Akt phosphorylation of T308 and S473 are independent events. Panel A.

<p>A bar diagram showing independence of phosphorylation of Akt at T308 (▪) and S473 (□) in kinase assays of the 1-LN cancer cells stimulated with α₂M* (50 pM/25 min) or insulin (200 nM/20 min). The bars are: (1) lipofectamine + buffer; (2) lipofectamine + α₂M* (50 pM/25 min); (3) Raptor dsRNA (100 nM/48 h) then α₂M*; (4) Rictor dsRNA (100 nM/48 h) then α₂M*; (5) lipofectamine + insulin; (6) Raptor dsRNA (100 nM/48 h), then insulin (200 nM/20 min); (7) Rictor dsRNA (100 nM/48 h) + insulin; and (8) scrambled dsRNA (100 nM/48 h) then insulin. Values are mean ± SE from three experiments and are expressed as fmol [³³P]-γ-ATP incorporated/mg cell protein. <b>Panel B.</b> Levels of p-Akt^T308 and p-Akt^S743 in cell lysates of 1-LN cells treated with α₂M* (50 pM/25 min) or insulin (200 nM/15 min). Representative immunoblots of p-Akt^T308 and p-Akt^S473 from three experiments of cells transfected with Raptor dsRNA or Rictor dsRNA as above are being shown.</p

MN10021 inhibits dermal vascular leak in a reverse passive Arthus reaction.

<p>Groups of 5 Hartley strain guinea pigs (male; ∼250 g) were injected s.c. with 1.0 ml of either saline or the indicated dose of MN10021. Eighteen hours later the flanks of the animals were shaved and both 6.25 and 12.5 µl of rabbit anti-BSA antiserum injected i.d. at each of two sites, one on each flank. The animals were then injected i.v. with 1.0 ml of PBS containing BSA (1.0 mg/ml) and Evans Blue dye (2.0 mg/ml). Six hours later the animals were euthanized by barbiturate overdose, the skin on the back removed, and the diameter of the extravasation of the Evans Blue dye measured for each site on the ventral surface of the skin. The values represent the mean ± SE of the 10 measurements obtained for each of the treatment conditions. ** p<0.01; * p<0.05; NS  =  not significant.</p

Anti-Inflammatory and Vasoprotective Activity of a Retroviral-Derived Peptide, Homologous to Human Endogenous Retroviruses: Endothelial Cell Effects

<div><p>Malignant and inflammatory tissues sometimes express endogenous retroviruses or their proteins. A highly-conserved sequence from retroviral transmembrane (TM) proteins, termed the “immunosuppressive domain (ID)”, is associated with inhibition of immune and inflammatory functions. An octadecapeptide (MN10021) from the ID of retroviral TM protein p15E inhibits <em>in vitro</em> release of pro-inflammatory cytokines and increases synthesis of anti-inflammatory IL-10. We sought to determine if MN10021 has significant <em>in vivo</em> effects. MN10021, prepared by solid-phase synthesis, was dimerized through a naturally-occurring, carboxy-terminal cysteine. <em>In vivo</em> anti-inflammatory activity was determined using a murine model of sodium periodate (NaIO₄)-induced peritonitis. <em>In vivo</em> vasoprotective effects were determined using: (1) a carrageenan-induced model of disseminated intravascular coagulation (DIC) in mice; (2) a reverse passive Arthus model in guinea pigs; and (3) vasoregulatory effects in spontaneously hypertensive rats (SHR). <em>In vitro</em> studies included: (1) binding/uptake of MN10021 using human monocytes, cultured fibroblasts, and vascular endothelial cells (VEC); (2) gene expression by RT-PCR of MN10021-treated VEC; and (3) apoptosis of MN10021-treated VEC exposed to staurosporine or TNF-α. One-tenth nmol MN10021 inhibits 50 percent of the inflammatory response in the mouse peritonitis model. Furthermore, 73 nmol MN10021 completely protects mice in a lethal model of carrageenan-induced DIC and inhibits vascular leak in both the mouse DIC model and a guinea pig reverse passive Arthus reaction. MN10021 binds to and is taken up in a specific manner by both human monocytes and VEC but not by cultured human fibroblasts. Surprisingly, orally-administered MN10021 lowers blood pressure in SHR rats by 10–15% within 1 h suggesting a direct or indirect effect on the vascular endothelium. MN10021 and derived octapeptides induce iNOS (inducible nitric oxide synthase) mRNA in VEC and nitrate in VEC cell culture supernatants and protect VEC from induced apoptosis or necrosis. However, pretreatment of VEC with nitro-L-arginine methyl ester (L-NAME), while inhibiting the release of nitrate, does not block the anti-apoptotic effect of MN10021 and derived octapeptides suggesting that their potent vasoprotective and anti-inflammatory activity is not nitric oxide dependent.</p> </div

