Integrin agonists as adjuvants in chemotherapy for melanoma.

PURPOSE: Metastatic melanomas are generally resistant to chemotherapy and radiation, even when wild-type for p53. These tumors often grow in small nests where many of the cells have little contact with extracellular matrix (ECM). Previous work showed that M21 melanomas undergo apoptosis in response to chemotherapy when cells are adherent to ECM but not in suspension. Thus, reduced integrin-dependent adhesion to ECM could mediate therapy resistance. The goal of this study was to test whether stimulation of integrin signaling could increase chemotherapeutic efficacy. EXPERIMENTAL DESIGN: Colony forming assays and survival assays were used to test the responses of melanoma lines in vitro. Severe combined immunodeficient mice with subcutaneous human melanomas received chemotherapy with or without reagents that stimulate integrin signaling; tumor volume was then monitored over time. RESULTS: Clonal growth assays confirmed that M21 cells showed reduced sensitivity to the chemotherapeutic drug 1-beta-D-arabinofuranosylcytosine (araC). When five additional primary melanoma lines were screened, 80% showed higher sensitivity when adherent compared with suspended. Subcutaneous M21 tumors in vivo showed minimal ECM between tumor cells. To evaluate the importance of integrin signaling in chemoresistance in this model, mice were treated with araC, with or without the multivalent snake venom disintegrin contortrostatin or the activating anti-beta1 integrin antibody TS2/16. Although araC, TS2/16, or contortrostatin alone had little effect on M21 tumor growth, combining araC with either integrin signaling reagents strongly reduced growth (P = 0001). CONCLUSIONS: Loss of integrin-mediated adhesion is rate limiting for therapeutic response in this model. Combining chemotherapy with reagents that stimulate integrin signaling may therefore provide a new approach to therapy.

Disintegrin-Integrin Binding for Attachment of Polymer Substrate to the Retina.

OBJECTIVE: We propose a novel attachment method for retinal tissue that utilizes silicone modified with bioactive molecules. DESIGN: This is an experimental study divided into an in vitro section performed in cadaveric pig eyes and an in vivo section performed in rabbits. SUBJECTS: During in vitro experiments 36 cadaveric pig eyes were used. During in vivo experiments 4 rabbits were used. METHODS: Different types of silicone went through a laser irradiation process to determine if binding sites for disintegrins could be created. Laser treated silicones that showed disintegrin binding were evaluated with in vitro testing for retina-silicone attachment. The best silicone binding in vitro was implanted into a rabbit's eye after a full vitrectomy was performed. Post-operative exams were done every two weeks to evaluate placement, attachment and sterilization method. After three months animals were euthanized and eye was enucleated for histology analysis. MAIN OUTCOME MEASURES: Attachment strength between silicone-disintegrin-retina, and signs of endophthalmitis during in vivo studies for biocompatibility purposes. RESULTS: A technique to successfully lase and produce an active area on the silicone surface was described. Scanning electron microscope (SEM) images were evaluated to assess physical ablation/debris field area on the surface, definition of edges, evenness, and symmetry of the lased area allowing us to select MED 4800 silicone family for further testing. Cell culture experiments showed disintegrin binding to the silicone active area. In vitro experiments with cadaveric eyes were performed to test retina-silicone attachment. MED 4860 showed strongest attachment to the retina and it was used during in vivo experiments. A sterilization protocol was tested and proved to be reliable for bioactive materials. Disintegrin coated silicone showed attachment in 2 of 4 rabbits during the 3-month implant period. The adhesion was persistent until reversed with plasmin. All rabbits were implanted for 3 months regardless of attachment, to test the feasibility of the sterilization method. None of the rabbits developed any type of eye infection during the implant period. CONCLUSION: We successfully lased and produced an active area on the silicone surface to allow disintegrin-silicone binding. Different silicones interact differently with the laser energy, and this is reflected in the strength of the silicone-disintegrin-retina attachment.

Vascular targeting and antiangiogenesis agents induce drug resistance effector GRP78 within the tumor microenvironment.

