PTEN and the pan phosphoinositide 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one (LY294002) exert significant control over tumor-induced angiogenesis and tumor growth in vivo. The LY294002 compound is not a viable drug candidate due to poor pharmacologic variables of insolubility and short half-life. Herein, we describe the development and antitumor activity of a novel RGDS-conjugated LY294002 prodrug, termed SF1126, which is designed to exhibit increased solubility and bind to specific integrins within the tumor compartment, resulting in enhanced delivery of the active compound to the tumor vasculature and tumor. SF1126 is water soluble, has favorable pharmacokinetics, and is well tolerated in murine systems. The capacity of SF1126 to inhibit U87MG and PC3 tumor growth was enhanced by the RGDS integrin (AvB3/A5B1) binding component, exhibiting increased activity compared with a false RADS-targeted prodrug, SF1326. Antitumor activity of SF1126 was associated with the pharmacokinetic accumulation of SF1126 in tumor tissue and the pharmacodynamic knockdown of phosphorylated AKT in vivo. Furthermore, SF1126 seems to exhibit both antitumor and antiangiogenic activity. The results support SF1126 as a viable pan PI3K inhibitor for phase I clinical trials in cancer and provide support for a new paradigm, the application of pan PI3K inhibitory prodrugs for the treatment of cancer. [Cancer Res 2008;68(1):206-15]

Herein we report that, despite the similarity of Rac2 to Rac1 (92% amino acid identity), macrophages derived from Rac2؊/؊ mice, which continue to express Rac1, display a marked defect in ␣ v ␤ 3 /␣ v ␤ 5 and ␣ 4 ␤ 1 integrindirected migration measured on vitronectin and fibronectin fragments (FN-H296), respectively. In contrast, mouse embryo fibroblasts derived from the Rac2 knockout mice utilize Rac1 for migration via ␣ v ␤ 3 /␣ v ␤ 5 and ␣ 4 ␤ 1 . The genetic reconstitution of bone marrowderived macrophages (BMM) with Rac2 restores the integrin-dependent migration of Rac2-deficient macrophages on vitronectin (VN) and FN-H296. The levels of GTP-Rac2 generated upon specific integrin engagement in wild type macrophages parallels the phenotypic defect observed in Rac2-deficient macrophages; i.e. FN-H296,In a COS7 cell system, the expression of Syk kinase alone is sufficient to convert the ␣ 4 ␤ 1 migration response to Rac2 dependence. Therefore, we present the first evidence that the ␣ 4 ␤ 1 receptor in blood cells has evolved a Syk-Rac2 signaling axis to transmit signals required for integrin-directed migration suggesting that Syk kinase in part encodes myeloid Rac2 specificity in vivo.Cell migration is an essential process during development and wound healing. During cell migration coordination between membrane traffic, cell substrate adhesion, and actin polymerization and reorganization is required for protrusion of the leading edge. Actin cytoskeletal reorganization is regulated by Rho family GTPases and, with a contribution from the endocytic cycle, serves to extend the forward edge of mammalian cells (1, 2). The Rho family GTPases have been recognized as the regulators of signal transduction pathways that mediate distinct actin cytoskeleton changes required for cell migration (3, 4). The Rho family of small GTPases (including Rho, Rac, and Cdc42) comprises a complex group of at least 15 proteins critically involved as molecular switches in a large number of biochemical events in many cell types (5-7). Rac activation results from a combination of reduced association with GDP dissociation inhibitors and/or enhanced exchange of GDP to GTP promoted by guanine nucleotide exchange factors. Activated GTP-bound Rac proteins then transduce signals to downstream effector proteins. Finally, through association with GTPase-activating proteins, the GTP-bound small GTPase returns to an inactive GDP-bound form by hydrolysis of the bound GTP. Importantly, the events by which cell surface receptors orchestrate the activation of Rac are likely dependent upon the upstream activation of distinct protein-tyrosine kinases and the recruitment of specific adapter proteins to specific subcellular locals to transmit specific signals.One feature of the Rac proteins is their capacity to regulate the architecture of the actin cytoskeleton (3, 8 -11). Dynamic rearrangement of the actin cytoskeleton is key for morphological changes observed under conditions of adhesion to the extracellular matrix and for cell migration. The structure and ...