α₂M*-induced protein synthesis and its sensitivity to rapamycin in 1-LN prostate cancer cells.

<p><b>Panel A.</b> α₂M* concentration-dependent protein synthesis. <b>Panel B.</b> Modulation of α₂M*-induced-protein synthesis in 1-LN cells. The bars are: (1) buffer; (2) α₂M* (50 pM); (3) wortmannin 30 nM/20 min then α₂M* (50 pM); (4) LY294002 (20 µM/20 min) then α₂M*; (5) Rapamycin (100 nM/20 min), then α₂M*; (6) Actinomycin D (5 µg/ml/15 min) then α₂M*. The values in <b>Panel A</b> and <b>B</b> are the mean ± SE from four independent experiments. Values significantly different at 5% level for α₂M*-treated cells marked by an asterisk (*).</p

Effect of MN10021 on blood leukocyte and platelet counts in murine DIC model.

<p>Each of 4 groups of 5 CD-1 mice (male, 6–8 weeks of age) was injected i.p with 1.0 ml of either saline (closed circles) or 1.0 mg (245 nmol) MN10021 in saline (open circles). Eighteen hours later all mice were injected i.p. with 1.0 ml of 0.45% carrageenan. At each of the indicated times one group of 5 mice for each treatment was euthanized, an anti-coagulated blood sample obtained by cardiac puncture, and total white blood cell (WBC) (panel A) or platelet (PLT) (panel B) counts performed on an automated hematology analyzer. In both panels the dotted line represents the average value obtained from the blood samples of 10 untreated CD-1 mice. The data plotted are the mean values ± SE. Error bars were calculated for all values and if not visible are contained within the symbol.</p

Phosphorylation of Akt at T308 and S473 in cells stimulated with α₂M*.

