Search

Src, a non-receptor tyrosine kinase frequently overexpressed and highly activated in malignancies, has been associated with a poor patient prognosis. The aim of the present studies was to examine the impact of an Src inhibitor (saracatinib) on a highly metastatic murine sarcoma cell line (KHT).Phosphorylation of Src and downstream effectors was determined using Western immunoblotting. Cell cycle was analyzed by flow cytometry using propidium iodide DNA staining, migration and invasion in modified Boyden chambers, activated MMP-9 by gel zymography, and visualization of pSrc and pFAK by confocal immunofluorescence. The number of KHT lung nodules in saracatinib-treated mice was compared to controls.Saracatinib inhibited major pathways in the metastatic cascade in vitro, including Src and FAK activation. Functions required for metastasis, such as migration and invasion, were reduced when cells were exposed to 0.5 μM and 1.0 μM saracatinib, respectively (p0.0001). Pretreatment of KHT cells with either 1 μM or 5 μM saracatinib prior to tail vein injection decreased lung colonies in mice from 13.0 to 5.0 (p0.05) and less than 1.0 (p0.01), respectively.These findings suggest that Src inhibition by saracatinib may reduce the metastatic activity of tumor cells.

VEGF is the key player in tumor angiogenesis. In the current study, the impact of VEGF expression on the response of tumors to the VEGFR2 associated tyrosine kinase inhibitor vandetanib was evaluated.Human colon carcinoma (HT29) and murine squamous carcinoma (SCCVII) clonal cell lines expressing varying levels of VEGF were established and their response to vandetanib was assessed in tissue culture and as solid tumors.Vandetanib treatment had no effect on tumor cell clonogenic cell survival in vitro but dosesor=10 nM significantly reduced endothelial cell migration. In vivo, tumors derived from cell clones expressing high levels of VEGF displayed significantly enhanced angiogenesis and more aggressive growth. An intradermal angiogenesis assay was used to demonstrate that a 4-day treatment with vandetanib (50 mg/kg/day) was able to significantly inhibit blood vessel growth induced by both parental and high VEGF-expressing tumor cell clones. In the HT29 tumor model, treatment response to vandetanib (50 mg/kg/day, Monday-Friday for 2 weeks) was greatest in xenografts derived from the highest VEGF-expressing cell clones. A similar trend was noted in the SCCVII tumor model. The present findings indicate that vandetanib therapy effectively counteracted the aggressive feature of tumor growth resulting from VEGF over-expressing tumor cells and suggest that such tumors may be particularly well suited for anti-VEGF interventions.

This study evaluated the therapeutic efficacy of combining vascular disrupting agents with antiangiogenic agents.Human clear cell renal carcinoma (Caki-1) tumors were established in nude mice. Treatments consisted of Avastin (2 mg/kg) administered twice a week; CA4P (100 mg/kg) or OXi4503 (25 mg/kg) administered 3 times a week for a period of 2 weeks; or a combination of Avastin and CA4P or OXi4503. Tumor response was assessed by growth delay.The tumor growth delays were 8, 6, and 18 days for Avastin, CA4P, and OXi4503, respectively. When the two therapies were combined, there was a significantly greater tumor response than what was achieved with single-agent treatments. For example, Avastin plus CA4P led to a growth delay of 13 days, and 27 days for Avastin plus OXi4503.Vascular-directed therapies that include both antiangiogenic and vascular disrupting therapeutics can result in significantly enhanced antitumor effects.

4-1BB (CD137) is a member of the tumor necrosis factor receptor superfamily. It interacts with 4-1BB ligand (4-1BBL) and delivers a costimulatory signal for T cell activation. The immune response induced by 4-1BB monoclonal antibodies (mAbs) has been shown to have a marked augmentation of tumor-selective cytolytic T cell activity.The antitumor efficacy of agonistic 4-1BB mAbs (BMS-469492, Bristol-Meyer Squibb), used alone or in combination with radiation therapy, was evaluated in murine lung (M109) and breast (EMT6) carcinoma models.Treatment with BMS-469492 led to only a modest growth retardation in M109 tumors (3 days, p0.05), but a significant growth delay in EMT6 tumors (12.5 days, p0.05). When BMS-469492 was administered in conjunction with single dose radiation therapy (5, 10 or 15 Gy), enhanced tumor responses were noted only at the highest evaluated radiation dose (15 Gy). In contrast, in the EMT6 model, BMS-469492 treatment resulted in enhanced antitumor effects at all radiation doses. In addition, the response of EMT6 tumors to fractionated radiotherapy also was significantly increased when BMS-469492 was included in the treatment protocol.These results suggest that monoclonal antibodies against 4-1BB may not only be efficacious in cancer immunotherapy, but may also have utility when applied in combination with conventional anticancer treatments, such as radiation therapy.