The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some Rap1a−/− embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a −/− mice. However, macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a−/− mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events.

Dysregulation of the phosphatidylinositol-3-kinase (PI3K) pathway in a wide range of tumors has made PI3K a consensus target to inhibit as illustrated by more than 15 inhibitors now in clinical trials. Our previous work, built on the early pioneering multikinase inhibitor LY294002, resulted in the only PI3K vascular-targeted PI3K inhibitor prodrug, SF1126, which has now completed Phase I clinical trials. This inhibitor has properties that impart more in vivo activity than should be warranted by its enzymatic potency, which in general is much lower than other clinical stage PI3K inhibitors. We embarked on the exploration of scaffolds that retained such properties while simultaneously exhibiting an increased potency toward PI3K. This work resulted in the discovery of the 5-morpholino-7H-thieno[3,2-b]pyran-7-one system as the foundation of a new compound class of potential PI3K inhibitors having improved potency toward PI3K. The synthesis and cancer stem cell-based activity of these compounds are reported herein.

Fcγ receptor–mediated phagocytosis is a complex process involving the activation of protein tyrosine kinases, events that are potentially down-regulated by protein tyrosine phosphatases. We used the J774A.1 macrophage cell line to examine the roles played by the protein tyrosine phosphatase SHP-1 in the negative regulation of Fcγ receptor–mediated phagocytosis. Stimulation with sensitized sheep red blood cells (sRBCs) induced tyrosine phosphorylation of CBL and association of CBL with CRKL. These events were completely or partially abrogated by PP1 or the heterologous expression of dominant-negative SYK, respectively. Heterologous expression of wild-type but not catalytically inactive SHP-1 also completely abrogated the phagocytosis of IgG-sensitized sRBCs. Most notably, overexpressed SHP-1 associates with CBL and this association led to CBL dephosphorylation, loss of the CBL-CRKL interaction, and the suppression of Rac activation. These data represent the first direct evidence that SHP-1 is involved in the regulation of Fcγ receptor–mediated phagocytosis and suggest that activating signals mediated by SRC family kinases SYK, CBL, phosphatidyl inositol-3 (PI-3) kinase, and Rac are directly opposed by inhibitory signals through SHP-1.

Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G 1 /S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV-induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs Cells exposed to UV irradiation are predominantly arrested in S-phase rather than at the G 1 /S boundary while repair occurs (1). The molecular mechanism of damage-induced Sphase arrest is not known; however, the effects of UV irradiation during S-phase on subsequent cell cycles are magnified in repair-deficient cells (2), indicating that these effects may be initiated by DNA damage itself. In contrast, in vitro replication experiments with cytosolic extracts from UV-damaged cells strongly indicate that UV-induced inhibition of replication is not due to a blockade of replication by DNA damage itself; rather, irradiation probably induces a mechanism that inhibits DNA replication (3, 4). It is not known how DNA damage induces the inhibition of DNA replication and yet permits DNA repair; however, proteins such as replication protein A (RPA 1 ; also known as human single-stranded DNA-binding protein) and proliferating cell nuclear antigen (PCNA) are involved in both processes (5-10) and may play a role in differential regulation. Earlier in vitro studies suggested that PCNA interacts with UV-induced protein, p21Cip1/Waf1 , which inhibits PCNA's function in DNA replication but not in repair (11-13). PCNA also interacts with GADD45 and MyD118, which are induced upon growth arrest and DNA damage, supporting a role for PCNA in damage-induced cell cycle arrest (14,15).RPA is a heterotrimeric single-stranded DNA-binding protein (70-, 34-, and 11-kDa subunits) originally identified as an essential factor for the replication of SV40 DNA (6, 9, 10). In addition to its role in replication, RPA is also required for DNA repair (5,16,17) and genetic recombination (18 -20), suggesting a possible role in regulation. In replication, RPA interacts with SV40 T-antigen and DNA polymerase ␣-primase complex, which probably mediates unwinding of SV40 origin-containing DNA (21-29). In addition, RPA stimulates polymerase ␣, ␦, and ⑀, which suggests its potential role in the elongation stage (30, 31). The middle subunit of RPA is phosphorylated in a cell cycle-dependent manner (32) and also by UV and ionizing radiation (3, 33). DNA-PK is responsible for the hyperphosphorylation of the 34-kDa subunit of RPA (34, 35); however, the in vivo observations with yeast and mammalian systems suggest additional involvement of other kinases, such as ataxia-telangiectasia mutant (ATM) (36,37). The observation that damageinduced RPA phosphorylation interferes with its interaction with p53 and DNA-PK su...

