T-cell malignancy caused by the human T-lymphotrophic virus type I (HTLV-I), and its therapeutic outcome still remains extremely poor. Therefore, novel therapeutic strategies are needed to improve treatment outcomes. In this study, we elucidated the therapeutic potential of targeting the anti-apoptotic Bcl-2 family proteins for the treatment of ATLL using ABT-737 (Abbott Laboratories, Abbott Park, IL, USA), a small molecule inhibitor of Bcl-2, Bcl-XL and Bcl-w. We first validated the rationale of this study by immunohistochemically assessing the expression of Bcl-2 family proteins in 25 lymph-node specimens derived from ATLL patients. The Bcl-2 and/or Bcl-XL proteins were expressed in 80% of specimens. We next examined the cytotoxicity of ABT-737 against three ATLL cell lines by the Colorimetric method. ABT-737 significantly inhibited growth of MT-1, MT-2 and HuT 102 cells with concentrations of 50 percent inhibition of 2.4, 0.23 and 0.008μM at 72 h, respectively. We then tried to clarify the mechanism of growth inhibition induced with ABT-737 using MT-1 and MT-2 cells. ABT-737 induced apoptosis in both cells with cleavage of caspases 9 and 3, and PARP. ABT-737 also induced apoptosis in fresh tumor cells derived from patients with ATLL. We also tested if ABT-737 enhances the cytotoxicity induced by conventional chemotherapeutic agents or novel agents. The interactions between them were evaluated using the Chou-Talalay method. ABT-737 synergistically enhanced the cytotoxicity and apoptosis induced by doxorubicin, vincristine or etoposide and bortezomib or suberolyanilide hydroxamic acid, which are current key drugs and promising candidates for the treatment of ATLL, respectively. Finally, we investigated the growth inhibition of ABT-737 in ATLL-xenografted mice. The mean tumor volume and weight, and mean serum level of soluble interleukin-2 receptor α at day 21 of the treated mice with ABT-737 (100mg/kg/day) were significantly lower than those of vehicle-treated mice. Moreover, massive induction of apoptosis was observed in tumors treated with ABT-737 by the TUNEL assay. These results suggest that the use of ABT-737, either alone, or in combination with other conventional cytotoxic and novel drugs, represents a promising novel targeted approach to overcome drug resistance and to improve patient outcome in ATLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4524.

Malnourished Malawian patients presenting with large Wilms tumours have a decreased vincristine clearance rate

Introduction In developing countries, patients with a Wilms’ tumour often present late with a high degree of malnutrition and large tumours. We investigated whether this affects vincristine pharmacokinetics. Methods Patients newly diagnosed with Wilms’ tumour in Malawi and the UK were included. We documented anthropometric parameters, nutritional status and tumour size. Vincristine (1.50 mg/m 2) was administered as part of the standard chemotherapy regimen. Vincristine plasma concentrations were measured at several time points by liquid chromatography–mass spectrometry. Vincristine pharmacokinetic parameters (clearance and area under the curve) were calculated by non-compartmental analysis. Results Eleven Malawian and 8 UK patients were included. Mean Z-score of (corrected) weight for height was significantly lower in the Malawian patients than in the UK patients (−2.3 versus 0.42, p < 0.0001). Mean tumour weight at diagnosis was significantly larger in Malawian patients (2.8 kg versus 0.7 kg, p = 0.007). Mean vincristine log Clearance was lower in Malawian as compared to UK patients (2.2 versus 2.6 ml/min, p = 0.001). Mean log AUC values were higher in Malawian than in UK patients (3.8 versus 3.5 μg/ml min, p = 0.003). This difference is reflected in the, on average, 1.98-fold larger vincristine AUC values for Malawian patients. The difference in AUC values was statistically significantly explained by nutritional status ( p = 0.043). Conclusion Malnourished patients in Malawi exhibited lower vincristine clearance rates and thus higher AUC values than a comparable patient population with a better nutritional status in the UK. In malnourished patients, dose reductions may need to be considered to prevent an increased incidence and severity of toxicity.

Noninvasive Contrast-enhanced US Quantitative Assessment of Tumor Microcirculation in a Murine Model: Effect of Discontinuing Anti-VEGF Therapy

To determine, by using contrast material-enhanced ultrasonography (US), how quickly renal tumors grafted in mice begin to revascularize after stopping bevacizumab treatment. All experiments were approved by the regional ethics committee. A human tumor cell line SK-NEP-1 was grafted at day 0 in the left kidney of 50 nude mice. Forty-two mice developed tumors and longitudinal follow-up was performed on 32 surviving mice. From day 13, 14 controls received biweekly saline; 11 mice received biweekly bevacizumab until day 35 (continuous); and seven received biweekly bevacizumab until day 22, then biweekly placebo until day 35 (discontinued). Contrast-enhanced US was performed on days 13, 14, 22, 27, and 35. Once the injected contrast material distribution reached an equilibrium phase, high-acoustic pressure pulses were applied to destroy microbubbles in the capillary bed in the imaged plane. Reperfusion was monitored, and time-signal intensity (SI) curves were obtained from the linear average of SIs in intratumoral and matched-depth renal cortex regions of interest. A kinetic parameter calculated from reperfusion curves reflects local perfusion, normalized with respect to adjacent renal cortex perfusion. Normalized perfusion obtained from each group was compared with that from the other groups and with necrosis percentages and microvascular density assessed histologically at day 35. Comparisons were made by using analyses of variance and Tukey-Kramer tests. The lowest excised mean tumor weights (+/- standard deviation) corresponded to the longest bevacizumab-treatment duration: 1.4 g +/- 1.1 (continuous-treatment) compared with 2.3 g +/- 2.1 (discontinued) and 3.7 g +/- 1.9 (control) (P = .01). On day 35, the respective control and continuously treated groups had comparable and significantly larger necrotic areas: 37% +/- 14 and 32% +/- 17 larger than the discontinued-treatment group (15% +/- 9; P < .05). Normalized perfusion increased significantly with time (P = .02) in the discontinued-treatment group after therapy ceased (day 22). Noninvasively measured contrast-enhanced US parameters demonstrated tumor revascularization after stopping antiangiogenic therapy in this murine tumor model.

