Levi Fried

Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice

Hemangiomas are the most common type of tumor in infants. As they are endothelial cell-derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T-transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/-, and Ang2-/- mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/- cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/- cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function.

The reactive oxygen-driven tumor: relevance to melanoma

In melanoma, at least four major signaling abnormalities have been described. They include β‐catenin deregulation (mutation/mislocalization), p16 loss, MAP kinase activation, and Akt activation. In this review, we discuss the role of the fourth pathway, known as the reactive oxygen driven tumor. The role of reactive oxygen in tumorigenesis is likely to relate to virtually all forms of cancer, and lends itself to specific therapies. These include blockade of reactive oxygen, resulting in decreased activation of NF‐κB, which should sensitize tumors to chemotherapy and radiation. The phenotype of the reactive oxygen driven tumor can be monitored using available markers already in use in most hospital laboratories.

Anti-invasive adjuvant therapy with imipramine blue enhances chemotherapeutic efficacy against glioma.

A novel compound, Imipramine Blue, enhances efficacy of chemotherapy in a single treatment against a rodent astrocytoma model in vivo by inhibiting cell invasion. Stopping the Invasion A hallmark of brain cancer (glioma) is its diffuse nature, where the cancer cells migrate easily through the soft tissue, well away from the tumor site. By comparison, solid tumors, such as those found in breast, lung, and prostate, are more compact with defined margins. It is this cancerous invasion that makes gliomas so difficult to treat surgically and chemotherapeutically. Now, Munson and colleagues describe the synthesis and application of a new small molecule called Imipramine Blue (IB) to stop migrating cancer cells in their tracks. When combined with the anticancer drug doxorubicin (DXR), IB can help prolong the lives of diseased animals. Munson et al. first tested the anti-invasive properties of IB in vitro in both human and rat glioma cell lines and in patient-derived neurospheres. Compared to untreated controls, IB was able to prevent cell outgrowth and invasion, without being toxic to the healthy cells. Similarly, IB packaged in liposomes for easy delivery (“nano-IB”) did not damage tissues or cause inflammation when injected into healthy rats—an important consideration for moving toward human testing. The authors then administered nano-IB to rats with an aggressive form of glioma (RT2) that shows key features of human brain cancer. Animals were treated with two doses of nano-IB and then killed for tumor analysis. Because IB inhibits invasion, the treated tumors were more compact than the controls. When the nano-IB was combined with a potent chemotherapeutic, liposomal DXR, 100% of the diseased rats were able to survive for 180 days, without any signs of regrowth by day 200. By comparison, only 33% of the animals treated with DXR lived that long. No untreated animals survived beyond 50 days. Munson and colleagues also explored the mechanism behind IB’s anti-invasive properties, arriving at a molecular pathway that disrupts actin fiber formation; that is, cells are rendered completely unable to migrate without their intracellular actin machinery. By combining with a chemotherapeutic already used in the clinic, this invasion-stopping compound is poised for testing in more animal models, with the hopes of Food and Drug Administration approval. The invasive nature of glioblastoma (GBM) represents a major clinical challenge contributing to poor outcomes. Invasion of GBM into healthy tissue restricts chemotherapeutic access and complicates surgical resection. Here, we test the hypothesis that an effective anti-invasive agent can “contain” GBM and increase the efficacy of chemotherapy. We report a new anti-invasive small molecule, Imipramine Blue (IB), which inhibits invasion of glioma in vitro when tested against several models. IB inhibits NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase–mediated reactive oxygen species generation and alters expression of actin regulatory elements. In vivo, liposomal IB (nano-IB) halts invasion of glioma, leading to a more compact tumor in an aggressively invasive RT2 syngeneic astrocytoma rodent model. When nano-IB therapy was followed by liposomal doxorubicin (nano-DXR) chemotherapy, the combination therapy prolonged survival compared to nano-IB or nano-DXR alone. Our data demonstrate that nano-IB–mediated containment of diffuse glioma enhanced the efficacy of nano-DXR chemotherapy, demonstrating the promise of an anti-invasive compound as an adjuvant treatment for glioma.