Recombinant adeno-associated viral vectors serotype 2 (rAAV2) can transduce several tissues with high efficiency.The transduction efficiency of rAAV2 in pancreatic and colon cancer was compared to that of recombinant adenovirus (rAd) in vitro and in vivo using green fluorescent protein (GFP).With the exception of SU.86.86, the percentage of GFP-positive cells was below 10% at a multiplicity of infection (MOI) of 100 for rAAV2. At the same MOI, almost 100% of cells expressed GFP when rAd was used. However, the transduction efficiency for rAA V2 was comparable to that of rAd when coinfected with wt adenovirus, leading to a dramatic increase in the amount of double-stranded rAAV2 DNA. Similar results were obtained in vivo. While widespread GFP expression was readily detected in all xenografts injected with rAd, only one section of all tumors injected with rAAV2 contained GFP-positive cells.The results of this study indicate that rAAV2 might be useful for an ex vivo approach in cancer gene therapy, but it does not seem to be feasible for the in vivo treatment of malignant tumors.

Vascular disrupting agents (VDAs) are designed to cause a rapid and selective shutdown of the established tumor vasculature, which leads to secondary ischemic tumor cell death.We examined the efficacy of a novel VDA, MN-029, in the rodent KHT sarcoma model.A significant reduction in the functional vessel number was observed after intraperitoneal injection of MN-029 at a dose of 100 mg/kg. Histological evaluation showed extensive necrosis (approximately 90%) by 24 h. MN-029 treatment to the tumor-bearing mice also resulted in a dose-dependent tumor cell killing. When used in combination with radiation or cisplatin chemotherapy, a 100 mg/kg dose of MN-029 significantly enhanced tumor killing compared to that seen with radiation or cisplatin alone.The results demonstrated that MN-029 could cause rapid vascular shutdown in solid tumors, dose-dependent secondary tumor cell killing, and effective enhancement of the antitumor effects of radiation and cisplatin chemotherapy.

Vascular endothelial growth factor (VEGF) is one of the most important factors involved in tumor angiogenesis.Antisense phosphorothiolate oligodeoxynucleotides (PS-ODNs) were used to reduce VEGF production while the small molecule PD0203359-0002 (PD203359) was used to inhibit VEGF/bFGF receptor tyrosine kinase activity.PD203359 exposure was found to profoundly impair the growth of human endothelial cells (HMVEC-L) at doses 20-fold less than those affecting human renal cell carcinoma (Caki-1) cell growth. In vivo, treatment with PD203359 inhibited tumor cell-induced angiogenesis and resulted in a significant tumor growth delay. Treatment with VEGF antisense PS-ODNs also significantly increased the time for tumors to grow to five times the starting size. Most importantly, when the PD203359 and VEGF antisense treatments were combined, a greater antitumor response than could be achieved with either therapy alone was observed.Simultaneously targeting VEGF production and VEGF receptor signaling enhances the anticancer efficacy of either therapy alone.

Silencing of the viral CMV immediate early enhancer promoter can be a problem in certain cell types when engineering stable cell lines.We compared the efficacy of the CMV promoter to the promoter of the elongation factor-1 alpha (EF-1 alpha) for the generation of stable colon carcinoma cell lines (HT-29). Green fluorescent protein (GFP) expression cassettes were delivered by recombinant adeno-associated virus (AAV) which is known for its ability to stably transduce cells. Stable cell lines were characterized in vitro by FACS and in vivo after HT-29 clones were grown as xenografts in nude mice.Stable HT-29 clones with97% of all cells homogeneously expressing GFP were generated with the EF-1 alpha promoter. In contrast in clones carrying the CMV promoter, only up to 60% of the cells were GFP-positive with expression levels varying widely between cells. Superinfection with wild-type adenovirus induced GFP expression in more than 90% of the cells indicating that the CMV promoter was silenced. In vivo the tumors carrying the EF-1 alpha promoter were homogeneously GFP-positive, whereas the CMV promoter gave rise to a scattered pattern of GFP expression.This study underlines the importance of the promoter for the generation of stable cell lines. In addition it demonstrates that recombinant AAV can effectively be used as a gene delivery system for this purpose.