<p><b>Panel A.</b> A bar diagram showing α₂M*-induced phosphorylation of Akt at T308 (▪) and S473 (□) in kinase assays of mTOR immunoprecipitates. The bars and lanes in the immunoblot are: (1) buffer; (2) α₂M* (50 pM/25 min); (3) LY294002 (20 µM/25 min) then α₂M*; (4) rapamycin (100 nM/20 min) then α₂M*. Values are the mean ± SE from four independent experiments and are expressed as fmol [³³P]-γ-ATP incorporated/mg cell protein. Values significantly different at the 5% level for α₂M*-treated cells are indicated by an asterisk (*). <b>Panel B.</b> A bar diagram showing α₂M*-induced phosphorylation of Akt at T308 (▪) and S473 (□) in kinase assays of Rictor immunoprecipitates. The bars and lanes in immunoblot are: (1) buffer; (2) α₂M* (50 pM/25 min); (3) LY294002 (20 mM/25 min) then α₂M* and (4) Rapamycin (100 nM/20 min). Values are the mean ± SE from four independent experiments and are expressed as fmol [³³P]-γ-ATP incorporated per mg cell protein. Values significant different at 5% level for α₂M*-treated cells are indicated by an asterisk (*). <b>Panel C.</b> A bar diagram showing α₂M*-induced phosphorylation of Akt at T308 (▪) and S473 (□) residues in kinase assays of mTOR immunoprecipitates and its modulation subsequent to suppression of GRP78 expression by RNAi. The bars and lanes in immunoblot are: (1) lipofectamine + buffer; (2) lipofectamine + α₂M* (50 pM/25 min); (3) GRP78 dsRNA (100 nM/48 h) then α₂M*; (4) scrambled dsRNA (100 nM/48 h) then α₂M* (50 pM/25 min). Values are the mean ± SE from four independent experiments and are expressed as fmol [³³P]-γ-ATP incorporated per mg cell protein. Values significantly different for α₂M* and scrambled dsRNA + α₂M*-treated cells are indicated by an asterisk (*). <b>Panel D.</b> A bar diagram of Akt phosphorylation of S473 in kinase assays and a p-Akt^S473 immunoblot showing α₂M*-induced phosphorylation of Akt S473 in Rictor immunoprecipitates of 1-LN cells and its modulation by silencing GRP78 expression by RNAi. The bars are: (1) lipofectamine + buffer; (2) lipofectamine + α₂M* (50 pM/25 min); (3) GRP78 dsRNA (100 nM/48 h) then α₂M*; (4) scrambled dsRNA (100 nM/48 h) and (5) antibody against the carboxyl-terminal domain in GRP78 (3 µg/ml/60 min) then α₂M. Immunoblot of p-Akt^S473 under bar 5 is not being shown since this has been reported earlier (18). Values are from four independent experiments and are expressed as fmol [³³P]-γ-ATP incorporated per mg cell protein. Values significantly different for α₂M* and scrambled dsRNA + α₂M*-treated cells are indicated by an asterisk (*). <b>Panel E.</b> A bar diagram showing α₂M*-induced increased phosphorylation of Akt at S473 in kinase assays of Rictor immunoprecipitates of 1-LN (▪) and DU-145 cells (□). The bars are: (1) buffer; (2) α₂M* (50 pM/25 min); (3) Rapamycin (100 nM/15 min) then α₂M* (4) LY294002 (25 µM/20 min) then α₂M* and (5) antibodies directed against the carboxyl-terminal domain of GRP78 (3 µg/ml/60 min) then α₂M*. Values are the mean ± SE from three experiments and are expressed as fmol [³³P]-γ-ATP incorporated/mg cell protein. Values significantly different at the 5% level for α₂M*-treated cells are indicated by an asterisk (*). <b>Panel F.</b> Immunoblot showing α₂M*-induced increased protein levels of p-Akt^S473 in Rictor immunoprecipitates of DU-145 prostate cancer cells but not in PC-3 cells. The lanes in the immunoblots are: (1) buffer-treated and (2) α₂M* (50 pM/25 min)-treated.</p

A model summarizing the results of this study.

<p>The abbreviation CTGRP78 Ab refers to antibodies directed against the COOH-terminal domain of GRP78. S6K is S6-Kinase.</p

Effect of silencing Raptor expression by RNAi on phosphorylation of S6 kinase and 4EBP1 in 1-LN cells stimulated with α₂M* and insulin.

