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1.  Autoantibodies Against Cell-Surface GRP78 Promote Tumor Growth in a Murine Model of Melanoma 
Melanoma research  2011;21(1):35-43.
Autoantibodies that react with GRP78 expressed on the cell-surface of many tumor cell lines occur in the sera of patients with prostate cancer, melanoma, and ovarian cancer. These autoantibodies are a negative prognostic factor in prostate cancer, and when purified, stimulate tumor cell proliferation in vitro. It is unclear, however, whether these IgGs are merely a biomarker, or if they actually promote tumor growth in vivo. We immunized C57Bl/6 mice with recombinant GRP78 and then implanted the B16F1 murine melanoma cell line as flank tumors. We employed the antisera from these mice for in vitro cell signaling and proliferation assays. The immunodominant epitope in human cancer patients was well represented in the antibody repertoire of these immunized mice. We observed significantly accelerated tumor growth, as well as shortened survival in GRP78-immunized mice as compared to controls. Furthermore, antisera from these mice, as well as purified anti-GRP78 IgG from similarly immunized mice, stimulate Akt phosphorylation and proliferation in B16F1 and human DM6 melanoma cells in culture. These studies demonstrate a causal link between a humoral response to GRP78 and the progression of cancer in a murine melanoma model. They support the hypothesis that such autoantibodies are involved in the progression of human cancers and are not simply a biomarker. Because GRP78 is present on the surface of many types of cancer cells, this hypothesis has broad clinical and therapeutic implications.
PMCID: PMC3116076  PMID: 21164368
Autoantibodies to GRP78; B16 Melanoma; Cell-surface GRP78; syngeneic tumor model
2.  The cAMP-activated GTP exchange factor, Epac1 Upregulates Plasma Membrane and Nuclear Akt Kinase Activities in 8-CPT-2-O-Me-cAMP-Stimulated Macrophages: Gene silencing of the cAMP-activated GTP exchange Epac1 prevents 8-CPT-2-O-Me-cAMP activation of Akt activity in macrophages* 
Cellular signalling  2008;20(8):1459-1470.
cAMP regulates a wide range of processes through its downstream effectors including PKA, and the family of guanine nucleotide exchange factors. Depending on the cell type, cAMP inhibits or stimulates growth and proliferation in a PKA-dependent or independent manner. PKA-independent effects are mediated by PI 3-kinases-Akt signaling and EPAC1 (exchange protein directly activated by cAMP) activation. Recently, we reported PKA-independent activation of the protein kinase Akt as well co-immunoprecipitation of Epac1 with Rap1, p-AktThr-308, and p-AktSer-473 in forskolin-stimulated macrophages. To further probe the role of Epac1 in Akt protein kinase activation and cellular proliferation, we employed the cAMP analog 8-CPT-2-O-Me-cAMP, which selectively binds to Epac1 and triggers Epac1 signaling. We show the association of Epac1 with activated Akt kinases by co-immunoprecipitation and GST-pulldown assays. Silencing Epac1 gene expression by RNA interference significantly reduced levels of Epac1 mRNA, Epac protein, Rap1•GTP, p-ERK1/2, p-B-Raf, p110α catalytic subunit of PI 3-kinase, p-PDK, and p-p70s6k. Silencing Epac1 gene expression by RNA interference also suppressed 8-CPT-2-O-Me-cAMP-upregulated protein and DNA synthesis. Concomitantly, 8-CPT-2-O-Me-cAMP-mediated upregulation of AktThr-308 protein kinase activity and p-AktThr-308 levels was prevented in plasma membranes and nuclei of the cells. In contrast, silencing Epac1 gene expression reduced AktSer-473 kinase activity and p-AktSer-473 levels in plasma membranes, but showed negligible effects on nuclear activity. In conclusion, we show that cAMP-induced Akt kinase activation and cellular proliferation is mediated by Epac1 which appears to function as an accessory protein for Akt activation.
PMCID: PMC2519236  PMID: 18495429
Cyclic AMP generation in macrophages; 8-CPT-2-O-Me-cAMP and cyclic AMP-dependent regulation in macrophages; Akt protein kinase activation; Epac1 and Akt protein kinase activation
3.  Interaction between TCL1 and Epac1 in the activation of Akt kinases in plasma membranes and nuclei of 8-CPT-2-O-Me-cAMP-stimulated macrophages* 
Cellular signalling  2007;20(1):130-138.