Secreted Frizzle-Related Protein 2 Stimulates Angiogenesis via a Calcineurin/NFAT Signaling Pathway

Secreted frizzle-related protein 2 (SFRP2), a modulator of Wnt signaling, has recently been found to be overexpressed in the vasculature of 85% of human breast tumors; however, its role in angiogenesis is unknown. We found that SFRP2 induced angiogenesis in the mouse Matrigel plug assay and the chick chorioallantoic membrane assay. SFRP2 inhibited hypoxia induced endothelial cell apoptosis, increased endothelial cell migration, and induced endothelial tube formation. The canonical Wnt pathway was not affected by SFRP2 in endothelial cells; however, a component of the noncanonical Wnt/Ca2+ pathway was affected by SFRP2 as shown by an increase in NFATc3 in the nuclear fraction of SFRP2-treated endothelial cells. Tacrolimus, a calcineurin inhibitor that inhibits dephosphorylation of NFAT, inhibited SFRP2-induced endothelial tube formation. Tacrolimus 3 mg/kg/d inhibited the growth of SVR angiosarcoma xenografts in mice by 46% (P = 0.04). In conclusion, SFRP2 is a novel stimulator of angiogenesis that stimulates angiogenesis via a calcineurin/NFAT pathway and may be a favorable target for the inhibition of angiogenesis in solid tumors.

Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes

Alterations in mitochondrial structure and function are a hallmark of cancer cells compared to normal cells and thus targeting mitochondria has emerged as an novel approach to cancer therapy. The mitochondrial thioredoxin 2 (Trx2) system is critical for cell viability, but its role in cancer biology is not well understood. Recently some cationic triphenylmethanes such as brilliant green (BG) and gentian violet were shown to have antitumor and antiangiogenic activity with unknown mechanisms. Here we demonstrate that BG killed cells at nanomolar concentrations and targeted mitochondrial Trx2, which was oxidized and degraded. HeLa cells were more sensitive to BG than fibroblasts. In HeLa cells, Trx2 down-regulation by siRNA resulted in increased sensitivity to BG, whereas for fibroblasts, the same treatments had no effect. BG was observed to accumulate in mitochondria and cause a rapid and dramatic decrease in mitochondrial Trx2 protein. With a redox Western blot method, we found that treatment with BG caused oxidation of both Trx1 and Trx2, followed by release of cytochrome c and apoptosis-inducing factor from the mitochondria into the cytosol. Moreover, this treatment resulted in an elevation of the mRNA level of Lon protease, a protein quality control enzyme in the mitochondrial matrix, suggesting that the oxidized Trx2 may be degraded by Lon protease.

Mammalian Target of Rapamycin (mTOR) is Activated in Cutaneous Vascular Malformations in Vivo

BACKGROUND Vascular malformation signaling pathways are the least understood out of all cutaneous endothelial lesions. The overexpression of Akt is known to cause vascular malformations in endothelial cells of mice. Since there are no Akt inhibitors approved for clinical use, we examined phosphorylated S6 expression, a downstream target of Akt. Phosphorylated S6 indicates potential sensitivity to rapamycin. METHODS AND RESULTS Immunohistochemistry for phospho-s6k against phospho-S6 ribosomal protein was performed on specimens of vascular malformations taken from Sturge- Weber patients. Of the specimens, 70.8% were immunopositive for phospho-s6k. CONCLUSION Endothelial expression of Akt is responsible for tumor responsiveness to rapamycin. We demonstrate that expression of phosphorylated S6 is elevated in specimens. Our findings provide a rationale for clinical trials of rapamycin on Sturge-Weber or Klippel-Trenaunay-Weber patients.