Therapeutic targeting of the tumor vasculature that destroys preexisting blood vessels of the tumor and antiangiogenesis therapy capitalize on the requirement of tumor cells on an intact vascular supply for oxygen and nutrients for growth, expansion and metastasis to the distal organs. Whereas these classes of agents show promise in delaying tumor progression, they also create glucose and oxygen deprivation conditions within the tumor that could trigger unintended prosurvival responses. The glucose-regulated protein GRP78, a major endoplasmic reticulum chaperone, is inducible by severe glucose depletion, anoxia, and acidosis. Here we report that in a xenograft model of human breast cancer, treatment with the vascular targeting agent, combretastatin A4P, or the antiangiogenic agent, contortrostatin, promotes transcriptional activation of the Grp78 promoter and elevation of GRP78 protein in surviving tumor cells. We further show that GRP78 is overexpressed in a panel of human breast cancer cells that has developed resistance to a variety of drug treatment regimens. Suppression of GRP78 through the use of lentiviral vector expressing small interfering RNA sensitizes human breast cancer cells to etoposide-mediated cell death. Our studies imply that antivascular and antiangiogenesis therapy that results in severe glucose and oxygen deprivation will induce GRP78 expression that could lead to drug resistance.

Chemical surface modification of parylene C for enhanced protein immobilization and cell proliferation.

To introduce the adhesion site of proteins and/or cells on parylene C (PC)-coated medical devices that can be used as implantable biosensors or drug delivery capsules, the PC surfaces were initially modified by the Friedel-Crafts acylation reaction to generate active chlorines. These chlorines were then employed to initiate the atom transfer radical polymerization of tert-butyl acrylate (TBA) and form a polymer brush layer of polyTBA on PC; the acrylate groups in the polymer brushes were hydrolyzed to carboxylic acid groups and further activated into succinimidyl ester groups via the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide coupling reaction. The PC surface grafted with polymer brushes and activated by succinimide showed efficient attachment of proteins, including gelatin, contortrostatin (CN) and bovine serum albumin (BSA), all at high density on the PC surface. The CN density on the surface was evaluated for both monolayer and polymer brush-based coatings. Based on fluorescence measurements, the polymer brush gives a 60-fold higher surface protein density than the monolayer-based system. Gelatin was used as a model protein and covalently coated onto the modified PC surface for cell culture study. Substrates with gelatin coating showed a significantly higher cell attachment and proliferation in 7 days cultures as compared to the uncoated substrates. In addition, a conventional photolithography technique was coupled with the surface chemistry to successfully pattern the BSA labeled with fluorescein isothiocyanate on the modified PC surfaces.

Phase II clinical trial of bevacizumab and low-dose metronomic oral cyclophosphamide in recurrent ovarian cancer: a trial of the California, Chicago, and Princess Margaret Hospital phase II consortia.

PURPOSE: Vascular endothelial growth factor (VEGF) plays an important role in the biology of ovarian cancer (OC). Inhibitors of VEGF suppress tumor growth in OC models. Metronomic chemotherapy, defined as frequent administration of low doses of cytotoxic chemotherapy, suppresses tumor growth, possibly by inhibiting angiogenesis. A phase II trial was conducted to evaluate the antitumor activity and adverse effects of bevacizumab and metronomic oral cyclophosphamide in women with recurrent OC. PATIENTS AND METHODS: Patients with measurable disease and prior treatment with a platinum-containing regimen were eligible. Up to two different regimens for recurrent disease were allowed. Treatment consisted of bevacizumab 10 mg/kg intravenously every 2 weeks and oral cyclophosphamide 50 mg/d. The primary end point was progression-free survival at 6 months. Plasma levels of VEGF, E-selectin, and thrombospondin-1 were obtained serially. RESULTS: Seventy patients were enrolled. The probability of being alive and progression free at 6 months was 56% (+/- 6% SE). A partial response was achieved in 17 patients (24%). Median time to progression and survival were 7.2 and 16.9 months, respectively. The most common serious toxicities were hypertension, fatigue, and pain. Bevacizumab-related toxicities included four episodes of gastrointestinal perforation or fistula, two episodes each of CNS ischemia and pulmonary hypertension, and one episode each of gastrointestinal bleeding and wound healing complication. There were three treatment-related deaths. Levels of VEGF, E-selectin, and thrombospondin-1 were not associated with clinical outcome. CONCLUSION: The combination of bevacizumab and metronomic cyclophosphamide is active in recurrent OC. Further study of this combination is warranted.