Epac1 is a cAMP-stimulated guanine exchange factor that activates Rap1. The protein product of the T cell leukemia 1 (TCL1) proto-oncogene binds to Akt enhancing its kinase activity. TCL1 and Epac promote cellular proliferation because of their activating effects on Akt. Employing macrophages, we have studied the mechanisms whereby these proteins function in the regulation of Akt kinase activity. Cells were treated with 8-CPT-2-O-Me-cAMP, a cAMP analog which acts selectively and specifically via Epac1. Epac1 co-immunoprecipitated with TCL1 in plasma membrane and nuclear fractions of 8-CPT-2-O-Me-cAMP-stimulated macrophages. Interaction of TCL1 and Epac1 was also observed in a [125I]GST-Epac1 pulldown assay. A two-threefold increase in AktThr-308 and AktSer-473 protein kinase activities and their phosphoprotein levels was observed in TCL1 immunoprecipitates of plasma membranes and nuclei of the treated cells. Elevated AktThr-308 protein kinase activity and its phosphoprotein levels were significantly reduced in TCL1 immunoprecipitates of plasma membranes of 8-CPT-2-O-Me-cAMP treated cells where Epac1 gene expression was silenced. In contrast, AktSer-473 protein kinase activity and its phosphoprotein levels were reduced only in plasma membranes. Our studies suggest that a ternary complex of TCL1, Epac1, and Akt forms in activated macrophages both promoting Akt activation and regulating intracellular distribution of Akt.
PMCID: PMC2259443  PMID: 17993260
Cyclic AMP generation in macrophages; 8-CPT-2-O-Me-cAMP and cyclic AMP-dependent regulation in macrophages; Akt protein kinase activation; Epac-1 and TCL1 interaction; protein kinases Akt in nuclei and plasma membranes
4.  Probing the Stability of Native and Activated Forms of α2-macroglobulin 
α2-macroglobulin (α2M) is a 718 kDa homotetrameric proteinase inhibitor which undergoes a large conformational change upon activation. This conformational change can occur either by proteolytic attack on an ~40 amino acid stretch, the bait region, which results in the rupture of the four thioester bonds in α2M, or by direct nucleophilic attack on these thioesters by primary amines. Amine activation circumvents both bait region cleavage and protein incorporation, which occurs by proteolytic activation. These different activation methods allow for examination of the roles bait region cleavage and thioester rupture play in α2M stability. Differential scanning calorimetry and urea gel electrophoresis demonstrate that both bait region cleavage and covalent incorporation of protein ligands in the thioester pocket play critical roles in the stability of α2M complexes.
PMCID: PMC2344126  PMID: 17996290
α2M; α2-macroglobulin; α2-macroglobulin and proteinase incorporation; α2-macroglobulin and non-proteolytic incorporation of lysozyme; α2-macroglobulin stabilization
5.  Incorporation of Low Molecular Weight Molecules into α2- Macroglobulin by Nucleophilic Exchange* 
α2-Macroglobulin (α2M) is a proteinase inhibitor that functions by a trapping mechanism which has been exploited such that the receptor-recognized, activated form (α2M*) can be employed to target antigens to antigen-presenting cells. Another potential use of α2M* is as a drug delivery system. In this study we demonstrate that guanosine triphosphate, labeled with Texas red (GTP-TR) formed complexes with α2M* following activation by proteolytic or non-proteolytic reactions. Optimal incorporation occurred with 20μM GTP-TR, pH 8.0 for 5h at 50°C. NaCl concentration (100mM or 200mM) had little effect on incorporation at this pH or temperature, but was significant at sub-optimum temperature and pH values. Maximum incorporation was 1.2 mol GTP-TR/mol α2M*. PAGE analysis showed that 70-90% of the GTP-TR is bound in a SDS/2-mercaptoethanol resistant manner. Guanosine, adenosine, and imidazole competed with GTP-TR to form complexes with α2M*.
PMCID: PMC1905847  PMID: 17428443
α2-macroglobulin and drug delivery; α2-macroglobulin and non-proteolytic incorporation of nucleosides; α2-macroglobulin and incorporation of GTP-Texas red
6.  Changes in oligosaccharide chains of autoantibodies to GRP78 expressed during progression of malignant melanoma stimulate melanoma cell growth and survival1 
Melanoma research  2011;21(4):323-334.