The physiological recruitment of circulating lymphocytes from the blood into secondary lymphoid tissues is an essential homeostatic mechanism for the immune system because it allows lymphocytes to encounter efficiently both their specific cognate antigen and the regulatory cells with which they need to interact, to initiate, maintain, and terminate immune responses appropriately. This constitutive lymphocyte trafficking is mediated by high endothelial venules (HEVs), which are present in secondary lymphoid tissues other than the spleen. There is growing evidence that lymphocyte trafficking across HEVs involves at least three steps, namely, (i) tethering/rolling, (ii) arrest/firm adhesion/intraluminal crawling, and (iii) transendothelial migration (TEM). Although the mechanisms underlying the first two steps have been determined relatively well, the mechanism regulating TEM is only partially understood. In particular, the molecular mechanism driving lymphocyte movement from the apical to the basolateral surface of the endothelial cells (ECs) of HEVs remains ill defined. This step is crucial for successful lymphocyte extravasation, and is thus an important target for therapeutic intervention in various immunological diseases. Here, we review the molecular mechanisms governing lymphocyte-HEV interactions, and highlight possible roles for two HEV proteins, i.e., nepmucin/CD300g and autotaxin, in lymphocyte TEM.

AIM:To investigate the correlation between the antifibrotic effect of baicalin and serum cytokine production in rat hepatic fibrosis. METHODS:Forty male Sprague-Dawley rats were divided randomly into four groups: normal control group, model group, baicalin-treated group, and colchicine-treated group. Except for the normal control group, all rats in the other groups were administered with carbon tetrachloride to induce hepatic fibrosis. At the same time, the last two groups were also treated with baicalin or colchicine. At the end of the 8 wk, all animals were sacrificed. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), transforming growth factor (TGF)-b1, tumor necrosis factor (TNF)-a, interleukin (IL)-6 and IL-10 were measured. Liver index, hepatic hydroxyproline content and the degree of liver fibrosis were also evaluated. RESULTS:The levels of ALT, AST and liver index in the baicalin-treated group were markedly lower than those in the model group (ALT: 143.88 ± 14.55 U/L vs 193.58 ± 24.35 U/L; AST: 263.66 ± 44.23 U/L vs 404.37 ± 68.29 U/L; liver index: 0.033 ± 0.005 vs 0.049 ± 0.009, P < 0.01). Baicalin therapy also significantly attenuated the degree of hepatic fibrosis, collagen area and collagen area percentage in liver tissue (P < 0.01). Furthermore, the levels of serum TGFb1, TNF-a and IL-6 were strikingly reduced in the baicalin-treated group compared with the model group, while the production of IL-10 was up-regulated: (TGF-b1: 260.21 ± 31.01 pg/mL vs 375.49 ± 57.47 pg/mL; TNF-a: 193.40 ± 15.18 pg/mL vs 260.04 ± 37.70 pg/mL; IL-6: 339.87 ± 72.95 pg/mL vs 606.47 ± 130.73 pg/mL; IL-10: 506.22 ± 112.07 pg/mL vs 316.95 ± 62.74 pg/mL, P < 0.01). CONCLUSION:Baicalin shows certain therapeutic effects on hepatic fibrosis, probably by immunoregulating the imbalance between profibrotic and antifibrotic cytokines.