Novel chemoembolization using calcium‐phosphate ceramic microsphere incorporating TNP‐470, an anti‐angiogenic agent

The purpose of the present study was to develop a new method of chemoembolization to improve the therapeutic effectiveness and safety profile of cancer treatment. A chemoembolization approach was designed for human solid tumors using resorbable calcium-phosphate ceramic microspheres loaded with an agent anti-angiogenic to tumor vasculature in vivo. The human uterine sarcoma cell line FU-MMT-3 was used in this study because this tumor is aggressive and also exhibits a poor response to radiotherapy or any chemotherapy currently used. The calcium-phosphate ceramic microspheres loaded with TNP-470, an anti-angiogenic agent, were injected into FU-MMT-3 xenografts in nude mice three times per week for 8 weeks. The treatment using TNP-470-loaded microspheres suppressed tumor growth, compared to treatment with TNP-470 alone, microspheres alone, and the control. The mean tumor weight after treatment using TNP-470-loaded microspheres was significantly lower than that after treatment with microspheres alone. These ceramic microspheres were remarkably embolized in tumor microvessels as well as in the feeding arteries and a significant reduction of intratumoral vascularity was also demonstrated following treatment with TNP-470-loaded microspheres. Severe loss of body weight was not observed in any mice treated with the TNP-470-loaded microspheres, compared to treatment with TNP-470 alone. These results suggest that targeting tumor vasculature in human uterine sarcoma using calcium-phosphate microspheres might be more effective and safer than the treatment that employs anti-angiogenic agent alone. This new chemoembolization method incorporating an anti-angiogenic agent may contribute to the effective treatment of locally advanced or recurrent solid tumors.

Targeting Bcl-2 Family Proteins in Adult T-Cell Leukemia/Lymphoma: In Vitro and In Vivo Effects of a Novel Bcl-2 Family Inhibitor ABT-737.

Abstract 1685Poster Board I-711Adult T-cell lymphoma/leukemia (ATLL) is a T-cell malignancy caused by human T-lymphotrophic virus type I (HTLV-I), and its therapeutic outcome is still remains very poor. Therefore, novel therapeutic strategies are needed to improve patient outcome. In this study, we elucidated the therapeutic potential to target anti-apoptotic Bcl-2 family proteins for the treatment of ATLL by using ABT-737 (Abbott Laboratories, Abbott Park, IL, USA), a small molecule inhibitor of Bcl-2, Bcl-XL and Bcl-w. We first validated the rationale of this study by assessing the expression of Bcl-2 family proteins among 25 lymph-node specimens derived from ATLL patients by using immunohistochemistory. Both or either of Bcl-2 and Bcl-XL proteins was highly expressed in 80% of specimens. We next examined the cytotoxicity of ABT-737 against ATLL cell lines. ABT-737 significantly inhibited growth of MT-1, MT-2 and HuT 102 cells with a concentration of 50 percent inhibition (IC50) at 72 h of 2.4, 0.23 and 0.008μM, respectively. We then elucidated the mechanism of growth inhibition induced by ABT-737 using MT-1 and MT-2 cells. ABT-737 induced apoptosis in MT-1, MT-2 cells with cleavage of caspase 9, 3 and PARP. ABT-737 also induced apoptosis in fresh tumor cells derived from patients with ATLL. We next elucidated the potential of ABT-737 to enhance the cytotoxicity induced by conventional chemotherapeutic agents. The interaction between them was evaluated using the Chou-Talalay method by determining the combination index. ABT-737 synergistically enhanced the cytotoxicity and apoptosis induced by either of doxorubicin, vincristine or etoposide, which is a current key drug to treat ATLL. Most importantly, ABT-737 significantly inhibited tumor growth of in vivo ATLL model using SCID mice inoculated by HuT 102 cells subcutaneously. The mean tumor volume, weight and serum level of soluble interleukin-2 receptor á of ABT-737 (100mg/kg/day)-treated mice were significantly lower than those of vehicle-treated mice after treatment for 21 days. Moreover, massive induction of apoptosis in tumors treated by ABT-737 was observed by immunofluorescent TUNEL assay. These results suggest that ABT-737 used either alone or in combination with conventional cytotoxic drugs, represents a promising novel targeted approach to overcome drug resistance and improve patient outcome in ATLL. DisclosuresNo relevant conflicts of interest to declare.