Lovastatin Inhibits VEGFR and AKT Activation: Synergistic Cytotoxicity in Combination with VEGFR Inhibitors

BACKGROUND: In a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR). Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR) and EGFR share similar activation, internalization and downstream signaling characteristics. METHODOLOGY/PRINCIPAL FINDINGS: The VEGFRs, particularly VEGFR-2 (KDR, Flt-1), play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM), also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC) and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses. CONCLUSIONS/SIGNIFICANCE: These results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors

Alteration of EGFR Spatiotemporal Dynamics Suppresses Signal Transduction

The epidermal growth factor receptor (EGFR), which regulates cell growth and survival, is integral to colon tumorigenesis. Lipid rafts play a role in regulating EGFR signaling, and docosahexaenoic acid (DHA) is known to perturb membrane domain organization through changes in lipid rafts. Therefore, we investigated the mechanistic link between EGFR function and DHA. Membrane incorporation of DHA into immortalized colonocytes altered the lateral organization of EGFR. DHA additionally increased EGFR phosphorylation but paradoxically suppressed downstream signaling. Assessment of the EGFR-Ras-ERK1/2 signaling cascade identified Ras GTP binding as the locus of the DHA-induced disruption of signal transduction. DHA also antagonized EGFR signaling capacity by increasing receptor internalization and degradation. DHA suppressed cell proliferation in an EGFR-dependent manner, but cell proliferation could be partially rescued by expression of constitutively active Ras. Feeding chronically-inflamed, carcinogen-injected C57BL/6 mice a fish oil containing diet enriched in DHA recapitulated the effects on the EGFR signaling axis observed in cell culture and additionally suppressed tumor formation. We conclude that DHA-induced alteration in both the lateral and subcellular localization of EGFR culminates in the suppression of EGFR downstream signal transduction, which has implications for the molecular basis of colon cancer prevention by DHA

Detection of immunotoxicity using T-cell based cytokine reporter cell lines ("Cell Chip")

Safety assessment of chemicals and drugs is an important regulatory issue. The evaluation of potential adverse effects of compounds on the immune system depends today on animal experiments. An increasing demand, however, exists for in vitro alternatives. Cytokine measurement is a promising tool to evaluate chemical exposure effects on the immune system. Fortunately, this type of measurement can be performed in conjunction with in vitro exposure models. We have taken these considerations as the starting point to develop an in vitro method to efficiently screen compounds for potential immunotoxicity. The T-cell lymphoma cell line EL-4 was transfected with the regulatory sequences of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma or actin fused to the gene for enhanced green fluorescent protein (EGFP) in either a stabile or a destabilised form. Consequently, changes in fluorescence intensity represent changes in cytokine expression with one cell line per cytokine. We used this prototype "Cell Chip" to test, by means of flow cytometry, the immunomodulatory potential of 13 substances and were able to detect changes in cytokine expression in 12 cases (successful for cyclosporine, rapamycin, pentamidine, thalidomide, bis(tri-n-butyltin)oxide, house dust mite allergen (Der p I), 1-chloro-2,4-dinitrobenzene, benzocaine, tolylene 2,4-diisocyanate, potassium tetrachloroplatinate, sodium dodecyl sulphate and mercuric chloride; unsuccessful for penicillin G). In conclusion, this approach seems promising for in vitro screening for potential immunotoxicity, especially when additional cell lines besides T-cells are included

Development of the "Cell Chip": a new in vitro alternative technique for immunotoxicity testing

Predictive testing of immunotoxicity associated with chemical compounds is complicated and cannot be accomplished with a single test. As most of the existing tests for immunotoxicity employ experimental animals, there is an increasing need for alternative tests in vitro. We have developed a new system for in vitro immunotoxicity testing, which employs changes in cytokine expression observed in vitro as an endpoint indicating potential for perturbation of the immune system in vivo. This system named "fluorescent cell chip" (FCC) is based on a number of genetically modified cell lines that regulate the expression of a transgene coding for fluorescent protein enhanced green fluorescent protein (EGFP) in a similar way as they regulate expression of IL-1beta, IL-2, IL-4, IFN-gamma, IL-10, TNF-alpha, and beta-actin. Morphological and functional features of selected cell lines expressing EGFP under the control of cytokine promotors were compared with maternal cell lines and this comparison showed that critical functional features of the maternal cell lines were preserved in EGFP expressing cells. Two chemicals with known immunotoxic activities, cyclosporine A and potassium tetrachloro-platinate(II), mediated compound-specific pattern of inhibition and activation of reporter gene expression. Thus, the "fluorescent cell chip" has demonstrated potential for application as a predictive screening test for immunomodulatory activities of chemicals. The major advantage of this approach is the possibility to apply this test in high throughput screening of high number of compounds for their well defined biological activit

Single molecule imaging and FLIM show different structures for high and low-affinity EGFRs in A431 cells

Epidermal growth factor (EGF) receptor (EGFR) modulates mitosis and apoptosis through signaling by its high-affinity (HA) and low-affinity (LA) EGF-binding states. The prevailing model of EGFR activation—derived from x-ray crystallography—involves the transition from tethered ectodomain monomers to extended back-to-back dimers and cannot explain these EGFR affinities or their different functions. Here, we use single-molecule Förster resonant energy transfer analysis in combination with ensemble fluorescence lifetime imaging microscopy to investigate the three-dimensional architecture of HA and LA EGFR-EGF complexes in cells by measuring the inter-EGF distances within discrete EGF pairs and the vertical distance from EGF to the plasma membrane. Our results show that EGFR ectodomains form interfaces resulting in two inter-EGF distances (∼8 nm and < 5.5 nm), different from the back-to-back EGFR ectodomain interface (∼11 nm). Distance measurements from EGF to the plasma membrane show that HA EGFR ectodomains are oriented flat on the membrane, whereas LA ectodomains stand proud from it. Their flat orientation confers on HA EGFR ectodomains the exclusive ability to interact via asymmetric interfaces, head-to-head with respect to the EGF-binding site, whereas LA EGFRs must interact only side-by-side. Our results support a structural model in which asymmetric EGFR head-to-head interfaces may be relevant for HA EGFR oligomerization

The First Step of Adenovirus Type 2 Disassembly Occurs at the Cell Surface, Independently of Endocytosis and Escape to the Cytosol

Disassembly is a key event of virus entry into cells. Here, we have investigated cellular requirements for the first step of adenovirus type 2 (Ad2) disassembly, the release of the fibers. Although fiber release coincides temporally with virus uptake, fiber release is not required for Ad2 endocytosis. It is, however, inhibited by actin-disrupting agents or soluble RGD peptides, which interfere with integrin-dependent endocytosis of Ad2. Fiber release occurs at the cell surface. Actin stabilization with jasplakinolide blocks Ad2 entry at extended cell surface invaginations and efficiently promotes fiber release, indicating that fiber release and virus endocytosis are independent events. Fiber release is not sufficient for Ad2 escape from endosomes, since inhibition of protein kinase C (PKC) prevents Ad2 escape from endosomes but does not affect virus internalization or fiber release. PKC-inhibited cells accumulate Ad2 in small vesicles near the cell periphery, indicating that PKC is also required for membrane trafficking of virus. Taken together, our data show that fiber release from incoming Ad2 requires integrins and filamentous actin. Together with correct subcellular transport of Ad2-containing endosomes, fiber release is essential for efficient delivery of virus to the cytosol. We speculate that fiber release at the surface might extend the host range of Ad2 since it is associated with the separation of a small fraction of incoming virus from the target cells