<p>Panel A. Bar diagram showing levels of Raptor in cells transfected with Raptor dsRNA and stimulated with α₂M* or insulin. The bars are: (1) lipofectamine + buffer; (2) lipofectamine + α₂M* (50 pM/25 min); (3) lipofectamine + insulin (200 nM/20 min); (4) scrambled dsRNA (100 nM/48 h) + insulin; (5) Raptor dsRNA (100 nM/48 h); (6) Raptor dsRNA (100 nM/48 h) then α₂M*; (7) Raptor dsRNA (100 nM/48 h) then insulin (200 nM/20 min); (8) rapamycin (100 nM/20 min) then insulin. Values are mean ± SE from three to four independent experiments and are expressed as arbitrary fluorescence units. Values significantly different at 5% level for α₂M*, insulin and scrambled dsRNA treated cells are indicated by an asterisk (*). A representative immunoblot of Raptor from three to four experiments along with its protein loading control actin is shown below the bar diagram. Panel B. A bar diagram showing mTORC1 activation in cells treated with α₂M* and insulin and effect of transfection with Raptor dsRNA on its activation as measured by assaying phosphorylation of S6-Kinase by autoradiography. The bars and lanes in autoradiograph are: (1) lipofectamine + buffer; (2) lipofectamine + α₂M* (50 pM/25 min); (3) Raptor dsRNA (100 nM/48 h); (4) Raptor dsRNA + α₂M*; (5) lipofectamine + insulin (200 nM/20 min); (6) scrambled dsRNA (100 nM/48 h) + insulin; (7) Raptor dsRNA (100 nM/20 min) then insulin; (8) rapamycin (100 nM/20 min) then α₂M*; (9) LY294002 (20 mM/20 min) then insulin; (10) rapamycin (100 nM/20 min) then insulin. A representative autoradiograph of three experiments of S6-Kinase phosphorylation is shown below the bar diagram. Values are the mean ± SE from three experiments and are expressed in arbitrary units. Values significantly different at 5% levels for α₂M*, insulin and scrambled dsRNA treated cells are indicated by an asterisk (*). Panel C. A bar diagram showing levels of S6-Kinase phosphorylated at T389 (▪); T229 (see grey square) and T 235/236 (□) in cells stimulated with α₂M* or insulin and transfected with Raptor dsRNA. The bars are as in Panel A. Representative immunoblots of p-S6-Kinase^T389, p-S6-Kinase^T229 and p-S6-Kinase^T235/236 of three experiments along with its protein loading control is shown below the bar diagram. Values are the mean ± SE from three experiments and are expressed in arbitrary fluorescence units. Values significantly different at 5% level for α₂M*, insulin or scrambled dsRNA are indicated by an asterisk (*). Panel D. A bar diagram showing phosphorylation levels of 4EBP1, in cells treated with α₂M* and insulin or transfected with Raptor dsRNA. The bars are as in Panel A. A representative immunoblot of p-4EBP1 of three experiments along with its protein loading control is shown below the bar diagram. Values are the mean ± SE from three experiments and are expressed in arbitrary fluorescence units. Values significantly different at 5% level for α₂M*, insulin and scrambled dsRNA-treated cells are indicated by an asterisk (*).</p

Induction by MN10021 and analogs of mRNA for iNOS in HUVEC.

<p>A. HUVEC were grown to 80–90% confluence in 12-well tissue culture plates using defined media provided by Cambrex and supplemented with 10% FBS. The cells were allowed to become quiescent by incubating them overnight in the basal media (without growth factors) supplied by Cambrex and supplemented with 1% FBS. Triplicate wells were incubated with either media alone or media containing 50 µM MN10021 for 3 h at 37°C, the wells washed with warm PBS, and the cells lysed. Total RNA was isolated, cDNA prepared, and RT-PCR performed as described in Materials & Methods. Primers for human iNOS and β-actin were obtained from R & D systems. B. HUVEC were prepared as described under Methods, grown to 80–90% confluence in 12-well tissue culture plates, and incubated overnight in basal medium (no growth factors) containing 1% FBS in order to make them quiescent. Triplicate wells were incubated for 3 h at 37°C with 50 µM of one of four different 8-amino acid long analogs of MN10021. DUK0001: NH₂-GLDLLFLK-COOH; DUK0004: acetyl-GLDLLFLK-acetyl; DUK0005: acetyl-GLDLLFLK-NH₂; DUK0006: NH₂-GLDLLFLK-NH₂. The wells were washed with warm PBS, and the cells lysed. Total RNA was isolated, cDNA prepared, and RT-PCR performed as described in Materials & Methods. Primers for human iNOS and β-actin were obtained from R & D systems. C. HUVEC were prepared and tested as described in panel A. The sequence of DUK0007 is acetyl-GLDLLYLK-NH₂ which differs from that of DUK0005 in that it has a Tyr at position 6 in place of a Phe.</p

Effect of retroviral peptides on lethality associated with carrageenan-induced DIC in mice.

<p>A. Groups of 10 CD-1 mice (male, 6–8 weeks of age) were injected i.p. with 0.25 ml of PBS containing either 1 mg of either MN10021 or MN20050. Eighteen hours later the mice were injected i.p. with 1 ml of 0.45% carrageenan and survival was monitored over 48 h. The results shown are representative of >5 independent studies. B. Groups of 10 CD-1 mice (male, 6–8 weeks of age) were injected i.p. with the indicated doses of MN10021. Eighteen hours later the mice were injected i.p. with 1 ml of 0.45% carrageenan and survival was monitored over 48 h.</p