A correlation between expression of the glucose-regulated protein of 78 kDa (GRP78) in malignant melanoma tumors and poor patient survival is well established. In this study, in addition to demonstrating the expression of GRP78 in tumor tissue, we investigated the immune response against GRP78 in a group of patients with different progression stages of malignant melanoma. Furthermore, we analyzed the glycosylation status of GRP78 IgG autoantibodies at these stages and evaluated their capacities to affect the Akt signaling pathway and unfolded protein response signaling mechanisms, all known to promote malignant melanoma cell proliferation and survival. We found that progression of disease correlates not only with enhanced expression of GRP78 in the tumor but also with an increase in GRP78 autoantibody serum titers in these patients. We also found that the glycosylation status of anti-GRP78 IgG changes as the disease progresses. The anti-GRP78 IgG is abnormally glycosylated in the Fc region and asymmetrically glycosylated in the Fab region. We demonstrate that hyperglycosylated anti-GRP78 IgGs stimulate cell proliferation via Akt signaling pathways. They also mimic the effects of α2M* on the up-regulation of GRP78 and XBP-1, ATF6α, and IRE1α as ER stress biomarkers and show no effect on expression or activation of caspases 3, 9 or 12. In conclusion, the anti-GRP78 IgG autoantibodies down-regulate apoptosis and activate unfolded protein response mechanisms which are essential to promote melanoma cell growth and survival.
PMCID: PMC4158745  PMID: 21597391
melanoma; glycosylation; autoimmunity; GRP78; cancer progression
7.  Model of Tumor Dormancy/Recurrence after Short-Term Chemotherapy 
PLoS ONE  2014;9(5):e98021.
Although many tumors regress in response to neoadjuvant chemotherapy, residual tumor cells are detected in most cancer patients post-treatment. These residual tumor cells are thought to remain dormant for years before resuming growth, resulting in tumor recurrence. Considering that recurrent tumors are most often responsible for patient mortality, there exists an urgent need to study signaling pathways that drive tumor dormancy/recurrence. We have developed an in vitro model of tumor dormancy/recurrence. Short-term exposure of tumor cells (breast or prostate) to chemotherapy at clinically relevant doses enriches for a dormant tumor cell population. Several days after removing chemotherapy, dormant tumor cells regain proliferative ability and establish colonies, resembling tumor recurrence. Tumor cells from “recurrent” colonies exhibit increased chemotherapy resistance, similar to the therapy resistance of recurrent tumors in cancer patients. Previous studies using long-term chemotherapy selection models identified acquired mutations that drive tumor resistance. In contrast, our short term chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that can resume growth after drug removal. Studying unique signaling pathways in dormant tumor cells enriched by short-term chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.
PMCID: PMC4028269  PMID: 24845582
8.  Resveratrol Worsens Survival in SCID Mice With Prostate Cancer Xenografts in a Cell-Line Specific Manner, Through Paradoxical Effects on Oncogenic Pathways 
The Prostate  2012;73(7):754-762.
Resveratrol increases lifespan and decreases the risk of many cancers. We hypothesized resveratrol will slow the growth of human prostate cancer xenografts.
SCID mice were fed Western diet (40% fat, 44% carbohydrate, 16% protein by kcal). One week later, human prostate cancer cells, either LAPC-4 (151 mice) or LNCaP (94 mice) were injected subcutaneously. Three weeks after injection, LAPC-4 mice were randomized to Western diet (control group), Western diet plus resveratrol 50 mg/kg/day, or Western diet plus resveratrol 100 mg/kg/day. The LNCaP mice were randomized to Western diet or Western diet plus resveratrol 50 mg/kg/day. Mice were sacrificed when tumors reached 1,000 mm3. Survival differences among groups were assessed using Cox proportional hazards. Serum insulin and IGF axis were assessed using ELISAs. Gene expression was analyzed using Affymetrix gene arrays.
Compared to control in the LAPC-4 study, resveratrol was associated with decreased survival (50 mg/kg/day—HR 1.53, P = 0.04; 100 mg/kg/day—HR 1.22, P = 0.32). In the LNCaP study, resveratrol did not change survival (HR 0.77, P = 0.22). In combined analysis of both resveratrol 50 mg/kg/day groups, IGF-1 was decreased (P = 0.05) and IGFBP-2 was increased (P = 0.01). Resveratrol induced different patterns of gene expression changes in each xenograft model, with upregulation of oncogenic pathways E2F3 and beta-catenin in LAPC-4 tumors.
Resveratrol was associated with significantly worse survival with LAPC-4 tumors, but unchanged survival with LNCaP. Based on these preliminary data that resveratrol may be harmful, caution should be advised in using resveratrol for patients until further studies can be conducted.
