D. Lynne Smith

Malignant mesothelioma growth inhibition by agents that target the VEGF and VEGF-C autocrine loops.

Malignant mesothelioma (MM) is a locally aggressive tumor that originates from the mesothelial cells of the pleural and sometimes peritoneal surface. Conventional treatments for MM, consisting of chemotherapy or surgery give little survival benefit to patients, who generally die within 1 year of diagnosis. Hence, there is an urgent need for the development of alternative therapies. Vascular endothelial growth factor (VEGF) is an autocrine growth factor for MM. The closely related molecule, VEGF‐C, is also implicated in malignant mesothelioma growth. VEGF‐C and its cognate receptor VEGFR‐3 are co‐expressed in mesothelioma cell lines. A functional VEGF‐C autocrine growth loop was demonstrated in mesothelioma cells by targeting VEGF‐C expression and binding to VEGFR‐3. The ability of novel agents that reduce the levels of VEGF and VEGF‐C to inhibit mesothelioma cell growth in vitro was assessed. Antisense oligonucleotide (ODN) complementary to VEGF that inhibited VEGF and VEGF‐C expression simultaneously specifically inhibited mesothelioma cell growth. Similarly, antibodies to VEGF receptor (VEGFR‐2) and VEGF‐C receptor (VEGFR‐3) were synergistic in inhibiting mesothelioma cell growth. In addition, a diphtheria toxin‐VEGF fusion protein (DT‐VEGF), which is toxic to cells that express VEGF receptors was very effective in inhibiting mesothelioma cell growth in vitro. These results indicate that targeting VEGF and VEGF‐C simultaneously may be an effective therapeutic approach for malignant mesothelioma.

Human Herpesvirus-8-Transformed Endothelial Cells Have Functionally Activated Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor

Kaposis sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposis sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, -2, and -3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression.

Phase I Study of Antisense Oligonucleotide Against Vascular Endothelial Growth Factor: Decrease in Plasma Vascular Endothelial Growth Factor With Potential Clinical Efficacy

PURPOSE Vascular endothelial growth factor antisense (VEGF-AS) is an antisense oligonucleotide that targets VEGF, inhibiting angiogenesis and tumor cell proliferation. This study established the safety, biologic effects, and pharmacokinetics of VEGF-AS in 51 patients with advanced malignancies. METHODS VEGF-AS was administered as a 2-hour infusion daily for 5 consecutive days for only one cycle on the first four dose levels, and then administered daily for 5 days every other week for up to 4 months on subsequent levels. Pharmacokinetics, tumor response, and the effect on plasma VEGF levels were determined. RESULTS The maximum-tolerated dose was 200 mg/m2. Dose-limiting toxicities included grade 4 fever, and pulmonary embolism in one patient each at 250 mg/m2. Mild anemia, fever, fatigue, and gastrointestinal complaints were the most common adverse events. VEGF-AS t(1/2beta) (beta-phase terminal half-life of drug concentration) was 2.25 hours (range, 1.97 to 2.95 hours). Mean plasma VEGF-A (P = .002) and VEGF-C (P = .01) levels decreased 24 hours postinfusion, with a trend towards greater decreases at higher dose levels. At the maximum-tolerated dose, five of six patients demonstrated reductions in plasma VEGF. Clinical responses included complete remission in one patient with AIDS-Kaposis sarcoma, a mixed but dramatic response in one patient with cutaneous T-cell lymphoma, and prolongation of progression-free survival compared with that obtained on the immediate prior regimen in six patients (12%) with renal cell, bronchoalveolar, small cell lung, thyroid, and ovarian carcinomas, and chondrosarcoma, respectively. CONCLUSION VEGF-AS was well tolerated, with biologic effects and preliminary evidence of clinical efficacy.

Natural killer cell cytolytic activity is necessary for in vivo antitumor activity of the dipeptide L-glutamyl-L-tryptophan

A dipeptide, L‐glutamyl L‐tryptophan (L‐glu‐L‐trp), was identified in a screen for immunomodulators in the soluble fraction of the thymus. L‐glu‐L‐trp inhibits tumor growth in mice without showing direct cellular toxicity in a variety of human tumor cell lines. L‐glu‐L‐trp antitumor activity in vivo requires the presence of natural killer (NK) cells. Defective trafficking of cytoplasmic granules caused by the Lyst mutation also resulted in loss of antitumor activity of the dipeptide. The effect of L‐glu‐L‐trp on tumor growth in mice with targeted gene mutations demonstrated the absolute requirement for perforin for antitumor activity. The requirement of 2 major modulators of NK cell activity, gamma interferon (IFNγ) and interleukin (IL)‐12, were also tested. L‐glu‐L‐trp had full antitumor activity in IFNγ knockout mice, but had significantly diminished activity in IL‐12 knockout mice. These data show that L‐glu‐L‐trp antitumor activity in mice is dependent on cytolytic cell activity of NK or NKT cells. L‐glu‐L‐trp in vivo regulates NK cell function independent of IFNγ but partly dependent on IL‐12.

Paclitaxel Induces Apoptosis in AIDS-Related Kaposi's Sarcoma Cells.

Paclitaxel is a microtubule stabilizing drug that causes dividing cells to arrest and then undergo apoptosis. It also has antiangiogenic activity because it alters cytoskeletal structure, affecting migration and invasion. Paclitaxel is an effective treatment for AIDS-related Kaposi’s sarcoma (KS). KS is a tumor in which there is marked proliferation of endothelial cells in addition to the tumor cells, which themselves share many markers with activated (proliferating) endothelial cells.We sought to determine the mechanism by which paclitaxel exerts its anti-KS tumor effects. In vitro, KS cells are very sensitive to paclitaxel, with half-maximal growth inhibition observed at 0.8 nM. Inhibition of migration of KS cells was also observed at nanomolar concentrations of the drug. Paclitaxel induced cell cycle arrest with an accumulation of cells in sub-G1.This was accompanied in vitro by various events typical of apoptosis: phosphorylation of two anti-apoptotic proteins Bcl-2 and Bcl-xL , release of cytochrome c into the cytoplasm, cleavage and activation of caspase-3. In vitro results were borne out by studies of KS tumor xenografts in nude mice. Paclitaxel (10 mg/kg) inhibited tumor growth by 75% over 21 days. Histological examination of the tumors revealed a decrease in proliferative index, a decrease in the number of mitotic figures and an increase in apoptotic cells compared to tumors from untreated mice.