Tris (Dibenzylideneacetone) Dipalladium, a N-Myristoyltransferase-1 Inhibitor, Is Effective against Melanoma Growth In vitro and In vivo

Purpose: Melanoma is a solid tumor that is notoriously resistant to chemotherapy, and its incidence is rapidly increasing. Recently, several signaling pathways have been shown to contribute to melanoma tumorigenesis, including constitutive activation of mitogen-activated protein kinase, Akt, and Stat-3. The activation of multiple pathways may account in part for the difficulty in treatment of melanoma. In a recent screen of compounds, we found that an organopalladium compound, Tris (dibenzylideneacetone) dipalladium (Tris DBA), showed significant antiproliferative activity against melanoma cells. Studies were carried out to determine the mechanism of action of Tris DBA. Experimental Design: Tris DBA was tested on efficacy on proliferation of human and murine melanoma cells. To find the mechanism of action of Tris DBA, we did Western blot and gene array analyses. The ability of Tris DBA to block tumor growth in vivo was assessed. Results: Tris DBA has activity against B16 murine and A375 human melanoma in vivo. Tris DBA inhibits several signaling pathways including activation of mitogen-activated protein kinase, Akt, Stat-3, and S6 kinase activation, suggesting an upstream target. Tris DBA was found to be a potent inhibitor of N-myristoyltransferase-1, which is required for optimal activity of membrane-based signaling molecules. Tris DBA showed potent antitumor activity in vivo against melanoma. Conclusion: Tris DBA is thus a novel inhibitor of N-myristoyltransferase-1 with significant antitumor activity and is well tolerated in vivo. Further preclinical evaluation of Tris DBA and related complexes is warranted.

Targeted therapy of oral hairy leukoplakia with gentian violet

Oral hairy leukoplakia (OHL) is a common oral manifestation of HIV infection. Clinically, these lesions appear as white plaques on the edges of the tongue. Pathophysiologically, these lesions occur because of infection of oral epithelium with Epstein-Barr virus (EBV). No universally effective therapy exists for OHL. We have previously shown that EBV infection and EBV viral products induce the generation of reactive oxygen. We have also demonstrated that the Food and Drug Administration-approved over-the-counter medication gentian violet is a potent inhibitor of reactive oxygen species. We thus chose to treat a patient with biopsy-proven OHL with topical gentian violet. Gentian violet solution was applied topically to the tongue of a patient with OHL. Complete clinical resolution was noted after three treatments. Treatment with topical gentian violet resulted in resolution of the lesions. Further studies with larger numbers of patients are required. The application of gentian violet can be used as a method to OHL treatment. Gentian violet is an inexpensive and safe therapy and, given that it inhibits reactive oxygen, this old therapy is now a targeted novel therapy.

Efficacy of rapamycin in scleroderma: A case study

Scleroderma is a common autoimmune disorder with no effective therapy. Current concepts of scleroderma include the hypothesis that scleroderma results from excess conversion of endothelial cells to fibroblast like cells, called endothelial mesenchymal transformation. This process is thought to be mediated by cytokines including transforming growth factor beta (TGFb), which causes increased collagen synthesis, resulting in fibrosis, the hallmark of the disease. In vitro studies have hypothesized that rapamycin may be of benefit in scleroderma due to antagonism of collagen synthesis. Given that rapamycin has antiangiogenic activities, inhibits wound healing, and prevents the synthesis of collagen in vivo, we tried rapamycin in a patient with scleroderma. We observed rapid improvement in skin stiffness and mobility. Our results provide the rationale for larger clinical trials of rapamycin in scleroderma and other fibrotic disorders.

Identifying new small molecule anti-invasive compounds for glioma treatment.

Glioblastoma is a disease with poor survival rates after diagnosis. Treatment of the disease involves debulking of the tumor, which is limited by the degree of invasiveness of the disease. Therefore, a treatment to halt the invasion of glioma is desirable for clinical implementation. There have been several candidate compounds targeting specific aspects of invasion, including cell adhesions, matrix degradation, and cytoskeletal rearrangement, but they have failed clinically for a variety of reasons. New targets against glioma invasion include upstream mediators of these classical targets in an effort to better inhibit invasion with more specificity for cancer. Included in these treatments is a new class of compounds inhibiting the generation of reactive oxygen species by targeting the NADPH oxidases. These compounds stand to inhibit multiple pathways, including nuclear factor kappa B and Akt. By conducting a screen of compounds thought to inhibit these pathways, a new compound to halt invasion was found that may have a beneficial effect against glioma, based on recent publications. Further, there are still limitations to the treatment of glioblastoma regardless of the discovery of new targets and compounds that should be addressed to better the therapies against this deadly cancer.