PMCID: PMC3628095  PMID: 23192356
resveratrol; prostate cancer; xenograft; IGF-1; E2F3; beta-catenin
9.  The Effect of Carbohydrate Restriction on Prostate Cancer Tumor Growth In a Castrate Mouse Xenograft Model 
The Prostate  2012;73(5):449-454.
PMCID: PMC3594433  PMID: 23038057
prostate cancer; diet; low-carbohydrate; castration; insulin; IGF-1
10.  Carbohydrate Restriction, Prostate Cancer Growth, and the Insulin-Like Growth Factor Axis 
The Prostate  2008;68(1):11-19.
Recent evidence suggests carbohydrate intake may influence prostate cancer biology. We tested whether a no-carbohydrate ketogenic diet (NCKD) would delay prostate cancer growth relative to Western and low-fat diets in a xenograft model.
Seventy-five male SCID mice were fed a NCKD (84% fat–0% carbohydrate–16% protein kcal), low-fat (12% fat–72% carbohydrate–16% protein kcal), or Western diet (40% fat–44% carbohydrate–16% protein kcal). Low-fat mice were fed ad libitum and the other arms fed via a modified-paired feeding protocol. After 24 days, all mice were injected with LAPC-4 cells and sacrificed when tumors approached 1,000 mm3.
Despite consuming equal calories, NCKD-fed mice lost weight (up to 15% body weight) relative to low-fat and Western diet-fed mice and required additional kcal to equalize body weight. Fifty-one days after injection, NCKD mice tumor volumes were 33% smaller than Western mice (rank-sum, P = 0.009). There were no differences in tumor volume between low-fat and NCKD mice. Dietary treatment was significantly associated with survival (log-rank, P = 0.006), with the longest survival among the NCKD mice, followed by the low-fat mice. Serum IGFBP-3 was highest and IGF-1:IGFBP-3 ratio was lowest among NCKD mice while serum insulin and IGF-1 levels were highest in Western mice. NCKD mice had significantly decreased hepatic fatty infiltration relative to the other arms.
In this xenograft model, despite consuming more calories, NCKD-fed mice had significantly reduced tumor growth and prolonged survival relative to Western mice and was associated with favorable changes in serum insulin and IGF axis hormones relative to low-fat or Western diet.
PMCID: PMC3959866  PMID: 17999389
prostatic neoplasms; diet; carbohydrate; fat; ketogenesis; insulin; IGF-1
11.  Carbohydrate restriction and lactate transporter inhibition in a mouse xenograft model of human prostate cancer 
BJU international  2012;110(7):1062-1069.
To determine if a no-carbohydrate ketogenic diet (NCKD) and lactate transporter inhibition can exert a synergistic effect on delaying prostate tumour growth in a xenograft mouse model of human prostate cancer.
120 nude athymic male mice (aged 6–8 weeks) were injected s.c. in the flank with 1.0 x 105 LAPC-4 prostate cancer cells.
Mice were randomized to one of four treatment groups: Western diet (WD, 35% fat, 16% protein, 49% carbohydrate) and vehicle (Veh) treatment; WD and mono-carboxylate transporter-1 (MCT1) inhibition via α-cyano-4-hydroxycinnamate (CHC) delivered through a mini osmotic pump; NCKD (84% fat, 16% protein, 0% carbohydrate) plus Veh ; or NCKD and MCT1 inhibition.
Mice were fed and weighed three times per week and feed was adjusted to maintain similar body weights.
Tumour size was measured twice weekly and the combined effect of treatment was tested via Kruskal – Wallis analysis of all four groups. Independent effects of treatment (NCKD vs. WD and CHC vs. Veh) on tumour volume were tested using linear regression analysis.
All mice were killed on Day 53 (conclusion of pump ejection), and serum and tumour sections were analysed for various markers. Again, combined and independent effects of treatment were tested using Kruskal – Wallis and linear regression analysis, respectively.
There were no significant differences in tumour volumes among the four groups (P=0.09).
When testing the independent effects of treatment, NCKD was significantly associated with lower tumour volumes at the end of the experiment (P=0.026), while CHC administration was not (P=0.981). However, CHC was associated with increased necrotic fraction (P<0.001).
Differences in tumour volumes were observed only in comparisons between mice fed a NCKD and mice fed a WD.
MCT1 inhibition did not have a significant effect on tumour volume, although it was associated with increased necrotic fraction.
PMCID: PMC3371292  PMID: 22394625
prostate cancer; carbohydrate; ketogenic; lactate
12.  The Effects of Varying Dietary Carbohydrate and Fat Content on Survival in a Murine LNCaP Prostate Cancer Xenograft Model 
Numerous dietary factors elevate serum levels of insulin and insulin-like growth factor I (IGF-I), both potent prostate cancer mitogens. We tested whether varying dietary carbohydrate and fat, without energy restriction relative to comparison diets, would slow tumor growth and reduce serum insulin, IGF-I, and other molecular mediators of prostate cancer in a xenograft model.
Experimental Design
Individually caged male severe combined immunodeficient mice (n = 130) were randomly assigned to one of three diets (described as percent total calories): very high-fat/no-carbohydrate ketogenic diet (NCKD: 83% fat, 0% carbohydrate, 17% protein), low-fat/high-carbohydrate diet (LFD: 12% fat, 71% carbohydrate, 17% protein), or high-fat/moderate-carbohydrate diet (MCD: 40% fat, 43% carbohydrate, 17% protein). Mice were fed to maintain similar average body weights among groups. Following a preliminary feeding period, mice were injected with 1 × 106 LNCaP cells (day 0) and sacrificed when tumors were ≥1,000 mm3.
Two days before tumor injection, median NCKD body weight was 2.4 g (10%) and 2.1 g (8%) greater than the LFD and MCD groups, respectively (P < 0.0001). Diet was significantly associated with overall survival (log-rank P = 0.004). Relative to MCD, survival was significantly prolonged for the LFD (hazard ratio, 0.49; 95% confidence interval, 0.29–0.79; P = 0.004) and NCKD groups (hazard ratio, 0.59; 95% confidence interval, 0.37–0.93; P = 0.02). Median serum insulin, IGF-I, IGF-I/IGF binding protein-1 ratio, and IGF-I/IGF binding protein-3 ratio were significantly reduced in NCKD relative to MCD mice. Phospho-AKT/total AKT ratio and pathways associated with antiapoptosis, inflammation, insulin resistance, and obesity were also significantly reduced in NCKD relative to MCD tumors.
These results support further preclinical exploration of carbohydrate restriction in prostate cancer and possibly warrant pilot or feasibility testing in humans.
PMCID: PMC3774034  PMID: 19470786
13.  Low-Carbohydrate Diets and Prostate Cancer: How Low Is “Low Enough”? 
Previous studies indicate that carbohydrate intake influences prostate cancer biology, as mice fed a no-carbohydrate ketogenic diet (NCKD) had significantly smaller xenograft tumors and longer survival than mice fed a Western diet. As it is nearly impossible for humans to consume and maintain NCKD, we determined whether diets containing 10% or 20% carbohydrate kcal showed similar tumor growth as NCKD. A total of 150 male severe combined immunodeficient mice were fed a Western diet ad libitum, injected with the human prostate cancer cell line LAPC-4, and then randomized 2 weeks later to one of three arms: NCKD, 10% carbohydrate, or 20% carbohydrate diets. Ten mice not injected were fed an ad libitum low-fat diet (12% fat kcal) serving as the reference in a modified-paired feeding protocol. Mice were sacrificed when tumors reached 1,000 mm3. Despite consuming extra calories, all mice receiving low-carbohydrate diets were significantly lighter than those receiving a low-fat diet (P < 0.04). Among the low-carbohydrate arms, NCKD-fed mice were significantly lighter than the 10% or 20% carbohydrate groups (P < 0.05). Tumors were significantly larger in the 10% carbohydrate group on days 52 and 59 (P < 0.05), but at no other point during the study. Diet did not affect survival (P = 0.34). There were no differences in serum insulin-like growth factor-I or insulin-like growth factor binding protein-3 at sacrifice among the low-carbohydrate arms (P = 0.07 and P = 0.55, respectively). Insulin was significantly lower in the 20% carbohydrate arm (P = 0.03). LAPC-4 xenograft mice fed a low-carbohydrate diet (10–20% carbohydrate kcal) had similar survival as mice consuming NCKD (0% carbohydrate kcal).
PMCID: PMC3757152  PMID: 20716631
Extracellular GRP94 (gp96) can initiate both innate and adaptive immune responses through interactions with antigen presenting cell surface receptors. Following the identification of CD91 as a receptor functioning in the cross-presentation of GRP94-associated peptides, scavenger receptors SR-A and SREC-I were demonstrated to function in GRP94 cell surface binding and endocytosis, lending controversy to the assignment of CD91 as the unique GRP94 endocytic receptor. To assess CD91 function in GRP94 surface binding and endocytosis, these parameters were examined in murine embyronic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via RNAi or eliminated by genetic disruption of the CD91 locus. Reduction or loss of CD91 expression abrogated the binding and uptake of the CD91 ligand receptor-associated protein (RAP); surface binding and uptake of an N-terminal domain of GRP94 (GRP94.NTD) was unaffected. GRP94.NTD surface binding was markedly suppressed following treatment of MEF cell lines with heparin, the sulfation inhibitor sodium chlorate, or heparinase II, demonstrating that heparin sulfate proteoglycans can function in GRP94.NTD surface binding. The role of CD91 in the cross-presentaton of GRP94-associated peptides was examined in the DC2.4 dendritic cell line. In DC2.4 cells, which express CD91, GRP94.NTD-peptide cross-presentation was insensitive to RAP or activated α2-macroglobulin and occurred primarily via a fluid phase uptake pathway. In summary, these data clarify conflicting data on CD91 function in GRP94 surface binding, endocytosis and peptide cross-presentation and identify HSPGs as novel GRP94 cell surface binding sites.
PMCID: PMC3717329  PMID: 21048103
15.  Anti-Inflammatory and Vasoprotective Activity of a Retroviral-Derived Peptide, Homologous to Human Endogenous Retroviruses: Endothelial Cell Effects 
PLoS ONE  2012;7(12):e52693.
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 in vitro release of pro-inflammatory cytokines and increases synthesis of anti-inflammatory IL-10. We sought to determine if MN10021 has significant in vivo effects. MN10021, prepared by solid-phase synthesis, was dimerized through a naturally-occurring, carboxy-terminal cysteine. In vivo anti-inflammatory activity was determined using a murine model of sodium periodate (NaIO4)-induced peritonitis. In vivo 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). In vitro 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.
PMCID: PMC3527569  PMID: 23285152
16.  Receptor-Recognized α2-Macroglobulin Binds to Cell Surface-Associated GRP78 and Activates mTORC1 and mTORC2 Signaling in Prostate Cancer Cells 
PLoS ONE  2012;7(12):e51735.
Tetrameric α2-macroglobulin (α2M), a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α2M (α2M*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α2M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells.
Employing immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies.
Stimulation of cells with α2M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, AktT308, and AktS473 in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α2M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α2M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α2M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of AktS473 phosphorylation and levels of p-AktS473 in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α2M*-induced phosphorylation of AktS473 phosphorylation in Rictor immunoprecipitates.
Binding of α2M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.
PMCID: PMC3522726  PMID: 23272152
17.  The Modified POSAS: A Novel Approach to Defining Pathologic and Non-Pathologic Scarring 
Plastic and reconstructive surgery  2011;127(1):242-247.
Scarring is a highly prevalent and multifactorial process, yet no studies to date have attempted to distinguish pathologic from non-pathologic scarring.
This article defines and proposes methods of classifying pathologic scarring as it pertains to clinical presentation.
We propose a new scar scale that incorporates pain and functional impairment.
The modified POSAS scar assessment scale is the first of its kind to factor in the functional deficits, pain and pruritus of scarring into measurements of associated morbidity. This has great potential in evaluating patient response to treatment and analyzing clinical outcomes.
PMCID: PMC3059548  PMID: 21200219
18.  Antibodies against the voltage-dependent anion channel (VDAC) and its protective ligand hexokinase I in children with autism 
Journal of neuroimmunology  2010;227(1-2):153-161.
Autistic children show elevated serum levels of autoantibodies to several proteins essential for the function of normal brains. The voltage-dependent anion channel, VDAC, and hexokinase-I, a VDAC protective ligand, were identified as targets of this autoimmunity in autistic children. These autoantibodies were purified using immunoaffinity chromatographic techniques. Both antibodies induce apoptosis of cultured human neuroblastoma cells. Because VDAC and hexokinase-I are essential for brain protection from ischemic damage, the presence of these autoantibodies suggests a possible causal role in the neurologic pathogenesis of autism.
PMCID: PMC2943043  PMID: 20576296
autism; autoimmunity; voltage-dependent anion channel; hexokinase-I; apoptosis
19.  Murine LRP is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis1 
C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically-deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared to LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep red blood cells suboptimally-opsonized with IgG or complement, compared to cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared to normal mouse serum, and addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.
PMCID: PMC2663906  PMID: 18566402
Macrophage; phagocytosis; apoptosis; cell surface molecules; complement
20.  An inhibitor of the F1 subunit of ATP synthase (IF1) modulates the activity of angiostatin on the endothelial cell surface1 
The Journal of biological chemistry  2004;280(3):1740-1745.
Angiostatin binds to endothelial cell (EC)-surface F1-F0 ATP synthase, leading to inhibition of EC3 migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin-mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is Inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F1 ATP synthase and inhibited F1-dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. While exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared to angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin-mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase, and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.
PMCID: PMC1201548  PMID: 15528193
angiostatin; angiogenesis; ATP synthesis; cell surface-associated ATP synthase; endothelial cells; Inhibitory Factor 1; F1-F0 ATP synthase
21.  LMP-420, a small-molecule inhibitor of TNF-alpha, reduces replication of HIV-1 and Mycobacterium tuberculosis in human cells 
Co-infections of human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (M. Tb) are steadily increasing and represent a major health crisis in many developing countries. Both pathogens individually stimulate tumor necrosis factor-alpha (TNF) release from infected cells and TNF, in turn, enhances the replication of each. A recent report on a Phase I clinical trial suggested that etanercept (soluble TNF receptor) might be beneficial in treating HIV/M. Tb co-infected patients. We sought to determine if a small molecule inhibitor of TNF synthesis and activity could block replication of either organism and thus be a potential adjunct to existing drugs targeting these agents.
LMP-420, a novel anti-inflammatory agent that inhibits TNF, was tested for HIV-1 inhibition both alone and in combination with AZT (3' -azido-3-deoxythymidine). LMP-420 alone was tested against M. Tb. HIV-1 infected human peripheral blood mononuclear cells (PBMC) or M. Tb-infected human alveolar macrophages (AM) were treated with a single dose of LMP-420 and viral or bacterial replication determined after 7 or 5 days respectively. Viral replication was determined from supernatant p24 levels measured by ELISA. M. Tb replication was determined by bacterial culture of macrophage lysates. LMP-420 alone inhibited HIV replication over 7 days with an IC50 of ~300 nM. Combination of LMP-420 with AZT doubled the level of HIV inhibition observed with AZT alone. LMP-420 alone inhibited the replication of virulent M. Tb by >80%, more than that observed with anti-TNF antibody alone.
Inhibition of TNF with inexpensive, small-molecule, orally-active drugs may represent a useful strategy for enhancing the activity of currently-available antiviral and anti-M. Tb agents, particularly in those areas where co-infections with these pathogens act to synergistically enhance each other.
PMCID: PMC1448187  PMID: 16573838
22.  Streptokinase Promotes Development of Dipeptidyl Peptidase IV (CD26) Autoantibodies after Fibrinolytic Therapy in Myocardial Infarction Patients 
Dipeptidyl peptidase IV (DPP IV) (CD26) plays a critical role in the modulation and expression of autoimmune and inflammatory diseases. We recently reported that sera from patients with rheumatoid arthritis and systemic lupus erythematosus contained low levels of DPP IV and high titers of anti-DPP IV autoantibodies of the immunoglobulin A (IgA) and IgG classes and found a correlation between the low circulating levels of DPP IV and the high titers of anti-DPP IV autoantibodies of the IgA class. Since streptokinase (SK) is a potent immunogen and binds to DPP IV, we speculated that patients with autoimmune diseases showed higher DPP IV autoantibody levels than healthy controls as a consequence of an abnormal immune stimulation triggered by SK released during streptococcal infections. We assessed this hypothesis in a group of patients suffering from acute myocardial infarction, without a chronic autoimmune disease, who received SK as part of therapeutic thrombolysis. Concomitant with the appearance of anti-SK antibodies, these patients developed anti-DPP IV autoantibodies. These autoantibodies bind to DPP IV in the region which is also recognized by SK, suggesting that an SK-induced immune response is responsible for the appearance of DPP IV autoantibodies. Furthermore, we determined a correlation between high titers of DPP IV autoantibodies and an augmented clearance of the enzyme from the circulation. Serum levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) increased significantly after 30 days of SK administration, while the levels of soluble IL-2 receptor remained unchanged during the same period, suggesting a correlation between the lower levels of circulating DPP IV and higher levels of TNF-α and IL-6 in serum in these patients.
PMCID: PMC130130  PMID: 12414758
23.  Catabolic Pathways for Streptokinase, Plasmin, and Streptokinase Activator Complex in Mice 
Journal of Clinical Investigation  1982;70(2):412-423.
The catabolic pathways of streptokinase, plasmin, and activator complex prepared with human plasminogen were studied in mice. 125I-streptokinase clearance occurred in the liver and was 50% complete in 15 min. Incubation with mouse plasma had no effect on the streptokinase clearance rate. Complexes of plasmin and α2-plasmin inhibitor were eliminated from the plasma by a specific and saturable pathway. Competition experiments demonstrated that this pathway is responsible for the clearance of injected plasmin. Streptokinase-plasminogen activator complex formed with either 125I-plasminogen or 125I-streptokinase cleared in the liver at a significantly faster rate than either of the uncomplexed proteins (50% clearance in <3 min). Streptokinase incubated with human plasma also demonstrated this accelerated clearance. p-Nitrophenyl-p′-guanidinobenzoate-HCl or pancreatic trypsin inhibitor-treated complex cleared slowly compared with untreated complex independent of which protein was radiolabeled. Significant competition for clearance was demonstrated between α2-macroglobulin-trypsin and activator complex only when the plasmin(ogen) was the radiolabeled moiety. Large molar excesses of α2-plasmin inhibitor-plasmin failed to retard the clearance of activator complex. Hepatic binding of streptokinase-plasmin, in liver perfusion experiments, was dependent upon prior incubation with plasma (8-10% uptake compared to a background of ∼ 2.5%). Substitution of human α2-macroglobulin for plasma also resulted in binding when the incubation was performed for 10 min at 37°C (7.5%). Electrophoresis experiments confirmed the transfer of 0.8 mol plasmin/mol α2-macroglobulin when activator complex was incubated at 37°C with α2-macroglobulin for 40 min. Streptokinase transfer from activator complex to α2-macroglobulin was negligible. The in vivo clearance of activator complex is proposed to involve active attack of the complex on the α2-macroglobulin “bait region,” resulting in facilitated plasmin transfer. Dissociated streptokinase is rapidly bound and cleared by sites in the liver.
PMCID: PMC371250  PMID: 6178757
24.  The Subunit Structure of Normal and Hemophilic Factor VIII 
Journal of Clinical Investigation  1973;52(9):2198-2210.
Human factor VIII from normals and hemophiliacs was partially purified by ethanol and polyethylene glycol precipitations. Final purification was achieved by gel filtration on 2 or 4% agarose or ion exchange chromatography on diethylaminoethyl cellulose. Comparable amounts of highly purified protein were obtained from normal and hemophilic plasma following the agarose chromatography step. Highly purified factor VIII was not dissociated by 6 M guanidine hydrochloride or 1% sodium dodecyl sulfate. However, when reduced by β-mercaptoethanol and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a single subunit species with an estimated 195,000 molecular weight was found for both normal and hemophilic factor VIII. By sedimentation equilibrium analysis, the normal factor VIII subunit was homogeneous and had an estimated molecular weight of 202,000. The subunit polypeptides from normal or hemophilic factor VIII contained carbohydrate. Each was homogeneous by isoelectric focusing. Immunodiffusion of purified normal and hemophilic factor VIII against rabbit antiserum to purified normal human factor VIII showed a single line of precipitation. Very low concentrations of purified human thrombin initially increased the activity of normal factor VIII about threefold and then progressively destroyed activity by 3 h. Only minimal activation occurred with hemophilic factor VIII. Both the activation and inactivation of normal and hemophilic factor VIII were unaccompanied by detectable changes in subunit molecular weight. These findings may have implications for the definition of the molecular defect in hemophilic factor VIII.
PMCID: PMC333021  PMID: 4199414
25.  Mechanism of ancrod anticoagulation 
Journal of Clinical Investigation  1972;51(11):2841-2850.
Fibrin formed in response to ancrod, reptilase, or thrombin was reduced by β-mercaptoethanol and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was found that ancrod progressively and totally digested the α-chains of fibrin monomers at sites different than plasmin; however, further digestion of fibrin monomers by either reptilase or thrombin was not observed. Highly purified ancrod did not activate fibrin-stabilizing factor (FSF); however, the reptilase preparation used in these experiments, like thrombin, activated FSF and thereby promoted cross-link formation. Fibrin, formed by clotting purified human fibrinogen with ancrod, reptilase, or thrombin for increasing periods of time in the presence of plasminogen, was incubated with urokinase and observed for complete lysis. Fibrin formed by ancrod was strikingly more vulnerable to plasmin digestion than was fibrin formed by reptilase or thrombin. The lysis times for fibrin formed for 2 hr by ancrod, reptilase, or thrombin were 18, 89, and 120 min, respectively. Evidence was also obtained that neither ancrod nor reptilase activated human plasminogen. These results indicate that fibrin formed by ancrod is not cross-linked and has significantly degraded α-chains: as expected, ancrod-formed fibrin is markedly susceptible to digestion by plasmin.
PMCID: PMC292433  PMID: 4263497

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